Cellular Roles of PKN1
Neil Torbett
This thesis is submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
University College University of London
August 2003
London Research Institute Cancer Research UK 44 Lincoln’s Inn Fields London W C2A 3PX ProQuest Number: U642486
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Protein Kinase Novel 1 (PKN1), which in part resembles yeast PKCs, has been shown to be under the control of Rho GTPases and 3-Phosphoinositide Dependent Kinase 1. Initial studies tested the hypothesis that Rho-PKNI inputs control PKB phosphorylation. Despite a demonstrable intervention in Rho function, no evidence was obtained that indicated a role for Rho-PKN1 in PKB control.
In seeking a cellular role for PKN1, it was found that GPP tagged PKN1 has the ability to translocate in a reversible manner to a vesicular compartment following hyperosmotic stress. PKN1 kinase activity is not necessary for this translocation and in fact the PKN inhibitor HA1077 is also shown to induce PKN1 vesicle accumulation. PKN1 translocation to these vesicles is dependent on Rad (and not Rho) activation although the GTPase binding HRIabc domain is not sufficient for this recruitment. The PKN1 kinase domain however localises constitutively to this compartment and it is demonstrated that this behavior is selective for PKNs. Associated with vesicle recruitment, PKN1 is shown to undergo activation loop phosphorylation and activation. It is established that this activation pathway involves PDK1, which is shown to be recruited to this PKN1 positive compartment upon hyperosmotic stress.
Further studies employing PKN 1-KO MEF cells place PKN1 upstream of the MKK4-JNK pathway in response to hyperosmotic stress. This PKN1 requirement is shown to be a selective hyperosmotic-induced response and to be specific for JNK, not affecting the closely related ERK1/2 and P38 MAPK pathways. Taken together these findings present a pathway for the selective, hyperosmotic-induced. Rad dependent PKN1 translocation, that leads to its endocytosis and PDK1-dependent activation. The role of PKN1 appears to be to facilitate MKK4 activation and its own catalytic activity is indicated to play critical role in this context. This provides a distinctive insight into PKN1 and its specificity of action. Acknowledgements
I would like to thank Peter for giving the opportunity to work in his lab and for being a great boss, his boundless enthusiasm is inspirational! I am particularly grateful to Adele Casamassima for being so supportive throughout my time in the lab, and Will Hughes whose guidance and patience when starting out was a big help. Thanks also to Richard, Phil and Tony who keep the lab running so smoothly and to all the CR-UK support services.
I have had a brilliant time in the lab over the past four years. I would like to thank all members of the Parker lab both past and present and in particular I would like to mention; Jyoti, Frank, Yoli, Ben, Jo, Jeff, Adrian, Fanny, Shipy, Shaby and Johanna, for all being so entertaining and making the lab a great place to be.
I thank all my friends for helping me to make the most of London, especially Karen for her support and encouragement! Finally I would like to thank Mum, Dad, my Grandparents and brother Richard without whom, this would not have been possible, they have always supported me.
IV Table of Contents
Title page Abstract il Acknowledgements iv Table of contents v List of figures x Abbreviations xiv
Chapter 1 Introduction
1.1 Signal transduction 1
1.1.1 Phosphorylarion; kinases and phosphatases 1
1.1.2 Protein-Protein interactions 2
1.2 Mitogen activated protein kinase (MAPK) cascades 4
1.2.1 Extracellular signal-regulated kinase (ERK) 7
1.2.2 c-jun NHg-terminal kinase (JNK) 7
1.2.3 p38 MAP kinase 9
1.2.4 Yeast MAP-kinases 9
1.3 Lipid signalling 11
1.3.1 Phosphoinositides 11
1.3.2 Phosphoinositide 3-kinase (PI3-kinase) 11
1.3.3 PI4 and PI5 kinases 14
1.3.4 Phosphoinositide phosphatases 14
1.3.5 Phosphoinositide binding modules 15
1.4 GTPases 17
1.4.1 Heterotrimeric G proteins 17
1.4.2 Small GTPases 19
1.4.2.1 Small GTPase mutants and inhibitory toxins 20
V 1.4.2.2 Rho 21
1.4.2.3 Cdc42 and Rac 22
1.4.2.4 Rho GTPases and membrane trafficking 24
1.5 AGO Kinases 25
1.5.1 Activation loop phosphorylation 25
1.5.2 Autophosphorylation site phosphorylation 27
1.5.2 Hydrophobic site phosphorylation 27
1.5.4 Protein kinase B (PKB) 29
1.5.5 Protein kinase C (PKC) 29
1.5.5.1 In vivo PKC studies 34
1.5.5.2 Yeast PKC 34
1.6 Protein Kinase N (PKN) 36
Chapter 2 Materials and Methods
2.1 Materials 43
2.1.1 Cell types 43
2.1.2 Buffers 43
2.1.3 Primary antibodies 45
2.1.4 Secondary antibodies 46
2.1.5 Pharmacological agents 47
2.1.6 DMA constructs 47
2.2 Methods 48
2.2.1 Molecular biology 48
2.2.1.1 PCR reactions 48
2.2.1.2 Ligation reactions 48
2.2.1.3 Transformation of E.Coli 49
2.2.1.4 Plasmid DNA preparation 49
2.2.1.5 DMA sequencing 49
2.2.1.6 Construction of GFP-PKN1, GFP-PKN2, DS-Red-PKN1 and DS-Red-PKN2 49
2.2.2 Cell culture and transfection 50
VI 2.2.2.1 Hyperosmotic stress 50
2.2.3 Microscopy 51
2.2.4 Polyacrylamide gel electrophoresis (PAGE) 51
2.2.5 Western blotting 52
2.2.6 C3-GST Protein Purification and cell loading 52
2.2.6.1 Preparation of FY810-P.Lys electro-competent cells 52
2.2.6.2 Transformation of FY810-P.Lys electro-competent cells 53
2.2.6.3 Purification of C3-GST 53
2.2.6.4 Cell loading with C3-GST 54
2.2.6.5 Ribosylation assay 54
2.2.7 R a d activation 55
2.2.8 Phosphorylation of GFP-PKN1 55
2.2.9 GFP-PKN1 kinase assay 55
2.2.10 Cell Fractionation 56
2.2.11 PKN3 Antibody generation 57
2.2.11.1 Peptide coupling 57
2.2.11.2 Affinity purification 57
Chapter 3 PKN and the PKB pathway
3.1 Introduction 59
3.2 Lack of a Rho/PKN input into PKB Serine 473 phosphorylation 61
3.3 The H R Iabc of PKN1 can inhibit PKB 8473 phosphorylation; rescue by R a d but 65
not Rho
3.4 Co-expression of RacV12 and PKN2 induces expression from the pBG plasmid 68
3.5 Discussion 70
VII Chapter 4 PKN1 is a stress responsive Rad effector
4.1 Introduction 72
4.2 PKN1 localisation is responsive to hyperosmotic stress 74
4.3 Hyperosmotic-induced PKN1 vesicles are not early endosomes or acidified 81
compartments but are associated with actin
4.4 Hyperosmotic-induced PKN1 vesicle recruitment is dependent on Rad and not Rho 87
4.5 The regulatory HR1 domain is insufficient for vesicle recruitment whereas the 92
catalytic domain of PKN1 is constitutively vesicular
4.6 Discussion 97
Chapter 5 PKN1 is activated by hyperosmotic stress
5.1 Introduction 102
5.2 PKN1 activity is not required for vesicle recruitment 103
5.3 PKN1 undergoes activation loop phosphorylation and activation in response to 105
hyperosmolarity
5.4 Density fractionation reveals the translocation of endogenous PKN1 and PKN2 in 113
response to hyperosmlarity and with kinase inhibition
Kinase reactions with immuno-purified PKN1 and cell lysate do not reveal
5.5 hyperosmotic-induced PKN 1 substrates 115
5.6 Discussion 117
VIII Chapter 6 PKN1 involvement in the hyperosmotic-induced JNK pathway
6.1 Introduction 121
6.2 GFP-PKN1 overexpresslon and the stress responses 123
6.3 PKN2 and PKNP levels are unaffected in PKN1-KO MEFs 125
6.4 PKB, p38 and ERK1/2 responses to hyperosmotic stress are not compromised in 127
PKN 1-KO- MEFs
6.5 PKN1 is required for efficient hyperosmotic-induced JNK and MKK4 activation 130
6.6 PKN1 activity contributes to hyperosmotic-induced JNK activation 136
6.7 Discussion 143
Chapter 7 Discussion
7.1 Overview 147
7.2 PKN1 Regulatory controls 149
7.3 PKN1 activation 151
7.3.1 The PKN-PKB question 152
7.4 PKN1 and the JNK cascade 154
7.5 Future Directions 157
References 160
IX List of Figures
Chapter 1
1.1 MAP-kinase phosphorelay systems 5
1.2 Receptor tyrosine kinase signalling and Protein-Protein interactions 6
1.3 Phosphoinositide metabolism 12
1.4 The GTPase cycle 18
1.5 Sequence alignment of key phosphorylation sites of the AGC kinases 26
1.6 PKC superfamily domain structure 31
1.7 Model for classical and novel PKC activation 32
1.8 PKN signalling 41
Chapter 3
3.1 Purification of C3-GST and GST protein 62
3.2 6 hours of pretreatment with C3-GST in NIH3T3 cells inhibits Rho. 63
3.3 Serum and growth factor stimulation PKB S473 phosphorylation 64
unaffected by Rho inhibition
3.4 Overexpression of PKN1 HR1 domain inhibits GST-PKB S473 phosphorylation 66
3.5 R a d can recover HR1 induced inhibition of GST-PKB S473 phosphorylation 67
3.6 Co-expression of PKN2 and RacV12 stabilises expression from the GST expression 69
plasmid.
Chapter 4
4.1 PKN1 has a punctate cytoplasmic distribution and is unchanged by agonist 75
stimulation
4.2 PKN1 translocates to large vesicles in response to hyperosmotic stress. 76
This translocation response is specific over other stress conditions
4.3 GFP-PKN1, DS-RED-PKN1, myc-PKNI and sorbitol hyperosmotic media display 78 the same hyperosmotic-induced translocation response.
4.4 Hyperomsotic-induced PKN1 translocation is selective over PKCe but common with 79
PKN2
4.5 Reversible translocation of GFP-PKN1 in response to hyperosmotic stress. 80
4.6 Hyperosmotic-induced PKN1 vesicles do not co-localise with early endosomes or 82
acidified compartments
4.7 PKN1 is associated with the with actin cytoskeleton, hyperosmotic stress induces 83
the down-regulation of stress fibres
4.8 Disruption of actin polymerisation traps PKN1 in actin patches. Hyperosmotic- 85
Induced PKN1 vesicle accumulation is independent from actin.
4.9 Hyperosmotic-induced PKN1 vesicle accumulation is independent from 86
microtubules.
4.10 Hyperosmotic-induced of PKN1 vesicle recruitment is independent from Rho 88
4.11 Rad dependent hyperosmotic-induced PKN 1 translocation 89
4.12 R a d activation in response to hyperosmotic stress 91
4.13 R a d binding is not sufficient for GFP-PKN1 translocation 93
4.14 The kinase domain of PKN 1 is associated with hyperosmotic-induced PKN 1 94
vesicles.
4.15 The kinase domain of PKN1 localises to vesicles selectively over kinase domain of 96
PKCa
Chapter 5
5.1 PKN1 activity is not required for PKN1 vesicle recruitment. 104
5.2 PKN1 undergoes activation loop Phosphorylation in response to hyperosmotic 106
stress.
5.3 The catalytic activity of GFP-PKN1 increases with hyperosmotic stress. 107
5.4 LY294002 sensitive GFP-PDK1 recruitment to DS-RED-PKN1 hyperosmotic 109
induced vesicles.
5.5 PDK1 is recruited to PKN1 kinase domain containing vesicles under control 110
conditions and upon hyperosmotic stress.
5.6 PKB does not localise to hyperosmotic-induced PKN1 vesicles 112
XI 5.7 Density fractionation of NIH3T3 cells. Endogenous PKN1 and PKN2 moves to 114
increasingly dense fractions upon hyperosmotic stress and treatments of HA1077
and, Staurosporine.
5.8 Incubation of immunopurified GFP-PKN1 with NIH3T3 cell lysate reveals several 116
PKN1 dependent heat stable substrates, no hyperosmotic-induced PKN1
substrates are detected.
Chapter 6
6.1 Transient overexpression of GFP-PKN1 has not effect on hyperomsotic induced 124
PKB degradation, JNK or p38 phosphorylation
6.2 PKN2 and PKN3 expression is unaffected in PKN1-KO MEF cells 126
6.3 Hyperosmotic-induced dephosphorylation of PKB S473 is detected in both WT and 128
PKN1-K0 MEFs
6.4 Hyperosmotic-induced p38 and ERK1/2 phosphorylation is unaffected in PKN1 KO 129
MEF cells
6.5 Inhibition of hyperosmotic-induced JNK and MKK4 phosphorylation in PKN 1-KO 131
MEF cells
6.6 MKK4, JNK,p38 and ERK1/2 activation is unaffected in PKN1 -KO MEFs following 132
UV stress
6.7 MKK4, JNK,p38 and ERK1/2 activation is unaffected in PKN1-K0 MEFs following 134
TN Fa stimulation
6.8 Cyclohexamide pretreatment does not inhibit prolonged ERK1/2 and JNK 135
hyperosmotic-induced phosphorylation.
6.9 Hyperosmotic-induced MKK4, JNK and ERK1/2 activation are partially inhibited by 137
LY294002
6.10 Hyperosmotic-induced MKK4 activation is inhibited by HA1077 138
6.11 Basal and hyperosmotic-induced phospho c -Jun levels are reduced in PKN 1-KO 140
MEF cells
6.12 GFP-PKN1 potentiates hyperosmotic-induced phospho c-Jun in WT and PKN 1-KO 141
MEF cells
6.13 GFP-PKN1 -KR does not block no prevent hyperosmotic-induced c-Jun 142
XII phosphorylation
Chapter 7
7.1 Model for the hyperosmotic-induced PKN1 response 148
XIII Abbreviations
ATP Adenosine 5’ -triphosphate BSA Bovine serum albumin DAG Diacylglycerol DNA Deoxyribonucleic acid DIT Dithiothreitol ECL Enhanced chemiluminescence EDTA Ethylene diamine tetra-acetic acid ES Embryonic stem (cells) PCS Foetal calf serum GDP Guanosine 5’-diphosphate GST Glutathione S-transferase GTP Guanosine 5’-triphosphate GTPase GTP hydrolase h hours HEPES N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulphonic acid) IPTG Isopropyl-p-D-thiogalactopyranoside KDa Kilo dalton LY294002 2-(4-morpholinyl)-8-phenyl-4H-1 Benzopyran-4-one M Molar m mili micro MAPK Mitogen activated protein kinase MEFs Mouse embryonic fibroblasts MBP Myelin Basic protein Mins Minutes
XIV n nano OD Optical density PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PCR Polymerase chain reaction PDK1 Ptdins (3,4,5)Pg-dependent kinase -1 PH Pleckstrin homology (domain) Ptdins (3,4,5)P3 Phosphatidylinositol (3,4,5) triphosphate Ptdins (4,5) Pg Phosphatidylinositol (4,5) bisphosphate PI3K Phosphatidylinositol 3-kinase PKB Protein kinase B PKC Protein kinase C PKN Protein kinase N PLC Phospholipase C PLD Phospholipase D rpm revolutions per minute PRK Protein kinase C related kinase SDS Sodium dodecyl sulphate Sec Seconds SH2/3 Src homology 2/3 domain v/v volume/volume w/v weight/volume
XV Chapter 1
Introduction
1.1 Signal transduction
Signal transduction pathways provide the means by which cells respond to their surroundings and elicit distinct functions. A key theme relating to how cell signalling mechanisms achieve such responsiveness is specificity. This is achieved through discrete protein-protein and protein-lipid interactions. The resulting modifications of these components form pathways or cascades that are responsible for highly specialised cellular functions. Defective signal transducers have been widely demonstrated to result in a multitude of diseases. The targeting of signalling components using small molecular weight inhibitors allows the intervention in signalling cascades and can therefore be of therapeutic benefit. Such drugs, for example Gleevec, are in fact already in clinical use (see recent review (Fabbro and Garcia-Echeverria, 2002)). The elucidation of signalling pathways along with their associated functions is clearly critical to understanding the full potential and consequences of such therapy in refining these approaches. The work in this thesis concerns one such signalling module. By way of introduction, a general commentary on relevant protein modifications and signalling pathways follows.
1.1.1 Phosphorylation; kinases and phosphatases
Phosphorylation is one of the most widely studied post-translational modifications and was first described as the reversible modification of phosphorylase kinase. This was shown to not only involve a kinase, but also a phosphatase, the function of which is to act antagonistically and catalyse dephosphorylation (Riley et ai, 1968).
1 In mammalian cells, protein kinases covalently attach y-phosphate groups to the hydroxyl side chain of either serine, threonine or tyrosine residues of specific protein substrates. The completion of the human genome project has enabled the elucidation of the complete human “kinome” which has recently been shown to comprise over 500 genes (Manning et al., 2002).
The dynamic nature of protein phosphorylation as a reversible modification to allow the rapid transduction of signals requires an “off” switch to counteract the “on” signals provided by protein kinases; this role is fulfilled by phosphatases. Phosphatases operate as distinct holoenzyme complexes comprised of various regulatory and catalytic subunits that confer distinct cellular localisation and substrate specificity. Protein phosphatases are reviewed (Cohen, 1989; Depaoli- Roach et ai, 1994).
Phosphorylation has widely been shown to confer activity on substrate proteins, however it is also known to render certain substrates inactive as is the case with Src (Kmiecik and Shalloway, 1987). This ability to act in a stimulatory and/or inhibitory manner on proteins is often conferred by the phosphate group in manipulating protein-protein interactions.
1.1.2 Protein-Protein interactions
Interacting domains of proteins provide recognition and target proteins to specific cellular locations. These modules thereby confer discrete modes of action on specific proteins such as the substrate specificity of kinases.
Src homology 2 (SH2) domains, first identified in the tyrosine kinase Src, along with phospho-tyrosine binding (PTB) domains, bind to regions that contain short stretches of amino acids which contain phospho-tyrosine residues. These are present within a number of signalling molecules such as PLC-y, SHP-2, the p85 subunit of PI3-kinase and the adaptor proteins Nek and Grb2 (Pawson and Nash, 2003). In terms of their responsiveness to phosphorylated tyrosine residues, SH2 and PTB domains highlight the capacity of phosphorylation as a mechanism for exerting control over signalling cascades.
Src homology 3 (SH3) domains, also identified in Src, bind to proline rich regions, examples include those found in the signalling intermediates SOS and POSH. Of particular interest to this study, PKN2 (see section 1.6) contains such a proline rich region and has been shown to bind the SH3 containing adaptor protein Nek (Quilliam etal., 1996).
Other protein-protein interaction motifs include: PDZ domains, WW domains, WD- 40 domains and 14-3-3 domains (see recent review (Pawson and Nash, 2003)). These are variously used in recognition of protein motifs including for example phospho-serine residues. These recognition modules serve to permit assembly of higher order signalling complexes that drive specificity and efficiency in relaying signals. Examples of these are described in the following section. 1.2 Mitogen activated protein kinase (MAPK) cascades
Perhaps the best characterised signalling pathways are the mitogen activated protein kinase (MARK) cascades. The first MARK to be discovered was initially described as a 42KDa protein that upon tyrosine and threonine phosphorylation becomes a serine/threonine kinase (Ray and Sturgill, 1988). This protein is now more commonly known as extracellular signal-regulated kinase (ERK), while the term MAR kinase refers to the wider superfamily.
In multi-cellular organisms three parallel MAR kinase pathways exist, all sharing common elements; ERK, c-Jun NHg-terminal kinase (JNK) and p38. Related components of these pathways operate as multi-level phospho-relay systems and comprise three sequentially activated serine/threonine kinases. MAR kinases are substrates for MARK kinases (MARKK). MARKKs are dual specificity kinases that recognise ThrXTyr motifs within the activation loop of MARKs, the “X” residue is glutamate for ERKs, proline for JNKs and glycine for the p38s. MARKKs are in turn substrates for MARK kinase kinases (MARKKK). The ERK, JNK and p38 signalling cascades are summarised in Figure 1.1.
The activation of MARKs by receptor tyrosine kinase (RTK) mediated signalling is well established, this cascade also highlights the role of protein-protein interactions in triggering these pathways. The RTK stimulated activation of the ERK1/2 cascade is illustrated in Figure 1.2. Upon ligand binding to a receptor, such as the platelet derived growth factor receptor (RDGF), dimérisation and transphosphorylation of receptors is followed by the binding of GRB2 via its SH2 domain to phosphorylated tyrosine residues on the receptor. GRB2 binds directly to the Ras nucleotide exchange factor SOS via an SH3 domain. The recruitment of SOS to the membrane results in Ras activation and the subsequent stimulation of the ERK cascade (see review (Rawson and Nash, 2000)). Stimulus
RasGTP Rad TRAF6 Activator TAB 1/2. r MAPKKK c-Raf1 MEKK1
i- j" ■
MAPKK MEKl MKK4 MKK3/6
MAPK
Substrate c-Jun
Fig 1.1 MAP-kinase phospho-reiay systems.
MAP kinase signalling components are organised according to their levels in the phospho-relay cascade; Activator, MAPKKK, MAPKK, MAPK and substrate. Members of the ERK, JNK, and p38 MAPK pathways are given and for many of the pathway constituents shown, multiple family members exist. Ligand ^
las Raf GDP
SOS MEKl GRb2
ERK1
phospho-tyrosine Transcription
SH2 domain
Si|{^SH3 domain
Fig 1.2 Receptor tyrosine kinase signaiiing and Protein-Protein interactions.
A schematic representation of receptor tyrosine kinase signalling. Receptor dimérisation and autophosphorylation is followed by the recruitment of SH2 and/or SH3 containing adaptor molecules and subsequent signalling events. As well as growth factor stimulated activation, the MAPKs are well known to be activated by a range of environmental stresses. These responses are critical for the ability of cells to sense and adjust to their surroundings. Such stresses include changes in the surrounding osmolarity, temperature and production of reactive oxygen species (by, for example, aerobic metabolism). The highly conserved MAP- kinases are known to be key elements in responding to the harmful effects of these stress conditions. By transducing signals from the cell surface these pathways lead to the stimulation of protective responses (for review see (Kyriakis and Avruch, 2001)). This aspect of MAPK signalling, in particular osmotic stress, is relevant to the work described in this thesis.
1.2.1 Extracellular signal-regulated Kinase (ERK)
The ERK family comprises five proteins (ERK1-5) although ERK5 does have marked differences in structure. ERK1 and 2 are involved in the regulation of cell division and also post mitotic functions, they are activated by a range of stimuli including; growth factors, cytokines and heterotrimeric G protein coupled receptor activation. Mutations in Ras that activate ERK1 and 2 are commonly found in tumours, this activation is thought to increase proliferation. Indeed recent evidence has highlighted BRAF (part of the ERK cascade) mutations to be critical in the development of a large proportion of melanomas (Davies etal., 2002). Inhibitors of the ERK1 and 2 pathway therefore represent potential anti-cancer agents (see review (Johnson and Lapadat, 2002)).
1.2.2 c-Jun NHg-terminal kinase (JNK)
The JNKs along with p38 MAP kinases (described below), form the stress activated protein kinase group (SAPK). JNKs comprise three genes (JNK1-3) and alternative splicing has been shown to produce 10 isoforms, all of between 46 and 55 kDa, although the significance of this splicing is not fully understood (Gupta et al., 1996). JNKs are activated by inflammatory cytokines and a variety of stress conditions and activation is achieved by phosphorylation of both Thr and Tyr residues by MKK4 and/or MKK7. While MKK7 is thought to be primarily activated by cytokines and MKK4 by stress conditions, the preferential phosphorylation of a Tyr residue for MKK4 and a Thr residue for MKK7 would suggest that these MKKs may act cooperatively (Fleming etal., 2000). MKK4 and MKK7 are activated by a variety of MAPK Kinase Kinases. To date,13 MKKKs have been described that regulate JNK activity and this diversity of JNK activators is thought to enable multiple levels of stimulus specificity. A good example of such specificity is demonstrated for the MAPK Kinase Kinase, TAK1 which has been shown to be essential for JNK activation in response to LPS. In contrast, multiple MAPK Kinase Kinases (TAK1, MLK2, ASK1 and MEKK1) were found to be required for MKK4 activation under hyperosmotic stress conditions (Chen etal., 2002).
Recent studies have highlighted the role of scaffolding proteins in the activation of the JNK pathway. JNK interacting protein (JIP) is known to scaffold MKK7 and JNK, indeed JIP1 knockout mice demonstrate an in vivo and in vitro requirement for JIP1 under specific stress conditions (Whitmarsh etal., 2001). JIP1 however does not interact with the other JNK upstream kinase, MKK4. The retention of MKK4 signalling through JNK might explain the lack of penetrance upon the JNK pathway found in JIP1 knockout mice (Yasuda etal., 1999). The protein, “plenty of SH3 domains” (POSH) has been shown to scaffold MKK4 and JNK in promoting apoptosis (Xu etal., 2003), and JNK stress-activated protein kinase-associated protein 1 (JSAP1) is also thought to have a role in JNK scaffolding (Ito etal., 1999). As discussed in section 1.4.2.3, the scaffolding protein POSH also binds the small GTPase Rac and regulation of JNK signalling by Rac is well established.
