Health Science Campus
FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Sciences
Regulation of Folate Receptor Raft Recycling
Submitted by: Hala Elnakat
In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences
Examination Committee
Major Advisor: Manohar Ratnam, Ph.D.
Academic Advisory Committee:
Han-Fei Ding, Ph.D.
Sonia Najjar, Ph.D.
Kandace Williams, Ph.D.
Robert Trumbly, Ph.D.
Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D.
Date of Defense: January 4, 2007
Regulation of Folate Receptor Raft Recycling
Hala Elnakat
The University of Toledo
2007 DEDICATION
A mes parents, que j’aime énormement.
ii ACKNOWLEDGMENTS
I would like to thank my major advisor, Dr. Manohar Ratnam, for his guidance, his patience and his optimism when all else fails.
I also would like to thank my advisory committee: Dr. Sonia Najjar, Dr. Robert
Trumbly, Dr. Han-Fei Ding, and Dr. Kandace Williams for their support and suggestions.
I would like to thank Dr. William Gunning for the many hours he spent helping me use the electron microscope.
I would like to thank Dr. David Giovannucci for his time and help with the calcium ionophore experiment.
I would like to thank Dr. Venkatesha Basrur for performing and analyzing the mass spectrometry data for us.
I would like to thank Dr. Khew-Voon Chin for the replication-deficient adenoviruses expressing Ad5-type adenovirus vectors containing the cDNA for wild type
PKCs (α, βII, δ and ε) and kinase negative mutants (DN-PKCα and DN-PKCβII). I would like to thank Dr. Kevin Pan for the PKCβI wild type construct. I would like to thank Dr. Darlene Dartt (Harvard Medical School) for the constitutively active myristoylated PKCα adenovirus.
I would like to thank Dr. Hongjuan Cui and Sandra Beach for their help and guidance with the retroviral and lentiviral infection systems.
I would like to thank all my colleagues George, Huiling, Ayman, Marcella, Remi,
Karen, Thuyet, Hong and Juan for being great people to work with.
iii Last but not least, I would like to thank Mariana Stoeva and Jenny Zak for always being there for me.
iv TABLE OF CONTENTS
DEDICATION...... ii
ACKNOWLEDGEMENTS...... iii
TABLE OF CONTENTS...... v
INTRODUCTION ...... 1
LITERATURE...... 4
MATERIALS AND METHODS...... 29
RESULTS ...... 45
DISCUSSION...... 75
SUMMARY...... 80
BIBLIOGRAPHY...... 81
ABSTRACT...... 112
v INTRODUCTION
The two human folate receptor (FR) isoforms, FRα and FRβ share 71% amino acid sequence homology (Lacey et al., 1989; Ratnam et al., 1989) and have a glycosyl- phosphatidylinositol (GPI) anchor attached to the carboxyl-terminal end of the proteins at serine 234 and asparagine 230, respectively (Yan and Ratnam, 1995). The third isoform,
FRγ lacks a GPI anchor due to the lack of an efficient signal for GPI modification (Shen et al., 1994, 1995). Like most GPI-anchored proteins, FRα and FRβ are located in special membrane microdomains referred to as rafts (Mayor et al., 1994; Wu et al., 1997) that are insoluble in cold non-ionic detergents and are additionally characterized by a concentration of cholesterol, glycosphingolipids and other signaling proteins (Edidin,
2003; Rajendran and Simons, 2005; Vereb et al., 2003). Caveolae are also detergent resistant membrane complexes that could be distinguished from rafts mainly by the presence of a coat of caveolin on the cytosolic surface of the membrane (Liu et al.,
1997a; Rothberg et al., 1992) and mostly the absence of GPI-anchored proteins.
Quantitative fluorescence microscopy data and kinetic studies in monkey kidney epithelial cells (MA104) have shown that FR quantitatively recycles, within minutes, between the cell surface and endocytic compartments via a Cdc42-regulated endocytic pathway (Kamen et al., 1988, 1989; Rothberg et al., 1990; Sabharanjak et al., 2002).
Furthermore, FR recycling in these cells is modulated by phorbol-12-myristate-13-acetate
(PMA) or other protein kinase C (PKC) activators which inhibit the internalization step of FR, thereby resulting in an increase in cell surface pool of FR (Smart et al., 1994).
1 This increase is abolished by incubating the cells with inhibitors specific for classical
PKCs prior to the addition of PMA (Kamen and Smith, 2004).
Diacylglycerol and PMA both activate classical and novel PKCs by binding to a
highly conserved cysteine-rich regulatory motif located in the amino-terminal end of the
kinase. As a result of this activation, different PKC isoforms have been visualized in live
cells to rapidly translocate from the cytoplasm to the plasma membrane (Almholt et al.,
1999; Ng et al., 1999; Ohmori et al., 1998; Sakai et al., 1997). Different activated PKC
isoforms can bind to specific receptors for activated C kinase (RACKs) which act as
shuttling proteins to the intracellular target sites of the kinase. Prolonged treatment with
PMA results in the ubiquitination of the protein which targets it for degradation by the
proteasomes (Lee et al., 1997; Leontieva and Black, 2004).
Since rafts play a major role in cell signaling, understanding how raft recycling
and reorganization are altered by physiological signals and various drug treatments offers
a means to modulate immune responses, malignant cell growth and cell death (Kaneko et
al., 1997; Mollinedo and Gajate, 2006; Simons and Toomre, 2000). In the specific case of
FR, one of the potential clinical significance of targeting raft recycling to internalize the
receptor is to increase the uptake of folate compounds and novel antifolates by
macrophages in rheumatoid arthritis and by ovarian or endometrial FR- positive tumor
cells (Buist et al., 1995; Mantovani et al., 1994; Nagai et al., 2006; Nagayoshi et al.,
2005; Theti et al., 2003; Veggian et al., 1989; Wu et al., 1999).
In this study, we investigated the molecular mechanism by which FR recycling is regulated by PMA since its effects on physiological processes are known to stimulate cell
2 signaling through the second messenger diacylglycerol. We first attempted to identify
which isoform(s) mediates the phorbol ester effect in MA104 cells especially since both
classical and novel PKCs are activated by PMA. Our data suggest that PKCα, targeted to specific membrane microdomains, is one of the key players in mediating the phorbol ester effect on FR recycling in these cells. In order to identify other proteins that might also be involved in FR recycling, we purified FR-rich rafts using an immobilized biotinylated folate probe. Among the proteins identified, annexin II was required for the internalization of FR rafts and RACK1 mediated the effect of PMA on FR recycling in
MA104 cells. Our hypothesis is that the population of FR rafts on the cell surface is increased by activators of PKCα as a result of targeting of PKCα to rafts by RACK1 and
phosphorylation/inhibition of annexin II. These detailed studies provide a comprehensive
molecular picture of the effect of PMA on FR recycling in MA104 cells.
3 LITERATURE
The Glycosyl-phosphatidylinositol-anchored Folate Receptor (GPI-anchored FR)
Folate receptor is bound to the plasma membrane via a GPI-anchor. The signal for
the addition of a GPI anchor which is located in the C-terminal end of a newly translated polypeptide is cleaved off in the endoplasmic reticulum and replaced with a GPI moiety
by a transamidase that covalently binds the ethanolamine of the GPI moiety to the new C- terminal end of the precursor protein destined to the plasma membrane (Bangs et al.,
1985, 1986; Ferguson et al., 1986). The GPI signals of different proteins share a number
of characteristics that mainly include an uncharged amino acid representing the GPI
attachment (ω) site, separated by a spacer of 8 to 12 amino acids from a sequence (10 to
20 amino acids) that most importantly has a hydrophobic core of 6 to 8 amino acids
(Cross, 1990; Ferguson and Williams, 1988; Moran et al., 1991) (Figure 1). Site-directed
mutagenesis experiments of amino acids in the carboxyl-terminal regions of FRα and
FRβ (Yan and Ratnam, 1995) allowed the characterization of the signal peptides of these
two human isoforms that share 71% amino acid sequence identity with each other (Lacey
et al., 1989; Ratnam et al., 1989). The third human FR isoform, FRγ is a secretory protein
due to the lack of an efficient signal for GPI modification (Shen et al., 1995). Folate
receptor α contains 257 amino acids (Lacey et al., 1989) and has a ω site at Ser234
separated by a spacer of nine amino acids from the hydrophobic domain (Yan and
Ratnam, 1995). As for FRβ, the ω site consists of Asn230 followed by 25 amino acid
residues (Yan and Ratnam, 1995).
4
GPI-signal sequence
(A)
(B) GPI- Anchored protein GPI-anchor Newly synthesized protein
Figure 1: Schematic diagrams of the GPI signal sequence and the reaction leading to the addition of a GPI-anchor to a newly translated protein. (A) Diagram of a newly synthesized protein with emphasis on the GPI signal which includes an uncharged amino acid representing the GPI attachment (ω) site, separated by a spacer of 8-12 amino acids from a hydrophobic core sequence of 6-8 amino acids. (B) The signal for the addition of a GPI-anchor located in the C-terminal end of a newly translated protein is cleaved off in the endoplasmic reticulum and replaced with a GPI moiety by a transamidase. The conserved GPI-anchor core typically consists of an ethanolamine phosphate, 3 mannose residues, one glucosamine and a phosphatidylinositol that attaches it to the membrane of the endoplasmic reticulum.
The physical and biochemical properties of GPI-anchored proteins include insolubility in
cold non-ionic detergents and usually sensitivity to bacterial phosphatidylinositol-specific
phospholipase C (PI-PLC) digestion even though some PI-PLC resistance is acquired if
the inositol ring of the GPI moiety has a palmityl group (Field et al., 1991; Roberts et al.,
1988a,b).
5 Membrane Microdomains
Membrane Rafts
Membrane rafts were thought of as “liquid-ordered plasma membrane microdomains that are characterized by a concentration of cholesterol, glycosphingolipids, GPI-anchored proteins and signaling proteins and that may, in some cells, cover up to half of the cell surface” (Edidin, 2003; Rajendran and Simons, 2005;
Vereb et al., 2003). Due to controversies in the field of rafts, up until this year, there was no consensus agreement on one definition for these membrane microdomains. After the recent Keystone Symposium of Lipid Rafts and Cell function, a new definition for rafts has emerged stating that: “Membrane rafts are small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions” (Pike, 2006). These glycosphingolipid- enriched membrane fractions (GEMs) (Rodgers et al., 1994) are believed to serve as platforms for signal transduction as well as units of protein recycling between the cell surface and endocytic compartments (Sabharanjak et al., 2002) or the Golgi (Nichols et al., 2001) through unique non-clathrin mediated pathways (Mayor et al., 2006).
After membrane rafts were originally conceptualized (Simons and van Meer,
1988), the idea took further hold around the experimental observation of a protein and
lipid complex that was insoluble in cold non-ionic detergent (Brown and London, 2000;
Brown and Rose, 1992). Owing to that property, rafts also have been referred to as
detergent resistant membrane complexes (DRMs) or detergent insoluble glycolipid-
6 enriched domains (DIGs) (Simons and Ikonen, 1997). A wide range of detergents have
been used to retrieve putative raft components (Chamberlain, 2004; Schuck et al., 2003).
Both FR and caveolin exist in such DRMs.
Caveolae
Plasmalemmal vesicles or caveolae, which are observed as cell surface
invaginations (Bruns and Palade, 1968; Yamada, 1955), are sometimes regarded as a
special type of lipid rafts. They also are enriched in cholesterol and glycosphingolipids as
well as proteins involved in signal transduction but are essentially characterized by a coat
of caveolin on their cytosolic surface (Liu et al., 1997a; Rothberg et al., 1992). Some cell types such as lymphocytes (Fra et al., 1994), neuroblastoma cells (Gorodinsky and
Harris, 1995), Fischer rat thyroid cells (Zurzolo et al., 1994) and the apical membrane of enterocytes (Danielsen and van Deurs, 1995) lack caveolae. In these cells, several GPI- anchored proteins such as Thy-1, Decay accelerating factor (DAF), the cellular isoforms of the prion protein (PrPc) and the transferrin-like iron-binding protein are present in
DRMs devoid of caveolin. Actually, most GPI-anchored proteins localize to raft
microdomains but there is morphological and functional evidence for the presence of at least some GPI- anchored proteins in caveolae such as CD59 (Sprenger et al., 2006). It has been also proposed that GPI-anchored proteins may exchange between rafts and caveolae upon activation (Mayor et al., 1994; Schnitzer et al., 1995). The earlier data
localizing FR with caveolin (Anderson et al., 1992; Rothberg et al., 1990) were
7 subsequently attributed to an artifact of the methodology due to cross-linking of the antibody probes (Mayor et al., 1994; Wu et al., 1997).