The most well described target of JNK is c-Jun which is part of the activator protein 1 (AP-1) complex and is important for transcriptional responses. JNK was shown to
8 bind c-Jun and phosphorylate the Ser-63 and Ser-73 residues, thereby promoting transcriptional activity (Derijard etal., 1994). Other JNK substrates include ATF2, ELK-1, p53 and c-Myc. Signals mediated by JNK are known to regulate a plethora of cellular responses including; cell proliferation, tumorigenesis, embryonic development and apoptosis (Dunn etal., 2002).
2.1.3 p38 MAP kinase
The p38 MAP kinase family comprises four isoforms (a,p,Y and 6). The p38 MAP kinases are activated by a variety of stress conditions such as hyperosmotic stress, heat shock and UV. They are also activated by inflammatory cytokines, hormones and heterotrimeric G protein coupled receptor activation. The importance of this family was highlighted by the identification of p38 as the target of a class of anti inflammatory drugs which block the expression of interleukin (IL-1) and tumour necrosis factor a (TNFa) upon stimulation with bacterial lipopolysaccharide (LPS) (Lee etal., 1994). The p38 MAP kinases are known to regulate the expression of many cytokines and are therefore also thought to be involved in asthma and autoimmunity (see recent reviews (Clark etal., 2003; Johnson and Lapadat, 2002 )).
2.1.4 Yeast MAP-kinases
The high degree of conservation within MAP-kinase pathways from yeast to man has made yeast a valuable model system, particularly for the study of stress activated signalling. This conservation was highlighted by the fact that human stress activated JNK and p38 MAP-kinases can partially substitute for the related S.cerevisiae high osmolarity glycerol (H0G1) pathway in mediating the yeast response to osmotic stress (Galcheva-Gargova etal., 1994; Han etal., 1994). The S.cerevisiae MAP-kinase system, while more simplified when compared with the mammalian MAP-kinase pathway, retains the basic elements of the cascade. In the case of the H0G1 pathway two MAPKKKs (Ssk2 and Ssk22) phosphorylate the MAPKK Pbs2, which in turn phosphorylates the MAPK H0G1. It has been shown however that unlike mammalian stress activated MAP-kinases, the H0G1 pathway is insensitive to stress conditions other than high osmolarity (Brewster et al., 1993; Schuller eta!., 1994).
1 0 1.3 Lipid Signaliing
The triggering of signalling cascades is often influenced by the availability and cellular location of specific phosphoinositides. This section will discuss these lipids, the kinases and phosphatases responsible for their metabolism and the protein domains which mediate specific lipid interactions.
1.3.1 Phosphoinositides
Phosphoinositides are composed of a glycerol backbone with fatty acids at positions 1 and 2, and an inositol 1-phosphate on position 3. Where the inositol ring contains no additional phosphates it is referred to as phosphatidylinositol (Ptdlns) (Figure 1.3 A). The inositol ring can however be specifically phosphorylated on positions 3,4 and 5, and the kinases responsible for these modifications are referred to as phosphoinositide PI3, PI4 and PI5 kinases respectively. The production of Ptdlns (3,4)Pg and Ptdlns (3,4,5)P3 is known to be induced rapidly upon agonist stimulation (Stephens et ai, 1993) and is associated with a variety of signalling events.
1.3.2 Phosphoinositide 3-kinase (PI3-kinase)
The importance of PI3-kinases is highlighted by their implication in cell growth, proliferation, survival, differentiation and cytoskeletal changes (see review (Vanhaesebroeck and Alessi, 2000)). There are three classes of PI3 kinases, all of which generate specific inositol lipids by phosphorylating phosphatidylinositol lipids at the D-3 position of the inositol ring (Figure 1.3 A). In conjunction with other lipid kinases, PI3-kinases can produce 3 lipid products; Ptdlns3P, Ptdlns (3,4)Pg and Ptdlns (3,4,5)P3; such lipid metabolism is illustrated in Figure 1.3 B. Class II PI3-
11 OH OH
OH
OH OH
PI3K
B Ptdlns(3,5)P2
PI4K Ptdlns(3)P ^ Ptdlns(3,4)P2 Ptdlns(3,4,5)P3
; I >k À PI3K PI3K PI3K
PI4K PI5K PLC Ptdlns Ptdlns(4)P > Ptdlns(4,5)P2 lns(4,5)P3 DAG
PI5K PI5K (type II) Ptdlns(5)P
Fig 1.3 Phosphoinositide metabolism.
A.The structure of phosphatidylinositol, relative positions on the inositol ring are numbered. B. A schematic representation of phosphoinositide metabolism, highlighting the role of PI3-kinase, PI4-kinase, PI5-kinase and Phospholipase 0 in lipid metabolism.
12 kinases are also able to produce PtdlnsSP and in addition Ptdlns (3,4)Pg from Ptdlns (4)P while class III PI3-kinases phosphorylate Ptdlns to form Ptdlns3P receptors (for review see (Vanhaesebroeck and Alessi, 2000)). The production of
Ptdlns ( 3 ,4 ,5)P3 performed by class 1 PI3-kinases, is of particular relevance to the studies described here. This section will therefore focus on Class 1 PI3-kinases.
Class 1 PI3-kinases can be further divided into class and class 1g PI3-kinases. While class are linked to receptor tyrosine kinases, class 1g are linked to heterotrimeric G-protein coupled receptors (for review see (Vanhaesebroeck and Alessi, 2000)). Class 1 PI3-kinases are heterodimers of; for class 1^, 110 kDa catalytic and 85 kDa regulatory proteins and for class 1g, pi lOy catalytic and 101 kDa regulatory proteins. For class 1^ PI3-kinase, the regulatory subunit mediates activation of the catalytic subunit. This is achieved by direct interaction with phosphotyrosine residues on growth factor receptors via the SH 2 domain of the regulatory subunit, in a similar manner to that described for the RTK stimulated MAPK activation in section 1.2. Active PI3-kinase is known to catalyse the
production of Ptdlns( 3 ,4 ,5)P3 from Ptdlns(4,5)Pg which is thought to be its preferred substrate (Stephens etal., 1998). This induces the accumulation of pleckstrin
homology (PH) domain containing proteins such as PDK 1 and PKB to these sites
of Ptdlns( 3 ,4 ,5)P3 production on membranes for which certain PH domains have a high affinity (PH domains are discussed in section 1.3.5). This leads to the modified functions of these PH domain containing proteins and often leads to the activation of subsequent downstream effectors, ultimately mediating their associated cellular responses.
The small molecule inhibitors LY294002 and wortmannin are widely used as tools to study PI3-kinase function. Both bind to the catalytic domains of PI3-kinases and
exhibit IC 50 values of IpM and 5nM respectively against class 1 PI3-kinase (Domin etal., 1997; Vlahos etal., 1994).
13 1.3.3 PI4 and PIS kinases
A PI4 kinase activity against PtdlnsSP to form Ptdlns( 3 ,4 )P2 has been described in platelets and a PI4 kinase activity against Ptdlns to form Ptdlns4P was shown to be stimulated by EGF treatment(Banfic etal., 1998). The PI4 kinases are divided into two classes; type II and type III, each of which include an ot and p member (see review (De Matteis etal., 2002)). In yeast, two PI4 kinases have described: Piki and Stt4p, which are homologous to type III PI4 kinase p and to type III PI4 kinase a respectively (Gehrmann and Heilmeyer, 1998).
To date there are three classes of PI5 kinase activity. Type I and type II PI5 kinases catalyse the formation of Ptdlns( 4 ,5)P2 . The preferred substrate of type I enzymes is thought to be Ptdlns4P (Anderson etal., 1999), however the type II enzymes were actually shown to phosphorylate PtdlnsSP at the D-4 position to produce Ptdlns( 4 ,5)P2 and may therefore require a change in nomenclature (Rameh etal., 1997). The type III PIS kinase activity was demonstrated in yeast to be Fabi and was shown to phosphorylate PtdlnsSP to form Ptdlns(S,S)P 2 (Cooke etal., 1998).
1.3.4 Phosphoinositide Phosphatases
Lipid phosphatases play a major role in controlling the PIS-kinase pathway. The lipid phosphatase, “phosphatase and tensin homolog deleted in from chromosome ten” (PTEN), dephosphorylates the 3 position of the inositol ring of Ptdlns( 3 ,4 ,S)P3 to form Ptdlns( 4 ,S)P2 , thereby acting antagonistically to PI3-kinase. The loss of heterozygosity of PTEN is widely found in cancer, the resulting uncontrolled PI3- Kinase signalling is therefore thought to contribute to cancer progression (Maehama and Dixon, 1999).
14 Several phosphatases are known to dephosphorylate the 5 position of the inositol
ring of Ptdlns( 3 ,4 ,5)P3 to produce Ptdlns( 3 ,4 )P2 . These include; SH2 containing inositol phosphates (SHIPs), GAP containing inositol 5-phosphatases (GIPs) and Sac domain containing inositol 5-phosphatases (SCIPS). Synaptojanini, a SCIP, was shown to be active against both Ptdlns( 4 ,5)P2 and Ptdlns( 3 ,4 ,5)P3 in vitro (Woscholski etal., 1997). Thus there are multiple routes of manipulation of
Ptdlns( 3 ,4 ,5)P3 available to modulate signalling events.
1.3.5 Phosphoinositide-binding modules
Of critical importance to phosphoinositide signalling is the link to signalling proteins. A number of conserved phosphoinositide binding domains have been discovered and these are known to preferentially bind different phophoinositide species. These domains are therefore able to confer localisation and activation on specialised pathways.
Many functionally diverse proteins contain pleckstrin homology (PH) domains, these are globular domains of around 100 amino acids. Nearly all PH domains require membrane association for their function (Bottomley etal., 1998). Examples of PH domain containing proteins include PDK1 and PKB and are discussed in sections 1.5.1 and 1.5.4.
The epsin amino-terminal homology domain (ENTH) is a phosphoinositide binding
domain recently shown to confer specific Ptdlns( 4 ,5)P2 binding to a number of proteins involved in endocytosis. This finding highlights the possibility that
Ptdlns( 4 ,5)P2 could be important for endocytic processes (Kay etal., 1999).
The FYVE domain is known to preferentially bind Ptdlns3P and this interaction has been shown to target FYVE domain containing proteins to endosomal compartments. Proteins which contain FYVE domains are typically involved in
15 membrane trafficking events such as: early endosomal antigen 1 (EEA1), Vac1, and Fab1 (see review (Cullen etal., 2001)).
The phox homology (PX) domain is found in a wide range of proteins from signalling molecules like phospholipase D (PLD) to vesicle trafficking proteins such as the human sorting nexin, SNX3. For SNX3, the PX domain has been shown to specifically bind Ptdlns3P and to target this protein to endosomal compartments (Xu etal., 2001). The issue of whether PX domains can function to target phosphoinositides other than Ptdlns3P remains to be addressed (see review (Cullen etal., 2001)).
1 6 1.4 GTPases
GTPases are responsible for guanine nucleotide hydrolysis and are divided into two main groups; heterotrimeric and small. While these proteins share a common enzymatic mechanism, it is well established that different GTPases operate within highly specialized signalling pathways. GTPases operate as molecular switches by cycling between “active” GTP bound forms and “inactive” GDP bound forms (Figure 1.4).
1.4.1 Heterotrimeric G proteins
Heterotrimeric G proteins comprise three subunits (a, p and y), which associate with the cytoplasmic domains of receptors with seven membrane spanning regions (serpentine receptors). These proteins transduce signals arising from stimulation of serpentine receptors and are implicated in a range of diverse processes (see review (Neves etal., 2 0 0 2 )).
There are 20 known Ga subunits, these contain a GTPase domain which is similar to that of the small GTPases. There are 6 Gp and 11 known Gy subunits, GPy subunits are tightly complexed and operate as obligate dimers. It is thought that GPy subunits interact with Ga subunits via an interface that covers the switch domain of the GTPase domain. Upon ligand binding to serpentine receptors, rearrangements in the receptor promote particular Ga domain(s) to exchange GDP for GTP (Bourne, 1997). This results in the loss of Gpy subunit binding and both functional subunits (Ga-GTP, free Gpy) activate downstream effectors (Hamm, 1998).
17 GDP GDP GTP
GEFs
Rho/Rac GTPase GTPase
GAPs
GDI
Targets
Cellular Responses
Fig 1.4 The GTPase cycle
Schematic representation of the GTPase cycle. Conversion between "active" GTP bound form and the "inactive" GDP bound form is performed by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) as indicated. Some Rho proteins bind guanine nucleotide exchange inhibitors (GDIs) which hold them in an inactive GDP bound state.
18 The Ga subunits are known to transduce signals to a variety of effectors including; adenyl cyclase, photoreceptor cGMP phosphodiesterase, phospholipase C-p and Bruton’s tyrosine kinase (Btk). The Gpy subunits are known to signal to; phospholipase C-p2, several ion channels and the kinases Raf1, Tsk and Btk (Hamm, 1998). Additionally Gpy subunits are known to bind the novel regulatory
PI3-kinase PI 01 subunit, this is thought to explain the increase in Ptdlns( 3 ,4 ,5)P3 upon stimulation of heterotrimeric G protein-linked receptors (Stephens etal., 1997).
1.4.2 Small GTPases
The Ras superfamily of small GTPases comprises over 70 mammalian members and includes; Ras, Rho, Rab, Arf and Ran families. Most of these proteins contain a C-terminal “CAAX” motif where the “X” confers a specific posttranslational modification on the cysteine residue, such as; prénylation, proteolysis or méthylation. For Rho this confers a geranylgeranylation modification since “X” is either leucine or phenyalanine whereas for Ras, farnesylation is specified.
This thesis is focussed on the PKN family of kinases which are known Rho family effectors. This section will therefore primarily discuss the Rho family of small GTPases.
The most widely studied Rho family GTPases include Rho (A, B, C isoforms), Rac (1,2,3 isoforms) and Cdc42 (Cdc42Hs and G25K), (see review (Bishop and Hall, 2000)). These proteins are widely thought to function by the disruption of auto- inhibitory intramolecular interactions of effector proteins, allowing exposure of functional domains. This is the case for kinase effectors such as the PKNs where Rho binding was shown to promote PDK1 interaction and catalytic activity and also
19 non-kinase effectors such as the scaffolding Cdc42 effector, WASP (Flynn etal., 2000; Kim etal., 2000).
Rho GTPases are regulated by guanosine nucleotide exchange factors (GEFs) which facilitate the exchange of GDP for GTP, and GTPase activating proteins (GAPs) which increase the rate of GTP hydrolysis of Rho GTPases (see Figure 1.4) (Lamarche and Hall, 1994; Van Aelst and D'Souza-Schorey, 1997). Rho GEFs contain a Dbl-homology (DM) domain which functions as the catalytic domain and also a PH domain which is thought to mediate membrane localisation. The PH domain has also been suggested to affect the activity of the Dbl homology domain (Soisson etal., 1998). Furthermore Rho and Rac are also thought to be modulated by guanine nucleotide dissociation inhibitors (GDIs) which bind Rho and Rac sequester GDP bound Rho/Rac, thereby inhibiting GDP-GTP exchange as illustrated in Figure 1.4 (Olofsson, 1999).
Rho GTPases have been associated with a variety of cellular functions although the major function is thought to be the regulation of the actin cytoskeleton (Hall, 1998). Additionally, however, Rho small GTPases have been shown to regulate several known biochemical pathways such as the serum response factor (SRF), nuclear factor kB (NF-kB) and the c-]un N-terminal kinase (JNK) and p38 mitogen- activated protein kinase pathways (Coso etal., 1995; Hill etal., 1995; Minden et al., 1995b; Perona etal., 1997). These pathways are associated with a plethora of cellular functions and highlight the importance of this class of proteins.
1.4.2.1 Small GTPase mutants and inhibitory toxins.
In dissecting the role of individual Rho-GTPases, activated and dominant negative mutants along with bacterial toxins have proved useful tools. For Rac, the amino acid substitution of Gly with Val at residue 1 2 (VI2 ) suppresses GTP hydrolysis and is therefore considered to confer constitutive activation (lhara etal., 1998).
2 0 Conversely the substitution of Asn for Thr on position 17 (N17) acts as a dominant negative by titrating endogenous GEFs (Feig, 1999).
Several bacterial toxins have also been employed for their ability to modify and inactivate Rho GTPases. The exoenzyme 03 transferase, isolated from Clostridium botuiinum, has been shown to ADP-ribosylate Rho proteins on the Asn41 residue thereby inactivating the Rho sub-family of Rho GTPases, in particular specificity has been demonstrated over Rac (Aktories and Just, 1995; Wilde etal., 2000). Toxin B, from Clostriduim difficile, has also been shown to inhibit Rho GTPases. Toxin B has a broader specificity and is thought to inhibit most Rho family GTPases (Aktories etal., 2000).
1.4.2.2 Rho
Rho is typically associated with actin stress fibers and Rho effector domains are thought to be different from those of Rac and Cdc42. The Rho effector domain. Homology region 1 (HR1), contains a leucine zipper like motif and is found in the Rho effectors PKN, Rhotekin and Rhophilin (see also section 1.6). However, the PKNs have also been shown to bind Rac, implying that such selectivity is not totally exclusive (Flynn etal., 1998). Non-HRI domain containing proteins have also been shown to bind Rho, for example ROCK and Kinectin (see review (Bishop and Hall, 2000)).
The well characterised Rho effectors ROCK and Dia are thought to be required for Rho induced actin stress fibre formation. Substrates of ROCK include MLC phosphatase which has been shown to stimulate actin-activated ATPase activity of myosin II, thereby promoting actomyosin filaments (Amano etal., 1996a; Bresnick, 1999; Kawano etal., 1999). Lim kinase (LIMK), another ROCK substrate, also promotes actin stress fibres by phosphorylating and inhibiting cofilin, this in turn stabilizes filamentous actin (Maekawa etal., 1999). The Rho effector Dia has been
21 shown to induce actin stress fibres in combination with ROCK, promoting the idea that both ROCK and Dia are necessary for stress fibre formation. Dia contains two formin homology domains (FH) which have multiple proline-rich motifs and allow interaction with the G-actin-binding protein profilin which promotes actin polymerisation (Wasserman, 1998; Watanabe etal., 1999).
1.4.2.3 Cdc42 and Rac
The conserved GTPase binding motif known as the CRIB (Cdc42/Rac interactive binding) domain is contained within several Rac and Cdc42 binding proteins including ACK, WASP and PAK (see review (Bishop and Hall, 2000)). Differences within the CRIB domain have revealed that these effectors have a different selectivity for Rac and Cdc42 binding. While ACK and WASP are specific Cdc42 targets, it remains unresolved whether the PAKs are targets for Cdc42, Rac or both. It should be noted that not all Cdc42 and Rac effectors mediate interaction via the CRIB domain, non-CRIB domain containing effectors include WAVE, POSH and CIP-4 (see review (Bishop and Hall, 2000)).
Rac is associated with lamellipodia formation and several Rac binding partners establish the role of Rac in cytoskeletal organisation. The Rac binding protein Partner of Rac (POR-1), was shown to be involved in Rac-induced Lamellipodia formation (Van Aelst etal., 1996). Another target of Rac is the lipid kinase PI-4-P5 Kinase, this GTP-independent interaction is thought to mediate actin filament uncapping, a requirement for actin assembly which was shown to be dependent on an increase in PIPg levels (Hartwig etal., 1995). WASP-like verprolin-homologous protein, known as WAVE, has also been shown to bind Rac and to localise to membrane ruffles. Mutants of WAVE have been shown to prevent Rac induced membrane ruffles (Miki etal., 1998b).
2 2 Cdc42 is most typically associated with the formation of filopodia (see review (Bishop and Hall, 2000)). Cdc42 binds N-WASP and contains several domains involved in protein-protein interactions. These include; an N-terminal PH domain, an SH2 domain, an actin monomer-binding WASP homology domain-2 (WH2) and an acidic C terminus which binds ARP2/3. N-WASP and Cdc42 overexpression have been shown to cause exaggerated Cdc42 effects such as large microspikes suggesting that they are both involved in the formation of filopodia (Miki etal., 1998a). Furthermore, since ARP2/3 binds actin monomers and is thought to act as a nucléation site for actin polymerisation, a mechanism for Cdc42 controlling the cytoskeleton is detailed (Welch, 1999).
Of particular relevance to this thesis, the overexpression of Rac and Cdc42 was shown to stimulate the activation of the stress activated JNK and p38 MAP kinase pathways (Coso etal., 1995; Minden etal., 1995a). Rac and Cdc42 have been shown to bind MLK1,2,3, MEKK1 and MEKK4, direct activators of the JNK pathway (Ranger etal., 1997; Teramoto etal., 1996), as well as the JNK scaffolding protein POSH (Xu etal., 2003). The Cdc42 and Rac effectors mediating p38 and JNK activation however remain unclear. A potential common target for Rac and Cdc42 is p21-activated kinase 1 (PAK1), since it was shown to induce both filopodia and membrane ruffles and to phosphorylate both LIMK and MLC kinase in vitro, two enzymes involved in these subcellular processes (Edwards etal., 1999; Sanders et al., 1999). The role of PAK1 in relation to p38 and JNK activation has therefore been investigated. PAK1 has been shown to enhance p38 activation in conjunction with both Cdc42 and Rac (Zhang etal., 1995). It was also reported that PAK1 could activate JNK (Brown etal., 1996). However it has also been suggested that Rac activation of p38 and JNK is independent from PAK1 (Westwick etal., 1997).
23 1.4.2.4 Rho GTPases and membrane trafficking
Small GTPases are known to be Involved in different stages of membrane trafficking (see review (Zerial and McBride, 2001)). While the Rab subfamily of small GTPases are perhaps most widely associated with membrane trafficking, the Rho subfamily have also been shown to be important for various membrane trafficking events. Rho associated trafficking events include; pinocytosis, phagocytosis, clathrin mediated endocytosis and exocytic pathways. These effects on vesicular traffic are thought to occur in part as a result of small GTPase effects on the actin cytoskeleton and the microtubule network. Cdc42 is thought to drive Arp2/3 mediated actin polymerisation at the vesicle surface and to act on different stages of endocytic pathways (Kroschewski etal., 1999). Rac is known to stimulate pinocytosis and phagocytosis and Rho is thought to act on vesicular traffic post endocytosis (Dharmawardhane etal., 2000; Ellis and Mellor, 2000; Leverrier and Ridley, 2001). There is also evidence that small GTPases influence microtubule mediated trafficking events since Rho was shown to bind kinectin and Rac was shown to bind tubulin (Best etal., 1996; Cook etal., 1998).
Important in small GTPase control of traffic is the integration with signalling molecules, for example activated Rac/Cdc42 in co-operation with p21-activated kinase (PAK) has been shown to stimulate pinocytosis (Dharmawardhane etal., 2000). Of particular relevance to the work of this thesis, PKN1 has also been shown to co-operate with RhoB to control EGF receptor traffic (Mellor etal., 1998). The role of controlling vesicle traffic places small GTPases central in transducing signals from the plasma membrane and resulting in transcriptional responses (see review (Ridley, 2001)).
24 1.5 AGC kinases
The “AGC” kinases comprise an extended superfamily of proteins including; PKA, PKG, PKC, PKB, p70^k and p90^^ (for definitions and review see http://pkr.sdsc.edu/html/index.shtml). These enzymes have a high degree of homology with respect to their kinase domain and are all modulated by phosphorylation. The need for activation loop, or T loop, phosphorylation to achieve optimal activity is a common feature of this group. Furthermore, there are two additional classes of phosphorylation that are associated with activation loop phosphorylation; autophosphorylation and hydrophobic site phosphorylation. The relative conservation of these sites among AGC kinases is illustrated in Figure 1.5.
1.5.1 Activation loop phosphorylation
The activation loop motif T(F/L)CGT is shared among AGC kinases and was first identified in PKCa where it was shown to be essential for catalytic activity (Cazaubon etal., 1994). Bacterial expression studies have shown that PKA autophosphorylation is sufficient for PKA activation loop phosphorylation, however for other AGC kinases such as PKBs, PKCs and PKNs this is not the case. Here the PI3-kinase pathway has been shown to trigger activation loop phosphorylation through 3-phosphoinositide dependent kinase 1 (PDK1) (Alessi etal., 1997b; Flynn etal., 2000; Le Good etal., 1998; Stephens etal., 1998).