Size of Rafts
Considerable controversy has surrounded the question about the size of lipid rafts in live cells, based on measurements of proximity of GPI-protein molecules (Kenworthy and Edidin, 1998; Kenworthy et al., 2000; Sharma et al., 2004) and disagreements on single particle tracking measurements (Subczynski and Kusumi, 2003). Nevertheless, biochemical and electron microscopic observations show that heterologous GPI-proteins, such as FR and placental alkaline phosphatase segregate into separate rafts (Wang et al.,
2002). Also, substantial evidence exists that raft size and protein composition are in a state of dynamic flux so that very small rafts of only a few molecules (Kenworthy et al.,
2000; Sharma et al., 2004) could coalesce to form functional units.
Methods Used to Purify DRMs
One of the earlier methods used to purify rafts relied on their biochemical properties that render them insoluble in cold (4°C) detergent (Brown and Rose, 1992).
Since then, multiple detergents have been tested in the quest for a gold standard for raft purification, ranging from the most commonly used Triton X-100, to CHAPS, Lubrol
WX, and to members of the polyoxyethylene ether series Brij58, Brij96 and Brij98
(Drevot et al., 2002; Madore et al., 1999; Schuck et al., 2003). Detergent resistant membrane complexes prepared from the same cell line with different detergents followed
8 by flotation on a sucrose density gradient are enriched with variable levels of cholesterol,
sphingolipids, saturated glycerophospholipids, and membrane proteins (Schuck et al.,
2003). While Triton X-100 and CHAPS usually have higher DRM selectivity than Brij98 at 4°C (Schuck et al., 2003), the latter allows the isolation of rafts at a physiological temperature, 37°C (Drevot et al., 2002). Even though purifying DRMs with detergents remains a very useful method, the detergents themselves can cause DRMs to form, aggregate or even lose some of their resident proteins, thereby resulting in misleading conclusions (Heerklotz, 2002; Madore et al., 1999; Mayor and Maxfield, 1995).
Therefore, additional lines of evidence using other techniques are required to further support the data.
The associations between rafts and signaling proteins have been demonstrated
both by methods that are dependent on detergent insolubility such as co-
immunoprecipitation (Rodgers et al., 1994; Solomon et al., 1996; Sprenger et al., 2006) and mass spectrometric detection of membrane proteins (Foster et al., 2003) as well as by detergent independent methods. The latter include the treatment of cells with drugs that alter the lipid composition of rafts by sequestering or depleting cholesterol or the addition of exogenous cholesterol, gangliosides or polyunsaturated fatty acids. Other detergent independent methods include confocal fluorescence microscopy (Liu et al., 1997a; Oh and Schnitzer, 2001), chemical crosslinking, in situ silica-coating of cell membranes (Liu et al., 1997a; Schnitzer et al., 1995) and fluorescence resonance energy transfer (FRET).
9 Signaling Proteins Associated with DRMs
A variety of signaling processes occur through rafts. Various protein components
of signaling complexes, including GPI-anchored proteins, receptors, non-receptor
tyrosine kinases and GTP-binding proteins (G proteins) occur as constitutive residents of
rafts or may translocate or become recruited to rafts upon activation by a signal (Simons
and Toomre, 2000). Both GPI-anchored and non-GPI anchored membrane proteins as
well as cytosolic proteins may be functional and/or structural components of such
signaling complexes. There is strong evidence that rafts mediate signaling through Thy-1
(Doherty et al., 1993), FcεRI receptor (Field et al., 1997), T-cell receptor (Janes et al.,
2000; Langlet et al., 2000), B-cell receptor (Cheng et al., 1999; Deans et al., 1998), EGF
receptor (Couet et al., 1997), insulin receptor (Mastick et al., 1995), ephrinB1 receptor
(Bruckner et al., 1999), neurotrophin (Bilderback et al., 1999), GFRα receptor (Tansey et
al., 2000), H-Ras (Roy et al., 1999) and integrins (Krauss and Altevogt, 1999; Wary et
al., 1998).
Caveolae also appear to mediate similar signal transduction processes (Anderson,
1998; Liu et al., 1997b; van Deurs et al., 2003) but there are some distinctive differences
in the distribution of signaling proteins between these two membrane microdomains. For example, it has been shown in different cell types that, among the heterotrimeric G protein subunits, Gi, Gs and Gβ predominantly localize in rafts instead of caveolae (Oh
and Schnitzer, 2001). In contrast, Gq preferentially localizes in caveolae without Gβγ;
however, in cells lacking caveolae Gq localizes in raft microdomains by default (Oh and
Schnitzer, 2001). A novel protein potentially involved in signaling (C8ORF2) was
10 predominantly found in caveolae unlike its other family member, stomatin that is raft-
associated (Sprenger et al., 2006). The GPI-anchored protein CD59 which also localizes with caveolin in endothelial cells has been implicated in EGFR signaling (Blagoev et al.,
2003; Sprenger et al., 2006).
In the specific case of FR, association with signaling proteins has been
demonstrated by reciprocal co-imunoprecipitation in detergent resistant membrane
fractions of IGROV1 ovarian carcinoma cells that lack caveolin (Miotti et al., 2000) and
by dual immunofluorescence confocal microscopy in MA104 kidney epithelial cells (Oh
and Schnitzer, 2001). These studies revealed the association of FR with the src family
member, p53-56 lyn and the Gαi-3, Gs, and Gi subunits of heterotrimeric G proteins. In addition, both p53-56 lyn and Gαi-3 were phosphorylated in an in vitro kinase assay with
immunoprecipitates of FR (Miotti et al., 2000). The src-protein tyrosine kinases and G proteins have been shown to frequently associate with other GPI-anchored proteins in lipid rafts such as GP2 (Parker et al., 2000), urokinase plasminogen activator receptor
(Bohuslav et al., 1995), Thy-1 (Solomon et al., 1996; Thomas and Samelson, 1992), DAF
(Gorodinsky and Harris, 1995; Shenoy-Scaria et al., 1992) and lipopolysaccharide receptor (Solomon et al., 1998; Stefanova et al., 1991). A signal motif in the N-terminus of src-family proteins and G proteins consisting of Met-Gly-Cys specifies the myristoylation of glycine and the palmitoylation of cysteine thereby targeting these proteins to rafts (Moffett et al., 2000; Rodgers et al., 1994; Shenoy-Scaria et al., 1994;
Wedegaertner et al., 1995; Zlatkine et al., 1997).
11 Role of Rafts in Signaling and Signal Modulation by Raft Recycling
Within the raft microenvironment, extracellular signals can clearly be transmitted,
either directly or indirectly, to downstream signaling proteins associated with the
cytosolic leaflet of the lipid bilayer. There are potential functional advantages of the raft
organization in signal transduction (Simons and Toomre, 2000), although some GPI-
anchored proteins do not need raft association for their signaling function (Economides et
al., 1995; Massague, 1996). Sequestration of activated signaling proteins in rafts can
allow the creation of a microenvironment that is conducive to the signaling process by
compartmentalizing the participating proteins (eg., kinases, phosphatases, palmitoylases
and depalmitoylases) and excluding others. This would lead to amplification of the signal
through relatively few activated receptors. Such a compartmentalized signaling could involve receptors that are constantly in association with rafts, receptors that oligomerize and partition into rafts upon activation by ligand or cross-linking proteins that could cause rafts to coalesce into a large signaling complex (Simons and Toomre, 2000). For example, during T-cell activation, rafts play a crucial role in the formation of the immunological synapses of several micrometers in diameter between T-cells and antigen presenting cells (APC) by the coalescing of rafts on the surface of T-cells (Katagiri et al.,
2001).
Internalization of DRMs
Internalization of rafts into endocytic compartments is an obvious means of
disassembling coalesced rafts (Simons and Toomre, 2000), but the consequence of GPI-
12 protein raft internalization on signaling function still has to be thoroughly investigated.
More attention has been focused on signal transduction in relation to the internalization of caveolae (Ikonen, 2001). The integrins (α2β1) are membrane receptors located in
DRMs that are internalized via caveolae in a PKCα-dependent manner (Upla et al.,
2004). Downregulation of PKCα activity following chronic activation by PMA may occur by delivery of activated PKCα to endosomes via caveolae (Prevostel et al., 2000).
In contrast, internalization of the EGF receptor through caveolae appears to result in
MAP kinase activation in the early/sorting endocytic compartment (Pol et al., 2000).
Internalization of raft proteins can occur via a number of different endocytic pathways depending on the raft-resident protein and the cell context (reviewed in Mayor and Riezman, 2004). Non-crosslinked GPI-anchored proteins are endocytosed into GPI- anchored protein-enriched early endosomal compartments (GEEC) in a Cdc42 regulated
manner that is independent of both caveolin and dynamin and recycled back to the
plasma membrane via recycling-endosomal compartments (Sabharanjak et al., 2002)
(Figure 2). Nonetheless, some GPI-anchored proteins are sorted to the recycling
endosomes or late endosomes (Fivaz et al., 2002). Finally, interleukin receptor-2 which
localizes in rafts is internalized by a clathrin independent pathway which involves
dynamin (Lamaze et al., 2001).
13
Figure 2: Endocytosis of GPI-anchored proteins (modified from Mayor and Riezman, 2004). GPI-anchored proteins are internalized via a Cdc42- dependent endocytic pathway into GEECs. Recycling endosomes will target these proteins back to the plasma membrane.
14 FR Raft Recycling
Kinetic studies in monkey kidney epithelial cells (MA104) have shown that FR
quantitatively recycles between the plasma membrane and endocytic compartments
within minutes (Kamen et al., 1988, 1989; Rothberg et al., 1990). The movement of FR
molecules was tracked using [3H]-labeled folic acid, which remains bound to the receptor while it recycles. The number of FR molecules bound to the cell membrane was
determined by exposure of MA104 cells to an acidic pH wash which results in the
dissociation of the receptor from its ligand. These studies also demonstrated that FR
recycling was dependent upon its GPI anchor (Ritter et al., 1995), membrane cholesterol
(Chang et al., 1992) and the actin cytoskeleton (Lewis et al., 1998a). Treatment of
MA104 cells with actin-disrupting agents such as cytochalasin D or latrunculin B results
in doubling of the external pool of FR as compared to untreated cells (Lewis et al.,
1998a). This increase is reversible if cytochalasin D is removed and FR is allowed to
reach steady-state again.
Clustering of FR was observed by using a monovalent, biotinylated folate affinity
labeling reagent on live cells under transport permissive conditions followed by fixing
and embedding the cells prior to probing with colloidal gold labeled streptavidin for
electron microscopy (Wu et al., 1997). Subsequently, FR clusters at the live cell surface
were demonstrated by chemical cross-linking (Friedrichson and Kurzchalia, 1998) and
fluorescence resonance energy transfer (Varma and Mayor, 1998). Several methods
including cell fractionation, electron microscopy and fluorescence microscopy have been
used to observe FR in endocytic compartments (Birn et al., 1993; Hjelle et al., 1991;
15 Mayor et al., 1998). Recycling of FR between the plasma membrane and intracellular
compartments occurs via a Cdc42-dependent endocytic pathway (Figure 2) (Sabharanjak
et al., 2002). Expression of a dominant negative form of Cdc42 but not the dominant-
active form results in a decrease in size and number of GEECs and in an increased
colocalization of GPI-anchored proteins with transferrin receptor.
Protein Kinase C (PKC)
An overview
Protein kinase C is the classical cellular receptor for the second messenger, diacylglycerol (DAG) as well as phorbol ester (Nishizuka, 1988; Ono et al., 1989). This is a key enzyme in signal transduction through phospholipase C coupled receptors that activate phospholipase C and result in the transient generation of DAG (reviewed in
Nishizuka, 1995). It has been established that maturation, regulation, stability and cellular localization of PKC also are governed by its phosphorylation by 3-phosphoinositide- dependent kinase (PDK-1) followed by autophosphorylation (Balendran et al., 2000;
Parekh et al., 2000; Toker and Newton, 2000). Mammalian PKCs are represented by a family of at least 10 different isozymes of serine-threonine kinases with variable tissue distribution and belong to one of three groups referred to as classical, novel and atypical
(Hug and Sarre, 1993; Nishizuka, 1988; Nishizuka, 1992). This division is mainly based on cofactor requirements and primary protein structures. PKCν (Hayashi et al., 1999) and
PKCμ (Johannes et al., 1994), which can be activated by DAG and phorbol ester were originally considered as being two additional members of the PKC family until further
16 studies showed that they are better classified as part of the protein kinase D family
instead (Rykx et al., 2003).
All the different PKC isozymes are characterized by an amino-terminal regulatory
domain and a carboxyl-terminal catalytic domain (Knopf et al., 1986; Nishizuka, 1992)
(Figure 3). Well defined motifs in the regulatory domain bind phospholipid cofactors and
Ca2+ and also constitute sites for specific, functionally important, protein-protein
interactions (Hug and Sarre, 1993; Schechtman and Mochly-Rosen, 2001). A common
highly conserved cysteine-rich regulatory motif, also known as the C1 domain acts as a
pseudosubstrate or autoinhibitory domain that, in the absence of cofactors, binds to the
substrate binding site to keep the enzyme inactive. In the case of both classical and novel
PKCs, phorbol ester and DAG both relieve this autoinhibition, apparently by binding to this C1 domain (Sharkey et al., 1984).
17
Figure 3: Schematic diagram of the domains of classical, novel and atypical PKCs (modified from Tan and Parker, 2003). All PKCs are characterized by an amino- terminal regulatory domain and a carboxyl-terminal catalytic domain.
Classical PKCs (cPKCs)
The members of this group, also referred to as conventional PKCs (α, βI, βII, and
γ) were initially cloned from a brain cDNA library and found to be activated by DAG or phorbol ester, phosphatidylserine and Ca2+ (Castagna et al., 1982; Inoue et al., 1977;
Nishizuka, 1988; Ryves et al., 1991). PKCβI and PKCβII result from alternative splicing from a single gene and only differ by 45% in the last 52 amino acids in their C-terminus end (Ono et al., 1987). Unlike PKCα and PKCβ, PKCγ has a narrow tissue expression
18 that is mainly limited to the central nervous system (Yoshida et al., 1988). Relatively
newer generations of PKC inhibitors are available to dissect the biological roles of
cPKCs from the other two groups of PKCs, and more importantly from the nPKCs.
However, research is currently underway to design “ideal” PKC inhibitors specific to
only one isoform as potential drugs against tumors or diseases overexpressing that
isoform. For example, the bisindolylmaleimide ruboxistaurin mesylate (RBX:
LY333531) has more than 60-fold more specificity for PKCβ than the other cPKCs
(Jirousek et al., 1996) and has recently been shown to improve the retinal hemodynamic
abnormalities induced by activation of PKCβ as a result of hyperglycemia in diabetic
patients (Aiello et al., 2006).
Classical PKCs have been visualized in live cells to translocate within a few
seconds from the cytoplasm to the plasma membrane upon activation by PMA (Almholt
et al., 1999; Ng et al., 1999; Sakai et al., 1997). In addition, both saturated and the
unsaturated fatty acids, oleic and arachidonic acids, also result in a rapid and reversible
translocation of a PKCγ-GFP fusion protein to the plasma membrane (Shirai et al., 1998).
Overexpression of PKC has been reported to accentuate the cellular responses due to phorbol ester treatment (Housey et al., 1988).
Long term treatment with PKC activators, such as PMA or bryostatin-1 results in
the degradation of cPKCs by at least two different pathways. In one pathway the active
kinase is ubiquitinated on the membrane and targeted for proteasomal degradation (Lee et al., 1997; Leontieva and Black, 2004). Alternatively, the active PKC can be internalized by caveolae and trafficked to the perinuclear region (Prevostel et al., 2000) where it is
19 first dephosphorylated then degraded by a proteasome-independent mechanism
(Leontieva and Black, 2004). Prolonged incubation with PMA for at least 24h leads to the
degradation of cPKCs but not the novel and atypical PKCs (Choi et al., 2006; Stewart
and O'Brian, 2005).
Novel PKCs (nPKCs)
Members of this second group, which includes PKCs δ, ε, η, and θ are also activated by DAG or phorbol ester but not by Ca2+. Phorbol-12-myristate-13-acetate
results in the activation and translocation of PKCδ to the plasma membrane where the
kinase remains for more than 1h after treatment (Ohmori et al., 1998). Moreover,
saturated fatty acids activate nPKCs and induce a rapid and reversible translocation of a
PKCε-GFP fusion protein to the plasma membrane except in the case of the unsaturated
fatty acids, linoleic and arachidonic acids, which target the kinase relatively slowly and
irreversibly to the perinuclear region (Shirai et al., 1998). Activation of some novel PKCs also has been reported to be mediated by antibody triggering membrane rafts to coalesce.
For example, the activation of T cells by anti-CD3 antibody, results in the targeting of
PKCθ to DRMs followed by the activation of NF-κB (Bi et al., 2001).
In addition to cPKCs and nPKCs, other phorbol ester receptors (α- and β-
chimaerins, Ras-GRP) exist, that lack kinase activity and are also quite distinct from PKC
in their domain structure and interactions with inhibitors and cellular proteins. The
various DAG/phorbol ester activated PKC isozymes have distinct and non-redundant
20 functions as observed on the basis of gene knockout studies and observed differences in
tissue distribution and cellular localization.
Atypical PKCs (aPKCs)
Even though this third group which includes PKCζ, the human PKCι and its
mouse ortholog PKCλ contains a C1 domain, it is unresponsive to DAG and phorbol
ester. Furthermore, it is not activated by Ca2+, but like all the other PKC isoforms, aPKCs
are responsive to phosphatidylserine. PKCζ has been shown to be phosphorylated in vivo
and in vitro by PDK-1 on a threonine residue in the activation loop of the kinase in a phosphoinositide-3 kinase (PI-3 kinase)-dependent manner (Chou et al., 1998; Dong et al., 1999; Le Good et al., 1998). PI-3 kinase also results in phosphorylation of PKCλ in the signaling pathways through epidermal growth factor and the platelet-derived growth factor (PDGF) (Akimoto et al., 1996). Furthermore, active Cdc42 plays a role in shuttling
PI-3 kinase activated PKCλ and PKCζ, from the nucleus to their site of action in the cytoplasm where they regulate Ras-dependent cytoskeletal changes such as stress fiber loss (Coghlan et al., 2000).
PKC Binding Proteins
A crucial aspect of the unique function of each PKC isozyme is its binding to
proteins that may or may not be its substrates, via non-substrate binding sites (Dempsey
et al., 2000; Ron and Kazanietz, 1999; Schechtman and Mochly-Rosen, 2001). Such
interactions are necessary for localization, shuttling, activation or inhibition of the
21 enzyme. The specific protein-protein interactions are dictated by the state of activation of the enzyme and cofactor requirement. Activated PKC can bind to receptors for activated
C kinase (RACKs) or to substrates that interact with C kinase (STICKs). Inactive PKC
can bind to A kinase anchoring protein (AKAPs).
RACK1
Among PKC isozymes, RACK1 specifically binds to and stabilizes activated PKC
βII and α and may cause a further increase in catalytic activity (McCahill et al., 2002;
Ron et al., 1994, 1995; Rotenberg and Sun, 1998; Stebbins and Mochly-Rosen, 2001). It
has been demonstrated that after RACK1 binds to activated PKCβII the two proteins co-
migrate to intracellular target sites (to the plasma membrane in human embryonal kidney
cells) (Ron et al., 1999); thus RACK1 acts as a shuttling protein. In addition to PKC, a
variety of other proteins can bind to RACK1 through its WD40 repeat motif (McCahill et
al., 2002). A number of peptide and peptidomimetic reagents have been developed that
block or promote protein binding to RACK1 at specific sites, by binding to either
RACK1 or to its binding partners such as specific PKC isozymes (McCahill et al., 2002;
Way et al., 2000). These agents may themselves activate PKC or prevent activation.
14-3-3
Several 14-3-3 protein isoforms (β, ε, γ, η, σ, τ, ζ) have been identified and share
a high degree of amino acid sequence homology between different species (reviewed in
Fu et al., 2000). The isoforms 14-3-3ε and 14-3-3 ζ have been shown to associate with
22 DRMs (Foster et al., 2003; Sprenger et al., 2006). The 14-3-3 proteins can bind to
different PKC isoforms and activate or inhibit them in vitro, in different cell lines and in
vivo (Acs et al., 1995; Dai and Murakami, 2003; Robinson et al., 1994; Van Der Hoeven
et al., 2000). Overexpression of 14-3-3τ decreased the PMA-induced membrane targeting
of PKCθ by 40% thereby inhibiting downstream signaling needed for the activation of
the interleukin-2 promoter in T cells (Meller et al., 1996). More recently, the
overexpression of 14-3-3ε has been shown to decrease the activity PKCα bound to insulin
receptor substrate-1 thus providing evidence for the potential role of 14-3-3ε as one of the
negative modulators of insulin signaling (Oriente et al., 2005).
PKC Substrates
Annexin II
Annexin II also known as lipocortin II, calpactin I heavy chain or p36 belongs to a
family of Ca2+-phospholipid binding protein composed of thirteen different proteins
(Gerke et al., 2005; Glenney, 1986; Gould et al., 1986). Binding of an Annexin II dimer
with two p11 (S100A10) proteins results in the formation of a complex that aggregates
opposing membranes in a Ca2+-dependent manner (Gerke and Moss, 2002; Lambert et
al., 1997; Rety et al., 1999). Annexin II also is involved in other membrane trafficking events such as organization of rafts, inhibition of lysosomal transport of fluid phase tracers, maintenance of the morphology of recycling endosomes and Ca2+-dependent
exocytosis (Chasserot-Golaz et al., 2005; Gerke et al., 2005).
23 Annexin II phosphorylation occurs on the N-terminus at serine or tyrosine
residues and is mediated by PKC or protein tyrosine kinases such as pp60src (Glenney,
1985; Glenney and Tack, 1985), respectively. The phosphorylation of annexin II on
tyrosine 23 occurs in response to certain growth factors such as PDGF and insulin but not
epidermal growth factor. Inhibition of protein tyrosine kinases using the tyrphostin AG-
18 in CHO-T cells stably expressing the human insulin receptor results in a decrease in annexin II phosphorylation by insulin in a dose dependent manner and in a 50% decrease
in the rate of IR internalization (Biener et al., 1996). The tyrosine phosphorylated annexin
II translocation to the plasma membrane upon an increase in cytosolic Ca2+ generated by
PDGF signaling in Swiss 3T3 mouse fibroblasts (Brambilla et al., 1991). The tyrosine
phosphorylated annexin II translocates to the plasma membrane upon an increase in
cytosolic Ca2+ generated by PDGF signaling in Swiss 3T3 mouse fibroblasts (Brambilla
et al., 1991). The targeting of annexin II to the plasma membrane is dependent on
tyrosine phosphorylation and binding with p11 (Deora et al., 2004).
Protein kinase C results in phosphorylation of annexin II on serines 11 and 25 in
vivo (Gould et al., 1986; Liu et al., 2003; Yan et al., 2006). Treatment of nasopharyngeal
carcinoma CNE1 cells with PMA or overexpression of Epstein-Barr virus-encoded latent
membrane protein 1 (LMP1) leads to an increase in annexin II serine phosphorylation
and translocation from the cytoplasm to the nucleus where it is hypothesized to play a
role in DNA synthesis (Yan et al., 2006). The translocation of annexin II to the nucleus in
prostate cancer LNCaP cells can be inhibited by site-directed mutagenesis of both serine
residues in the N-terminus of the protein to glutamic acid (Liu et al., 2003). The
24 accumulation of annexin II in the nucleus is linked to a decrease in proliferation of
LNCaP cells (Liu et al., 2003). Annexin II is downregulated in high grade prostatic intraepithelial neoplasia (Chetcuti et al., 2001; Lehnigk et al., 2005; Stewart et al., 2006) but is overexpressed in a number of other human malignancies such as pancreatic
(Esposito et al., 2006) and colorectal carcinomas (Emoto et al., 2001), glioblastomas
(Reeves et al., 1992; Roseman et al., 1994) and lung cancers (Brichory et al., 2001).
Effect of PKC Modulators on Recycling of Different Membrane Receptors
FR
In confluent MA104 cells, phorbol ester and other activators of PKC such as
mezerein, indolactam V and phorbol 12,13 dibutyrate inhibit the internalization step of
FR recycling, causing an increase in cell surface FR (Smart et al., 1994). Diacylglycerol
also inhibits internalization of FR but to a lower extent, which is probably linked to the
rapid hydrolysis of this compound and to the transient activation of PKC (Smart et al.,
1994). In addition, treatment of MA104 cells with a physiologic hormone, progesterone,
results in an increase in the external membrane-bound pool of FR in a dose dependent
manner (Smart et al., 1996). All these experiments were done using a radioactive folic
acid probe to track the movement of FR because unlike 5-methyltetrahydrofolate
(5CH3THF), the physiologic ligand of FR, it remains bound to the receptor and does not get released into the cytoplasm as a polyglutamated form (Kamen et al., 1988). Phorbol-
12-myristate-13-acetate increased the uptake rate of 5CH3THF by about 1.8-fold as
compared to control MA104 cells (Lewis et al., 1998b). Incubation of MA104 cells with
25 PKC inhibitors (Gö6976 and GF 109203X) specific to conventional PKCs abolishes the
increase in FR caused by PMA (Kamen and Smith, 2004).