PDK1 is a 63KDa serine/threonine kinase which was isolated as an activity responsible for PKBa activation loop phosphorylation. This activation was shown to be dependent on inclusion of Ptdlns( 3 ,4 )P2 or Ptdlns ( 3 ,4 ,5)P3 in the in vitro reaction (Alessi etal., 1997b) (Stephens etal., 1998). PDK1 has more recently
25 Activation ioop site 1 PKCa HIKIADFGMCKEHMMDGVTTR^FCGTPDYIAPE PKC6 HIKIADFGMCKENIFGENRAST^CGTPDYIAPE. PKCe HIKLTDYGMCKEGLGPGDTTSTFCGTPNYIAPE. PKNl YVKIADFGLCKEGMGYGDRTS TFCGTPEFLAPE PKBa HIKITDFGLCHEGIKDGATMKTFCGTPEYLAPE P7 0"'" HVKLTDFGLCKESIHDGTVTHTFCGTIEYMAPE
* * * * * * * * * * * * * *
Autophosphoryiation site Hydrophobic site i i PKCa FTRGQPVLTPP-DQLVIANID---QSDFEGFSYVNPQFV PKCÔ FLNEKPQLSFS-DKNLIDSMD---QTAFKGFSFVNPKYE PKCe FTSEPVQLTPD-DEDVIKRID---QSQFEGFEYTNPLLS PKNl FTGEAPTLSPPRDARPLTAAE---QAAFLDFDFVAGGC- PKBa FTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGTA P 7 ftj^q t p v d SPD-DSTLSESAN QVFLGFTYVAPSVL
Fig 1.5 Sequence aiignment of key phosphoryiation sites of the AGC kinases.
Activation loop, autophosphoryiation and hydrophobic sites among several AGC kinases are indicated. The stars highlight conservation of residues. The two stretches of amino acids are separated by approximately 125 residues.
26 been demonstrated to phosphorylate equivalent residues on many other AGC kinases including; p70®®\ PKA, and PKCs. The PH domain of PDK1 has a higher in v/Yro affinity for Ptdlns ( 3 ,4 ,5)P3 and Ptdlns( 3 ,4 )P2 than Ptdlns( 4 ,5)P2 , implying that PDK1 can be responsive to PI3-kinase activity (see review (Vanhaesebroeck and Alessi, 2000)).
The activation loop site of several AGC kinases has been shown to undergo phosphorylation upon stimulation by regulatory effectors. For example DAG stimulates T-loop phosphorylation of classical and novel PKCs (Le Good etal., 1998). The activation loop phosphorylation of PKN 1/2 is also subject to regulation by small GTPases (Flynn etal., 2000). These regulatory controls are thought to induce a conformational change in PKN which is consistent with the need for such a change to allow PDK1 docking and phosphorylation (Biondi etal., 2000).
1.5.2 Autophosphoryiation site phosphorylation
Initially described as an autophosphoryiation site on PKC pi (Thr 642) and on PKC a (Thr 638), this conserved AGC kinase motif is also known as the TP site or turn motif (Cazaubon and Parker, 1993). The occupation of this site has variable effects on function. For PKCp, mutation of this site led to the phosphorylation of proximal sites resulting in a fully active kinase (Newton, 1997). However, for PKCa this site is specifically thought to play a role in affecting the conformation of the kinase, thereby affecting the rate of dephosphorylation and inactivation (Bornancin and Parker, 1996).
1.5.3 Hydrophobic site phosphorylation
The highly conserved FSY motif is set within a hydrophobic sequence of the Vg region of the AGC kinase domain (see figure 1. 6) and has been shown to contribute to the activation and stabilisation of the kinase. Controversy surrounds
27 the regulation of this phosphorylation site with several reports suggesting that this is an autophosphoryiation site (Keranen etal., 1995; Toker and Newton, 2000a), whilst other studies on PKB suggested this is in fact a target for transphosphorylation (Alessi etal., 1997b). The mechanism of phosphorylation is unclear, although interestingly, this region can act as a binding site for PDK1 (Biondi etal., 2000).
The putative kinase that mediates hydrophobic site phosphorylation is referred to as 3-phosphoinositide dependent kinase 2 (PDK2).The hydrophobic kinase for AGC kinase family members remains elusive although several proteins have been suggested to play a role in phosphorylating this site. It has been suggested that integrin-linked kinase-1 (ILK1) may have a role in phosphorylation of the hydrophobic serine residue at amino acid 473 of PKB (S473). PI3-kinase activated ILK1 was proposed to enhance PKB S473 phosphorylation in NIH3T3 cells and also in vitro, although this is thought to be an indirect mechanism since a kinase inactive form of ILK1 also enhanced PKB S473 phosphorylation (Lynch etal., 1999).
An alternate hypothesis for the origin of PDK2 activity involves protein kinase N 2 (PKN2), which was identified as a potential PDK1 interacting partner by a yeast two- hybrid screen. A C-terminal PKN2 fragment termed PDK1 interacting fragment (PIP), was isolated and shown to convert PDK1 to a form that will phosphorylate PKB on S473 in vitro, raising the possibility that PDK2 is in fact a modified PDK1 (Balendran etal., 1999). However more recent work involving full length PKNs has cast doubt on this hypothesis and would seem to indicate that they play negative rather than positive roles on PKB phosphorylation (section 1.6). Candidate PDK2 activities are reviewed (Chan and Tsichlis, 2001).
2 8 1.5.4 Protein Kinase B (PKB)
PKB was originally identified by its homology to PKC and PKA and was also known as RAC1 (related to A and 0 kinases) (Bellacosa et al., 1991 ; Coffer and Woodgett, 1991; Jones etal., 1991). PKB is a major target of PI3-kinase lipid
products and is recruited to cell membranes by Ptdlns( 3 ,4 ,5)P3 via its Pleckstrin Homology (PH) domain where it is subsequently phosphorylated by PDK1. PKB has been shown to require phosphorylation at its activation loop site, (Thr 308) which is mediated by PDK1, and also at the hydrophobic site (Ser473) site for full activation, for which the upstream kinase or mechanism of phosphorylation remains elusive (see sections 1.5.1 and 1.5.3). Targets of PKB include Glycogen synthase kinase 3 (GSK3), the phosphorylation of which results in its inactivation and hence subsequent stimulation of glycogen synthesis (Cross etal., 1995).
PKB is of great interest since it is overexpressed in a range of cancer cell types and is known to have anti-apoptotic properties. Indeed one mechanism for this has been proposed since PKB was shown to phosphorylate the proapoptotic protein BAD. This phosphorylation event is thought to promote binding of BAD with the 14- 3-3 protein, rendering BAD non-functional (Downward, 1998; Franke etal., 1997; Sabbatini and McCormick, 1999). Other targets of PKB are also involved in promoting cell survival including: human caspase-9, forkhead transcription factors and IkB kinases (see review (Vanhaesebroeck and Alessi, 2000)).
1.5.5 Protein kinase 0 (PKC)
PKC was one of the first protein kinase families to be identified, originally reported as a histone kinase activity that is activated by proteolysis (Inoue etal., 1977). PKC was shown to be activated by phosphatidylserine (PS) and diacylglycerol (DAG) in a Ca^^ dependent manner. The tumour promoting phorbol esters (such as PMA) were also shown to activate PKC (Castagna etal., 1982; Nishizuka, 1984). To date
29 the PKC superfamily comprises twelve distinct genes and these have varying capacities for Ca^^ and DAG responsiveness (see figure 1 .6). However, it is likely that the number of gene products is higher than this as PKCp was shown to be alternatively spliced to give two proteins which differ at the C-terminus (Coussens etal., 1987).
PKC isoforms have been classified according to their enzymatic properties. The classical PKCs (cPKC) include a,p and y isoforms and are activated by phosphatidylserine in a calcium dependent manner, they are also activated by
DAG. The novel PKCs (nPKC) comprise ô, 0 ,e and r\ isoforms and while independent from calcium they are activated by DAG. The atypical PKCs (aPKCs) comprise i and ^ isoforms and are both calcium and DAG independent. The most recent PKC superfamily members to be identified are the Protein kinase Ns (PKN) (also known as protein kinase C- related kinase PRK). Human PKNs comprise at least three members (1,2, and 3) of which only 1 and 2 are fully cloned and characterised. These are activated independently from calcium and DAG although they share significant homology with the catalytic domain of other PKCs. They have been shown to be regulated by the Rho family of small GTPases and are discussed in more detail in section 1. 6 . The comparative domains structures of
PKC superfamily members are summarised (Figure 1. 6).
A generalised model of PKC activation has emerged in recent years and is illustrated in Figure 1.7. Classical and novel PKCs are recruited to the plasma membrane in response to increased levels of DAG through lipid hydrolysis by either phopholipase C (PLC) or indirectly via phospholipase D (PLD); this is mediated via the Cl domain. Classical PKCs additionally require Ca^^ binding which occurs via inositol 1,4,5-trisphosphate (IP3 ) induced calcium release and is mediated by the C 2 domain (Berridge, 1993). Cl and C 2 domains are discussed below. This co-factor binding to PKCs is thought to precede a conformational change resulting in the displacement of the autoinhibitory pseudosubstrate domain
30 cPKC (a,p,y)
$2-lik catalytic nPKC (ô,0 ,e,ri)
f cl catalytic j aPKC (i,Ç)
:2-lik#-^r catalytic PKN1
PKN2/3
0{b)^2-liÈ Pkc1 (cerevisiae)
Fig 1.6 PKC superfamily domain structure.
A schematic representation of PKN superfamily domain structure. Cl domains are indicated in green, 0 2 and 0 2 -like domains in grey, catalytic domains in red and the V5 regions in purple. THe HR1 regions are indicated in blue and the PKN2 proline rich region is in pink.
31 Agonists
RTK V5-kinase DAG PIP3 PLC Actr PDKl
Autophosphoryiation
Targets and cellular responses
T Cytosolic Ca^^
Fig 1.7 Model for classical and novel PKC activation.
PKCs are activated by agonist dependent production of second messengers DAG and Ca^' (classical) which bind 01 and 02 domains (classical) respectively in the context of membrane lipids. This relieves the autoinhibitory pseudosubstrate domain from the active site. This allows targeted phosphorylation at the activation loop site by PDKl, the hydrophobic site phosphorylation by the "V5 kinase" and subsequent autophosphoryiation resulting in a fully active PKO.
32 from the active site. These events facilitate PDK1 docking and multi-site phosphorylation and result in a fully functional kinase (see figure 1.7 and (Le Good etal., 1998; Parekh etal., 2000)).
Conventional and novel PKCs contain a Cl domain which is composed of two repeated zing-finger motifs (Hurley etal., 1997). This domain is thought to fold into a distinct structure unlike other zinc finger domains (Hommel etal., 1994). It has been shown that diacylglycerol (DAG) binds to the Cl domain and that the phorbol ester pH]phorbol-12,13-dibutyrate (PDBu) competes with DAG for Cl binding, hence DAG and PDBu are thought to have the same Cl domain point of contact (Kaibuchi etal., 1989; Ono etal., 1989) (Sharkey and Blumberg, 1985). The atypical PKCs, while unresponsive to phorbol esters, do however contain a single Cl zinc-finger motif.
Conventional PKCs contain a C 2 domain which is located C-terminal to the Cl domain. These are thought to bind phospholipid in a calcium dependent manner, indeed this has been shown for PKCp (Shao et al., 1996). While the calcium binding C2 domain is missing from the novel and atypical PKCs, these calcium independent PKCs contain a closely related region that is termed C2-like or ‘Vq’ in novel PKCs, a PB 1 domain in atypical PKCs and an ‘HR 2 ’ domain in PKNs. Although these regions lack aspartate residues that are required for calcium binding, it has been suggested that they could mediate phospholipid binding or protein-protein interactions (Mellor and Parker, 1998). The crystal structure of the
C2 -like domain of PKCÔ has been solved and was shown to be a C2-fold (Pappa et al., 1998).
PKCs have a wide range of substrates in vitro, however precise in vivo targets remain largely unresolved, particularly in relation to physiological outputs. Well characterised PKC substrates include MARCKS protein (myristoylated alanine-rich C-kinase substrate) and various STICKS (Substrates that interact with C kinases)
33 such as the adducins, syndecan-4 and PAR-3. As a consequence of the highly conserved catalytic domain among PKCs, isoform specific substrates are not distinguished in vitro. It is likely therefore that isoform specific binding partners serve to target individual classes of PKC to their precise in vivo substrates (Jaken and Parker, 2000).
1.5.5.1 In vivo PKC studies
Knockout PKC mice have been generated for most PKC isoforms and phenotypes have been found. For example, PKCe knockout mice present an attenuated response to interferon y stimulation and macrophage activation (Castrillo etal., 2001). PKCp knockout mice were shown to have a marked immunodeficiency resulting from impairment of B cell receptor signalling (Leitges etal., 1996). PKCÔ and PKC Ç knockout mice were also shown to have B cell defects (Martin etal., 2002; Mecklenbrauker etal., 2002). PKC0 knockout mice however were shown to be defective for T cell receptor signalling (Sun etal., 2000b). Mice deficient in PKCy were shown to be resistant to neuropathic pain syndrome after partial sciatic nerve section, implying a role for PKCy in the central nervous system (Malmberg et al., 1997). The possibility remains that PKC isoform redundancy can partially compensate for the normal PKC responses. Future studies involving multiple isoform knockout mice will answer more comprehensively the full extent of PKC involvement on cellular control.
1.5.5.2 Yeast PKC
The yeast S.cerevisiae contains one PKC homologue, Pkcl while S.pombe contains two PKCs; and Pcki and Pck2. These homologues show close structural resemblance to mammalian PKC and related PKNs as reviewed (Mellor and Parker, 1998). As indicated in figure 1.6, Pkcl contains a putative DAG sensitive
34 C1 domain and a C2-related domain. Also present are two HR1 repeats similar to those found in mammalian PKNs, these are thought to mediate binding of the yeast Rho homologue Rho1 (Nonaka etal., 1995).
The yeast Pkcl is known to activate the MAP kinase cascade involving BCK (MAPKKK), MKK1 and MKK2 (MAPK), and MpKI. This pathway is also controlled by Rhol, and has been linked to the control cell wall integrity (Arellano etal., 1999; I he etal., 1993; Levin etal., 1994). Interestingly, this control mechanism has been likened to the osmotic stress response (Alonso-Monge etal., 2001).
35 1.6 Protein Kinase N (PKN).
PKNs (Protein Kinase Novel, also known as PRKs (Mukai and Ono, 1994) (Palmer etal., 1994)) are a subfamily of serine/threonine kinases identified independently by molecular cloning, protein purification and polymerase chain reaction based screens for PKC related kinases. PKNs currently comprise three isoforms, PKN1, PKN2 and PKN3 (formerly known as PKN or PRK1, PRK2 and PKNp respectively). Two human isoforms are fully cloned and characterised; PKN1 and PKN2 which migrate on SDS-PAGE at 120 and 140 kDa respectively. PKN1 and PKN2 were found to be expressed ubiquitously (Mukai and Ono, 1994; Quilliam etal., 1996). However human PKN2 was also shown to have a distinct pattern of expression (Palmer etal., 1995b). PKN3, while undetected in adult tissue has been found abundantly in cancer cell lines (Oishi etal., 1999).
The C-terminal kinase domains of these proteins are closely related to those of PKC. At their amino termini they have a conserved repeated domain known as homology region 1 (HR1a,b,c) followed by a 02-related domain; overall these proteins have a domain organisation related to that of the yeast PKC-related proteins excepting the lack of a 01 domain (see (Mellor and Parker, 1998) and figure 1. 6 ). The HR1 domain was identified as a Rho interacting region and incorporates a leucine zipper-like motif (Amano etal., 1996b; Vincent and Settleman, 1997; Watanabe etal., 1996). Other examples of this domain have been identified such as the Rho binding regions of Rhotekin and Rhofilin (Watanabe etal., 1996). The C2-like domain does not confer calcium sensitivity to PKNs since it does not contain the critical aspartate residues needed, although it is thought to act as an autoinhibitory region (Mukai etal., 1995).
PKNs are activated by fatty acids and phospholipids in vitro’, arachidonic acid.
36 linoleic acid, cardiolipin, Ptdlns ( 4 ,5)P2 , Ptdlns ( 3 ,4 ,5)P3 and LPA have all been shown to activate PKNs in vitro although the in vivo significance of this remains unclear (Morrlce etal., 1994; Mukai and Ono, 1994; Palmer etal., 1995a; Peng et a!., 1996). PKN activation has also been shown to be under the Influence of small
GTPases. The Interaction of Rho with PKN 1 has been demonstrated to facilitate
PKN1 activation loop phosphorylation by 3-Phospholnosltlde Dependent Kinase 1 (PDK1). The same study demonstrated an in v/vo ternary complex of Rho-PKNI- PDK1 which was shown to be dependent on PI3-klnase activity and to be critical for the catalytic activation of PKN1 (Flynn etal., 2000).
The role of PDK1 In relation to PKN 1/2 was studied using PDK1 null embryonic stem (ES) cells (Balendran etal., 2000). These cells were found to have a much reduced level of PKN1 expression when compared with WT ES cells. The expression level of PKN2 was unaffected In PDK 1 null cells and some residual
PKN2 activation loop phosphorylation was detected when compared with WT ES cells. These findings Imply that for PKN1, PDK1 mediated activation loop phosphorylation may be critical for the stability of the protein. However, some residual PKN2 activation loop phosphorylation was found In PDK1 null ES cells.
The role of PKNs In relation to the downstream PDK1 effector PKB has also been the subject of debate. A C-termlnal PKN2 fragment was shown to convert PDK1 to a form that will phosphorylate PKB in vitro on the serine residue at amino acid 473 (S473) functioning as the elusive PDK2 activity (Balendran etal., 1999). This work raises the possibility that PKN signals can Influence PKB S473 phosphorylation thereby acting as a “PDK2” complex. However, a recent study has suggested that full length PKN1 and PKN2 may negatively regulate PKB phosphorylation (Wick et al., 2000). PKN2 cleavage during apoptosis has also been shown to Inhibit PKB phosphorylation (Koh etal., 2000). The role of PKNs In regulating other AGC kinases has also been Investigated since a C-termlnal PKN2 fragment was found to Inhibit PDK1 mediated PKCÇ phosphorylation (Hodgklnson and Sale, 2002).
37 Much like PKCs, in-vitro PKN substrates have been described, although the in-vivo significance of these remains unclear. PKN substrates include, MARCKS protein, vimentin, Tau, and CPI-17 in vitro (Hamaguchi etal., 2000; Kawamata etal., 1998; Matsuzawa etal., 1997; Palmer etal., 1996). Pharmacological inhibition of the PKNs is achievable since the ROCK inhibitors HA1077 and Y27636 have been described to inhibit PKN2 with IC 50 values of 4 |liM and 600nM respectively (Davies et al., 2 0 0 0 ), however these have not been exploited to correlate with potential PKN functions.
Perhaps the most striking structural difference between PKN1 and other PKNs is the proline rich region between the C2 and the catalytic domains of PKN2 and PKN3. PKN2 and PKN3 contain 1 and 2 proline rich sequences respectively which are thought to target SH3 domains. Indeed, PKN 2 has been shown to bind the SH3 domain containing molecules Nek and GRB4 (Braverman and Quilliam, 1999; Quilliam etal., 1996), whilst PKN3 was shown to bind the SH3 domain containing GRAF (Shibata etal., 2001). Since these adaptor proteins can bind directly to receptor tyrosine kinases, these interactions could conceivably couple PKN2 and PKN3 to receptor mediated events thereby conferring selective localisation and stimulus specificity.
PKNs have been implicated in several MAP-kinase signalling events; however a definitive role for PKNs in these pathways has not emerged. PKN2 was shown to bind MEK Kinase 2 (MEKK2) and this interaction was shown to be mediated through the proline rich region of PKN2, not present in PKN1. This interaction was shown to promote PKN2 activation although this effect was independent of MEKK2 activity (Sun etal., 2000a). Recent evidence has also placed PKN1 downstream of Rho, at the MAPK Kinase Kinase level of the p38y cascade leading to c-Jun activation (Marinissen etal., 2001). Further implicating PKN1 in the p38 pathway, PKN1 was shown to phosphorylate the novel p38 MAPK Kinase Kinase, MLTK
38 (Takahashi etal., 2003).
The loss of PKN function in Drosophila highlighted a defect in dorsal closure. This phenotype resembles the loss of function of the GTPase Rho1 and the Rac mediated JNK cascade. The study however, indicated that PKN1 was not operating within the JNK pathway but a parallel system that is also important for dorsal closure (Lu and Settleman, 1999).
The small GTPase, RhoB has been shown to target PKN 1 to an endosomal compartment where it is implicated in controlling the kinetics of epidermal growth factor (EGF) receptor traffic (Mellor etal., 1998) (Gampel etal., 1999). PKN1 was also found by electron microscopy, to be enriched in a variety of membrane compartments including ER-derived vesicles, late endosomes and multi-vesicular bodies (Kawamata etal., 1998), further implicating PKN1 in vesicle transport. Furthermore, PKN1 was shown also to bind and activate Phospholipase D (PLD) in
the presence of Ptdlns( 4 ,5)P2 in a manner independent of PKN 1 activity (Oishi et al., 2001). PLD has been demonstrated to be activated by EGF stimulation in endosomes (Hughes etal., 2002). These findings raise the possibility that PKN- PLD interactions may be part of vesicular trafficking events. Furthermore, PKN1 may act upstream of PLD in stimulating a variety of events directly and/or indirectly, such as classical and novel PKC activation of PLD mediated via lipid hydrolysis and the production DAG.
Several reports have indicated that PKNs play a role within cytoskeletal structures. This notion is consistent with PKNs being small GTPase effectors which are also well established as cytoskeletal regulating proteins (see section 1.4). PKN1 was shown to stimulate actin reorganisation (Dong etal., 2000) and the kinase inactive form of PKN2 has been shown to induce the disruption of the actin cytoskeleton (Vincent and Settleman, 1997). However, care does need to be taken when interpreting data concerning the overexpression of PKNs since these proteins may
39 well titrate Rho proteins and highlight Rho functions which are in fact mediated by Rho effectors other than PKNs. Further establishing the link between PKNs and the actin cytoskeleton, PKN1 was however shown to bind the actin crosslinking protein a-actinin (Mukai etal., 1997). Another potentially interesting PKN interaction concerns PKN2 which was shown to bind the phosphatase PTP-BL which has been implicated in cytoskeletal rearrangement. PKN2 and PTP-BL are found to co-localise in lamellipodia-like structures (Gross etal., 2001).
In highlighting a functional role for PKNs, PKN2 has been placed on a pathway controlling cell adhesion. A Rho mutant that fails to bind PKN2 was unable to recruit E cadherin and promote keratinocyte differentiation. Furthermore PKN2 was shown to enhance keratinocyte cell-cell adhesion and induce phosphorylation of the tyrosine kinase Fyn and also p and y catenin. This work implicates PKN2 as a direct mediator between Rho and Fyn signalling (Calautti etal., 2 0 0 2 ).
PKNs have also been implicated in signalling to the nucleus; PKN activation, known substrates and transcriptional responses are summarised in Figure 1. 8 . PKN1 was described to translocate to the nucleus in response to heat shock (Mukai etal., 1996). Furthermore, PKN1 was shown to drive c-fos expression from the serum responsive element (SRE) and also reporter expression from the ANF promoter which contains an SRE like sequence. This would be consistent with PKNs driving transcriptional responses downstream of Rho activation (Morissette etal., 2000). Additionally PKN1 was shown to drive stimulation of ATF2 and
MEF2A which acts on the c-jun promoter by signalling through ERK 6 and P38y (Marinissen etal., 2001). PKN1 has also been shown to bind the neuron specific Helix-Loop-Helix (bHLH) transcription factor, and to enhance the transactivation of
NDRF/NeuroD2 (Shibata etal., 1999). Recently PKN 1 was shown to induce transcriptional activation of the androgen receptor (Metzger etal., 2003). Taken together, these studies suggest that PKNs may have multiple roles in gene transcription events.
40 Rho GTPase
Active PKN T' Loop P Catalytic
HR1 PDK1
Substrates e.g. Marcks Vimentin Tau MKK3 CPI-17
p38y
c-Jun c-Fos NDRF/NeuroD2
Fig 1.8 PKN Signalling.
A summary of PKN signalling including a model for PKN activation, potential downstream substrates and transcriptional responses.
41 Current literature presents a model for the activation of PKNs. Other studies have identified several PKN binding partners and associated transcriptional responses, however, a definitive cellular role for the PKNs remains elusive. This work seeks to increase our understanding of PKN function by monitoring PKB as a candidate for Rho-PKN signals, and also by screening for potential PKN agonists.
42 Chapter 2
Materials and Methods,
2.1 Materials
All reagents were obtained from Sigma-Aldrich unless otherwise indicated. Cy3 anti mouse antibody were from Jackson Immunoresearch laboratories. AcrylaGel and Bis-AcrylaGel was from National Diagnostics. Ethanol, Methanol, TritonX-100 and Tween-20 were from BDH. Glutathione-Separose, ultra pure dNTPs, radioisotopes, protein standard markers, hyperfilm and EGL western blotting detection reagent were from Amersham-Pharmacia. PVDF membrane was from Millipore. Lipofectamine 2000 reagent. Optimum media, and agarose were from Gibco-BRL. Protein A, Protein G and restriction enzymes were from New England Biolabs. Phosphatidyl serine lipids were from Lipid Products. The Rad activation assay was purchased as a kit from Totam Biologicals. Actigel resin was from Sterogene.
2.1.1 Cell Types
NIH3T3 cells were from Cancer Research UK cell services, WT and PKN 1-KO primary mouse embryonic fibroblast (MEF) cells were generated by Dr Adele Cassamassima.
2.1.2 Buffers
Luria-Bertani (LB), PBS, Trypsin-versine, EDTA, Dulbecco's MEM (DMEM) were from CR-UK research services.