Treatment of malignant cells, which express much higher levels of membrane-
bound FR than MA104 cells, with PMA does not result in any detectable increase in FR
externalization (Kamen and Smith, 2004). These tumor cells appear to already be
“PMAed” since about 90% of the receptor is already on the cell surface. Therefore,
addition of the drug would not be expected to significantly change the distribution of the
receptor. The higher level of cell surface FR in actively growing MA104 cells and
malignant cells may reflect a higher level of appropriately targeted endogenous activated
PKC. To test this hypothesis, PKC inhibitors were incubated with various tumor cells and
still no change in FR distribution occurred (Kamen and Smith, 2004). Further
experiments are needed to elucidate the signaling mechanisms that govern FR recycling
in these cells and to rule out any role of PKC or lack of it.
TfR
Phorbol-12-myristate-13-acetate results in redistribution of the external and internal pools of TfR in a number of cell lines. Treatment of hepatoma cells (HepG2), human chronic myelogenous (K562) or promyelocytic leukemia (HL60) cells with PMA leads to the internalization of about half of the surface TfR as determined by binding of monoclonal anti-TfR antibodies or radiolabeled iodinated transferrin ([125I]-Tf) and by
resistance to proteolytic digestion (Fallon and Schwartz, 1986; Klausner et al., 1984; May
et al., 1984; Watts, 1985). The initial model proposed for TfR endocytosis was that ligand
26 binding or PMA treatment both constituted a signal for internalization of the otherwise static external TfR pool (Klausner et al., 1984). Subsequent experiments using a more sensitive detection assay than antibody labeling revealed that TfR recycling occurs continuously and has the same internalization rate whether ligand is bound to the receptor or not (Watts, 1985).
In contrast, incubation of Chinese hamster ovary (CHO) cells (McGraw et al.,
1988) or mouse tumor macrophage-like cells, J774 (Buys et al., 1984) with PMA results in externalization of TfR in a dose dependent manner. This increase in the external pool of TfR is due to an increase in the rate of exocytosis and is independent of phosphorylation of TfR on Ser-24 (McGraw et al., 1988).
An Opportunity to Seek New Drug Targets
GPI-protein rich rafts may be expected to play a role in several human diseases.
For example, GPI anchoring of the scrapie prion protein is necessary for its pathogenesis
(Kaneko et al., 1997) and it may be beneficial to target raft recycling to decrease the density of this protein on the cell surface. In the case of FR, targeting raft recycling to internalize the receptor would probably improve the uptake of folate compounds and novel antifolates by macrophages in rheumatoid arthritis or by FR-positive cancerous cells in ovarian and endometrial tumors (Buist et al., 1995; Mantovani et al., 1994; Nagai et al., 2006; Nagayoshi et al., 2005; Theti et al., 2003; Veggian et al., 1989; Wu et al.,
1999). One of the main advantages that render it possible to exploit FR as a tumor target is that the receptor expression in most proliferating normal tissues is restricted to the
27 luminal surface of certain epithelial cells that does not come in contact with the bloodstream. Moreover, not only do specific types of insidious malignant tumors overexpress FR, but also the receptor is accessible via the circulation and thus capable of binding the folate/antifolate drugs administered.
Furthermore, the major role of rafts in cell signaling implies that immune responses and malignant cell growth or cell death may be modulated by drugs that control raft recycling or reorganization. Edelfosine and aplidin are two antitumor drugs that result in clustering of Fas death receptors (CD95 or Apo-1) into lipid rafts which signals for apoptosis to begin (reviewed in Mollinedo and Gajate, 2006). Regulating the raft population on the cell surface could be one means of enhancing the cell’s sensitivity to external or internal signals and allowing cross-talk between signaling pathways.
28 MATERIALS AND METHODS
Chemicals and Reagents
LipofectamineTM 2000, Geneticin, penicillin/streptomycin/L-glutamine, human recombinant purified PKC proteins (α, β, γ, δ, ε), fetal bovine serum (FBS), Fura-2,AM,
Coomassie Brilliant Blue, Opti-MEM I reduced serum medium, folate-free RPMI
(FFRPMI), MEM and DMEM media were purchased from Invitrogen Life Technologies,
Inc. (Carlsbad, CA). Monoclonal and affinity purified antibodies raised against PKCs α,
βI, βII, γ, RACK1 karyopherin α2 and GAPDH were purchased from Santa Cruz
Biotechnologies (Santa Cruz, CA). Affinity purified rabbit anti-human annexin II was from BD Transduction Laboratories (San Jose, CA). Polyclonal rabbit anti-14-3-3 antibody was purchased from Zymed Laboratories Inc. (San Francisco, CA). Rabbit antiserum against FR was described previously (Ratnam et al., 1989). The reagents for
RT-PCR and real-time PCR were purchased from Applied Biosystems (Branchburg, NJ).
Hexadimethrine bromide “polybrene,” 2-mercaptoethanol, puromycin dihydrochloride, non-essential amino acids, holo-transferrin and protease inhibitors were purchased from
Sigma (St Louis, MO). Cyclopiazonic acid (CPA) was purchased from Acrōs Organics
(Pittsburgh, PA). Bradford reagent was purchased from Bio-Rad Laboratories (Hercules,
CA). Phorbol-12-Myristate-13-Acetate (PMA) was purchased from Calbiochem (La
Jolla, CA). Liquid scintillation fluid “EcoscintTM H” was purchased from National
Diagnostics (Atlanta, GA). Streptavidin magnetic beads were purchased from Roche
Diagnostics (Indianapolis, IN). [3′, 5′, 7, 9-3H]folic acid diammonium salt (43.2
29 Ci/mmol) and [125I]-diferric transferrin (80.0 Ci/mmol) were purchased from Moravek
Biochemicals (Brea, CA) and Perkin Elmer (Boston, MA), respectively.
DNA Expression Plasmids
The PKC α-EGFP and PKC βII-EGFP plasmids were purchased from Clontech
(Mountain View, CA). Protein kinase C βI cloned into the pEF1 mammalian expression plasmid was a kind gift from Dr Kevin Pan at the Medical University of Ohio.
To generate the pSUPER-PKCα and the pSUPER-control constructs, the custom oligonucleotides (5′- gatccccGGATGTGGTGATTCAGGATttcaagaga
ATCCTGAATCACCACATCCtttttggaaa-3′ and 3′- gggCCTACACCACTAAGTCCTA aagttctctTAGGACTTAGTGGTGTAGGaaaaaccttttcga -5′, position 1160 in the PKCα mRNA sequence accession number NM_002737) and (5′- gatccccGAGTGAGTTGCA
GGTTAGTttcaagagaACTAACCTGCAACTCACTCtttttggaaa-3′ and 3′- gggCTCA
CTCAACGTCCAATCAaagttctctTGATTGGACGTTGAGTGAGaaaaaccttttcga-5′) were respectively synthesized by Integrated DNA Technologies, Inc. (Coralville, IA)
(Brummelkamp et al., 2002). The 19 nucleotide PKCα target sequence and the control sequence are indicated in capital letters in the oligonucleotides. The annealed oligos were ligated into the pSUPER.Retro vector digested with BglII and HindIII and transformed using XL1-Blue competent cells. The DNA constructs were amplified using the Qiagen plasmid High-speed Maxiprep kit (Chatsworth, CA) and the sequences were verified by automated DNA sequence analysis using an H1 promoter primer.
30 Cell Culture
Unless otherwise mentioned, all the cell lines were grown in media supplemented
with fetal bovine serum (10% v/v), penicillin (100 units/ml), streptomycin (100 g/ml) and
L-glutamine (2mM) at 37°C in a 5% CO2 cell culture incubator. MA104 cells and CHO
cells purchased from American Type Culture Collection (Rockville, MD) were cultured
in FFRPMI. The recombinant CHO cell line stably expressing FR-β (CHO-FRβ) (Wu et
al., 1997) were maintained in the same media supplemented with L-Proline (14.5 mg/l).
The stable human-derived 293 GPG packaging cell line was cultured as
previously described (Ory et al., 1996) in DMEM supplemented with heat-inactivated
FBS (10%), puromycin (1 μg/ml), doxycycline (2 μg/ml) and geneticin (300 μg/ml). The
293 FT cells used for packaging lentiviral particles were kindly provided by Dr. Yeung
(University of Toledo). These cells were maintained in DMEM, heat inactivated FBS
(10%), geneticin (500 μg/ml) and non-essential amino acids (1%). HEK-293 cells were cultured in DMEM. All the cell lines used for packaging or generating viral particles were switched to FFRPMI 3 days before the transfection or infection started.
FR Recycling
MA104 cells were split as 2.25x105 cells per well in 6-well plates in FFRPMI and used for a FR recycling experiment on day 2. Briefly, the cells were incubated with 27nM of [3H]folic acid in serum free FFRPMI for 1h at 37°C. After washing the cells twice
with 37°C media to remove any unbound radioactivity, vehicle or 1μM PMA was added.
At the end of an incubation period of 30 min at 37°C, the media was aspirated and the
31 cells were washed with 1ml of acid buffer (10mM sodium acetate, pH=3.5/150mM NaCl)
for 1 min on ice. The acid wash was counted in scintillation liquid and represents the
amount of external [3H]folic acid bound. The cells were washed once with ice-cold PBS
(137mM NaCl, pH=7.4/ 27 mM KCl, 10mM Na2HPO4 and 2mM KH2PO4) and then
lysed with 0.1N NaOH to determine the amount of internalized [3H]folic acid. MA104 cells also were incubated with 20 times more unlabeled folic acid, relative to the amount of [3H]folic acid added, to ensure the specificity of uptake via FR. All samples were
measured in triplicates.
FR Internalization and Externalization
MA104 were plated in 6-well plates as 2.25 x 106 cells/well in FFRPMI and used
for a FR influx or efflux experiment on day 5. For FR internalization experiments, the
cells were first incubated with vehicle or 1μM PMA in serum-free FFRPMI for 30 min at
37°C. Then 27nM of [3H]folic acid were added in serum-free FFRPMI and incubated at
37°C for different length of time. At the end of the incubation period, the media was
aspirated and the cells were washed twice with ice-cold PBS and the amount of external
and internal [3H]folic acid bound was measured by liquid scintillation counting as
specified above.
For FR externalization experiments, cells were incubated with 27nM of [3H]folic acid in serum free FFRPMI for 1h at 37°C. Then vehicle or 1μM PMA was added and incubated for another 30 min at 37°C. The media was aspirated and the cells were washed once with ice-cold PBS and chilled on ice for 20 min. The cells were washed once with
32 1ml of acid buffer for 1 min followed by two ice-cold PBS washes. At time 0, 37°C
FFRPMI was added to each well and incubated for different length of time at 37°C. At
the end of each time point, the amount of external and internal [3H]folic acid bound was
measured as described under the “FR recycling” section.
In all of these experiments, samples were measured in triplicates and an excess of
cold folate was added to determine the non-specific background binding.
TfR Recycling
MA104 were plated in 6-well plates as 2.25 x 105 cells/well in FFRPMI were
used for a TfR recycling experiment on day 5 as previously described (Takeuchi et al.,
1992). Essentially, the cells were incubated with vehicle or 1μM PMA in serum-free
FFRPMI for 30 min at 37°C. Then the cells were washed once with ice-cold HEPES- buffer (20mM HEPES, pH=7.2 containing 0.15 M NaCl, 0.5 mM MgCl2 and 0.05 mM
125 CaCl2), chilled on ice for 20 min and incubated with 2nM [ I]transferrin (77 Ci/mmol)
for 1h on ice in HEPES-saline containing 0.3% (w/v) bovine serum albumin. The cells
were washed twice with ice-cold HEPES-buffer and 37°C serum free FFRPMI was added
to each well. The cells were incubated at 37°C for different amount of time, at the end of
which the media was counted with a gamma counter to obtain the amount of TfR that
recycled. Cell bound [125I]transferrin was released by adding acidic HEPES-buffer
(pH=2.0) for 4 min on ice. Finally, the cells were lysed with 1% TX-100 and 0.5% SDS
and the amount of internalized [125I]transferrin was measured. Non specific binding of
[125I]transferrin was determined in identical assays by adding 400-fold excess unlabeled
33 holo-transferrin along with the labeled compound. All samples were measured in
triplicates and counted with a gamma counter.