43 SDS Running Buffer 0.025M Tris-HCI 0.192M Glycine 0.1% (w/v) SDS
SDS Sample Buffer (4X) 1M Tris-HCI pH6.8 20% (w/v) SDS 10% Glycerol 0.1% DTT 0.01% (w/v) Bromophenol Blue
Transfer Buffer 26mM Tris-HCI 192mM Glycine 10% Methanol
TBST 150mM NaCI 20mM Tris-HCI ph7.5 0.1% Tween 20
44 2.1.5 Primary antibodies Name Origin Description Use 9E10 CR-UK monoclonal Mouse monoclonal specific W, IF services for Myc epitope HA Santa Cruz Rabbit polyclonal W,IF PRK1 mab Transduction Mouse monoclonal W Laboratories PRK2 mab Transduction Mouse monoclonal W Laboratories Phospho-PRKI PP lab (Flynn etal., Rabbit polyclonal purified W Thr-774 2000) by affinity chromatography. Phospho-PKB Cell Signalling Rabbit polyclonal W (P)Ser473 Technology PKB Cell Signalling Rabbit polyclonal W Technology Phospho-PKB Cell Signalling Rabbit polyclonal W (P)Thr308 Technology Phospho-P38 Cell Signalling Rabbit polyclonal W Thr180/Tyr182 Technology P-38 Cell Signalling Rabbit polyclonal W Technology Phospho-JNK Cell Signalling Rabbit polyclonal W Thr183/Tyr185 Technology JNK Cell Signalling Rabbit polyclonal W Technology Phospho-ERK1/2 Cell Signalling Rabbit polyclonal W Thr202/Tyr204 Technology PAN-ERK Cell Signalling Rabbit polyclonal W Technology
45 Phospho-c-Jun Cell Signalling Rabbit polyclonal W Ser63 Technology c-Jun Cell Signalling Rabbit polyclonal W Technology MKK4 Cell Signalling Rabbit polyclonal W Technology Phospho-MKK4 Cell Signalling Rabbit polyclonal W Thr261 Technology GPP Clontech Rabbit polyclonal W,IP DS-Red Clontech Rabbit polyclonal w, EEA1 Santa Cruz Goat polyclonal IF Tubulin Sigma Mouse monoclonal W PDK1 Dr Dario Alessi Sheep polyclonal W (Balendran etal., 1999) Actin Transduction Goat polyclonal W laboratories Rad Totam Biologicals Mouse monoclonal W,IP GST Transduction Mouse monoclonal W Laboratories
(W= Western, IF= Immunoflourescence, IP = Immunoprécipitation, P- = Phospho- specific antibody)
2.1.4 Secondary antibodies
Name Origin Description Use Mouse-HRP Amersham W Rabbit-HRP Amersham W Goat-HRP DAKO W Sheep-HRP Sigma W Cy3-anti mouse Jackson Laboratories IF
46 2.1.5 Pharmacological Agents
Drug Origin Target Working concentration HA1077 Calbiochem PKN 20\iU LY294002 Calbiochem PI3 Kinase lO^iM
2.1.6 DNA constructs
Plasmid Source Expression Vector C3-GST Dr Harry Mellor pGEX (Amersham) PKN1 Dr Harry Mellor (Flynn et pCDNA3 al., 1998) (Invitrogen) Myc-PKN 1 myc Dr Harry Mellor (Flynn et pCDNA3 al., 1998) Myc-PKN2 Dr Harry Mellor (Flynn et pCDNA3 al., 1998) GST-PKB Dr Brian Hammings pBC (EMBL X78316) Myc-Rad wt Dr Alan Hall pCDNA3 Myc-Rad N17 Dr Alan Hall pCDNA3 HA-HR1abc Dr Harry Mellor (Mellor et pCDNA3 al., 1998) GFP-PKN1 See section 2.2.1.6 pEGFP-C1 (clontech) GFP-PKN1-KR Dr Adele Casamassima pEGFP-01 (PP lab) GFP-PKN2 See section 2.2.1.6 pEGFP-CI DSRED-PKN1 See section 2.2.1.6 PDS-RED-C1 (Clontech) GFP-PKCe Dr Johanna Ivaska pEGFP-01 (Ivaska et al., 2002) GFP-PKCa kinase domain Dr Scott Parkinson (PP pEGFP-01 lab) Myc-PKNI-kinase domain Dr Harry Mellor(Mellor et pCDNA3 al., 1998) Myc-PKNI-Kinase domain-KR Dr Harry Mellor(Mellor et pCDNA3 al., 1998) GFP-PKB Dr Sandra Watton pEGFP-C1 (Watton and Downward, 1999)
47 2.2 Methods
2.2.1 Molecular biology
Standard molecular biology procedures such as agarose gel electrophoresis were as described (Maniatis etal., 1989). Restriction digests were carried out according to the manufacturer's instructions.
2.2.1.1 PCR Reactions
Polymerase chain reactions incorporated: 1 unit of Pfu Turbo in combination with the recommended buffer, 200p,M of dNTPs (from a lOmM stock of ultrapure dNTPs with respect to each deoxynucleotide triphosphate), IpM of each sense and antisense primer, and 50ng of double stranded DNA template to a final volume of 50|xl. The program used for PKN1 cloning into pEGFP and pDS-Red vectors (as described below) is as follows; 94°C for 5min followed by 30 cycles of 94°C dénaturation step for 45 seconds, a 65°C annealing step for 45 seconds, and an extension step of 68°C for 6.5min. PCR products were separated by agarose gel electrophoresis, the desired bands were excised and purified using the Qiagen QIAquick gel extraction kit.
2.2.1.2 Ligation reactions
Inserts and vectors were digested and separated using agarose gel electrophoresis. Bands were excised and purified using the QIAquick gel extraction kit from Qiagen. Ligation reactions typically incorporated 0.5|xl of T4 DNA ligase with the appropriate buffer and a ratio of 3:1 insert to vector to a volume of 10|xl.
48 2.2.1.3 Transformation of E.Co/i
Aliquots of E.coli DH5a competent cells (50pl) were incubated with 500ng of plasmid DNA (4|liI of ligation reaction) for 20min on ice. Cells were then heat shocked at 42°C for 45 seconds and then replaced on ice for 2min. LB media (1 ml) was added and cells incubated at 37°C for 1 hour. The bacterial inoculate (SOpI) was spread onto an LB plate containing the appropriate selection antibiotic; lOOpgml'^ ampicilin, and SOpgml'^ kanamycin and incubated overnight at 37°C.
2.2.1.4 Plasmid DNA preparation.
A colony from transformed DHSa cells was picked and grown in a 5ml culture of LB containing the relevant selection antibiotic. From this, small scale DNA purifications utilised the Qiagen Mini-prep kit. Large scale DNA purifications added the 5ml overnight culture as described above to 200ml of selecting LB and after a further overnight culture prepared DNA using the Qiagen Plasmid Maxi-prep kit. A ratio
QD260/OD280 of 1.8 from maxi prep prepared DNA was considered an acceptable DNA quality.
2.2.1.5 DNA sequencing
DNA sequencing was carried out using the ABI prism Dye Terminator kit.
2.2.1.6 Construction of GFP-PKN1, GFP-PKN2, DS-Red-PKNI and DS- Red-PKN2.
GFP-PKN1 and GFP-PKN2 constructs were generated by polymerase chain reaction using as templates PKN1 and PKN2 constructs respectively. PKN1 was amplified using the primer (1) 5'-
49 GCGCAAGCTTGCATGGCCAGCGACGCCGTGCAGAGTGAGCC-3' and (2) 5'- GCGCGGTACGTTAGCAGCCGGCGGCCACGAAGTCGAAGTCCAGGAAGGC-3' and for PKN2 (3) 5'-GGGGAAGGTTGGATGGGGTGGAAGGGGGAAGGG-3' and (4) 5'- GGGGGGTAGGTTAAGAGGAATGAGGAATGTAGTGAAAATGTGTGAAGATTTGG TGG-3'. Primers 1 and 2 incorporated a Hindi!I restriction site, primers 3 and 4 contained a Kpnl site. The resulting products contained the full length PKN sequences and were cloned into PGR blunt (Invitrogen). This was subsequently digested with Hindlll and Kpnl and cloned into pEGFP-G1 (Glontech) enabling the fusion of an N-terminal GPP tag in frame with the full length PKN1 and PKN2. The DS-Red PKN1 was constructed in the same manner as GFP-PKN1, using the pDS-Red-G1 vector from Glontech.
2.2.2 Cell Culture and transfection
NIH 3T3 cells were maintained in Dulbecco's modified Eagle’s medium and supplemented with 10% foetal calf serum (10%E4). Transient transfections were performed using Lipofectamine 2000 (Invitrogen). The lipid DNA mix was incubated for 6 h according to the manufacturer’s protocol and all subsequent manipulations (see text and figure legends) were performed 48 h post transfection.
2.2.2.1 Hyperosmotic Stress
Cells were subjected to hyperosmotic conditions for 30 minutes by the addition of Dulbecco's modified Eagle’s medium containing 0.4M sucrose and 25mM Hepes pH 7.2. Where indicated, sorbitol was substituted for sucrose at the same molarity. For recovery experiments, cells were shocked for 30 minutes and then medium was replaced with normosmotic medium. The average number of vesicles after 30min of hyperosmotic stress and subsequent 15 and 30 minutes of recovery were determined. From each condition, 10 GFP-PKN1 vesicle containing cells were
50 sampled. Images were taken as confocal sections through the centre of the cell and the number of vesicles from each image counted.
2.2.3 Microscopy
Cells were seeded on acid washed glass coverslips, transfection and stimulation was as indicated in figure legends. Cells were then washed and fixed in 4% paraformaldehyde for 15min. When processing for immunoflourescence, cells were permeablized with 0.2% (v/v) Triton X-100 for 5 min, washed and incubated in 1% (w/v) bovine serum albumin (BSA) for 20min. Coverslips were incubated with primary antibody containing 1% (w/v) BSA for 1 h. Cells were then washed and incubated with fluorescent dye-conjugated secondary antibody for 1 h, washed three times, the final wash in water. All washes and incubations were performed in Phosphate Buffered Saline (PBS) unless otherwise stated. Cells were mounted on glass slides under MOWIOL [lOOmM TrisHCI pH 8.8, 10% (w/v) MOWIOL 4-88 (Calbiochem) and 25% (v/v) glycerol] containing antiphotobleaching agent [2.5% (w/v) 1,4-diazabicyclo[2.2.2]octane (Sigma)]. Slides were examined using a confocal laser scanning microscope (Axioplan2 with LSM 510; Carl Zeiss Inc.) equipped with 63x/1.4 Plan-APOCHROMAT oil immersion objectives. GPP and Cy3 were excited with 488 and 543 nm lines of Kr-Ar lasers respectively and individual channels scanned in series to prevent bleed through. Each confocal image is representative of a given field of transfected cells. Images are representative of at least three separate experiments unless otherwise indicated. Each image represents a single 1.0pm T optical section.
2.2.4 Polyacrylamide gel electrophoresis (PAGE)
Sodium dodecyl sulphate (SDS) PAGE was performed according to (Laemmli, 1970) using Hoeffer gel apparatus. Routinely, 10% acrylamide running gels and 3% acrylamide stacking gels were used with the exception of Myelin Basic Protein
51 resolution where a 15% acrylamide running gel was used. Protein samples were typically prepared in a 4X Laemmli sample buffer (see buffers) unless otherwise stated and heated to 95° for lOmin. Molecular weight standards were run in parallel for all samples to determine apparent molecular weight. Proteins were visualised by either coomassie blue staining, western blotting followed by subsequent immunoblotting or where indicated gels were dried and autoradiographed.
2.2.5 Western blotting
Proteins were transferred electrophoretically onto polyvinylidene difluoride membrane (PVDF). Membranes were pre-incubated with TBS containing 3%BSA for one hour followed by overnight incubation with TBS containing 3%BSA and primary antibody typically 1/2000 dilution unless otherwise indicated. Membranes were then washed 3X15 min with TBS followed by incubation with the relevant secondary antibody (see secondary antibodies) in TBS containing 1%BSA to a dilution of 1/7000. Membranes were then washed 3X15min with TBS and visualised with enhanced chemiluminescence ECL according to the manufacturer’s guidelines, using hyperfilm.
2.2.6 C3-GST Protein Purification and cell loading
2.2.6.1 Preparation of FY81 0-P.lys electro-competent cells.
A 10ml culture of LB, inoculated with FY810 p./ys cells containing 34^ig/ml of chloramphenicol was incubated overnight at 37°C. Cells were pelleted and the supernatant discarded. Cells were then added to a 500ml LB culture with 34p.g/ml of chloramphenicol and incubated at 37°C until an ODgoo of 0.4 was reached. The culture was then incubated at room temperature until an ODgoo of 0.6 was achieved. The culture was cooled on ice for 30min and subsequently pelleted (20min 4200 rpm in a JA20 rotor at 2°C). Cells were then washed in 500ml ice cold
52 HgO and pelleted (20min 4200 rpm in a JA20 rotor at 2°C) twice. Cells were resuspended in 80ml 10% glycerol (in ice cold HgO) and pelleted. Cells were then resuspended in 1.5ml 10% glycerol, 50pl aliquots dispensed on dry ice and stored at -70°C.
2.2.6.2 Transformation of FY81 0-P.lys electro-competent cells.
Cells were added to electroporation cuvettes on ice for lOmin, 1-2pg of DNA was added and the mixture was subjected to electroporation (2.5KV, 25pF with pulse of 200 ohms). Cells were incubated at 37°C in LB, pelleted and spread onto an LB plate containing the appropriate selection antibiotic; lOOpgml ^ ampicilin, and SOpgml'^ kanamycin with 34pg/ml of chloramphenicol and incubated overnight at 37°C.
2.2.6 3 Purification of C3-GST
FY810 plys cells were transformed with the C3-GST plasmid, and used to establish a 5ml overnight culture containing ampicillin (lOOpg/ml) and chloroamphenicol (34pg/ml). This was used to inoculate 500ml of LB with the same antibiotic concentration and grown to an ODeoo of 0.6 while shaking at 37°C for approximately 3h. Protein synthesis was induced by the addition of 500pl 1M I PTC and followed by further shaking for 2.5h. Cells were harvested by centrifugation at 6000 rpm in a Beckman JA-20 rotor for 20min. The cell pellet was frozen in liquid nitrogen and then resuspended in 40ml of ice-cold extraction buffer (50mM Tris; pH7.5, 105mM NaCI, 0.1 mM PMSF, ImM EDTA, ImM DTT). The cells were then incubated on ice for 30min followed by the addition of 20% Triton X-100 to a final concentration of 1% and then sonicated for 20 seconds at 18 microns (using the MSB probe sonicator 150). The samples then underwent centrifugation at 12000 rpm for 30min in a Beckman JA-20 rotor. 250pl of Glutathione-Sepharose beads
53 equilibrated in extraction buffer were added to the supernatant and then incubated with agitation in the cold room for 30min. The beads were pelleted by centrifugation, and after 2X washes in wash buffer (50mM Tris; pH7.5, 105mM NaCI, 1 mM EDTA, 1 mM DTT), 03-GST was eluted from the beads by a final wash in buffer containing 10mM glutathione pH8.0. C3-GST protein samples were then aliquoted and stored at -70°C.
2.2.6.4 Cell loading with C3-GST
NIH3T3 cells were incubated with 03-GST at a concentration of 5|Lig/ml for Ghours. The effectiveness of cell loading was measured by phalloidin staining and in vitro ribosylation assay (see below).
2.2.6 5 Ribosylation assay
In vitro ADP-Ribosylation of Rho by 03-GST was essentially as described in (Aktories and Just, 1995). NIH3T3 cells were seeded (500,000 cells per 6cm dish), grown in10%FOS E4 and subsequently starved for 16 hours. Cells were then treated with 5pg/ml of 03-GST or GST protein as indicated for 6h, harvested in 400|il lysis buffer (20mM TrisHOI pH7.5, 0.25M Sucrose, 5mM MgOlg, ImM EDTA, ImM DTT, 0.5mM PMSF, 5mM Leupeptin, ImM Benzamidine) and sonicated for 20 seconds at 18 microns (MSE probe sonicator 150). A volume of 150|xl of lysate was added to 50pl of reaction buffer (400mM TrisHOI pH8, 40mM Thymidine,40mM Nicotinamide, 4mM DTT, 20mM MgOlg, 200pM NAD, lOpM pP]NAD) and incubated at 30°0 for 30min. Reactions were terminated by the addition of lOOpI SDS sample buffer. Samples were then processed and loaded onto a 12.5% PAGE gel. Gels were dried and exposed to the Storm (Molecular Dynamics) phospho-imager.
54 2.2.7 Rad activation
NIH 3T3 cells (500,000 cells per 6cm dish) were seeded and grown in 10%FCS E4 and subsequently starved for 16 hours with 0.05%FCS E4. Cells were then subjected to treatments of hyperosmotic stress as indicated in figure legends. The Rac activation assay was performed on cell extracts using reagents contained in the kit from Totam biologicals. This assay employs the p21 binding domain (PBD or CRIB domain) of p21 activated kinase 1 (PAK) as a GST fusion protein which selectively binds GTP-Raci in a “pull down” assay enabling the detection of activated R a d . All samples were prepared and processed according to the manufacturer's guidelines. Quantitation was obtained by image capture and densitometry using NIH™ image software.
2.2.8 Phosphorylation of GFP-PKN1
Cells were transiently transfected on 6cm^ plates with GFP-PKN1 and subjected to hyperosmotic stress as indicated. Cells were harvested in 400pl sample buffer and fractionated on a 10% SDS polyacrylamide gel. Activation loopThr-774 phosphorylation of PKN1 was assessed by western blotting using a phospho- specific polyclonal antibody with excess of dephospho-peptide as described (Flynn etal., 2000). Relative PKN1 phosphorylation was determined as a function of PKN1 protein levels defined using the PKN1 monoclonal antibody. GFP-PKN1 phosphorylation is taken from three duplicate experiments, where represented graphically, error bars denote the standard error of the mean.
2.2.9 GFP-PKN1 kinase assay
For each condition a IScm^ plate of cells was transfected with GFP-PKN1 and stimulated with hyperosmotic medium as indicated in the figure legend. Cells were harvested in a lysis buffer comprising: 20mM TrisHCI pH7.5, 0.5% (w/v) CHAPS, 1
55 complete tablet (protease inhibitor from Roche) per 50ml lysis buffer, 2mM EDTA, 150mM NaCI. Preclearance of cell lysates with pre-equilibrated protein G was followed by incubation with 9pg of GFP polyclonal antibody for 20 min at 4°C. Immunocomplexes were captured by incubation for 1h with protein G. Immunoprecipitates were washed three times with lysis buffer, once with 0.5M NaCI and a final wash in reaction buffer, 20mM Hepes pH 7.5, 10mM MgClg. For each reaction, 10p,l of immunopurified GFP-PKN1 was incubated in a 40|xl reaction mix of: 20mM TrisHCI pH 7.5, 10mM MgClg, 25pg bath sonicated phosphatidyl-L- serine, lOjig myelin basic protein, 5pM ATP and 1p,Ci [y^^P] ATP for 20 minutes at 30°C with agitation. Reactions were terminated by the addition of 40pil of sample buffer prior to fractionation on a 15% SDS-polyacrylamide gel, subsequently western blotting analysis was performed. To assess MBP phosphorylation, membranes (Immobilon-P from Millipore) were exposed to phospho-imager (STORM, Molecular Dynamics) prior to immunoblotting with anti PKN1. Relative specific activity was determined as a function of i mmu noreactive GFP-PKN1. The data presented is representative of three duplicate experiments and where represented graphically, error bars denote the standard error of the mean. Additional duplicate reactions incorporated 20pM HA1077 as a control for specific PKN activity (Amano etal., 1999) (Davies etal., 2000).
2.2.10 Cell Fractionation
Samples were subjected to velocity and equilibrium sucrose gradient centrifugation essentially as previously described (Tooze and Huttner, 1992). NIH3T3 cells were seeded onto 15cm^ plates and stimulated as indicated. Cells were washed with cold PBS and harvested in 1ml of homogenisation buffer (0.25M Sucrose, lOmM Hepes-KOH pH7.2, ImM EDTA pH7.5, ImM Mg Ac and 1 complete tablet per 50ml). Samples were homogenised using a cell cracker (EMBL) and spun at 3000 rpm for 7min using a bench-top centrifuge. The supernatant or (post-nuclear supernatant, PNS) was loaded onto a velocity gradient ranging from 0.3M to 1.2M
56 sucrose. This was spun using a Beckman JA20 rotor at 25000rpm for 18min. In all conditions, the top 3, 1ml fractions were then loaded onto an equilibrium gradient composed of; 1ml 1.2M, 2ml 1M, 2ml 0.8M and 1ml 0.4M sucrose. This was spun overnight at 250000rpm, 1 ml fractions were subsequently collected,!OO^iI of which was run on 10% SDS-PAGE gel and subjected to western analysis and immuoblotting as described earlier.
2.2.11 PKN3 Antibody generation
The PKN3 specific peptide “QQAAFRDFDFVSERFLEP” was used to generate rabbit polyclonal antibodies.
2.2.11.1 Peptide coupiing
Keyhole limpet haemocyanin (KLH) (0.6 ml of 21 mg/ml) was dialysed overnight against PBS. KLH (lOmg) was added to lOmg of peptide dissolved in 440|il of
PBS. Glutaraldehyde (1 0 |liI)was added and incubated for 15min incubation at room temperature, a further 5|liIof glutaraldehyde was added followed by another 15min incubation. Glycine (200pil of 1M pHG.O) was added to quench glutaraldehyde and this was diluted to a volume of 19.2ml with PBS from which 6 X 3.2ml aliquots were sent for rabbit immunisation.
2.2.11.2 Affinity purification
Actigel resin (5ml) was washed X3 with water by centrifugation at 3000rpm for 5min. Coupling buffer (5ml) containing 0.5ml coupling solution (actigel), 0.5ml of 1M PBS pH 6 (a pH away from the isoelectric point. Pi, of the peptide) and 5mg of peptide. This mixture was agitated for 4h followed by a 0.5M NaCI and buffer wash.
57 Serum (5ml) was mixed with PBS/0.02% (v/v) Tween 20 (5ml) and pre-cleared by centrifugation at SOOOrpm for 5min. Actigel resin (2ml) (see above) was added to each column used, pre-cleared serum was passed through the column X3 times. The column was washed with 20ml PBS/0.02% (v/v) Tween 20, 40ml 0.5M NaCI in PBS/0.02% (v/v) Tween 20 and finally a further PBS/0.02% (v/v) Tween 20. Antibodies were eluted with 0.1 M citrate pH2.5/ 0.02% Tween. Fractions of 600pl were collected, neutralised and tested by western for immuno-reactivity.
58 Chapter 3
PKN and the PKB pathway,
3.1 Introduction
As discussed in the introduction, regulation of PKN1 and PKN2 is distinct from other PKC superfamily members; they are both calcium and diacylglycerol independent. The PKN regulatory region contains a novel N-terminal domain termed homology region 1 (or HR1) which is comprised of three homologous sequences (HR1a, HR1b and HRIc) and has been shown to interact with the small GTPases Rho and Rad (Flynn etal., 1998; Vincent and Settleman, 1997). Other G-proteins may play a role in PKN regulation, indeed a novel GTPase, AWP1 has recently been shown to interact with PKN1 (Duan etal., 2000). The interaction of RhoA with PKN1 operates through binding to the HRIa and HRIb sequences of the HR1 domain. Binding to the HRIa region is GTP dependent while the interaction with HR1 b was shown to bind both GDP and GTP forms of RhoA (Flynn etal., 1998). This interaction has also been shown to facilitate the activation loop phosphorylation of PKN1 by 3-Phosphoinositide Dependent Kinase 1 (PDK1) (Flynn etal., 2000).
Another substrate for PDK1 is Protein kinase B (PKB). PKB has two specific phosphorylation sites that are required for full activation, these sites are on residues threonine 308 and serine 473 (Alessi etal., 1996). The T308 site has been shown to be directly phosphorylated by PDK1 (Alessi etal., 1997a; Alessi et al., 1997b; Stokoe etal., 1997). The S473 site is however more controversial in its regulation, it has been suggested that an as yet unidentified kinase, termed “PDK2”, is responsible for PKB S473 phosphorylation since PDK1 was found not to phosphorylate this site directly (Alessi etal., 1997b). Phosphorylation of the serine
59 473 has also been suggested to be an autophosphorylation site resulting from threonine 308 phosphorylation by PDK1 (Toker and Newton, 2000b).
Further work on the relationship between PKN2 and PDK1 has implicated PKB as a downstream target for PKN2. A C-terminal PKN2 fragment was shown to convert PDK1 to a form that will phosphorylate PKB on the serine residue at amino acid 473 (S473) in v/ïmfunctioning as the elusive PDK2 activity (Balendran eta!., 1999). This chapter seeks to test the hypothesis that Rho-PKN signals can influence PKB S473 phosphorylation thereby acting as a complex with PDK2 activity.