Calcium Release and Measurement
Intracellular calcium was measured as previously described (Giovannucci et al.,
2000; Perez et al., 1999). Briefly, MA104 cells split on glass coverslips were incubated at
37°C for 1h in a loading buffer (10mM HEPES, pH=7.4 containing 132mM NaCl, 5mM
KCl, 1.8mM CaCl2, 0.8mM MgCl2, 1 mM sodium pyruvate and 1% bovine serum
albumin) containing 10μM Fura-2/AM, a cell-permeable intracellular calcium indicator
that is excitable with ultraviolet light. At the end of the incubation period the cells were
washed twice and Ca2+ free Lockes (10mM HEPES, pH=7.2 containing 143mM NaCl,
5mM KCl, 1mM MgCl2 and 10mM glucose). Ratiometric imaging was done using a
Nikon TE2000-S microscope equipped with a Nikon Super Fluo 40x/NA= 1.30
epifluorescence oil-immersion objective (Melville, NY). Fura-2/AM-loaded cells were
locally superfused at a rate of 1 ml/min with Ca2+ free Lockes and excited at 340 or 380
±15 nm. The emission fluorescence was collected with a 510 ± 25 nm bandpass filter
(Chroma, Rockingham, VT). Intracellular Ca2+ imaging was analyzed using the TILL-
Photonics Polychrome IV digital fluorescence imaging system and VISIONsoftware
(New Milford, CT). Cyclopiazonic acid (CPA) (30μM) and EGTA (0.2mM) were
perfused into the chamber to release internal Ca2+ stores. CPA was added for a second
time to ensure that the internal Ca2+ stores were completely depleted.
34 Cells were incubated with 27 nM [3H]folic acid for 1h at 37°C, washed twice with
37°C FFRPMI in order to remove any unbound radioactivity and further incubated with
vehicle or (30μM CPA and 0.2mM EGTA) for 5 min at 37°C in Ca2+ free Lockes. Then the cells were washed once with 37°C Lockes and incubated with vehicle or 1μM PMA for 30 min at 37°C. The cell surface bound and the intracellular radioactivity were recovered by treatment at low pH or the addition of 0.1N NaOH, respectively. The subtracted non-specific association (< 2 percent) of [3H]folic acid was determined using
negative control samples that were treated with excess unlabeled folic acid. All samples
were measured in triplicates.
Adenovirus-mediated Gene Transfer to MA104 Cells
Replication-deficient adenoviruses expressing Ad5-type adenovirus vectors
containing the cDNA of wild type PKCs (α, βII, δ and ε) and kinase negative mutants
(DN-PKCα and DN-PKCβII) were a kind gift from Dr. K.V. Chin (University of Toledo)
(Matsumura et al., 2003; Oka et al., 2002). The constitutively active myristoylated PKCα
(PKCα-CA) was a kind gift from Dr. D. Dartt (Harvard Medical School) (Hodges et al.,
2004). The adenovirus carrying the green fluorescent protein (GFP) gene was designated
as GFP-Ad and used as a control virus to determine the infection efficiency. The
adenoviral stocks were amplified by infecting HEK-293 cells in 10 cm plates with
∼4x1015 infectious particles in FFRPMI containing heated FBS and (8μg/ml) of
polybrene. The cells were harvested 48h post-infection when they displayed cytopathic
effects, subjected to five consecutive freeze/thaw cycles to release adenoviuses trapped
35 inside the cells, and centrifuged at 3000 rpm for 20 min. The supernatant was aliquoted
and stored at -80°C. Finally, the adenoviral stocks were titered using the optical
absorbance method (Maizel et al., 1968).
MA104 cells were split in FFRPMI as (0.095x106 cells/well) in a 6-well plate on
day 0. On day 6, cells were infected for 1h with ∼1015 infectious particles per well in 1ml of serum free FFRPMI containing (8μg/ml) of polybrene. At the end of which, an equal volume of FFRPMI containing 20% heated FBS was added. The cells were cultured for another 48h before FR recycling experiments were done or cell lysates from the infected cells were analyzed by western blot. Using these conditions, the adenoviruses infected most of the cells (>90%) based on the fluorescence of the GFP-Ad infected cells visualized with a fluorescent microscope.
Packaging of Recombinant Lentiviruses and Infection of Cells
293 FT cells were transfected with MISSIONTM shRNA lentiviral constructs in
pLOK.1-puro for RACK1 and annexin II knockdown (Sigma-Aldrich) or a control
sequence (Table I) along with plasmids needed for lentiviral packaging (pMD2G,
pMDLg/pRRE and pRSV-Rev) using LipofectamineTM 2000 according to the vendor’s
protocol. Viruses were harvested at 48h and 72h post-transfection. Essentially, the
supernatant was centrifuged at 2500 rpm for 10 min at 4°C then filtered through a 0.45
μM sterile filter (Nalgene). MA104 cells were infected with lentivirus at 30% confluence.
Polybrene (8μg/ml) was added to increase the infection efficiency. Folate receptor
36 recycling experiments were done 72h post-infection of MA104 cells with the
recombinant lentivirus.
Table I. Sequence of the control sequence, RACK1 and annexin II knockdown sequences cloned into the pLOK.1-puro plasmid purchased from Sigma-Aldrich (MISSIONTM shRNA clones)
Gene/Target (Accession number) Sense strand sequence
Control (Non-target) 5′-CAA CAA GAT GAA GAG CAC CAA-3′
RACK1 (NM_006098) 5′-GAT GTG GTT ATC TCC TCA GAT-3′
Annexin II (NM_001002857) 5′-GCA GGA AAT TAA CAG AGT CTA- 3′
Packaging of Recombinant Retrovirus and Infection of MA104 Cells
The stable human-derived 293 GPG packaging cells were transfected with pSuper
constructs containing a control or a PKCα knockdown sequence (described under the
“DNA expression plasmids” section) using LipofectamineTM 2000 according to the
vendor’s protocol. The media was replaced 4h post-transfection and the following day
with fresh FFRPMI containing 10% heated FBS. The retroviral media was harvested at
48h and 72h post-transfection. Essentially, the virus was harvested by filtering the
harvested media through a 0.45 μM sterile filter (Nalgene). MA104 cells split on day 0 as
(0.095x106 cells/well) in FFRPMI containing heated FBS were infected on days 1 and 2 by adding the retroviral media and polybrene (8μg/ml) to increase the infection efficiency. On day 3, the media was replaced with fresh FFRPMI media. Folate receptor
37 recycling experiments were done 72h post-infection of MA104 cells with the retroviral
media.
Membrane Preparations
Membranes from confluent CHO, CHO-FRβ and MA104 cells were prepared as
previously described (Wang et al., 2002). Essentially, cells were washed on ice with acid
buffer to release any bound folates from FR followed by two PBS washes. The cells were
lysed in lysis buffer [1mM NaHCO3, pH=7.2 containing 2mM CaCl2, 5mM MgCl2, and
1mM phenylmethylsulfonyl fluoride (PMSF)] for 30 min on ice, homogenized with a dounce homogenizer and centrifuged for 10 min at 2,000 ×g at 4°C. The supernatants were spun at 30,000 ×g for 45 min at 4°C and the membrane pellet were washed with acid buffer followed by three PBS washes. Finally, the membranes were resuspended in
1× Hank’s balanced salt solution (HBSS) containing protease inhibitors (1mM PMSF,
2µg/ml Aprotinin, 2 µg/ml Leupeptin and 1 µg/ml Pepstatin A).
Affinity Purification of FR Rafts using Biotinylated Folic Acid
All the following steps were carried out on ice or at 4ºC unless specified otherwise. Streptavidin coated-magnetic particles (0.5mg) from Roche Diagnostics
(Indianapolis, IN) were washed twice with HBSS (containing 1% Triton X-100 and
1mg/ml BSA). Membranes (50 μg) from CHOK1 or CHO-FRβ cells were incubated with biotin-SS-folate (Fan et al., 1991; Wu et al., 1997) (1μM final concentration) for 30 min on a rotary shaker. An equal volume of HBSS (containing 2% Triton X-100 and 2mg/ml
38 BSA) was added to each sample and the prewashed streptavidin particles and rotated for another 4h. The samples were washed six times using a magnet stand with HBSS
(containing 1% Triton X-100 and 0.5 M NaCl) followed by three washes with HBSS only. Finally, 10 mM DTT in HBSS (containing 0.5% Triton X-100) was added to the samples in order to hydrolyze the disulfur bond in biotin-SS-folate thereby eluting the
FR-rich rafts.
The same steps were followed to purify FR-rich rafts from MA104 cells except that 200µg of membranes were used instead of 50 μg. Also, some samples were incubated with 1mM folic acid for 30 min prior to the addition of biotin-SS-folate in order to determine nonspecific binding to the probe.
Mass Spectrometry
The affinity purified FR rafts using biotinylated folic acid were electrophoresed on a 12% polyacrylamide-SDS gel and gel slices from each lane were digested with trypsin. The peptides were extracted with (60% acetonitrile: 0.1%TFA) followed by a reverse phase column separation step. The peptides were directly introduced into an ion- trap mass spectrometer equipped with a nano-spray source. Collision induced dissociation spectra were either manually interpreted or searched against an appropriate non-redundant database using TurboSEQUEST.
39 Electron Microscopy
MA104 cells were washed with HBSS then incubated in solution A (1x HBSS containing 30mM NaN3 and 1% BSA) for 30 min at room temperature. The cells were then probed with rabbit anti-FR antibody or rabbit pre-immune serum as negative control for 2h, washed with solution A four times and then incubated with 12nm colloidal gold- conjugated donkey-anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove,
PA) for 1.5h at room temperature. The cells were washed again four times with solution
A, fixed with 3% glutaraldehyde, washed with 0.2M sodium cacodylate (pH=7.2), embedded in LR-White medium and sectioned. The sections were first incubated in solution B (PBS containing 1% fish gelatin) for 15 min, then probed with goat anti-
RACK1 or goat anti-karyopherin α2 antibodies for 2.5h. Then, the sections were washed five times with solution B, followed by the incubation with 6-nm colloidal gold- conjugated donkey-anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove,
PA) for 1.5h. Again, the sections were washed five times with solution B, one high salt wash with 2.5M NaCl (pH=8.5), and four distilled water washes. The grids were examined with a CM-10 Philips electron microscope. In order to better visualize the gold particles, uranyl acetate and lead citrate poststaining was avoided.
RNA isolation and quantitative Real-Time RT-PCR
Total RNA from MA014 cells was prepared using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. Reverse transcription PCR (RT-PCR) followed by quantitative real-time PCR was used to measure endogenous mRNA levels for PKCs
40 α, βI, and βII and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Total RNA
(400 ng/sample) was reverse transcribed with random primers using the “high-capacity
cDNA archive kit” from Applied Biosystems. The reverse transcription product was
measured by quantitative real-time PCR using the Real-Time PCR master mix (Applied
Biosystems) in the 7500 Real-Time PCR System (Applied Biosystems). Essentially, 10
μl of the reverse transcribed RNA was mixed with 12.5 µL of PCR Mastermix (Applied
Biosystems), 0.5 µL each of the forward and reverse primers (9 μM) and 0.5 µL of the
TaqMan probe (2.5 μM). The primers and TaqMan probe for PKC α, PKC βI, and PKC
βII (Table II) were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA) as previously described (Guo et al., 2003). The primers and the TaqMan probe for the control GAPDH gene were purchased from Applied Biosystems. The PCR conditions
were 2 min at 50°C, followed by 10 min at 95°C, then 40 cycles each of 15 sec at 95°C
and 1 min at 60°C. Fluorescence data generated were monitored and recorded on a 7500
Real-Time PCR sequence detection system (Applied Biosystems). All samples were
measured in triplicate and normalized to GAPDH values. Different concentrations of
PKC α-EGFP, PKC-βII-EGFP and PKC-βI plasmids were used as positive controls for
the Taqman probes and to determine the relative level of mRNA of each of these
isoforms in MA104 cells. Finally, mRNA without any reverse transcriptase added during
the reverse transcription step was used as negative control for quantitative Real-Time RT-
PCR.
41 Table II. PKC α, βI and βII primers and Taqman probes used for quantitative Real-Time
RT-PCR (Guo et al., 2003)
Name Sense probe Probe Antisense (GenBank accession no)
CAGATCCTT 6-FAM-TCCTGATCCCA TGTAGAGCG PKC α ATGTGAAGC AGAACGAAAGCAAACA- GATGGTCTT (NM_002737) TGAAACTT TAMRA GGTT
CACCAGACA 6-FAM-GCCTACTGATA GAAGCCAGC PKC βI GCCTGTGGA AACTCTTCATCATGAAC AAATTCGTT (NM_002738) ACT TTGGA-TAMRA TTG
CTACTTAGC ACCAGGAAG 6-FAM-TCAGAATTCG PKC βII TCTTGACTTC TCATCAGGA AAGGATTTTCCTTTGTT (X07109) AGGTTTTAA ATATTGA AACTCTGAA-TAMRA AA
Cdc42 Activity Assay
MA104 cells treated with vehicle or 1μM PMA for 30 min at 37°C were assayed
for Cdc42 activity in whole cell lysates using a GST-fusion protein containing the p21-
binding domain (PBD) of human p21-activated kinase 1 (Pak1) to affinity pull-down
active Cdc42. The assay was done following the manufacturer’s protocol provided by
Stressgen (BC, Canada). Briefly, the cells were washed once with ice-cold PBS and lysis
buffer (freshly mixed with a cocktail of protease inhibitors) was added. The cells were
immediately scrapped off the plate, incubated for 5 min on ice and centrifuged at 16,000g
42 for 15 min at 4°C. The supernatant was added to a spin cup in which an immobilized
glutathione disc and 20 μg of GST-Pak1-PBD were already mixed. The spin cup was
vortexed, incubated for 1h at 4°C with gently rocking then spun at 7,200 g for 30 sec and
washed three times with the lysis/binding/wash buffer (provided in the kit). Finally, 50 μl
of 2x SDS sample buffer with 5% fresh β-mercaptoethanol were added to the resin,
boiled for 5 min at 100°C and centrifuged at 7,200 g for 2 min. For each of the cell
lysates assayed a positive control was performed by adding 10 mM GTPγS, which is a
non-hydrolysable form of GTP that irreversibly binds to Cdc42, and 10mM EDTA
followed by an incubation of 15 min at 30°C. The reaction was stopped by placing the
samples on ice and adding MgCl2 for a final concentration of 60 mM. All samples then
were assayed for Cdc42 by western blot using a mouse monoclonal anti-Cdc42 antibody.