6 0 3.2 Lack of a Rho/PKN input to PKB serine 473 phosphorylation
To investigate the putative interaction of PKN2 and PKB, the Rho sensitivity of PKB was tested by employing the Rho inhibitor C3 transferase which is known to specifically inhibit Rho over other GTPases such as Rad (Wilde etal., 2000). GST-C3 and GST (control) protein were purified from GST beads (Figure 3.1) and NIH3T3 cells were loaded with GST-C3 protein. The modification of Rho activity was measured in vitro using a post extraction ribosylation assay (Aktories and Just, 1995). Following 6 hours of pretreatment with GST-C3, a reduction in in vitro ribosylated Rho of approximately 80% could be detected, the result of two duplicate experiments (Figure 3.2 A). Since Rho controls actin dynamics, the effectiveness of cell loading GST-C3 in vivo was also measured by phalloidin staining. Following 6 hours of treatment with GST-03, a loss of stress fibre formation was observed, consistent with the ribosylation and loss of function of Rho (Figure 3.2 B).
Having established that Rho can be inactivated by cell loading with GST-03, the pattern of PKB 473 phosphorylation was observed in NIH-3T3 cells after either 6 hours of treatment with GST or GST-03. This was examined under conditions of serum starvation followed by stimulation with various agonists including; total serum, FGF, LPA, PDGF and EG F (Figure 3.3). No difference in the pattern of PKB 473 stimulation with respect to these agonists was detected between GST and GST-03 treatments indicating no Rho-PKN inputs into the regulation of the PKB 473 site.
61 C3-G ST GST
Total Lysate + + Beads + + Eluted protein + +
66KDa m m ^ »
42KDa
Fig 3.1 Purification of C3-GST and GST protein.
Fy810-Plys bacteria were transformed with C3-GST and GST plasmids. 03- GST and GST proteins were purified from Glutathione Sepharose beads as described in chapter 2. Total lysate, beads and eluted protein were run on SDS- PAGE and proteins visualised by coomassie staining.
62 4 5 C3-GST pretreatment + + in vitro C3-GST + +
m
B 6hGST 6h C3-GST
Fig 3.2 6hours of pretreatment with C3-GST In NIH3T3 cells inhibits Rho.
A. NIH3T3 cells were seeded onto 6 omoplates and subjected to in vitro ribosylation assay: (1) excluded C3-GST in vitro, (2-5) incorporated 03-GST in the reaction. Cells in (3) were harvested after 6h of 25^g GST pretreatment, (4) had 6h of 2|ig C3-GST pretreatment and (5) had 6h of 25pg C3-GST pretreatment prior to harvesting and ribosylation assay. B. NIH3T3 cells were treated for 6h with GST or for 6h with 03-GST prior to fixation (as described in chapter 2). Visualisation of the actin cytoskeleton was achieved by staining with Alexa-488 phalloidin. Images are representative of single 1 .Opm Z' optical sections and the scale bar is equivalent to 10|im.
63 6h 5jxg/ml C3-GST 6h 5|ig/ml GST
Stimulation (min) 0 2 5 1015 0 2 5 10 15
Serum
c o h' . ■mmmrn 'mm. o ig Q. (0 O LPA Q. CO
CO PDGF g CL EGF
Total PKB (EGF)
Fig 3.3 Serum and growth factor stimulation of PKB 8473 phosphorylation is unaffected by Rho inhibition.
NIH3T3 cells were seeded onto 6 crn^ plates, subjected toi 6 hours of serum starvation and 6 hours incubation with either 5|mg/ml 03-GST or 5^g/ml GST. Cells were stimulated with: 10% FCS, lOng/ml FGF, 500ng/ml LPA, 20ng/ml PDGF or lOOng/ml EGF as indicated prior to harvesting in Laemmli sample buffer. Samples were run on SDS-PAGE and subjected to western analysis.
64 3.3 The HRIabc domain of PKN1 can Inhibit PKB S473 phosphorylation; rescue by Rad but not Rho
Although the above experiments appear to exclude Rho dependent effects of PKN on PKB 473 phosphorylation, the possibility remains that other inputs (i.e. C3 insensitive) to the HRIabc domain may influence a PKN driven PKB response. To test this issue, we used the overexpression of this domain enabling the titration of HRIabc binding partners and monitored PKB phosphorylation. The co-expression of GST-PKB and HA tagged HRIabc shows an inhibition of PKB S473 response to serum stimulation (Figure 3.4). The loss of PKB S473 phosphorylation is also detected by co-expression of full length PKN1 with GST-PKB (Figure 3.5). HRIabc induced inhibition of PKB 473 phosphorylation can be recovered upon co expression with the constitutively active RacV12, but not active RhoB QL (Figure 3.5). This observation is consistent with GST-C3 experiments showing a lack of Rho involvement in PKB 473 phosphorylation. The upregulation of HRIabc depleted GST-PKB phosphorylation by the co-expression of RacV12 raises the question of whether Rad-PKN inputs can influence PKB 473 phosphorylation.
65 GST-PKB GST-PKB + HA-HR1
Serum Stimulation (min) 0 5 10 20 0 5 10 20
p-473 GST-PKB
Total GST-PKB
Fig 3.4 Overexpression of PKN1 HR1 domain inhibits GST-PKB 8473 phosphorylation.
NIH3T3 cells were seeded onto 6 omoplates, transfected with either GST-PKB or GST-PKB and HA-HR1 as indicated and subjected to i6 hours of serum starvation. Cells were stimulated with; 10% PCS for the time-points indicated prior to harvesting in Laemmli sample buffer. Samples were run on SDS-PAGE and subjected to western analysis.
66 1.2 T------
I 1 -
« OS £O a . r> 0.6 f œ ^ 0.4
Œ
10
3 4 10 5min 10% FCS + + GST-PKB + + + HA-HRl + + RhoBQl RaclV12 PKNl
p-473 GST-PKB
Total GST-PKB
Fig 3.5 Rad can recover HR1 Induced inhibition of GST-PKB 8473 phosphorylation.
NIH3T3 cells were seeded onto 6 omoplates, transfected with either GST-PKB, HA-HR1, RhoBQL, Rac1V12 and PKN1 as indicated and subjected to16 hours of serum starvation. Cells were stimulated for 5 min with 10% FCS as indicated prior to harvesting in Laemmli sample buffer. Samples were run on SDS-PAGE and subjected to western analysis. Band intensities were analysed using NIH image^"".
67 3.4 Co-expression of Rac1V12 and PKN2 induces expression from the pBC plasmid
To investigate the possible PKN involvement in Rad mediated PKB S473 phosphorylation, co-expression studies were performed involving GST-PKB, RacV12, HA-HR1abc, PKN1 and PKN2. Increasing plasmid concentrations of transfected HA-HR1abc or PKN1 combined with Racv12 and GST-PKB, resulted in a decrease in GST-PKB 473 phosphorylation and protein level. This could perhaps be an effect of increasing plasmid concentration. However, with increasing amounts of PKN2 plasmid in combination with RacV12 and GST-PKB, an increase in phosphorylation paralleled by an increase in overexpressed GST-PKB protein level is detected (Figure 3.6 A). Further investigation shows that co-expression of RacV12 and PKN2 induces increased expression of GST protein from the pBC plasmid as used for GST-PKB (Figure 3.6 B); this expression vector is driven by an SV40 promoter.
68 GST-PKB + + + + + + + + + + + + Racl-V12 + + + + + + + + + + + + HA-HRl (ng) - 1 2 5 ------PKNl([i.g) — — — — 0125 — — — — PKN2(^ig) ------0 1 2 5
PKB S473
Total PKB
B
GST-PKB + + + + ____ GST - - - - + + + + PCDNA3 + + - - + + - - Racl-V12 - - + + - - + + PKN2 +
GST-PKB — . — ^
GST H III— ■
Fig 3.6 Co-expression of PKN2 and Rac1V12 stabilises expression from the GST expression plasmid.
A. NIH3T3 cells were seeded onto 6 omoplates and transfected with either GST- PKB, Rac1-V12, and varying amounts of HA-HR1, PKN1 and PKN2 plasmids as indicated. B. NIH3T3 cells were seeded onto 6 omoplates and transfected with GST-PKB, PCDNA3, Rac1-V12 and PKN2 plasmids as indicated. Samples were harvested in Laemmli sample buffer, run on SDS-PAGE and subjected to western analysis.
69 3.5 Discussion
This chapter has adopted a candidate approach to investigate PKN targets and has focused on the contribution of Rho-PKN signals to PKB phosphorylation. It is demonstrated that pre-incubation of C3-GST for 6 hours is effective in the intervention of Rho function, however no difference in serum-induced PKB phosphorylation between GST and C3-GST treated cells was observed. The possibility remains that Rho-PKN dependent inputs into PKB could be driven under specific circumstances that otherwise may be obscured by a multitude of inputs controlling PKB 473 phosphorylation under serum stimulation. It was therefore pertinent to investigate the C3-GST sensitivity of specific growth factor stimulation time courses on PKB 473 phosphorylation. Given that such a wide range of stimulation conditions trigger the same pattern of PKB S473 phosphorylation regardless of Rho inhibition, we conclude that there are unlikely to be requirements for Rho-PKN inputs into this phosphorylation site.
It is interesting to note that the overexpression of HRIabc has the ability to deplete PKB 473 phosphorylation. Since Rho dependency on this phosphorylation site has been excluded, attention is drawn to the other HR1 binding GTPase, R a d . Indeed, Rad has been reported to stimulate PKB 473 phosphorylation by PI3 kinase activation in T cells (Genot etal., 2000). It is not possible to attribute the inhibition of PKB 473 phosphorylation by HR1 directly to the influence of PKN as the titration of HR1 binding partners, including Rad could compromise Rad mediated pathways that are unrelated to PKN. Resolving the question of PKN2 involvement in Rad mediated PKB phosphorylation has proved problematic given the effects on expression with the co-transfection of Rac1V12 and PKN2. These effects could be a result of increased transcriptional activity from the SV40 promoter within the pBC vector, driven by the co-operation of Rad and PKN2. If this is the case, the
70 effect of Rad and PKN2 on transcriptional activity may prove to be a useful readout for PKN2 activity and future experiments could seek to address this question. These studies could employ a luciferase reporter assay to investigate further the induction of gene expression.
Overall, data presented involving the monitoring of PKB 473 phosphorylation as a potential downstream target for PKN2 indicate no Rho-PKN inputs to this phosphorylation site. Overexpression of HRIabc is shown to inhibit PKB 473 phosphorylation and this was overcome by co-expression with Rad but not RhoA or RhoB. The precise role of PKNs in relation to PKB phosphorylation remains unclear, indeed recent co-expression studies along with data presented in chapter 6 suggest that PKNs may even play negative roles in PDK1 mediated PKB phosphorylation (Wick etal., 2000). While Rad may influence PKB 473 phosphorylation, and mediation by PKN is a possibility, a direct role for PKNs in PKB 473 phosphorylation has not been established rather it is all but excluded. The evidence that PKN1 does not act on the pathway to PKB indicated that other pathway(s) were likely to lie downstream. The next chapter seeks to adopt an alternative approach in establishing PKN functional roles by screening for potential PKN1 agonists.
71 Chapter 4
PKN1 is a stress responsive R ad effector.
4.1 Introduction
Evidence is accumulating that in order to elicit regulated responses to specific stimuli, signal transduction mechanisms are governed not only by their ability to perform specific functions. The context in terms of complex assembly and cellular location is of increasing interest.
To date, limited information is available regarding the localised control and functions of PKN1. It has been shown that PKN1 can be targeted by RhoB to endosomes and is thought to regulate epidermal growth factor receptor traffic (Gampel etal., 1999). Further studies have also provided evidence for the formation of a ternary complex (Rho-PKN1-PDK1) which is shown by the recruitment of PDK1 in the presence of PKN1 to RhoB containing endosomes (Flynn etal., 2000). PKN1 has also been linked to stress responses, the translocation of PKN1 to the nucleus from the cytoplasm upon heat stress has been shown implying a role for PKN1 in the transduction of signals resulting in transcriptional responses (Mukai etal., 1996).
The role of PKN1 with regard to the cytoskeleton is an interesting question given its control by Rho GTPases. It has been proposed that PKN1 is associated with the actin cytoskeleton, through binding to alpha-actinin (Mukai etal., 1997). A role for PKN1 in the assembly of microtubules through specific phosphorylation of tau, leading to the disruption of tubulin assembly has also been suggested (Taniguchi efa/.,2001).
72 This chapter addresses further the PKN1 localisation question by screening for potential agonists and using GFP-tagged PKN1 to monitor any gross changes in cellular distribution. This approach is taken to understand the context in which PKN1 is functionally active.
73 4.2 PKN1 localisation is responsive to hyperosmotic stress.
To observe the sub-cellular localisation of PKN1 and PKN2, GPP tagged versions of these proteins were constructed and sequenced. Full length PKN1 and PKN2 constructs were sub-cloned into the pEGFP-C1 vector to give an N-terminal GFP fusion protein as described in the materials and methods section. The expression of full length GFP-PKN1 and GFP-PKN2 is shown (Figure 4.1 A). For GFP-PKN1, based on a transfection efficiency of 30%, it is estimated that there is a 10 fold increase in PKN1 protein above endogenous levels.
GFP-PKN1 expressed in NIH3T3 cells displayed a punctate cytoplasmic localisation with an absence of nuclear localisation. The effect of a range of potential PKN1 agonists on the sub-cellular distribution of GFP-PKN1 was investigated including epidermal growth factor, insulin and LPA. No gross alterations in the distribution of PKN1 in response to these growth factor stimulations were detected. Images are shown 30min after growth factor stimulation and are representative from time-courses over a 2 hour period (Figure 4.1 B). The effect on GFP-PKN1 localisation of platelet derived growth factor (PDGF) and bombesin was also investigated with no gross changes observed (data not shown). The effect on PKN1 localisation under stress conditions was also investigated (Figure 4.2). Oxidative stress, temperature stress and hypo-osmotic stress conditions did not change the profile of GFP-PKN1 localisation. Notably however, cells treated with hyperosmotic media for 30 min displayed a dramatic change in localisation, with GFP-PKN1 accumulating in large cytoplasmic vesicular structures (Fig 4.2). Since the DS-Red construct is known to be an oligomeric chromophore, DS-Red-PKN1 was co-expressed with GFP-PKN1 to confirm that it shows the same localisation behaviour before and after hyperosmotic stress, indeed this was the case (Figure 4.3). While DS-Red- and GFP-PKN1 are both N-
74 terminal fusion proteins, a C-terminal myc-PKN1 was localised and upon hyperosmotic stress is shown to
75 Control GFP-PKN1
GFP-PKN1 PKN1
Control GFP-PKN2 GFP-PKN2 PKN2
B
Fig 4.1 PKN1 has a punctate cytoplasmic distribution and Is unchanged by agonist stimulation.
A.NIH3T3 cells were transiently transfected with GFP-PKN1 and GFP-PKN2 as indicated, samples were harvested in Laemmli sample buffer, run on SDS- PAGE and analysed by western blotting. B. NIH3T3 cells were transiently transfected with GFP-PKN1. Cells were either unstimulated (control) or subjected to growth factor stimulation. Images shown are representative of GFP-PKN1 transfected cells after 30min of; 100ng/ml EGF, 5^g/ml LPA and Insulin. The scale bar is equivalent to lOpim.
75 Control Oxidative Oligomycin
hyperosmotic 12°C 45°C
Fig 4.2 PKN1 translocates to large vesicles in response to hyperosmotic stress. This translocation response is specific over other stress conditions.
NIH3T3 cells were transiently transfected with GFP-PKN1. Cells were either unstimulated (control), or subjected to various stress conditions including; oxidative stress (H 2 O2 ), Oligomycin, hyperosmotic (0.4M sucrose) and temperature stress (12°C or 45°C). Images shown are representative GFP- PKN1 transfected cells after 30min of stress treatment. All images are a representative single 1 .O^m ‘Z’ optical section and the scale bar is equivalent to 10um.
76 undergo the same translocation response. The translocation response of GFP- PKN1 was shown under conditions of hyperosmotic sorbitol as well as hyperosmotic sucrose (Figure 4.3).
GFP-PKCe has been shown to accumulate in vesicular structures following chronic PKC inhibition in MEF cells (Ivaska etal., 2002). To investigate the selectivity of the PKN1 translocation in response to hyperosmotic stress, DS-Red PKN1 and GFP-PKCe were co-expressed. The ability of DS-Red PKN1 to translocate into vesicles upon hyperosmotic stress was unaffected by the presence of GFP-PKCe, furthermore PKCe did not locate to the DS-Red PKN1 containing structures (Figure 4.4 A), indicating that the observed behaviour of PKN1 in response to hyperosmotic stress is selective. The ability of a second PKN sub-family member to respond to hyperosmotic stress was also followed. GFP-PKN2 when unstimulated was largely cytoplasmic with some limited nuclear accumulation although it was noted to be excluded from the nucleoli (Figure 4.4 B). After hyperosmotic stress, GFP-PKN2 showed the same translocation pattern as GFP- PKN1 (Fig 4.4 B), indicating that the described behaviour is a conserved PKN response.
The dynamic nature of the hyperosmotic-induced structures was investigated by placing cells back into osmotically balanced media following SOmin of hyperosmotic stress. Cells containing GFP-PKN1 vesicles were randomly selected and sectional images taken by confocal microscopy. These vesicles were clearly dissipated on removal from hyperosmotic conditions; quantitation showed that the average number of vesicles per cell decreased in a time-dependent fashion after re-addition of osmotically balanced medium. It is evident therefore that the accumulation of vesicular PKN1 is a reversible process (Figure 4.5).
77 GFP-PKN1 DS-Red-PKN1 Merge
V m
Control Hyper
Sorbitol Hyper
CL ÛL LL 0
Fig 4.3 GFP-PKN1, DS-Red-PKN1, myc-PKN1 and sorbitol hyperosmotic media display the same hyperosmotic-induced PKN1 translocation response.
NIH3T3 cells were transiently transfected with GFP-PKN1, DS-Red-PKN1 and myc-PKN1 as indicated. Cells were either unstimulated (control) or subjected to a 30min hyperosmotic stress (hyper) prior to fixation. Where indicated, sorbitol hyperosmotic media was used in place of sucrose. All images are a representative single 1 .Oiim ‘Z’ optical section and the scale bar is equivalent to 10um.
78 GFP-PKCe DS-Red PKN1 merge
B Control Hyper
Fig 4.4 Hyperosmotic-induced PKN1 translocation is selective over PKCe but common with PKN2
A. NIH3T3 cells were transiently transfected with DS-Red-PKN1 and GFP- PKCe. B. NIH3T3 cells were transiently transfected with PKN2. Cells were either unstimulated (control) or subjected to a 30min hyperosmotic stress (hyper) as indicated prior to fixation. All images are representative of single 1 .O^m ‘Z’ optical sections and the scale bar is equivalent to 10^im.
79 140 - r -
120 -
100 -
30min 45min 60min
30min 45min 60min Hyperosmotic ^ Recovery
Flg 4.5 Reversible translocation of GFP-PKN1 in response to hyperosmotic stress.
NIH3T3 cells were transiently transfected with GFP-PKN1. The reversibility of the PKN1 stress response was recorded by monitoring cells after 30 min hyperosmotic stress and also 15 and 30 min of recovery in normal osmotic media post stress treatment. GFP-PKN1 vesicle containing cells were selected randomly for each time point and the average number of vesicles per cell section is indicated graphically. Error bars indicate the standard deviation given by 10 cells from each time-point. Representative cell sections from each time point are shown as an inset. All images are representative of single 1 .O^im ‘Z’ optical sections and the scale bar is equivalent to lO^m.
80 4.3 Hyperosmotic-induced PKN1 vesicles are not early endosomes or acidified compartments but are associated with actin.
In characterizing the PKN1 response to hyperosmotic stress, the nature of this compartment was investigated. Since PKN1 has been implicated in endosomal traffic control (Mellor etal., 1998), hyperosmotic-induced PKN1 vesicles were co- immunostained with early endosomal antigen 1 (EEA1), a marker for early endosomes. No co-localisation of PKN1 vesicles and EEA1 positive vesicles was observed (Figure 4.6 A). Another possibility is that these compartments could be degradative late endocytic compartments or lysosomes. In order to test this, GFP- PKN1 transfected cells were treated with a marker for acidic compartments, lysotracker™. Hyperosmotic-induced PKN1 vesicles did not co-localise with acidified compartments (Figure 4.6 B).
Small GTPases play an important role in cytoskeletal rearrangements and vesicle trafficking, their association with PKNs is discussed in chapter 1. The relationship between hyperosmotic-induced PKN1 vesicles and the cytoskeleton was therefore investigated. In the first instance the relationship of PKN1 with actin was monitored by immunostaining with FITC-phalloidin. Under control conditions, an association of PKN1 with actin stress fibres was detected (Figure 4.7). It is also shown that hyperosmotic-induced PKN1 vesicles show some association with actin stress fibres as indicated by arrows (Figure 4.7). Other examples of hyperosmotic- induced PKN1 vesicles do not however contain actin stress fibres (Figure 4.7). It is possible that the PKN1 vesicle formation is dependent on actin stress fibres, this was investigated by the disruption of actin polymerisation with cytochalasin D. It is observed that the disruption of actin traps some GFP-PKN1 within actin patches
81 GFP-PKN1 EEA1 merge
GFP-PKN1 Lvsotracker
Fig 4.6 Hyperosmotic-Induced PKN1 vesicles do not co-localise with early endosomes or acidic compartments.
A.NIH3T3 cells were transiently transfected with GFP-PKN1 and stained with anti EEA1. B. NIH3T3 cells were transiently transfected with GFP-PKN1 and treated with Lysotracker for 30min prior to stimulation. Cells were either unstimulated (control) or subjected to 30min hyperosmotic stress (hyper). The scale bar is equivalent to 10^m.
82 GFP-PKN1 Phalloidin merge h . i l i 4
40^
Fig 4.7 PKN1 is associated with the actin cytoskeleton, hyperosmotic stress induces the down-reguiation of stress fibres.
NIH3T3 cells were transiently transfected with GFP-PKN1. Cells were either unstimulated (control) or were subjected to 30min hyperosmotic stress (hyper) as indicated. Two hyperosmotic examples are given so as to highlight the heterogeneity observed.To visualise the actin cytoskeleton alongside GFP- PKN1, cells were stained with FITC-Phalloidin. The scale bar is equivalent to 10um.
83 (Figure 4.8). However upon hyperosmotic stress, PKN1 vesicle recruitment is not prevented and PKN1 vesicles appear distinct from actin patches.
The involvement of microtubules in the PKN1 hyperosmotic response was investigated by immunostaining with an anti-tubulin antibody. It is clear that under control conditions PKN1 does not co-localize with microtubules and that under hyperosmotic stress, GFP-PKN1 vesicles do not associate with the disorganised microtubule network (Figure 4.9). These observations are further supported by the disruption of microtubules with nocodazole. Treatment with nocoazole clearly disrupts the microtubule network and this is shown not to affect GFP-PKN1 localisation under control conditions of the ability of GFP-PKN1 to translocate to vesicles under hyperosmolarity (Figure 4.9).
84 GFP-PKN1 Phalloidin merge
V,-.,
Fig 4.8 Disruption of actin polymerisation traps PKN1 in actin patches. Hyperosmotic-induced PKN1 vesicle accumulation is independent from actin.
NIH3T3 cells were transiently transfected with GFP-PKN1. Cells were treated with 1 hour of 20.um Cytochalasin D or 30min of 20|^m Cytochalasin D followed by 30min hyperosmotic stress in the presence of Cytochalasin D, as indicated . To visualise the actin cytoskeleton alongside GFP-PKN1, cells were stained with FITC-Phalloidin. The scale bar is equivalent to 10^m.
85 GFP-PKN1 Tubulin merge
0 S -ao o o
0 s o •O Üo o
Fig 4.9 Hyperosmotic-induced PKN1 vesicle accumulation is independent from microtubuies.
NIH3T3 cells were transiently transfected with GFP-PKN1. Cells were either unstimulated (control) or stimulated with hyperosmotic stress for 30min (hyper), treated for 1 hour with 20^m Nocodazole or for 30min with 20um Nocodazole followed by 30min of hyperosmotic stress in the presence of Nocodazole. After fixation, cells were stained with a-tubulin antibody. The scale bar is equivalent to 10um.
86 4.4 Hyperosmotic-induced PKN1 vesicie recruitment is dependent on Rad and not Rho.
PKN has been shown to bind to and become activated by members of the Rho family of small GTPases via the regulatory HR1 domain and indeed has been shown previously to be recruited to an endosomal compartment by RhoB (Mellor et al., 1998). The involvement of Rho proteins in the PKN response to hyperosmotic stress was tested by employing the C3 toxin from clostridium botulinum. C3 toxin has been shown to specifically inhibit the Rho subfamily over other Rho family GTPases such as Rad (Wilde at ai., 2000). NIH3T3 cells were transfected with GFP-PKN1, then pre-loaded with 03-GST for 6 hours at Spg/ml followed by subjection to hyperosmotic stress. The effectiveness of 03-GST cell loading was measured by an in vitro ribosylation assay and also by monitoring the extent of stress fiber disruption assessed by phalloidin staining (see chapter 3). The accumulation of PKN containing vesicles was independent of 03-GST treatment, implying that Rho function is not critical for this response (Figure 4.10).
The small GTPase Rad has previously been implicated in the control of PKN2 (Vincent and Settleman, 1997) and very recently in response to hyperosmotic shock (Lewis at ai., 2002). Hence in the absence of a Rho-input, the potential involvement of Rad in the translocation of PKN1 was investigated. Myc tagged Rad was co-expressed with GFP-PKN1. Under control conditions GFP-PKN1 was cytoplasmic and Rad located to lamellipodia, the plasma membrane and to some vesicular structures (Figure 4.11). After 30 min of hyperosmotic stress Myc-Rad and GFP-PKN co-localised in vesicles (Figure 4.11). Co-expression of the dominant negative Myc-Rad-N17 with GFP-PKN1 resulted in no vesicular translocation of GFP-PKN1 after hyperosmotic stress (Figure 4.11). These observations imply that GTP loading of Rad is necessary for the hyperosmotic stress induced movement of GFP-PKN1. Rad has recently been shown to
87 GFP-PKN1 Phalloidin merge
O $
Fig 4.10 Hyperosmotic-induced PKN1 vesicle accumulation is independent from Rho.
NIH3T3 cells were transiently transfected with GFP-PKN1. Cells were either unstimulated (control), subjected to 30min hyperosmotic stress (hyper) or treated for 6hr with 5,ug/ml of 03-GST with and without hyperosmotic stress prior to fixation. To visualise the actin cytoskeleton alongside GFP-PKN1, cells were then stained with FITC-Phalloidin. The scale bar is equivalent to 10|im.
88 GFP-PKN1 myc-Rac1-wt merge
GFP-PKN1 Rad N17 merae
Fig 4.11 Rad dependent hyperosmotic-induced PKN1 translocation.
NIFI3T3 cells were transiently co-transfected with GFP-PKN1 and myc-tagged Rad or Rad N17. Cells were either unstimulated (control) or were subjected to 30min hyperosmotic stress (hyper) as indicated.The scale bar is equivalent to 10um.
89 become GTP loaded upon hyperosmotic stress in neutrophils (Lewis etaL, 2002). This was confirmed here by employing a Rad pull-down assay, using the CRIB domain of PAK which selectively binds GTP bound R a d . After 30min of hyperosmotic stress, a 1.5-fold increase in the GTP loading of endogenous Rad was detected (Figure 4.12).
90 1 2
GTP-Raci
R ad
Fig 4.12 Rad activation in response to hyperosmotic stress.
NIH3T3 cells were seeded on 6cm^ plates and the Rad activation assay was performed. Treatments were: unstimulated cells (1), unstimulated cells with exogenous GTPyS (2), 10min hyperosmotic stress (3) and 30 min hyperosmotic stress (4). One of two experiments performed in duplicate is shown; error bars indicate the range of duplicate observations.
91 4.5 The regulatory HR1 domain is insufficient for vesicle recruitment whereas the cataiytic domain of PKN1 is constitutively vesicular.
Rad, like Rho, binds to the HR1 domain of PKN proteins (Flynn etaL, 1998; Shibata etaL, 1996). Thus the impact of the regulatory HRIabc region on hyperosmotic stress induced vesicle recruitment was investigated through localisation studies of the ectopically expressed HA-tagged HRIabc domain of PKN1. This domain constitutively localised around the cell periphery under control conditions and after hyperosmotic stress did not change its distribution. HA- HRIabc was co-expressed with full length GFP-PKN1. The distribution of the HRIabc domain remained unchanged when co-expressed with GFP-PKN1 before and after hyperosmotic stress and the presence of HRIabc did not abolish GFP- PKN 1 vesicle recruitment (Figure 4.13).
To assess the potential contribution of other PKN domains in vesicle recruitment, the myc-PKNI kinase domain was transfected into NIH3T3 cells. Under control conditions, the myc-PKNI kinase domain already displayed a partially vesicular distribution and upon hyperosmotic stress, this vesicular localisation became more pronounced (Figure 4.14). To test whether the kinase domain locates to the same structures as the full-length protein, the myc-kinase domain was co-expressed with GFP-PKN1 ; after hyperosmotic stress they were found to co-localise in large vesicular structures (Figure 4.14).
The observation that the PKN1 kinase domain can by itself localise to vesicles, raises the possibility that the kinase domains of the highly related PKG isoforms could behave in a similar manner. To assess further the specificity of the observed PKN behaviour, the myc-PKNI kinase domain was co-expressed with the GFP-
92 GFP-PKN1 HA-HR1abc merge
Fig 4.13 Rad binding is not sufficient for GFP-PKN1 transiocation.
NIH3T3 cells were transiently co-transfected with GFP-PKN1 and HA tagged HRIabc. Cells were either unstimulated (control) or were subjected to 30min hyperosmotic stress (hyper) as indicated. The scale bar is equivalent to lO^m.
93 Control Hyper
myc-PKN1 Kin
GFP-PKN1 myc-PKN1 Kin merge
Fig 4.14 The kinase domain of PKN1 is associated with hyperosmotic- induced PKN1 vesicles.
NIH3T3 cells were transiently transfected with the myc-kinase domain of PKN1 Myc tagged kinase domain of PKN1 was also transiently co-transfected with GFP-PKN1. Cells were either unstimulated (Control) or subjected to 30min hyperosmotic stress (hyper) as indicated. The scale bar is equivalent to 10pm.
94 PKCa kinase domain. After hyperosmotic stress the kinase domain of PKN1 could be detected in vesicles and in these structures the kinase domain of PKCa was absent (Figure 4.15). These data indicate that vesicle targeting of PKN1 through the kinase domain follows distinct mechanisms when compared with a closely related kinase domain.
95 GFP-PKCaKin myc-PKN1 Kin merge
Fig 4.15 The kinase domain of PKN1 localises to vesicles selectively over the kinase domain of PKCa.
NIH3T3 cells were transiently co-transfected with the GFP-kinase domain of PKCa and the myc tagged kinase domain of PKN1. Cells were either unstimulated (control) or subjected to 30min hyperosmotic stress. The scale bar is equivalent to lOjim.
96 4.6 Discussion.
The results described here demonstrate that PKN1 is acutely regulated in a reversible manner by hyperosmotic stress. Hyperosmotic-induced PKN1 vesicles are shown to be negative for both early endosomes and acidified compartments, although they do show some relationship with actin. Actin and tubulin disruption is shown not to prevent GFP-PKN1 vesicle formation, indicating that these arise independently from these cytoskeletal components. The assembly of vesicular PKN1 is shown however to be triggered by activation of Rad and not Rho. However, Rad contacts to the regulatory HR1 domain of PKN1 alone are found to be insufficient for accumulation of vesicular PKN1. A distinct site of interaction between PKN1 and the described compartment appears to be required and this is consistent with the finding that the kinase domain itself is selectively targeted to vesicles.
Upon stimulation with serum and a range of growth factors such as EGF, Insulin, HGF, LPA and PDGF no difference in the pattern of cellular localisation was observed. This observation may place PKN function downstream of receptor mediated events at the plasma membrane. Alternatively, the extent of recruitment to putative receptor complexes could be limited by the concentration of receptors and hence obscured by the bulk of GFP-PKNs. Roles for PKN1 consequent to receptor mediated events are not excluded, they could be simply beyond the detection limits of the expression system used.
It is shown that PKN1 localisation does not change upon stimulation under several stress conditions. Notably PKN1 translocation to the nucleus upon heat shock was not observed although this has been previously reported (Mukai etaL, 1996). However, the observed translocation in response to hyperosmotic stress is of
97 particular interest with regard to PKNs. Yeast PKC homologues share structural similarity to PKNs and have been implicated in controlling cell wall integrity, this response has been compared with hyperosmotic stress (Alonso-Monge et al., 2001). The observed mammalian PKN1 response to hyperosmotic stress could therefore highlight a conserved functional role.
The GFP-PKN1 translocation response to hyperosmotic stress is also demonstrated with DS-Red-PKN1. This control experiment helps to confirm the PKN1 response and the use of a “red” PKN1 widens the scope of localisation studies. The issue of “tagging” is also addressed since the C-terminal myc tagged PKN1 is shown to translocate to vesicles upon hyperosmotic stress in a similar manner to the N-terminal GFP-PKN1. Initial localisation studies sought to address the nature of the PKN1 vesicular compartment. Markers for early endosomes and acidified compartments show that PKN1 vesicles are not positive for either of these compartments. The precise nature of the PKN1 vesicular compartment remains unresolved.
The nature of the PKN1 translocation was also investigated, it is shown that hyperosmotic-induced vesicles dissipate upon the re-addition of norm-osmotic media, demonstrating this response to be a reversible process. These observations add further specificity and provide an insight into the dynamics of this stimulus specific response.
Hyperosmolarity is known to stimulate the remodelling of the actin cytoskeleton (Lewis etaL, 2002). PKN1 is shown to partially co-localise with actin stress fibres when unstimulated. Under conditions of hyperosmotic stress PKN1 vesicles are shown to partially co-localise with actin. The idea that initial PKN1 vesicle accumulation is dependent on the actin cytoskeleton was tested by inhibition of actin polymerisation with cytochalasin and PKN1 vesicle formation is shown to occur independently of actin. Further investigation of cytoskeletal inputs to PKN1
98 translocation reveals that PKN1 localisation has no relationship with the microtubule network. The disruption of microtubuies with nocodazole also shows that PKN1 translocation occurs independently from microtubule disruption.
The regulatory GTPase binding HR1 domain of PKN1 distinguishes PKNs from other PKCs. It was considered that this region of PKN1 could be important for the hyperosmotic induced translocation of PKN1. This idea was tested by inhibiting the HR1 interacting protein Rho, with the ADP-ribosylation factor C3 toxin. C3 has been shown to specifically inhibit Rho over other GTPases (Wilde etaL, 2000). These experiments lead to the conclusion that Rho-GTP interaction is not essential for the behaviour of PKN1 in response to hyperosmotic shock.
Since Rad has also been reported to bind the HRIabc domain of PKN1 (Flynn et al., 1998), the possible role of this PKN effector was investigated. The ability of the dominant negative Rad to suppress PKN accumulation in the vesicular compartment indicates that Rad plays a key role in this hyperosmotic-induced entry event. It is shown that Rad becomes GTP loaded in response to hyperosmotic shock, and during the course of these studies, this finding was independently reported (Lewis etaL, 2002). However this role is permissive for subsequent events rather than sufficient, since the HRIabc domain of PKN1 is not sufficient for vesicle recruitment despite retaining the Rad (and Rho) interacting domain (Amano etaL, 1996b; Vincent and Settleman, 1997; Watanabe etaL, 1996).
The evidence thus indicates that Rad recruits PKN1 at the plasma membrane where HRIabc can also interact and that the PKN 1-Rad complex but not the HRIabc-Rad complex is then endocytosed. Since HRIabc was unable to prevent GFP-PKN 1 vesicle association, it is likely that either endogenous Rad remains in excess under these conditions or that GFP-PKN1 will associate with Rad and effectively remove it from (or compete with) the available HRIabc pool. Thus, while
99 the regulatory input of Rad into PKN1 might be essential in priming PKN1 for endocytosis/vesicle recruitment, it cannot be the sole device by which PKN1 vesicle association is mediated.
Selectivity of the PKN1 response to hyperosmolarity is demonstrated over PKCe. PKN2 does show the same translocation pattern in response to hyperosmolarity suggesting that this is however a common PKN response. However, the observation that the PKN1 kinase domain can by itself localise to vesicles raises the possibility that the kinase domains of highly related PKC isoforms could behave in a similar manner. The myc-PKNI kinase domain was co-expressed with the GFP-PKCa kinase domain. After hyperosmotic stress the kinase domain of PKN1 could be detected in vesicles; in these structures the kinase domain of PKCa was absent. These data indicate that vesicle targeting of PKN1 through the kinase domain is specific for this compartment and must follow distinct mechanisms when compared with a closely related kinase domain.
The finding that the PKN1 kinase domain is in part constitutively vesicular, coupled with the observation that full length PKN1 requires Rad activation, leads to the conclusion that Rad binding to PKN1 on hyperosmotic stress induces a conformational change exposing the catalytic domain and hence allowing vesicular recruitment. Consistent with this, it has been suggested that the interaction of Rho GTPases at the amino terminal HR1 motif acts to disrupt an autoinhibitory intramolecular interaction thereby allowing activation i.e. an open conformation (Kitagawa etaL, 1996).
In summary it is shown that PKN1 is a stress responsive kinase. The induced translocation is shown to be selective over a closely related PKC although common with PKN2. Many stress induced signaling cascades are generalised stress responses, however the described PKN1 response is specific for hyperosmotic stress. PKN1 translocation is shown to be dependent on Rad and not Rho
100 although it is indicated that a distinct contact within the kinase domain is required for this translocation. These findings detail a mechanism and context within which PKN1 is selectively regulated and can potentially elicit specific downstream events. Leading on from these observations, chapter 5 addresses the question of PKN1 activity, specifically is activation loop phosphorylation and catalytic activation regulated under hyperosmolarity and is linked to the translocation response outlined here.
101 Chapter 5
PKN1 is activated by hyperosmotic stress.
5.1 Introduction
The Rad dependent translocation of PKN1 under conditions of hyperosmotic stress is characterized in chapter 4. To further explore the role of PKN1 in relation to hyperosmolarity, the activation state of PKN1 under these conditions was investigated. Previous studies have investigated the mechanism of PKN1 translocation and the interaction of Rho with PKN1 was shown to facilitate PKN1 activation loop phosphorylation by 3-Phosphoinositide Dependent Kinase 1 (PDK1) (Flynn etaL, 2000). PDK1 was originally purified as an activity responsible for PKBa activation loop phosphorylation in a manner that is dependent on PI3 kinase activity (Alessi etaL, 1997b) (Stephens etaL, 1998). PDK1 has more recently been demonstrated to phosphorylate equivalent residues on many other AGO kinases including; pYO^^"", PKA, and PKCs (reviewed in (Vanhaesebroeck and Alessi, 2000)). The in v/Vo ternary complex of Rho-PKN1-PDK1 has been shown to be dependent on PI3 kinase activity and is critical for the catalytic activation of PKN1 (Flynn etaL, 2000). The idea that GTPase inputs into PKN1 drive catalytic activation raises the possibility that Rad dependent PKN1 translocation described in chapter 4 can stimulate catalytic activation of PKN1.
This chapter seeks to address the role of PKN1 activity in response to hyperosmotic stress. Related to this, the role of PKN1 activity in vesicle recruitment is also addressed by employing a kinase inactive mutant PKN1. It was anticipated that these studies will provide an insight into the context within which PKN1 is functionally active.
102 5.2 PKN1 activity is not required for vesicie recruitment
The influence of catalytic activity on hyperosmotic-induced PKN1 vesicle recruitment was investigated by employing the drug HA1077 which has been shown to inhibit both PKN1 and PKN2 (Amano etaL, 1999; Davies etaL, 2000). Pre-treatment of GFP-PKN1 transfected cells with HA1077 followed by hyperosmotic stress did not prevent PKN1 vesicle recruitment. Interestingly cells treated with HA1077 alone under norm-osmotic conditions displayed an accumulation of vesicular GFP-PKN1 (Figure 5.1 A). To further investigate the contribution of PKN1 activity, localisation studies were performed using the kinase- dead GFP-PKN-K644R mutant. When unstimulated this inactive PKN1 mutant shows a punctate cytoplasmic distribution and after hyperosmotic stress accumulates in vesicles (Figure 5.1 B) as observed for wt GFP-PKN1. DS-Red- PKN1 and GFP-PKN1-KR were co-expressed to investigate whether either form of PKN1 could behave in a dominant fashion with respect to vesicle recruitment. Both wt and kinase dead PKN1 were cytoplasmic in unstimulated cells and co-localised in vesicles upon hyperosmotic stress (Figure 5.1 C).
103 GFP-PKN B GFP-PKN-KR (HA1077)
A
GFP-PKN1-KR DS-Red PKN1 merge
Fig 5.1 PKN1 activity is not required for vesicie recruitment.
A. NIH3T3 cells were transiently transfected with GFP-PKN1 and treated with 20|aM HA1077 for 1hour (control) or pre-treated with 20^iM HA1077 for 30min followed by 30min hyperosmotic stress maintained with HA1077 (hyperosmotic). B.NIH3T3 cells were transiently transfected with GFP-PKN1-KR, unstimulated (control) or 30min hyperosmotic stress (hyperosmotic) treatments are shown as indicated. B. NIFI3T3 cell were co-transfected with GFP-PKN1-KR and DS-Red-PKN1, unstimulated (control) or 30min hyperosmotic stress (hyperosmotic) treatments are shown. The scale bar is equivalent to
104 5.3 PKN1 undergoes activation loop phosphorylation and activation in response to hyperosmolarity.
The HA1077 effects on the basal state and osmotic-induced distribution of PKN1 (Figure 5.2), imply that its activity is not required for recruitment to vesicles but could be required for vesicle turnover or exit. However it is not clear whether activation of PKN takes place in response to osmotic stress. Activation loop phosphorylation is required for optimum catalytic activity of PKN1 (Flynn etaL, 2000). Phospho-specific polyclonal antibodies were used to assess the effect of hyperosmotic shock on GFP-PKN1 activation loop phosphorylation. A two-fold increase in phosphorylation after 30 min of hyperosmotic stress was detected on GFP-PKN 1 (Figure 5.2). An increase in endogenous PKN1 phosphorylation after hyperosmotic shock was also detected. In the example shown, a two fold increase in endogenous PKN1 phosphorylation is detected upon hyperosmotic stress, however in other experiments, our phospho-PKN antibody was unable to detect endogenous PKN1.
Immunoprecipitated GFP-PKN1 from transiently transfected NIH3T3 cells, either prior to or post hyperosmotic shock, was used to determine directly the effect of osmotic stress on activity. After hyperosmotic shock, immunoprecipitated GFP- PKN 1 displayed a specific activity approximately two-fold above un-shocked GFP- PKN 1 (Figure 5.3). The possibility of associated kinases being responsible for this activity was precluded with parallel control assays incorporating the PKN inhibitor HA1077. This inhibitor has been reported to be specific for PKN1 over kinases which can associate with it (e.g. PDK1, see below) (Amano etaL, 1999; Davies et al., 2000). Immunoprecipitated GFP-PKN1 activity after hyperosmotic shock was reduced to background levels in the presence of 20|liM MAI 077. The fact that
105 Hyper
P-GFP-PKN1 P-PKN1 GFP-PKN1 PKN1
Fig 5.2 PKN1 undergoes activation loop phosphorylation in response to hyperosmotic stress.
NIH3T3 cells were transiently transfected with GFP-PKN1 and subjected to 30min hyperosmotic stress as indicated. Activation loop Thr-774 phosphorylation of PKN1 was assessed by western blotting using a phospho- specific polyclonal antibody. Immunoreactive endogenous and GFP-PKN1 are indicated in the figure. Relative PKN1 phosphorylation was determined as a function of PKN1 protein levels by western blotting and analysed using NIH image™. The relative GFP-PKN1 phosphorylation is taken from three experiments run in duplicate and error bars denote the standard error of the mean.
106 1 2 3 Il------Il------GFP-PKNl +
Hyper +
50|rM HA1077 + GFP-PKN1 TT ! - f MBP
s 0.8 Il i IÉ
Fig 5.3 The catalytic activity of PKN1 increases with hyperosmotic stress.
Immunopurified GFP-PKN1 isolated from transfected NIFI3T3 cells was incubated with 10mM IVIgCI^, 25^g phosphatidyl-L-serine, lO^ig myelin basic protein, and 50\iM ATP for 20 minutes at 30°C in a total reaction volume of 40|il. A bead control for the immunopurification is shown (1,2). Cells were either unstimulated (1-4) or subject to 30min hyperosmotic stress prior to immunopurification (5,6). All reactions were performed in parallel as duplicates. Additional duplicate reactions incorporated the PKN inhibitor 20}.iM FIAI 077. Relative specific activity was determined as a function of immunoreactive GFP- PKNl determined by western blotting from the same filter used for MBP phosphorylation. Band intensities were analysed using NIFI image™ and error bars represent the standard error (n=3).
107 HAÏ077 did not reduce un-shocked GFP-PKNl activity could mean that basal PKN1 activity is very low.
PDK1 has previously been shown to bind to and facilitate the activation loop phosphorylation of PKN1 (Flynn etaL, 2000). Given the finding that PKN1 is both phosphorylated and activated after osmotic stress, the potential involvement of PDK1 in the control of vesicular PKN1 was assessed. GFP-PDK1 was co expressed with DS-Red-PKN1. The localisation of both proteins was cytoplasmic in unstimulated cells, but after hyperosmotic stress, GFP-PDK1 and DS-Red-PKN1 were found to be co-localised in vesicles (Figure 5.4). PI3-kinase influences PDK1 via its PH domain (Alessi etaL, 1997a; Stokoe etaL, 1997) and the subsequent phosphorylation and activation of PKN1 (Flynn etaL, 2000). The effect on osmotic responses following pre-treatment with the PI3-Kinase inhibitor LY294002 was investigated. After hyperosmotic stress in the presence of LY294002, GFP-PDK1 was no longer recruited to DS-Red-PKN1 positive vesicles. Notably, DS-Red-PKN1 was still recruited to vesicles, however these were smaller after the pre-treatment with LY294002 (Figure 5.4).
Chapter 4 shows that PKN1 kinase domain is sufficient for vesicle recruitment, previous studies have described PKN1-PDK1 interactions that occur via the 0- terminal PI F (PDK1 interacting fragment) PKN region which has been shown to interact with a hydrophobic pocket of PDK1. This is thought to act as a “docking site” for PDK1 substrates (Biondi etaL, 2000). The nature of PDK1-PKN co recruitment was therefore investigated by co-expressing myc-PKNI kinase domain with GFP-PDK1. It is shown that PDK1 and PKN1 kinase domain co-localise in vesicles under control conditions and after hyperosmotic stress (Figure 5.5).
The widely studied downstream target of PDK1, PKB is also known to be modulated upon hyperosmolarity (Meier etaL, 1998). In the light of PDK1
108 GFP-PDK1 DS-Red PKN1 merge
CT) (D
Fig 5.4 LY294002 sensitive GFP-PDK1 recruitment to DS-Red-PKN1 hyperosmotic induced vesicles.
NIH-3T3 cells were transiently co-transfected with DS-Red-PKN1 and GPP- PDK1. Cells were unstimulated (control), treated with 30min hyperosmotic stress (hyperosmotic) and pre-treated for 20min with 10^m LY294002 as indicated. The scale bar is equivalent to 10|im.
109 GFP-PDK1 Myc-PKN1-kin merge
Fig 5.5 PDK1 Is recruited to PKN1 kinase domain containing vesicles under control conditions and upon hyperosmotic stress .
NIH-3T3 cells were transiently co-transfected with GFP-PDK1 and Myc-PKN1 kinase domain (Kin). Cells were unstimulated (control), or treated with 30min hyperosmotic stress (hyperosmotic). The scale bar is equivalent to 10|im.
110 recruitment to hyperosmotic-induced PKN1 vesicles, PKB localisation under these circumstances was investigated. It is shown that that GFP-PKB and DS-Red-PKN1 when co-expressed do not co-localise in hyperosmotic induced PKN1 vesicles. GFP-PKB was shown to be cytosolic with some nuclear accumulation as others have seen (Watton and Downward, 1999), this localisation did not change upon hyperosmotic stress (Figure 5.6).
I l l GFP-PKB DS-Red-PKN merge
c o Ü
(D & X
Fig 5.6 PKB does not localise to hyperosmotlc-lnduced PKN1 vesicles.
NIH-3T3 cells were transiently co-transfected with DS-Red-PKN1 and GFP- PKB. Cells were unstimulated (control) or treated with 30min hyperosmotic stress (hyperosmotic). The scale bar is equivalent to 10^m.
112 5.4 Density fractionation reveals the translocation of endogenous PKN1 and PKN2 In response to hyperosmolarlty and with kinase Inhibition.
To reveal the behaviour of endogenous PKN1 and PKN2 upon hyperosmotic stress, an attempt was made to isolate enriched hyperosmotic induced PKN1 vesicles by density fractionation. NIH 3T3 cells were stimulated with; hyperosmotic media, HA1077 and staurosporine. The PKN inhibitor HA1077 was shown in 5.1 to induce PKN1 vesicular accumulation, staurosporine was used as a second PKN inhibitor albeit a more broad-spectrum kinase inhibitor. Cells were processed according to the fractionation procedure set out in chapter 2 (Figure 5.7). It is shown that endogenous PKN1 is detected in fractions 3-7 under control conditions and fractions 3-8 after hyperosmotic stress. The translocation to higher density fractions is more pronounced with HA1077 treatment, as detected in fractions 3-10 and after staurosporine treatment in fractions 2-9. Endogenous PKN2 is detected principally in fractions 3-8 under control conditions and after hyperosmotic stress is detected in fractions 3-9. After HA1077, PKN2 is present in fractions 3-9 with an enrichment in fractions 7-9 and after staurosporine treatment PKN2 is present in fractions 3-10 with an enrichment in fractions 7-10. The most obvious translocation detected is the effect of staurosporine treatment on PKN2. However a broad distribution of both PKN1 and PKN2 still remains under all conditions tested.
113 Sucrose Molarity 0.3M 1.2M
Fraction 1 2 7 8 9 10 11
PKN1 Control Hyper MAI 077 Li: Stauro
PKN2 Control Hyper HA1077 Stauro
Fig 5.7 Density fractionation of NIH3T3 cells. Endogenous PKN1 and PKN2 move to increasingly dense fractions upon hyperosmotic stress and treatments of HA1077 and Staurosporine.