Preparation of Cell Lysates and Western Blots
Cell pellets were resuspended in lysis buffer (PBS containing 1% Triton X-100
and a cocktail of protease inhibitors) and incubated on ice for 30 min. The cell lysates
were centrifuged at 13000 rpm for 10 min. The protein concentration in the cell lysates
was measured using the Bradford assay reagent. Cell lysates (10-100 μg) were mixed with 4×SDS loading buffer. Samples were resolved by electrophoresis on 8-12%
polyacrylamide SDS gels and electrophoretically transferred to a nitrocellulose
membrane. The blots were first probed overnight at 4°C with the appropriate primary
antibodies and then with goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase. The bands were visualized by enhanced chemiluminescence. The
43 same membranes then were similarly re-probed with a primary mouse anti-GAPDH antibody as loading control.
Data Analysis
The results are plotted as a mean of triplicate values ± standard error. The statistical difference between two different groups is represented by a p value indicated in the figure legend were relevant. The p values were calculated using the ANOVA test and the software Statview.
44 RESULTS
Introduction
Lipid rafts are liquid-ordered plasma membrane microdomains that are
characterized by a concentration of cholesterol, glycosphingolipids, GPI-anchored
proteins and signaling proteins and that may, in some cells, cover up to half of the cell
surface (Edidin, 2003; Rajendran and Simons, 2005; Vereb et al., 2003). These units of the plasma membrane are believed to serve as platforms for signal transduction as well as units of protein recycling between the cell surface and endocytic compartments
(Sabharanjak et al., 2002) or the Golgi (Nichols et al., 2001) through unique non-clathrin mediated pathways. Rafts were biochemically identified as protein and lipid complexes that were insoluble in cold non-ionic detergent and known as detergent resistant
membrane complexes (DRMs) (Brown and London, 2000; Brown and Rose, 1992;
Chamberlain, 2004) but the presence or absence of certain raft proteins in DRMs could
result as an artifact of detergent treatment (Heerklotz, 2002; Heerklotz et al., 2003;
Zurzolo et al., 2003). Indeed, considerable controversy has for some time surrounded the
question of the nature and size as well as the very existence of lipid rafts in live cells in
situ, based on measurements of proximity of GPI-protein molecules (Kenworthy and
Edidin, 1998; Kenworthy et al., 2000; Sharma et al., 2004) and disagreements on single particle tracking measurements (Subczynski and Kusumi, 2003). There is also substantial evidence that raft size and protein composition are in a state of dynamic flux (Simons and
Toomre, 2000) so that very small rafts of only a few molecules (Kenworthy et al., 2000;
45 Sharma et al., 2004) could coalesce to form functional units. A consensus definition has recently been formalized for rafts that identifies them in the size range of 10-200 nm, as heterogeneous, highly dynamic and capable of stabilization by protein-protein and protein-lipid interactions between rafts (Pike, 2006). Caveolae or plasmalemmal vesicles, which are observed as cell surface invaginations, are regarded as a special type of lipid rafts that are also enriched in cholesterol and glycosphingolipids as well as proteins involved in signal transduction and are characterized by a coat of caveolin molecules on their cytosolic surface (Anderson, 1998). Most recent studies discount the presence of a significant amount of FR in caveolae but there is morphological and functional evidence for the localization of at least some GPI-anchored proteins in caveolae; it has even been proposed that GPI-anchored proteins may exchange between caveolin and non-caveolin rafts (Anderson, 1998). The recycling of raft associated GPI-anchored proteins between the cell surface and endocytic compartments has been shown to occur via a cdc42- regulated pinocytic pathway (Sabharanjak et al., 2002) but possible mechanisms by which raft recycling could be regulated are not clear.
The GPI-anchored FR has been used extensively as a tractable marker to study the
movement of rafts and lends itself to the studies of regulation of raft recycling addressed
in this study. The recycling of FR is also physiologically important in the intracellular
delivery of reduced folate coenzyme. Bart Kamen and colleagues have established that in
MA104 (monkey kidney epithelial) cells, FR quantitatively recycles, within minutes,
between the cell surface and intracellular compartments (Kamen et al., 1988, 1989;
46 Rothberg et al., 1990). The recycling of FR was dependent upon its GPI anchor (Ritter et
al., 1995) as well as membrane cholesterol (Chang et al., 1992) and the actin cytoskeleton
(Lewis et al., 1998a). The studies demonstrated that virtually every molecule of FR in the plasma membrane of MA104 cells is functional in this manner providing a compelling argument for the quantitative localization of FR in rafts in live cells. Clustering of FR was observed by using a monovalent, biotinylated folate affinity labeling reagent on live cells under transport permissive conditions followed by fixing and embedding the cells prior to probing with colloidal gold labeled streptavidin for electron microscopy (Wu et al., 1997). Subsequently, FR clusters at the live cell surface were demonstrated by chemical cross-linking (Friedrichson and Kurzchalia, 1998) and fluorescence resonance energy transfer (Varma and Mayor, 1998). Folate receptor has been observed in endocytic compartments by cell fractionation, electron microscopy and fluorescence microscopy (Birn et al., 1993; Hjelle et al., 1991; Mayor et al., 1998). Recycling of FR also has been shown to be Cdc42-dependent (Sabharanjak et al., 2002).
Studies by the Kamen group have demonstrated that the steady state distribution of
FR between the cell surface and intracellular compartments is altered upon treatment of
cells with phorbol ester resulting in an increase in the proportion of the receptor
molecules on the cell surface (Smart et al., 1994). This increase occurred due to
inhibition of the internalization step of FR recycling while the externalization step was
unaffected. Using PKC inhibitors, it was suggested that the phorbol ester effect was
mediated by a classical PKC (cPKC) (Kamen and Smith, 2004). Since phorbol ester
47 effects on physiological processes are known to simulate natural cell signaling through the second messenger diacylglycerol, we undertook to investigate in detail, the molecular mechanism by which FR recycling in MA104 cells is regulated by phorbol ester. We initially identified putative components of FR-rich rafts by taking advantage of our ability to use a folate ligand to affinity purify FR-rich DRMs, which are virtually free of caveolin (Wang et al., 2002) and subsequently established their functional roles in the regulation raft recycling. The studies provide a comprehensive molecular picture of phorbol ester action on the recycling of FR in MA104 cells. Our hypothesis is that the population of FR rafts on the cell surface is increased by activators of PKCα as a result of targeting of PKCα to rafts by RACK1 and phosphorylation/inhibition of raft-associated protein(s).
48
Results
Phorbol Ester Redistributes FR Molecules to the Cell Surface by Inhibiting Influx
The movement of FR molecules between the cell surface and intracellular compartments may be tracked using receptor bound [3H]-labeled folic acid, the non-
physiologic form of the vitamin (Kamen et al, 1988). This method takes advantage of the
fact that once bound, folic acid dissociates extremely slowly from the receptor and remains bound during the entire time that it recycles. It also takes advantage of the fact that the receptor molecules residing at the cell surface at any time may be identified by their ability to release the bound folic acid upon exposure to low extracellular pH at ~
4ºC. Thus, the ratio of the acid labile fraction of the receptor to the acid resistant fraction reflects the ratio of extracellular to intracellular FR. As previously reported, treatment of
MA104 cells with PMA caused a dose-dependent increase in the extracelluar fraction of
FR reaching the maximal effect at 0.1 μM PMA (Figure 4).
An alteration in the steady state distribution of FR in the cell could result from a
change in the rate of either its internalization or its externalization. Phorbol-12-myristate-
13-acetate did not significantly alter the externalization rate of FR (Figure 5A) but did
substantially decrease its internalization rate (Figure 5B). The decreased rate of
internalization accounts quantitatively for the increase in the steady state level of the cell
surface fraction of FR. These results are in agreement with previous studies of the
kinetics of FR recycling even though those studies were designed to examine the effect of
49 phorbol ester on caveolae (Smart et al., 1994). The recycling of the transferrin receptor, monitored using [125I]-labeled transferrin, was relatively unaffected by PMA (Figure 6).
Since the recycling transferrin receptor is localized in clathrin-coated pits, the results
indicate that the effect of PMA on FR recycling is specific to its membrane microdomain
in MA104 cells.
4
) ** 3
2
external/internal
( 1
R F 0 1234 PMA (µM) 0 0.01 0.1 1.0
Figure 4: PMA increases cell surface FR in a dose-dependent manner in MA104 cells. Cells were incubated with 27 nM [3H]folic acid for 1h at 37°C, washed twice with 37°C FFRPMI in order to remove any unbound radioactivity and further incubated with vehicle or 1μM PMA for 30 min at 37°C. The cell surface bound radioactivity (representing external FR) was removed by treatment at low pH, and the intracellular radioactivity (representing internal FR) was recovered by the addition of 0.1N NaOH. The subtracted non-specific association (< 2 percent) of [3H]folic acid was determined using negative control samples that were treated with excess unlabeled folic acid (data not shown). Each bar is the average of triplicate measurements. The star (*) denotes that the values are statistically different from the vehicle treated cells with (p<0.005).
50
A 30 Vehicle PMA
]fo H lic 3 20
10 acid (percent of loaded) of loaded) acid (percent
Externalization of [
0 0 2 4 6 8 10 12 14 16 Time in minutes
Figure 5: Effect of phorbol ester on externalization and internalization of FR. (A) MA104 cells were incubated with 27 nM [3H]folic acid for 1h at 37°C followed by the addition of vehicle or 1μM PMA for 30 min at 37°C. At the end of the incubation period, the cells were washed once with ice-cold PBS and chilled on ice for 20 min. Then the cells were washed once with acid buffer followed by two cold PBS washes. FFRPMI prewarmed to 37°C was then added to each well and the cells were incubated at 37°C for the indicated times. The amount of external [3H]folic acid bound was removed with a 1 min cold acid buffer wash.
51
B 6000 Vehicle PMA
4000 H]folic acid acid H]folic 3 cells) 6
2000 (cpm/10
Internalization of [ 0 0 2 4 6 8 10 12 14 16
Time in minutes
Figure 5: Effect of phorbol ester on externalization and internalization of FR (continued). In (B), MA104 cells were first incubated with vehicle or 1μM PMA for 30 min at 37°C. Then the media was aspirated and 27 nM [3H]folic acid was added to each well and incubated at 37°C for the indicated times. At the end of each time point, the cells were immediately washed twice with ice-cold PBS and low pH buffer to remove the cell surface bound radioactivity. The intracellular radioactivity which represents internal FR was recovered by the addition of 0.1N NaOH. For (A) and (B), the subtracted non- specific association (< 2 percent) of [3H]folic acid was determined using negative control samples that were treated with excess unlabeled folic acid (data not shown). Each bar is the average of triplicate measurements.
52
0.6 Media Vehicle / Total Vehicle Media PMA / Total PMA
0.4
I]Transferrin I]Transferrin
125 0.2
Recycled [
0 0 2 4 6 8 10 12 14 Time in minutes
Figure 6: Effect of phorbol ester on TfR recycling. MA104 cells were incubated with vehicle or 1μM PMA in serum free FFRPMI for 30 min at 37°C. Then the cells were washed once with ice-cold HEPES-buffer, chilled on ice for 20 min and incubated with 2nM [125I]transferrin for 1h on ice in HEPES-saline containing 0.3% (w/v) bovine serum albumin. The cells were subsequently washed twice with ice-cold HEPES-buffer and 37°C serum free FFRPMI was added to each well and incubated at 37°C for different length of time. At the end of each time point, the media was counted to determine the amount of TfR that recycled. Cell surface bound [125I]transferrin was released by adding acidic HEPES-buffer for 4 min on ice. Finally, the cells were lysed to measure the amount of internalized [125I]transferrin. The total amount of TfR represents the sum of the amounts of [125I]transferrin in the media, on the cell surface and inside the cells. Non specific binding of [125I]transferrin was determined in identical assays by adding 400-fold excess unlabeled holo-transferrin along with the labeled compound (data not shown). All samples were measured in triplicates and counted with a gamma counter.