NIH3T3 cells were either unstimulated (control) or subjected to 30min hyperosmotic stress (Hyper) and treatments of HA1077 (20piM) or Staurosporine (500nM) as indicated. Cells were processed according to the density fractionation procedure in chapter 2, run on SDS PAGE and subjected to western blot analysis.
114 5.5 Kinase reactions with immuno-purified PKN1 and ceii lysate do not reveal hyperosmotlc-lnduced PKN1 substrates.
Since density fractionation did not reveal total translocation of PKN1 upon hyperosmotic stress, the question of whether hyperosmotic-induced PKN1 can phosphorylate specific substrates was addressed. The increase in the catalytic activity of PKN1 after hyperosmotic stress is described in section 5.3. An attempt was made to utilize this increased activity in an in vitro reaction by using NIH-3T3 cell lysate to screen for specific PKN1 substrates. Immunoprecipitated GFP-PKN1 from transiently transfected NIH3T3 cells, either prior to or post hyperosmotic shock, was used to assess whether hyperosmotic induced PKN1 substrates could be visualised. Phosphorylated bands are shown to appear as a result of the incubation of immunoprecipitated PKN1, examples are indicated with arrows (Figure 5.8). These proteins are activated in a manner that is insensitive to pre heating the cell lysate at 65°C for 10 min. This treatment was included to inactivate endogenous kinases, these reactions therefore represent more stringent conditions for identifying specific PKN1 substrates. However, no hyperosmotic-induced candidate PKN1 substrates were detected.
115 1 r 1 r GFP-PKNl - + + + 4- + + Hyper - - - + + + + HA1077 - ---- + + Lysate A BAB AB AB
PKN1 auto 96KDa
» '"î
66KDa
Fig 5.8 Incubation of immunopurified GFP-PKN1 with NIH3T3 cell lysate reveals several PKN1 dependent heat stable substrates, no hyperosmotic-induced PKN1 substrates are detected.
Immunopurified GFP-PKN1 isolated from transfected NIH3T3 cells was incubated with 10mM IVIgCI^, 25^g phosphatidyl-L-serine, and SOpilVI ATP. Kinase reactions were incubated with NIH3T3 cell lysate (A) or NIH3T3 cell lysate pre-heated at 65°C for 10min. Reactions were performed for 20 minutes at 30°C. A bead control for the immunopurification is shown (1 ). Cells were either unstimulated (2) or subject to 30min hyperosmotic stress prior to immunopurification (3-4). Additional reactions incorporated the PKN inhibitor HA1077 (20^M) (4). PKN1 autophosphorylation is indicated (PKN1 auto) and PKN1 dependent substrates are indicated by arrows.
116 5.6 Discussion
It is shown that PKN1 activity is not required for hyperosmotic induced vesicle accumulation, although PKN1 is observed to undergo activation loop phosphorylation and catalytic activation under these conditions. This activation is paralleled by PDK1 recruitment to PKN1 vesicles under hyperosmolarlty, however PKB, another PDK1 substrate is shown not to be recruited to this compartment. Attempts have also been made to isolate enriched fractions of PKN1 vesicles and to screen for hyperosmotic-PKN 1 substrates.
The finding that the kinase inactive GFP-PKN1-K644R mutant localises to hyperosmotic-induced PKN1 vesicles demonstrates that intrinsic PKN1 catalytic activity is not required for vesicle recruitment. The PKN inhibitor, HA1077 does not prevent hyperosmotic induced PKN1 vesicle accumulation but in unstimulated cells does induce PKN1 vesicle accumulation. These findings indicate a role for PKN catalytic activity in either vesicle exit or turnover and maybe part of a constitutive process.
The activation loop phosphorylation of PKN1 has been shown to be a pre-requisite for PKN1 catalytic activity and to be under the influence of Rho GTPases (Flynn et al., 2000). Indeed both activation loop phosphorylation and catalytic activity are shown to increase under hyperosmolarity. These findings provide a context in which PKN1 is activated and can signal to downstream targets.
The recruitment of PDK1 to PKN1 vesicles is described and is shown to occur through the kinase domain of PKN1, this is likely to involve the FXXFDY motif described as a PDK1 docking site (Biondi eta!., 2002). This vesicular interaction is not responsible however for the accumulation of PKN1, since this still occurs when
117 PDK1 recruitment is blocked by the PI3 kinase inhibitor LY294002. Thus the kinase domain docking in the vesicle membrane must be determined by a distinct protein (or perhaps lipid) contact. The inhibitory effect of LY294002 on PDK1 recruitment to PKN1, indicates that PI3,4,5P3 is required to influence the conformation of PKN1 or that of PDK1 to facilitate complex formation. Both of these proteins have been shown to be influenced by PI3,4,5P3 however in the case of PKN1 no specificity was observed relative to the precursor lipid PI4,5Pg (Palmer et ai, 1995a). Hence it is likely that the role of PI3,4,5P3 is to enhance membrane occupancy of PDK1 through its PH domain (Alessi et ai, 1997a) and so facilitate recruitment to the membrane bound PKN1. The reduction in size of PKN1 positive vesicles on treatment of shocked cells with LY294002 suggests that the larger vesicles observed are a consequence of a PI3 kinase-dependent vesicle fusion event; this is a characteristic of homotypic endosome fusion (Jones and Clague, 1995).
A hyperosmotic stress response has been described previously for the Rad effector, p21-activated protein kinase y-PAK which binds to and is activated by R a d . y-PAK has been shown to translocate from a soluble to a particulate fraction and become activated in response to hyperosmolarity (Roig et ai, 2000). Interestingly it was demonstrated that the activation but not translocation of y-PAK was sensitive to wortmannin, suggesting a two-step mechanism for the y-PAK response to hyperosmotic stress. This parallels the situation described here for PKN1 where inhibition of PI3 kinase does not prevent vesicle accumulation of PKN1, while blocking recruitment of the upstream kinase PDK1. It has been shown previously that inhibition of PI3 kinase will block activation loop phosphorylation of PKN1 (Flynn et ai, 2000). Since PKN1 translocation is shown to be dependent on Rad and independent from PI3 kinase, it would seem likely that under hyperosmotic stress Rad can be GTP loaded independently from PI3 kinase activity. This highlights a perhaps very specialised mechanism of Rac activation given that PI3 kinase has been shown to be an upstream regulator of Rac (Han et ai, 1998).
118 The combined Rac1/PI3,4,5P3 regulatory input that facilitates PDK1 phosphorylation of PKN1 provides further evidence for the view that the specificity of PDK1 actions is driven by the co-association of regulatory inputs to target kinases (Vanhaesebroeck and Alessi, 2000). In this context it is notable that despite the requirement of PI3 kinase for PDK1 recruitment, PKB which is also recruited by 3-phosphoinositides, is not recruited to this hyperosmotic-induced compartment. This suggests that PKB is either actively removed or its affinity for PIP3 is weaker. Indeed the PH domain of PKB was reported to have a lower affinity for PIP3 when compared with PDK1 as reviewed (Vanhaesebroeck and Alessi, 2000).
The hyperosmotic-induced PKN1 compartment was further investigated biochemically by density fractionation. In attempting to isolate fractions that are enriched with PKN1 vesicles, it was anticipated if achieved, that these could be further exploited by 2D electrophoresis and mass spectrometry to identify other constituent proteins which could help explain the functional role of PKN1 in this setting. It is shown that endogenous PKN1 does translocate to more dense fractions (which are indicative of vesicular fractions) with hyperosmolarity and this is also observed for PKN2. The kinase inhibitors HA1077 and staurosporine induce a more robust translocation to higher density fractions. The finding that staurosporine induced the most dramatic change in the profile of PKN2 points to multiple kinase inputs in controlling vesicle turnover or exit. The extent of translocation however is not total and PKN distribution remains dispersed over many fractions. It could be that PKN1 association to this membrane bound compartment is a weak interaction and could be disrupted during lysis or processing. Future experiments to exploit this technique in isolating hyperosmotic induced PKN1 vesicle enriched fractions could involve the use of irreversible crosslinkers to stabilise protein-proteins and protein-lipid interactions such as DTSSP (Jung and Moroi, 1983). Alternatively these could involve “Spiking” by
119 transfection with vesicle associated proteins such as the overexpressed kinase domain of PKN1.
Since PKN1 catalytic activity is shown to increase under hyperosmolarity, PKN1 substrates were sought using immunoprecipitated PKN1 from cells harvested prior to or post hyperosmotic stress, incubated in a kinase reaction with NIH3T3 cell lysate. This approach is shown to reveal PKN1 specific substrates as is shown by the arrows in Figure 5.8. One of these is approximately BOKDa and previously the BOKDa protein MARCKs was shown to be a substrate of PKN by a similar technique (Palmer etal., 1996). However, no such candidate PKN1 substrates associated with hyperosmotically treated samples were detected. It could be that this is due to the lack of abundance of such proteins and hence beyond the detection limits of this assay. This approach could, however by re-examined as a method for the identification of PKN1 substrates.
In summary PKN1 is shown to be a stress responsive kinase. The selective translocation of PKN1 coupled with its subsequent activation, details a mechanism by which hyperosmotic shock can elicit a particular repertoire of responses through this kinase. The outputs for these events are examined in Chapter 6 which utilises PKN 1-KO mouse embryonic fibroblast cells to address this question.
120 Chapter 6
PKN1 involvement in the hyperosmotic-induced
JNK pathway.
6.1 Introduction
The acute translocation to vesicles and associated activation of PKN1 in response to hyperosmotic stress has been detailed in chapters 4 and 5. Hyperosmotic stress is a potent activator of several signalling cascades which include stress activated protein kinase (SAPK or JNK) (Galcheva-Gargova etal., 1994), p38 (Han eta!., 1994) and extracellular signal-regulated kinases (ERKs) (Matsuda etal., 1995). In contrast PKB is downregulated by hyperosmotic stress via dephosphorylation of its regulatory Thr308 and Ser473 phosphorylation sites (Meier etal., 1998).
Precedent has also been set for the involvement of PKCs in the hyperosmotic stress response since classical and novel PKG activation is suggested to be a requirement for hyperosmotic-induced ERK activation (Zhuang etal., 2000). The yeast PKCs, which closely resemble PKNs, are known to activate the MAP kinase cascades involving BCK (MAPKKK), MKK1 and MKK2 (MAPK), and MpKI and have been linked to the control of cell wall integrity (Arellano etal., 1999; I he etal., 1993; Levin etal., 1994). This control mechanism has been likened to the osmotic stress response (Alonso-Monge etal., 2001). Mammalian PKNs have also been linked to MAP kinase responses since PKN1 was shown to be on a pathway involving p38y (Marinissen etal., 2001). PKN1 has more recently been shown to phosphorylate the novel MAPK kinase kinase, MLTK, which is also on the p38 MAPK pathway (Takahashi etal., 2003). Furthermore, PKN2 has been shown to
121 bind MEKK2, although this was independent of PKN1 (Sun etal., 2000a). The related p38, JNK, and ERK MAP kinases therefore represent good candidate pathways in which PKN1 may operate under hyperosmotic stress.
This chapter seeks to address the involvement of PKN1 in stress activated MAP kinase pathways and the hyperosmotic-induced PKB response. Initially these studies involve the transient overexpression of PKN1 and monitoring of these pathways. Subsequent studies also employ primary PKN1 knockout mouse embryonic fibroblast (PKN1-K0-MEF) cells in comparison with wild type (WT) MEFs. The absence of PKN1 should reveal any PKN1 dependency in these stress-activated cascades.
122 6.2 GFP-PKN1 overexpression and the stress responses
Hyperosmotic stress is known to activate the stress activated protein kinase (JNK) (Galcheva-Gargova etal., 1994), p38 MAP kinase pathway (Han etal., 1994) and is known to down-regulate PKB (Meier etal., 1998). It is shown that transient overexpression of GFP-PKNl does not appear to influence the basal activity nor hyperosmotic-induction of these pathways (Figure 6.1). Since any differences here could be obscured by low transfection efficiency, an alternative loss of function approach was adopted. This involved primary PKN1-K0 versus WT MEF cells.
123 Hyperosmotic Stress + + GFP-PKNl + +
GFP-PKN1 > PKN1 >
PKB
JNK-P
...... p38-P
p38
Fig 6.1 Transient overexpression of GFP-PKNl has no effect on hyperosmotic-induced PKB degradation, JNK or p38 phosphorylation.
NIH-3T3 cells were transiently transfected with GFP-PKN1. Cells were unstimulated or subject to 30min hyperosmotic stress as indicated. Samples were harvested in Laemmli sample buffer, run on SDS-PAGE followed by western blot analysis. The p38 protein serves as a loading control.
124 6.3 PKN2 and PKNS levels are unaffected in PKN1-K0 MEFs.
The impact of PKN1 deletion on PKN2 and PKN3 expression levels was investigated by comparing PKN1-K0-MEF cells with WT-MEFs. PKN3 polyclonal antibodies were generated as described in the materials and methods section and used in western analysis (Figure 6.2 A). This antibody is shown to cross-react with PKN1, as confirmed by lack of immuno-reactivity in PKN1 KO cells, see figure. Indeed the peptide sequence used for the generation of the PKN3 antibody shares significant homology with PKN1 (Figure 6.2 B). Both PKN3 and PKN1 signals were competed by immunoblotting PKN3 antibody in the presence of excess PKN3 peptide. It is evident that no differences in PKN3 protein levels were detected between WT and PKN 1-KO MEF cells. Commercial PKN2 monoclonal antibodies were used to asses PKN2 levels (Figure 6.2 A), and it is clear that PKN2 levels are not altered in PKN 1-KO MEF cells.
125 + PKNS peptide
WT PKN 1-KO WT PKN 1-KG
PKNS PKN1 PKNS I PKN2
B PKNl QAAFLDFDFVAGGC--- PKN3 QAAFRDFDFVSERFLEP
■k -k -k -k k k k k k
Fig 6.2 PKN2 and PKN3 expression is unaffected in PKN1-K0 MEF cells.
A.Wt and PKN1-K0 primary MEF cell extracts were harvested in Laemmli sample buffer, run on SDS-PAGE and analysed by western blotting. Membranes were incubated with PKNS antibody, PKNS antibody with competing peptide and PKN2 antibody as indicated. B. Alignment of peptide sequence used to generate PKNS antibody with PKN1 sequence, stars indicate conserved residues.
126 6.4 PKB, p38 and ERK1/2 responses to hyperosmotic stress are not compromised in PKN1-K0 MEFs.
PKB is known to be desphosphorylated and degraded upon hyperosmotic stress (Meier etal., 1998). PKB S473 phosphorylation was monitored in WT and PKN1- KO cells after 16 hours of serum starvation followed by stimulation with hyperosmotic stress (Figure 6.3). It was observed that PKB S473 dephosphorylation is detected in both WT and PKNl-KO MEFs following hyperosmotic stress. It is also clear however, that resting PKB S473 phosphorylation levels in PKNl-KG MEFs are elevated, this experiment has since been repeated and confirmed in conjunction with Dr A. Casamassima.
Both p38 and ERK1/2 are activated in response to hyperosmotic stress and their activation was monitored in PKNl-KO MEF cells (Figure 6.4). It is clear that there is no detectable difference in the pattern of p38 and ERK1/2 activation between WT and PKNl-KO -MEF cells. A sustained activation in response to hyperosmotic stress from ten minutes onwards is shown for both p38 and ERK in PKNl-KO and WT MEF cells.
127 WT PKN 1-KO Hyperosmotic Stress (min) 0 5 10 30 0 5 10 30 p-473
Total PKB ' m - ■ m
Fig 6.3 Hyperosmotlc-lnduced déphosphorylation of PKN 8473 Is detected In both WT and PKN1-K0 MEFs.
Wt and PKNl-KG primary MEF cells were subjected to hyperosmotic stress as indicated. Samples were harvested in Laemmli sample buffer, run on SDS- PAGE and analysed by western blotting.
128 WT PKN1-K0 Hyperosmotic ------Stress (min) 0 10 30 60 90 0 10 30 60 90
Tubulin
pERK1/2 > ^ ^ .^1,.___ ^ ■
Tubulin >► ^
Fig 6.4 Hyperosmotic-induced p38 and ERK1/2 phosphorylation is unaffected in PKN1-K0 MEF cells.
Wt and PK N l-KO primary MEF cells were subjected to hyperosmotic stress as indicated. Samples were harvested in Laemmli sample buffer, run on SDS- PAGE and analysed by western blotting.
129 6.5 PKN1 is required for efficient hyperosmotic-induced JNK and MKK4 activation.
The stress activated protein kinase or JNK pathway is established as being activated by a variety of stress stimuli including hyperosmotic stress (Derijard etal., 1994; Galcheva-Gargova etal., 1994). An increase in phosphorylation of the major JNK isoforms (46Kda and 54Kda) is detected in response to hyperosmolarity and this response is shown to be dramatically reduced in PKNl-KO MEF cells (Figure 6.5). It is also noted that the phospho JNK western shows other bands which are modulated by hyperosmolarity and which are inhibited in PKN1-K0-MEF cells. Since JNK proteins are known to produce many splice variants (Gupta et al., 1996), it is likely that these are indeed other JNK proteins. The JNK pathway was further investigated by monitoring the phosphorylation status of a JNK activator or MAPK kinase namely, MKK4 (Sanchez etal., 1994). MKK4 is also known to be activated following hyperosmotic stress, (Moriguchi etal., 1995) and elevated phosphorylation of MKK4 in response to hyperosmolarity is detected in a similar profile to JNK induction (Figure 6.5). Hyperosmotic-induced MKK4 phosphorylation is also dramatically reduced in PKNl-KO MEF cells in comparison with WT-MEF cells.
It has been suggested that different MAPK kinase Kinases are responsible for JNK activation in a stimulus specific manner (Chen etal., 2002). The selectivity of PKNl dependent JNK activation was therefore investigated. Following UV stress, a known JNK activator, (Derijard etal., 1994) MKK4 and JNK activation is detected in both WT and PKNl-KG MEF cells with no detectable difference (Figure 6.6). p38 and ERK1/2 responses to UV stress are also unaffected in PKNl-KG MEF cells. TNF stimulation is also know to stimulate JNK activation (Sanchez etal., 1994). It is
130 Hyperosmotic PKN 1-KO Stress (min) 0 10 30 60 90 0 10 30 60 90
p-JNK — (46 and 54kda)
Tubulin
P-MKK4
MKK4
Fig 6.5 Inhibition of hyperosmotic-induced JNK and MKK4 phosphorylation in PKN1-K0MEF cells.
Wt and PKNl-KG primary MEF cells were subject to hyperosmotic stress as indicated. Samples were harvested in Laemmli sample buffer, run on SDS-PAGE and analysed by western blotting.
131 WT PKN 1-KO
Post-UV (min) 0 C 15 30 60 120 0 0 15 30 60 120
P-MKK4 ---- >
Total MKK4 p-JNK
Total JNK p-p38
Tubulin
P-ERK1/2
Fig 6.6 MKK4, JNK, p38 and ERK1/2 activaton is unaffected in PKN1-K0 MEFS following UV stress.
Wt and PKNl-KG primary MEF cells were treated with UV stress (dose of 30 J/m^). Samples were harvested post UV treatment at the time point indicated in Laemmli sample buffer, run on SDS-PAGE and analysed by western blotting.
132 also shown that TNF-induced JNK, ERK1/2, and p38 phosphorylation are unaffected by the absence of PKN1 (Figure 6.7).
Hyperosmotic stress can induce a range of transcription factor responses resulting in gene expression changes and alterations in the activation state of signalling pathways. The possibility that hyperosmotic-induced JNK phosphorylation could arise from changes in gene induction resulting from the activation of parallel pathways such as p38, was investigated by inhibiting protein synthesis using cycloheximide (Figure 6.8). It is shown that pretreatment with cycloheximide does not inhibit hyperosmotic-induced JNK or ERK1/2 activation, although cycloheximide pretreatment did induce some JNK phosphorylation without stress. Cycloheximide pre-treatment in PKN1-K0-MEF cells also elevated resting levels of JNK phosphorylation. Cycloheximide did not however affect the compromised JNK activation with respect to hyperosmolarity in PKN1-K0-MEF cells.
133 TNFa WT PKN1-K0 (min) 0 10 30 60 0 10 30 60
P-MKK4
Total MKK4 p-JNK (46 and 54kda)
Total JNK
Tubulin — ^ ^
P-ERK1/2 ^
Fig 6.7 MKK4, JNK, p38 and ERK1/2 activation is unaffected in PKN1-K0 MEF cells following TNFa stimulation.
Wt and PKN1-K0 primary MEF cells were treated with 20ng/ml TNFa as indicated. Samples were harvested in Laemmli sample buffer, run on SDS- PAGE and analysed by western blotting.
134 W T PK N 1-K 0 30min 50^ig/ml cycloheximide - - + + + - - - + + +
Hyperosmotic 0 60 90 0 60 90 0 60 90 0 60 90 Stress (min)
p-JNK (46 and 54kda)
Tubulin
P-ERK1/2
Tubulin
Fig 6.8 Cycloheximide pre-treatment does not inhibit the prolonged phosphorylation of ERK1/2 and JNK following hyperosmotic stress.
Wt and PKN1-K0 primary MEF cells were treated with cycloheximide (50^ig/ml) for SOmin where indicated and/or then subjected to hyperosmotic stress for the time-points shown. Samples were harvested in Laemmli sample buffer, run on SDS-PAGE and analysed by western blotting.
135 6.6 PKN1 activity contributes to hyperosmotic-induced JNK activation.
Previous work has demonstrated PKN1 activity to be dependent on PI3-kinase (Flynn etal., 2000). It is also shown in chapter 5 that PKN1 undergoes catalytic activation following hyperosmotic stress. The role of PKN1 activity under hyperosmolarity with respect to MKK4 and JNK induction was therefore investigated by employing the PI3-kinase inhibitor, LY294002, and the ROCK inhibitor HA1077 which has been shown to inhibit PKN1 directly (Amano etal., 1999).
Pretreatment with LY294002 shows some inhibition of hyperosmotic-induced MKK4 and JNK phosphorylation (Figure 6.9), however this effect is much less than that seen in PKN1-K0-MEF cells. Direct inhibition of PKN1 catalytic function by pretreatment with HA1077 inhibited hyperosmotic-induced MKK4 phosphorylation in WT MEF cells and further inhibited the low level of MKK4 phosphorylation induced in PKN1-K0-MEF cells (Figure 6.10). However, hyperosmotic-induced JNK phosphorylation was largely unaffected in WT MEF cells, a partial inhibition of JNK phosphorylation in PKN1-K0 MEF cells is detected. These studies are consistent with recent studies of by Dr A. Casamassima from the laboratory.
The most well characterised target for JNK is c-Jun (Derijard etal., 1994). The role of PKN1 in hyperosmotic-induced c-Jun phosphorylation was monitored by immunostaining for phospho c-Jun in WT and PKN1-K0 MEF cells. It was apparent that phospho c-Jun levels increased dramatically upon hyperosmotic stress in WT-MEF cells, consistent with JNK induction. In PKN1-K0 MEF cells, basal levels of phospho c-Jun are reduced compared with WT MEF cells. Upon hyperosmotic stress, increased phospho c-Jun levels are detected in PKN1-K0-
136 W T PK N1-K 0
20min + + + - - - + + + LY294002
Hyperosmotic 0 60 90 G 60 90 0 60 90 0 60 90 Stress (min)
P-MKK4
Tubulin
p-JNK (46 and 54kda)
Tubulin
P-ERK1/2
Tubulin
Fig 6.9 Hyperosmotic-induced MKK4, JNK, and ERK1/2 activaton are partially inhibited by LY294002.
Wt and PKN1-K0 primary MEF cells were treated with 10^iM LY294002 for 20min and/or then subjected to hyperosmotic stress as indicated. Samples were harvested in Laemmli sample buffer, run on SDS-PAGE and analysed by western blotting.
137 W T PK N 1-K 0
HA1077 + + + -- - + 4-4-
Hyperosmotic Stress (min) 0 60 90 0 60 90 0 60 90 0 60 90
P-MKK4
p-JNK (46 and 54kda)
Tubulin
Fig 6.10 Hyperosmotic-induced MKK4 activation is inhibited by MAI 077.
Wt and PKN1-K0 primary MEF cells were treated with 20^iM HA1077 for 1h and/or then subjected to hyperosmotic stress as indicated. Samples were harvested in Laemmli sample buffer, run on SDS-PAGE and analysed by western blotting.
138 MEF cells, however these levels are lower compared with WT MEF cells (Figure
6.11).
The influence of PKN1 in driving c-Jun activation was further investigated by transiently transfecting WT and PKN1-K0 MEF cells with GFP-PKN1 and the kinase inactive GFP-PKN1-KR mutant. It is shown that in WT MEF cells, under basal conditions, GFP-PKN1 does not increase phospho c-Jun levels, however under hyperosmotic stress, GFP-PKN1 transfectants appear to have higher levels of phospho c-Jun (Figure 6.12). In PKN1-K0 MEF cells, GFP-PKN1 has no effect on basal phospho c-Jun levels although upon hyperosmotic stress, GFP-PKN1 transfectants have increased levels of phopsho c-Jun (Figure 6.12). Upon transfection of the kinase dead GFP-PKN1-KR mutant, it is shown that GFP-PKN1 KR does not prevent hyperosmotic-induced phospho c-Jun induction in WT MEF cells. However in PKN1-K0 MEF cells, GFP-PKN1-KR does not potentiate phospho c-Jun induction (Figure 6.13).