53
Ca2+ Also Modulates FR Distribution in MA104 Cells
Since Ca2+ plays an important role in cell signaling in concert with diacylglycerol, the effect of the calcium ionophore CPA, was tested on steady state FR distribution and on its redistribution by PMA (Figure 7). CPA induced a quantitative release of intracellular Ca2+ stores (Figure 7A) which resulted in a doubling of the cell surface fraction of FR (Figure 7B) simulating the effect of PMA. Treatment with PMA following
CPA resulted in a relatively smaller further increase in externalization of the receptor
(Figure 7B). This result suggests that the PMA effect may be mediated by the mobilization of intracellular Ca2+.
(A) 2000
CPA CPA
340nm /380 Ratio
1000 0 100 200 300 400 500 600 Time in secondes
Figure 7: Effect of intracellular calcium release on FR distribution in MA104 cells. (A) MA104 cells were loaded with Fura-2/AM for 1h and changes in intracellular Ca2+ were recorded as a function of time after the chamber was perfused with CPA (30μM) and EGTA (0.2mM) at the time points indicated by the arrows. A representative plot (of at least 20 individual cell recordings) of the CPA-induced changes in intracellular Ca2+ in a single cell as a function of time.
54
(B) 6 Vehicle b 1μM PMA c 4
a 2 Folate receptor Folate receptor
(external/internal)
0 CPA -+
Figure 7: Effect of intracellular calcium release on FR distribution in MA104 cells (continued). In (B), cells were incubated with 27 nM [3H]folic acid for 1h at 37°C, washed twice with 37°C FFRPMI in order to remove any unbound radioactivity and further incubated with vehicle or (30μM CPA and 0.2mM EGTA) for 5 min at 37°C in Ca2+ free Lockes. Then the cells were washed once with 37°C Lockes and incubated with vehicle or 1μM PMA for 30 min at 37°C. The cell surface bound radioactivity (representing external FR) was removed by treatment at low pH, and the intracellular radioactivity (representing internal FR) was recovered by the addition of 0.1N NaOH. The subtracted non-specific association (< 2 percent) of [3H]folic acid was determined using negative control samples that were treated with excess unlabeled folic acid (data not shown). Each bar is the average of triplicate measurements. The letters denote that “b” and “c” are statistically different from “a” with (p<0.005).
55
PKC α is the Only Classical PKC Expressed in MA104 Cells
Phorbol-12-myristate-13-acetate activates both classical and novel PKCs. The
ability of CPA to induce externalization of FR supports a previous suggestion using PKC inhibitors that the phorbol ester effect on FR recycling may be mediated by a cPKC since
this class of PKCs is typically Ca2+ -dependent. However, it is also possible that the
calcium ionophore effects were independent of PKC activation by phorbol ester and that
the PKC inhibitors produced non-specific effects. To directly test the role of individual
cPKCs in the action of phorbol ester on FR recycling, it was first necessary to identify the
possible candidate cPKCs in MA104 cells. Cell lysates together with purified
recombinant classical and novel PKCs (antibody specificity controls) were probed on
western blots using antibodies against the individual cPKCs (Figure 8). A strong band
was obtained for PKCα in the cell lysate whereas none of the cPKCs isoforms including
βI, βII, or γ could be detected. Since the antibody for PKCβI was cross-reactive with
PKCγ, the absence of PKCβI/βII was further confirmed by real time RT-PCR which only revealed the expression of mRNA for PKCα (Table III). It may be noted that the Taqman
probes for PKCs βI and βII used in Table III were > 8-fold more sensitive to their
respective target genes than the PKCα probe. Based on these results the likely isoform of
PKC that mediates phorbol ester effects in MA104 cells is PKCα.
56
MA104 α βI βII γ δ ε η θ PKCα
PKCβI
PKCβII
PKCγ
Figure 8: PKCα is the only classical PKC that can be detected in MA104 cells. Total cell lysates from MA104 cells (100 μg/well) and 5ng of each of the classical and novel PKC purified recombinant proteins (PKCs α, βI, βII, γ, δ, ε, η, and θ) were subjected to western blot analysis using anti-PKCs α, βI, βII and γ antibodies.
Table III. PKCs α, βI and βII mRNA levels in MA104 cells as determined by quantitative Real-Time RT-PCR
Ct values by quantitative real-time RT-PCR
PKC Standard 0.001 ng plasmid† MA104 RNA sample
α 20.2±0.8 21
βI 15.4±0.4 Negative*
βII 16.3±0.2 Negative*
(†) The standard plasmids used for PKCs α, βI and βII are the mammalian expression vectors PKCα-EGFP, PKCβI in pEF1 and PKCβII-EGFP, respectively. (*) Negative represents a Ct cycle value that is equal to the one obtained from the negative control sample which consists of mRNA samples of MA104 cells that had no reverse transcriptase added during the reverse transcription step.
57
PKCα Regulates FR Recycling
Next several experiments were conducted to examine a possible function for
PKCα in regulating FR recycling. Overexpression of PKCα using an adenoviral vector
resulted in an increase in PMA-induced FR externalization (Figure 9A); in contrast, a
dominant-negative form of PKCα but not PKCβII abrogated the PMA effect (Figure 9B)
similar to knocking down PKCα (Figure 9C). Further, down-regulation of PKCα by
prolonged (48h) exposure to PMA restored the original steady-state distribution of FR
(Figure 9D) as well as the rate of FR internalization (Figure 10).
(A) 44 Vehicle b 0.1μM PMA GFP PKCα 3 3 a PKCα 22
GAPDH Folate receptor Folate receptor 1 (external/internal) (external/internal)
0 GFP PKCα
Figure 9: Effect of PKCα on FRα recycling in MA104 cells. (A) MA104 cells were infected with GFP-Ad or PKCα-Ad and FRα recycling was determined 48h later. The cells were incubated with 27 nM [3H]folic acid for 1h at 37°C, washed twice with 37°C FFRPMI in order to remove any unbound radioactivity and further incubated with vehicle or 1μM PMA for 30 min at 37°C. The cell surface bound radioactivity (representing external FR) was removed by incubation with ice-cold acid buffer and the intracellular radioactivity (representing internal FR) was recovered by the addition of 0.1N NaOH. Cell lysates from the same experiment were also analyzed by western blot using PKCα antibody and the same blots were re-probed with a mouse anti-GAPDH antibody to ensure the uniformity of sample loading. The letters denote that “b” is statistically different from “a” with (p<0.005).
58
(B) 33 Vehicle GFP DN-βII DN-α 1μM PMA PKCα tor 2
PKCβII
11 Folate recep
(external/internal) (external/internal) GAPDH
0 GFP DN-PKCβII DN-PKCα
(C) 3 Vehicle 1μM PMA Control KD tor 2 PKCα
GAPDH
Folate recep 1 (external/internal) (external/internal)
0 Control KD-PKCα
Figure 9: Effect of PKCα on FRα recycling in MA104 cells (continued). In (B), the same experiment was done except that MA104 cells were infected with GFP-Ad, DN- PKCβII or DN-PKCα. The western blots were additionally probed with PKCβII antibody. In (C), MA104 cells were infected with retrovirus containing a scrambled sequence or a specific sequence to knockdown PKCα. The recycling of FRα was determined 72h post-infection as described in (A).
59
(D) 6 Vehicle 1μM PMA Veh PMA 4 PKCα
GAPDH
Folate receptor 2 (external/internal) (external/internal)
0 Vehicle PMA
Figure 9: Effect of PKCα on FRα recycling in MA104 cells (continued). In (D), MA104 cells were split as 0.225x106 cells per well in FFRPMI. The cells were treated with vehicle or 1μM PMA on day 2 and further cultured for another 48h at the end of which FRα recycling was determined as described under (A). In all cases (A) through (D), the subtracted non-specific association (< 2 percent) of [3H]folic acid was determined using negative control samples that were treated with excess unlabeled folic acid (data not shown). Each bar is the average of triplicate measurements.
60
12000 Vehicle (V48) PMA (V48) Vehicle (P48) c PMA (P48) lic a id 8000 ]fo H 3 cells)
6
(cpm/10 4000
Internalization of [
0 0 1 2 3 4 5 6 7 8 9 10 11
Time in minutes
Figure 10: Effect of PKCα downregulation by prolonged PMA treatment on FR internalization in MA104 cells. The cells were treated for 48h with vehicle or 1μM PMA (labeled as V48 and P48 respectively in the figure legend) in order to downregulate cPKCs before FR influx was measured. Following 48h of treatment, the cells were incubated with vehicle or 1μM PMA for 30 min at 37°C in FFRPMI with no serum, then the media was aspirated and 27 nM [3H]folic acid was added to each well and further incubated at 37°C for the indicated times. At the end of each time point, the cells were immediately washed twice with ice-cold PBS and low pH buffer to remove the cell surface bound radioactivity. The intracellular radioactivity which represents internal FR was recovered by the addition of 0.1N NaOH. The subtracted non-specific association (< 2 percent) of [3H]folic acid was determined using negative control samples that were treated with excess unlabeled folic acid (data not shown). Each bar is the average of triplicate measurements.
61
PKCδ has a Dominant-negative Effect on FR Externalization in MA104 Cells
In testing the effect of overexpressing several PKCs, a curious observation was
that PKCδ inhibited FR externalization by PMA (Figure 11). This dominant negative effect of PKCδ was not observed for other PKCs tested including PKCs βII and ε (Figure
11). This observation indicates that the PKCα mediation of FR recycling by phorbol ester
may be affected by the complement of PKCs in a given cell type.
44 Vehicle 1μM PMA 33
22
Folate receptor Folate receptor 1 (external/internal) 1
00 GFP PKCβII PKCδ PKCε
Figure 11: PKCδ has a dominant-negative effect on FR externalization in MA104 cells. MA104 cells were infected with GFP-Ad, PKCβII-Ad, PKCδ-Ad and PKCε-Ad and FRα recycling was determined 48h post-infection. The cells were incubated with 27 nM [3H]folic acid for 1h at 37°C, washed twice with 37°C FFRPMI in order to remove any unbound radioactivity and further incubated with vehicle or 1μM PMA for 30 min at 37°C. The cell surface bound radioactivity and the intracellular radioactivity were measured as described under “FR recycling” in the “Materials and Methods” section.
62
FR-rich DRMs are Physically Associated with Cellular Effectors of PKC Action
To identify proteins with a known functional relationship with PKCα in FR rafts as well as candidate proteins involved in raft recycling, FR-rich DRMs were affinity purified using an immobilized biotinylated folate probe and the purified protein complexes resolved by denaturing electrophoresis. Since this approach required an appropriate control to exclude background proteins that were not specifically associated with FR, parental FR-negative CHO cells (CHO-K1 cells) and recombinant CHO cells stably expressing FRβ (CHO-FRβ) were used (Figure 12A). The purified complex contained FR (Figure 12B). All of the proteins in the preparations obtained from the parental and recombinant cells were subjected to mass spectrometry and the proteins that were specifically associated with FR were identified in this manner (Table IV). Most of the proteins identified in Table IV have previously been identified in association with lipid rafts and include cytoskeletal proteins (actin and tubulin) (Brdickova et al., 2001;
Foster et al., 2003), heterotrimeric G-proteins (Miotti et al., 2000; Oh and Schnitzer,
2001), annexin II (Gerke et al., 2005) and KCIP-1 (Foster et al., 2003). The apparent association of receptor for activated C kinase 1 (RACK1) as well as annexin II and
KCIP-1 with FR-rich DRM aroused our interest because they represent either PKC binding proteins or PKC substrates. Though RACK1 is present in cells in both soluble and detergent-insoluble particulate forms this protein had not previously been reported to be associated with membrane rafts.
63 Since all the functional studies of FR recycling conducted in this study are in
MA104 cells expressing endogenous FRα, the CHO cell experiments were extended to
MA104 cells specifically to confirm the association of RACK1, annexin II and KCIP-1
with FR-rich DRM in these cells. Toward this end, FR-rich rafts were affinity purified using biotinylated folate from MA104 cell membranes and probed using antibodies to
FR, RACK1 and KCIP-1; all three proteins were present in the FR-rich DRM isolated
from MA104 cells (Figure 12C). However, these proteins were not detectable in
preparations in which an excess amount of folic acid was used to block specific
association of FR with the biotinylated folate during the purification (Figure 12C)
indicating the specificity of the association of RACK1 and KCIP-1 with FR.