139 WT-MEF PKN1- KO-MEF
Fig 6.11 Basal and hyperosmotic-induced phospho c-Jun levels are reduced in PKN1-K0 MEF ceils.
Wt and PKN1-K0 primary MEF cells were unstimulated (control) or subjected to 1 hour hyperosmotic stress as indicated. Cells were stained with anti-phospho S63 c-Jun antibodiy. The scale bar is equivalent to
140 WTMEF GFP-PKN1-WT Phos-c-Jun merge
PKN1-K0-MEF GFP-PKN1-WT Phos-c-Jun merge
Fig 6.12 GFP-PKN1 potentiates hyperosmotic-induced phospho c-Jun in Wt and PKN1-K0 MEF cells.
Wt and PKN1-K0 primary MEF cells were transiently transfected with GFP- PKN1 and were unstimulated (control) or subject to 1 hour hyperosmotic stress as indicated. Cells were stained with anti-phospho S63 c-Jun antibody. The scale bar is equivalent to 10^m. 141 WTMEF GFP-PKN1-KR Phos-c-Jun merge
c o Ü
PKN1-K0-MEF GFP-PKN1-KR Phos-c-Jun merge
Fig 6.13 GFP-PKN1-KR does not block nor prevent hyperosmotic-induced c-Jun phosphorylation.
Wt and PKN1-K0 primary MEF cells were transiently transfected with GFP- PKN1-KR and were unstimulated (control) or subject to 1 hour hyperosmotic stress as indicated. Cells were stained with anti-phospho S63 c-Jun antibody. The scale bar is equivalent to 10^m.
142 6.7 Discussion
This chapter set out to establish a role for PKN1 in classical stress responses given the data presented in chapter 5, which outlined the hyperosmotic-induced activation of PKN1. The transient overexpression of GFP-PKN1 was shown not to affect hyperosmotic-induced p38, JNK phosphorylation, or PKB down-regulation. However it is also clear that only limited transfection efficiency is achieved and that another approach is needed to investigate further the role of PKN1 in relation to these known stress activated cascades.
PKN1-KO-MEF cells, generated in the lab by Dr A. Casamassima, were employed as another approach to study the involvement of PKN1 in stress responses. PKN1- KO-MEF cells are shown to express similar levels of other PKNs compared with WT MEF cells. Since no compensation in terms of up-regulated PKNs is detected in these primary MEF cell cultures, these cells are considered valid tools for dissecting the role of PKN1.
Data presented in this chapter show that PKN1 is not required for hyperosmotic- induced PKB dephosphorylation. Since it has been shown that PKB dephosphorylation in response to hyperosmotic stress can be prevented by phosphatase inhibitors (Meier etal., 1998), it is possible that any influence of PKN1 on PKB phosphorylation is masked by phosphatase induction. Interestingly, PKN1 would appear to have some role in PKB control given elevated steady state levels of PKB-S473 phosphorylation in PKN1-KO-MEF cells. The mechanism by which PKN1 exerts a negative influence over S473 phosphorylation was not investigated, however this could conceivably involve competition for access to the upstream kinase shared by both PKN1 and PKB, namely PDK1. The absence of PKN1 could alter levels of free PDK1 and increase the incidence of PKB-PDK1 complexes.
143 Alternatively, PKN1 may directly have a negative influence on PKB phosphorylation, indeed it has previously been suggested that PKNs can inhibit PDK1 mediated PKB phosphorylation (Wick etal., 2000).
It is shown that the hyperosmotic-induced JNK is impaired in PKN1-KO-MEF cells. Given that the absence of PKN1 results in defective JNK and MKK4, the possibility is raised that PKN1 could function at the MAP3K level or higher within this cascade. The findings that p38, and ERK1/2 MAP kinase hyperosmotic responses are intact in PKN1-KO-MEF indicate that PKN1 operates selectively through the JNK pathway over parallel MAP kinase signalling events.
MKK4 is known to bifurcate MAP kinase signalling pathways by directly activating both JNK and p38 pathways (Derijard etal., 1995). Results presented here however indicate that PKN1 may engage MKK4-JNK signals independently from p38 since in PKN1-K0-MEFs, p38 induction is unaffected. Such selectivity within these pathways has been described since Rad was shown to regulate the JNK response to hypertrophic stimuli independently from p38 activation (Clerk et al., 2001). The data described indicates that PKN1 could be important in conferring specificity to MKK4-JNK induction, whilst other MAPK kinases control p38 activation under these conditions.
The JNK pathway is known to have at least two MAPK kinases, namely MKK4 and MKK7 and several MAPK kinase kinases such as TAK1, MLK1, and ASK1. It has been shown that MAP3 kinases can work on a stimulus specific basis (Chen et al., 2002) and it is thought that these integrate signals from upstream activators such as R a d . Data presented here indicate that PKN1 controls operating on the JNK pathway lie upstream of MKK4 and are specific for hyperosmotic stress over other JNK activators such as UV stress and TNF stimulation. Direct evidence for the selective MKK4 activation of JNK pathway has come from MKK4-K0-MEF cells that are deficient in JNK signals in response to IL-1, TNFa, hyperosmotic stress
144 and anisomycin (Ganiatsas etal., 1998). Also in this study, p38 responses were inhibited in MKK4-K0-MEF cells upon stimulation with IL-1, TNFa and anisomycin, however the p38 response was intact upon hyperosmotic stress. These findings support the notion that signalling complexes are assembled under specific circumstances, in this case resulting in conferring specificity to MKK4 and the level of cross talk between MAP kinase signalling. Results described here therefore indicate that PKN1 may play a role in specifying the hyperosmotic-induced MKK4- JNK pathway.
It is concluded that the hyperosmotic-induced JNK phosphorylation arises from direct activation of the JNK cascade rather than the induction of gene expression since the inhibition of protein synthesis is shown not to block hyperosmotic-induced JNK phosphorylation. Cycloheximide treatment is however shown to stimulate JNK phosphorylation in WT-MEF cells and to a lesser extent in PKN1-KO-MEF cells. These results imply that PKN1 may have a direct role in JNK pathway signalling under conditions of hyperosmolarity and also under JNK signalling triggered by cycloheximide.
The described PKN1 requirement for hyperosmotic-induced MKK4-JNK activation, together with the finding that PKN1 is activated under hyperosmolarity, (Torbett at a/., 2003) raises the possibility that PKN1 may be a direct MAPK kinase kinase or have an important catalytic role in MKK4 complex assembly. It is also possible that PKN1 dependent MKK4-JNK activation could be the result of a scaffolding role for PKN1 within this cascade. PI3-Kinase inhibition using the drug LY294002 results in the partial inhibition of MKK4 and JNK in wt cells and no difference was detected in PKNI-KO-MEFs (Figure 6.8). Direct PKN1 inhibition with HA1077 resulted in reduced MKK4 activation in WT MEFs and residual MKK4 activation observed in PKNI-KO-MEFs is further diminished with HA1077 treatment. Partial inhibition of JNK induction with HA1077 is also detected in PKNI-KO-MEFs (Figure 6.9). These findings imply that PKN1 activity may contribute to PKN1 dependent hyperosmotic-
145 induced MKK4-JNK activation. The fact that these inhibitor treatments do not reduce WT-MEF MKK4 or JNK induction to levels seen in PKN1-KO-MEF cells could imply that either PKN1 plays a scaffolding role in this context or that catalytic inhibition is not fully achieved. However the observation that MKK4 and JNK activation is further inhibited by HA1077 in PKN1-K0-MEFs is suggestive of a potential catalytic role for other PKNs such as PKN2 or PKN3. This idea is supported by the finding that PKN2 activity is sensitive to HA1077. Taken together, the data points to a catalytic rather than a scaffolding role for PKN1 in MKK4-JNK hyperosmotic induction.
Consistent with the notion that PKN1 operates within the JNK cascade under conditions of hyperosmolarity, it is shown that PKN1-K0 MEF cells have a reduced capacity for c-Jun induction under hyperosmotic stress. Furthermore, the re- introduction of PKN1 is shown to restore the ability of PKN1-K0 cells to activate c- Jun after hyperosmotic shock. The observation that the kinase dead GFP-PKN1- KR does not restore c-Jun activation provides further evidence for PKN1 catalytic involvement in the JNK cascade. Since PKN1-KR does not prevent c-Jun induction in WT MEF cells, it is indicated that PKN1-KR does not behave in a dominant fashion over endogenous PKN1.
Overall, the results presented in this chapter establish a requirement for PKN1 in the hyperosmotic-induced MKK4-JNK-c-Jun pathway. The role of PKN1 with respect to MKK4-JNK induction is shown to be selective over parallel MAP kinase pathways and is also demonstrated to be stimulus specific. PKN1 activity is shown to be important for this MKK4-JNK response indicating a direct catalytic role for PKN1 at or upstream from the MAPK kinase kinase level of the cascade. The identification of PKN1 as a critical component of this MAP kinase response provides a new dimension to PKN1 and JNK signalling.
146 Chapter 7
Discussion
7.1 Overview
A screen for potential PKN agonists has revealed that PKN1 is regulated by hyperosmolarity. The characterised PKN1 response to hyperosmotic stress combines both translocation and activation and a PKN1 requirement for the classically stress activated MKK4/JNK pathway is also established. It is demonstrated that both the PKN1 translocation response and PKN involvement in the JNK cascade behave in a stimulus specific manner. The PKN1 requirement for JNK activation is shown to operate independently from other stress activated cascades and the PKN1 translocation behaviour is demonstrated to be specific over the related PKCe. A model for the PKN1 response to hyperosmolarity is illustrated in figure 7.1.
147 148 7.2 PKN1 Regulatory controls
It is intriguing that previous studies have principally focused on Rho as a PKN activator (Amano etal., 1996b; Flynn etal., 1998; Shibata etal., 1996; Watanabe etal., 1996), whereas this study has found no dependence on Rho for the PKN1 response to hyperosmolarity. However several studies have also found Rac-PKN interactions (Amano etal., 1996b; Flynn etal., 1998; Vincent and Settleman, 1997; Watanabe etal., 1996). Taken together the possibility remains that PKN1 is a target of both Rho and Rac small GTPases.
Evidence is presented here for the Rac mediated PKN1 response to hyperosmolarity and is supported by several observations. Rac is shown to co- localise in vesicles and the dominant negative Rac N17 is shown to prevent PKN1 vesicle accumulation. This provides direct evidence for the Rac dependence of PKN1 translocation. Rac is also demonstrated to become GTP-loaded upon hyperosmotic stress as recent studies have found (Lewis et al., 2002). This would provide a mechanism for subsequent PKN induction. Furthermore, PKN1 dependent MKK4-JNK activation under hyperosmolarity is established and this pathway is well characterised to be a Rac dependent cascade (Coso etal., 1995; Minden etal., 1995a).
One alternate hypothesis regarding PKN1 regulation is that there is a dynamic of Rho/PKN and Rac/PKN triggered responses. Perhaps under hyperosmolarity, the activation of Rac predominates and may actually downregulate Rho. Such a mechanism has been shown where Rac mediated production of reactive oxygen species (ROS) results in the downregulation of Rho by inhibiting the p190Rho-GAP (Nimnual etal., 2003). In subverting Rho-PKN signals, hyperosmotic stress would enable Rac-PKN responses to be elicited. This might be tested initially by monitoring the dynamics of Rho and Rac GTP loading under hyperosmolarity.
149 Many examples of Rac acting downstream of PI3-kinase signalling have been demonstrated and this mechanism is thought to operate through Rac GEFs. A good example is the recently described Rac GEF, P-REX1. Like other Rac GEFs P-REX1 contains a PH domain and is activated by Ptdlns(3,4,5)P3, a product of PI3-kinase activation (Welch etal., 2002). The dominant negative Rac N17 is shown here to inhibit hyperosmotic-induced PKN1 translocation. However the inhibition of PI3-kinase is shown not to inhibit hyperosmotic-induced PKN1 vesicle association. These findings would therefore indicate that the Rac-PKN translocation response to hyperosmotic stress is PI-3 kinase independent. It is possible that Rac could actually be triggering PI3-kinase pathways, thereby promoting PDK1 recruitment to the PKN1 positive compartment. Indeed, while the mechanism of Rac induced PI3-kinase activation is unclear, Rac has been shown to bind the p85 regulatory subunit of PI3-kinase (Zheng etal., 1994). The evidence for Rac signalling both downstream and upstream of PI3-kinase has been recently reviewed (Welch etal., 2003).
The mechanism of PKN1 vesicle association has not been fully elucidated, although some indications as to the PKN domain responsible for this association are given. The regulatory HRIabc domain, when localised, is not vesicular while the kinase domain is constitutively vesicular. It is therefore concluded that PKN1 vesicle association is mediated by interactions with the kinase domain. PDK1 is known to interact with PKN1 via the kinase domain of PKN1 and is also present on PKN1 vesicles. However, it is shown that PI3-kinase inhibition can dissociate PDK1 from these PKN1 positive compartments. It is therefore likely that an as yet unidentified PKN1 kinase domain interacting component(s) (“X”) mediates this association (a model for this assembly is illustrated in figure 7.1).
150 7.3 PKN1 activation
The incubation of Rho-GTP with PKN1 has been shown to increase the phosphorylation and catalytic activity of PKN1 (Amano etal., 1996b; Vincent and Settleman, 1997; Watanabe etal., 1996). It is shown that PKN1 undergoes activation loop phosphorylation and catalytic activation upon hyperosmolarity. Consistent with PKN1 activation within the PKN1 vesicle compartment, the PKN1 activator, PDK1, is also recruited. The behaviour of PKN1 described here supports the view that the allosteric input through the amino-terminal HR1 domain is required for complex formation with and subsequent phosphorylation by PDK1.
The role of hyperosmotic-induced PKN1 activation within the vesicular compartment is of interest. Hyperosmolarity is a known apoptotic stimulus (Edwards etal., 1998; Morales etal., 2000) and PKNs have been shown to undergo caspase cleavage in response to apoptotic stimuli and under ischemic conditions (Cryns etal., 1997; Sumioka etal., 2000). However, cleavage of PKN1 under osmotic stress has not been observed. The responses defined here indicate that in fact the behaviour of PKN1 observed under hyperosmotic conditions reflects an underlying constitutive process. The partial vesicular localisation of the kinase domain in the absence of hyperosmotic shock shown in chapter 4 and the observation that HA1077 induces some PKN1 vesicle accumulation described in chapter 5, suggests that this is a constitutive trafficking pathway up-regulated by hyperosmotic shock. The findings indicate that the PKN1 response to hyperosmolarity is not simply targeting PKN1 for degradation but that PKN1 activity is involved in the turnover or exit from this vesicular compartment.
The reversibility of hyperosmotic-induced vesicle formation is described in chapter 3. It would be interesting to monitor whether this reversal correlates with a reversal in PKN1 activation loop phosphorylation and catalytic activation. Perhaps this could
151 be controlled by the recruitment and/or dissociation of PDK1. The reversal of hyperosmotic-induced PKN1 activation could also be controlled by recruitment of inactivating phosphatases and the effect of phosphatase inhibition on this system would be of interest.
7.3.1 The PKN-PKB question
The PKN1 translocation, activation and PDK1 recruitment in response to hyperosmotic stress is established here. Given that PKB is a well characterised PDK1 substrate, the potential involvement of PKB under conditions of hyperosmolarity was investigated. Consistent with previous reports, it is shown that PKB is dephosphorylated on hyperosmotic stress (Meier etal., 1998). The localisation of PKB translocation under hyperosmolarity was also monitored, it is shown that PKB is not recruited to PKN1 vesicles. The findings gathered from PKN-PKB studies in chapter 3 indicate that Rho-PKN signals do not positively regulate PKB phosphorylation. In fact recent studies have indicated a negative regulatory role for PKNs on PKB phosphorylation (Koh etal., 2000; Wick etal., 2000). These studies, combined with finding that PKB phosphorylation levels are higher in PKN 1-KO MEF cells together indicate a negative influence from PKN on PKB phosphorylation.
The observation that PKBS473 resting phosphorylation is higher in PKN1 KO- MEFs raises some interesting questions regarding the influence of PKNs on PKB regulation. This could conceivably involve competition for access to the upstream kinase namely PDK1, shared by both PKN1 and PKB, which would be consistent with our findings that PDK1 is shown to be recruited to the PKN1 vesicular compartment upon hyperosmotic stress. This could affect the stability of PKB and render it more susceptible to degradation. Indeed PDK1 has been shown to affect the stability of AGC kinases given that in PDK1 -/- ES cells, PKN protein levels are compromised (Balendran etal., 2000). The question of how this could relate to
152 other AGC kinases is an interesting point, indeed a C-terminal fragment of PKN2 was shown to inhibit PDK1 mediated PKCÇ phosphorylation (Hodgkinson and Sale, 2002). These findings would also be consistent with a model of competition for access to PDK1 among AGC kinases in general.
153 7.4 PKN1 and the JNK cascade
The role of PKN1 in classically activated stress pathways was investigated and PKN1 dependence on the hyperosmotic induction of the JNK pathway is described. The question of whether PKN1 is directly activating JNK, perhaps as a MAPKKK or upstream kinase, or is functioning to scaffold components of this pathway is critical to understand PKN1 function. The idea that PKN1 may have a catalytic influence on a MAP-kinase pathway has recently been suggested since PKN1 was shown to phosphorylate in vitro the p38 pathway MAPKKK, MLTK. The kinase inactive form was shown to inhibit a hyperosmotic-induced mobility shift of MLTK (Takahashi et a!., 2003). The results presented here indicate that PKN1 may have a catalytic contribution to the JNK cascade since the PKN1 inhibitor HA1077 was found to inhibit MKK4 induction, the effect was not total although this discrepancy could be due to the effectiveness of in vivo PKN1 catalytic inhibition. Furthermore, the kinase inactive form of PKN1 was found not to potentiate phospho c-Jun staining in MEF cells as was the case with WT-PKN1. While these studies imply a catalytic role for PKN1 in JNK cascade activation, a scaffolding role has not been excluded. Further studies could seek to address these questions by asking whether PKN1 can directly phosphorylate MKK4 or upstream kinases of the JNK cascade. Co- immunoprecipitation experiments to determine whether PKN1 can bind JNK pathway kinases such as MKK4 and/or scaffolding proteins such as POSH, JIP, and JSAP (Ito etal., 1999; Xu etal., 2003; Yasuda etal., 1999) will also be informative with regard to the precise nature of the PKN1 role.
The question of what aspect of PKN1 is necessary and sufficient to engage productive JNK signals could be addressed by transfection studies in PKN 1-KO and WT MEF cells. The kinase domain of PKN1 was shown in chapter 4 to be constitutively vesicular, this observation raises the possibility that it may, in lacking an autoinhibitory mechanism, be able to signal to downstream effectors in a
154 constitutive manner. It would therefore be interesting to ask whether the transfection of PKN1 kinase domain could trigger MKK4/JNK activation under basal conditions. If so, the extent of PKN1 activity involved in JNK signalling could be addressed by employing the kinase dead kinase domain in the same experiment.
It has been shown that PDK1 and Rac are both recruited to the PKN1 induced compartment. Given that this pathway seems to be required for JNK signalling the possibility is raised that these vesicles function as signalling modules. One key question for future studies is to address whether the stimulus specificity of the translocation response correlates with and/or confers stimulus specificity on PKN1 dependent signalling through the JNK pathway. This would need to establish a direct link between hyperosmotic-induced PKN1 vesicle recruitment and JNK pathway constituents. Co-localisation studies could be performed with GFP-PKN1 and constituent members of the JNK cascade such as MKK4 and JNK alongside JNK scaffolding proteins such as JIP, POSH and JSAP (Ito etal., 1999; Xu etal., 2003; Yasuda etal., 1999).
The JNK scaffolding protein JIP1 is known to bind kinesin motors. This may provide a link between vesicle trafficking events such as those observed with hyperosmotic-induced PKN1 translocation and related signalling events such as the triggering of the JNK pathway. The role of trafficking processes in regard to JNK signalling has been reviewed (Goldstein, 2001). The coupling of trafficking and signalling processes may explain how these signals are transmitted from the plasma membrane to the nucleus.
It is possible that other PKNs may operate in a similar manner to that described for PKN1. It is shown that PKN2 displays the same translocation response to that of PKN1. Furthermore the PKN inhibitor HA1077 is shown to inhibit residual MKK4 activation in PKN 1-KO-MEF cells. Since what appears to be a highly stimulus
155 specific and pathway selective role for PKN1 is described, the question of whether different stimuli and perhaps related MAP-kinase pathways are regulated by other PKNs and/or related PKCs is raised. This idea is supported from the finding that hyperosmotic-induced ERK activation was found to be dependent on classical and novel PKC activation (Zhuang etal., 2000). Further supporting this theory, recently PKCÔ was shown to be required for JNK activation, operating through the MAPKKK, MKK7 pathway in response to DNA damaging agents (Yoshida etal., 2002). To investigate this potential cross-over of function, small interfering RNAs could be employed for PKN2, PKN3 and other PKC superfamily members. As has been demonstrated, this technique is effective in preventing expression of targeted proteins (Elbashir etal., 2001). These studies would enable the dissection of the stimuli and MAP-kinase pathways within which individual isoforms operate.
156 7.5 Future Directions
The nature of the induced PKN1 vesicular compartment described here is not resolved. Immunostaining indicates that this is not an early endosomal compartment, nor an acidified compartment. However, the effect of PISkinase inhibition on the induced PKN 1-positive compartment, would be consistent with this being part of an endocytic pathway, where inhibition of PISkinase arrests homotypic fusion (Jones and Clague, 1995). To gain a greater understanding of the precise size and morphology of the vesicles, electron microscopy studies could be performed. This would provide greater resolution and magnification as compared with light microscopy techniques.
Future studies might focus on isolating this compartment and the optimisation of the density fractionation protocol would be a powerful approach to enable the identification of proximal targets for PKN1 and the nature of the PKN1 compartment. This could involve the transfection of constituent components of hyperosmotic-induced PKN1 vesicles, including the kinase domain of PKN1 or perhaps a constitutively activated Rac. These proteins may help achieve greater stability of the PKN1 vesicle association, for example the constitutively activated Rac would prevent GTP hydrolysis upon cell lysis and in doing so may give stability to the complex. As also indicated from the preliminary experiments in chapter 5, kinase inhibitors may prove useful tools in stabilising PKNs on membrane compartments. In purifying PKN1 enriched vesicle fractions, subsequent 2D gel and mass spectrometry analysis may identify novel components such as hyperosmotic-induced binding partners and substrates for PKN1.
The JNK cascade is well known to stimulate a variety of transcription factors resulting in a range of cellular responses such as apoptosis, cell proliferation and tumourigenesis (Dunn etal., 2002). In seeking to understand transcriptional
157 outcomes of PKN1-JNK responses, gene chip technology could be employed. The comparison of total RNA samples from hyperosmotically stressed WT and PKN1- KO MEF cells may highlight differences in gene induction and could point to precise functional roles for this pathway.
The role of the JNK pathway in relation to cell death has been an active area of research with both pro and anti apoptotic roles for JNK reported, these have been recently reviewed (Lin, 2003). Since hyperosmolarity has been shown to be an apoptotic trigger (Edwards etal., 1998; Morales etal., 2000), it would be of interest to assess a role of PKN1 within the JNK pathway in relation to cell death. This could be achieved by performing apoptotic assays on hyperosmotically shocked PKN1-K0 and WT-MEF, such studies have demonstrated involvement for other components of the JNK pathway in cell death (Xu et al., 2003).
A potentially pathogenic property of the JNK pathway is that of invasion, indeed invasiveness has been associated with increased JNK activity. This process has been linked to focal adhesion kinase (FAK) signalling and cells lacking FAK were shown to lack invasive properties. Downstream signalling from FAK in this process was shown to involve the Rac-MKK4-JNK pathway (Hsia etal., 2003). It had been proposed that this pathway involves PAK as an intermediate signalling molecule between Rac and MKK4 (Almeida etal., 2000). Given the studies described in this thesis, it would be interesting to determine any contribution from PKN1 into the invasive process.
A physiological situation where osmotic gradients are important is in the kidney where water re-absorption is a key function. Aquaporin-1 (AQP1) is a water channel that is induced by hyperosmolarity. The activation of ERK, p38 kinase, and JNK pathways were shown to be involved in hyperosmotic-induced AQP1 expression in mlMCD-3 cells (Umenishi and Schrier, 2003). It would be interesting, perhaps to ask whether such induction is impaired in PKN 1-KO cells derived from
158 the kidney. Further studies looking at physiological consequences of loss of PKN could involve water deprivation experiments. In monitoring changes in urine composition, any defect in the ability to reabsorb water would be revealed. Long term studies could involve the generation of PKN2 and PKN3 KO-mice. Assuming viability, multiple crosses of these animals could reveal the total PKN contribution and level of redundancy among these proteins to physiological situations as well as stress activated and other signalling cascades.
In conclusion, a role for PKN1 in an important signalling cascade is demonstrated. Translocation and activation of PKN1 in a vesicular compartment points to a functional role for these vesicles in eliciting downstream signalling. The identification of complex components within these compartments and associated downstream transcriptional responses will help to further understand the function of this hyperosmotic-induced pathway.
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