(A) (B)
}FR β
Figure 12: Identification of proteins associated with FR rafts by mass spectrometry. (A) A scan of one of the gels analyzed by mass spectrometry. The gel stained with Coomassie Brilliant Blue shows the proteins in the DRMs that were affinity purified from a detergent-insoluble membrane fraction prepared from CHO-K1 and CHO-FRβ cells, using Biotin-SS-folate attached to streptavidin-coated magnetic particles. The folate- bound complexes were specifically eluted from the beads using dithiothreitol. In (B), the DRMs from CHO-K1 and CHO-FRβ cells were analyzed by western blot using FR antibody.
64
1 2 (C) FRα
RACK1
KCIP-1
Figure 12: Identification of proteins associated with FR rafts by mass spectrometry. (continued). In (C), FR-rich rafts purified from MA104 membranes (lane 1) were analyzed by western blot using antibodies for FR, RACK1 and KCIP-1. The same MA104 membranes were also incubated with 1mM folic acid for 30 min prior to the addition of biotin-SS-folate in order to determine nonspecific binding to the probe (lane 2).
Table IV: Proteins associated with FR rafts identified by mass spectrometry
Proteins detected in the folate ligand affinity purified DRM from CHO-FRβ membranes*
14-3-3 protein δ/ε/ζ (KCIP-1, PKC inhibitor protein-1) PKC related proteins RACK1(Receptor for Activated C Kinase 1) Annexin II
γ Actin Cytoskeletal Proteins Tubulin α1 chain
Gβ4 (Transducin β4) G proteins Gαi-2
* The table lists the proteins identified on the basis of matching peptide sequences and theoretical mass. Non-specific proteins, identified as those obtained from the negative control (FR-negative CHO parental cells) are not listed.
65
Additional Evidence for the Association of RACK1 with FR-rich Membrane
Domains
The association of RACK1 with FR-rich DRMs favors the notion that FR rafts may represent the physical site for membrane recruitment and action of PKCα in phorbol
ester-induced regulation of their recycling. To more directly test whether RACK1 is
associated with FR rafts, electron microscopy was used to visualize possible
colocalization of FR and RACK1 in situ in MA104 cells (Figure 13). Folate receptor rafts
were first cross-linked with an FR-specific rabbit antibody to enable visualization of FR
rafts in the form of large clusters of 12nm colloidal gold particles conjugated to the
secondary antibody. RACK1, which is intracellular, was visualized by probing with an
anti-RACK1 antibody and 6nm gold-conjugated secondary antibody in sections, post-
embedding. The use of LR White embedding medium (for antibody permeability) did not
allow the use of reagents to clearly stain the cell membrane; however, the cell surface is
visible in the electron micrographs (Figure 13). RACK1 appeared to frequently associate
with FR (Figure 13). Using a definition for co-localization of FR and RACK1 as the
occurrence of a cluster of at least three 12nm gold particles with at least three 6nm gold
particles within a distance of either 40nm or 10nm, a much greater proportion of FR
clusters colocalized with RACK1 compared to the control, karyopherin α2, which is a
cytoplasmic/nuclear shuttle protein (Table V).
66
Figure 13: Colocalization of FRα and RACK1 in MA104 cells. Cells were probed in the presence of 30 mM NaN3 with rabbit anti-FR antibody followed by 12-nm colloidal gold-conjugated donkey-anti-rabbit IgG. The cells were subsequently fixed with 1% glutaraldehyde, embedded in LR White medium and sectioned. The sections were probed with goat anti-RACK1 antibody followed by 6-nm colloidal gold-conjugated donkey- anti-goat IgG. The arrows indicate clusters of 12-nm gold and the arrowheads indicate clusters of 6-nm gold.
67
Table V. Quantitative analysis of associations° between clusters* of FR and RACK1 or FR and karyopherin on the cell surface of MA104 cells by electron microscopy.
Number of RACK1 clusters (6nm gold) associated with FR clusters 17/31 (12nm gold) at ≤ 40nm (percent) (54.8%)
Number of karyopherin α2 clusters (6nm gold) associated with FR 2/34 clusters (12nm gold) at ≤ 40nm (percent) (5.8%)
Number of RACK1 clusters (6nm gold) associated with FR clusters 5/31 (12nm gold) at ≤ 10nm (percent) (16.1%)
Number of karyopherin α2 clusters (6nm gold) associated with FR 0/34 clusters (12nm gold) at ≤ 10nm (percent) (0 %)
* Clusters are defined as the occurrence of ≥ 3 gold particles separated by ≤ 20nm.
° Association between clusters is defined at two levels of stringency, as the occurrence of two clusters separated by ≤ 40nm or by ≤ 10nm.
68
Membrane Microdomain Localization of PKCα is Crucial for the Phorbol Ester
Effect on FR Recycling
To test whether localization in rafts was necessary for activated PKCα to mediate
the phorbol ester effect on FR recycling, a constitutively active mutant PKCα (PKCα-
CA) was ectopically expressed in MA104 cells. Unlike the wild-type enzyme, PKCα-CA
is myristoylated and consequently targeted in a ligand-independent manner to the plasma
membrane but not specifically to lipid rafts (Hodges et al., 2004). In contrast to the
observed increase in the PMA induction of FR externalization by ectopic overexpression of wild-type PKCα (Figure 9A), overexpression of PKCα-CA acted in a dominant- negative manner to abrogate the PMA effect on FR distribution (Figure 14). This result indicates that appropriate targeting of the activated PKCα to a specific membrane microdomain is necessary for it to modulate FR recycling.
69
(A) 6 Vehicle 0.1 μM PMA
4
2 Folate receptor Folate receptor (external/internal) (external/internal)
0 GFP PKCα-CA
(B)
PKC α -CA GFP PKCα
GAPDH
Figure 14: Membrane microdomain localization of PKCα is crucial for the phorbol ester effect on FR recycling. (A) MA104 cells were infected with GFP-Ad or PKCα-CA and FRα recycling was determined 48h post-infection. The cells were incubated with 27 nM [3H]folic acid for 1h at 37°C, washed twice with 37°C FFRPMI in order to remove any unbound radioactivity and further incubated with vehicle or 1μM PMA for 30 min at 37°C. The cell surface bound and intracellular radioactivity which represent external and internal FR respectively were recovered as described in the “Materials and Methods” section. The subtracted non-specific association (< 2 percent) of [3H]folic acid was determined using negative control samples that were treated with excess unlabeled folic acid (data not shown). Each bar is the average of triplicate measurements. (B) Cell lysates from the same experiment also were analyzed by western blot using PKCα antibody and the same blots were re-probed with a mouse anti-GAPDH antibody to ensure the uniformity of sample loading.
70
Annexin II and RACK1 have Functional Roles in the Steady State Recycling of FR
or its Regulation by Phorbol Ester
Annexin II is a substrate of PKCα that plays an important role in membrane
fusion during vesicular transport and RACK1 is crucial for the activation of PKCα.
Therefore, the functionality of endogenous annexin II and RACK1 in FR recycling was
tested by siRNA knockdown (Figure 15B). The steady state distribution of FR was
unaltered by knocking down RACK1 (Figure 15A) but knocking down annexin II
resulted in externalization of FR, similar to the effect of PMA (Figure 15A). In contrast,
whereas there was not an appreciable further externalization of FR by PMA when
annexin II was knocked down, knocking down RACK1 essentially abrogated the PMA
effect (Figure 15). Furthermore, the partial knockdown of annexin II (Figure 15C) results
in a decrease in the influx rate of ∼50% within one minute of uptake of [3H]folic acid
(Figure 15D).
The results in Figure 15 suggest RACK1 is not involved in determining the steady
state distribution of FR but does mediate the PMA effect on FR recycling. Conversely,
annexin II is needed for internalization of FR rafts and maintaining the steady state distribution of the receptor.
71
(A) 6 Vehicle 1μM PMA
44
22 Folate receptor Folate receptor (external/internal) (external/internal)
00 Control Annexin II-KD RACK1-KD
(B)
Annexin II KD RACK1 KD Control
Annexin II
RACK1
GAPDH
Figure 15: Effect of annexin II and RACK1 knockdown on FR recycling in MA104 cells. (A) MA104 cells were infected with lentiviral particles containing a control sequence or sequences to specifically knockdown RACK1 or annexin II. The next day, the media was replaced and the cells were cultured for another 48h. The recycling of FRα was determined 72h post-infection. The cells were incubated with 27 nM [3H]folic acid for 1h at 37°C, washed twice with 37°C FFRPMI in order to remove any unbound radioactivity and further incubated with vehicle or 1μM PMA for 30 min at 37°C. The cell surface bound and intracellular radioactivity which represent external and internal FR respectively were recovered as described in the “Materials and Methods” section. Each bar is the average of triplicate measurements. (B) Cell lysates from the same experiment were also analyzed by western blot using annexin II and RACK1 antibodies and the same blots were re-probed with a mouse anti-GAPDH antibody to ensure the uniformity of sample loading.
72
Control Annexin II KD (C) Annexin II
GAPDH
(D) 50 Control (Veh)
40 Control (PMA) Annexin II KD (Veh) Annexin II KD (PMA) 30
H]folic acid H]folic acid 3 20
10 (percent of total bound) Internalized [ Internalized
0 0 1 2 3 4 5 6 7 8 9 10 Time in min
Figure 15: Effect of annexin II and RACK1 knockdown on FR recycling in MA104 cells (continued). In (C) and (D) MA104 cells split on day 1 as 0.225x106 cells per well in a 6-well plate were infected with lentiviral particles containing a control sequence to knockdown annexin II 48h later. The cells were lysed on day 6 and analyzed by western blot using annexin II and GAPDH antibodies in (C). In (D) FR internalization was measured as described under the “Material and Methods” section. The data are plotted as the percentage of [3H]folic acid relative to the total radioactivity bound. All samples were measured in triplicates.
73 Inhibition of FR Internalization by PMA is not Associated with Inactivation of
Cdc42
Since FR recycling has been shown to occur through a Cdc42-dependent pinocytic pathway, and since Cdc42 is a downstream substrate for phosphorylation by
PKCα, it was of interest to test whether the effect of phorbol ester on FR recycling was mediated by inactivation of Cdc42. Figure 16 shows that PMA increases the activity of
Cdc42 as determined by the whole cell lysate pull-down assay done using the Pak1-PBD binding domain. This result would suggest that since Cdc42 activity did not decrease upon phorbol ester treatment that it is not likely a mediator of the inhibition of FR raft internalization.
Veh PMA Cdc42
GTPγS
Cdc42
Figure 16: Effect of phorbol ester on Cdc42 activity and FR-rich raft recycling in MA104 cells. MA104 cells were treated with vehicle or 1μM PMA for 30 min at 37°C and then assayed for Cdc42 activity. The cell lysates were centrifuged at 16,000g for 15 min at 4°C. The supernatant was added to a spin cup in which an immobilized glutathione disc and 20 μg of GST-Pak1-PBD were already mixed and incubated for 1h at 4°C with gently rocking then washed three times. Finally, 2x SDS sample buffer with 5% fresh β-merceptoethanol were added to the resin, boiled for 5min and centrifuged. For each of the cell lysates assayed a positive control was performed in which both 10 mM EDTA and 10 mM GTPγS (a non-hydrolysable form of GTP that irreversibly binds to Cdc42) were added for 15 min at 30°C. The reaction was stopped by placing the samples on ice and adding MgCl2 for a final concentration of 60 mM. All samples then were assayed for Cdc42 by western blot using a mouse monoclonal anti-Cdc42 antibody.
74 DISCUSSION
The recycling and signaling aspects of rafts have been studied quite extensively
but separately. The results of this study offer a mechanistic link between cell signaling
and raft recycling. Phorbol ester, an agonist of cPKCs and diacylglycerol, their natural
and relatively unstable agonist is assumed here to prolong the redistribution of FR rafts
for a duration that enables methods designed to accurately measure the distribution of the
receptor. The molecular mechanistic data described above for the effect of phorbol ester
on FR distribution in the cell combined with known molecular interactions in PKC-
mediated signaling may be used to put in perspective, physiological processes involved in
at least one regulatory mechanism of raft recycling (Figure 17). In this scheme, the
second messenger diacylglycerol, generated due to a cell surface signal that activates
membrane phospholipase C (PLC) (reviewed in Nishizuka, 1995), activates PKCα
together with RACK1 to target it to rafts. Intracellular Ca2+ that is mobilized by activation of PLC (reviewed in Rebecchi and Pentyala, 2000) or induced by CPA also contributes to the activation of PKCα. Whereas PKCα and RACK1 are not engaged in the steady-state recycling of FR rafts, annexin II associated with the rafts is required for their internalization. Phosphorylation of annexin II by activated PKCα (Gould et al., 1986; Liu et al., 2003; Yan et al., 2006) inhibits raft internalization to increase the raft population on cell surface. The activation of Cdc42, which is required for raft recycling
(Sabharanjak et al., 2002) and is an indirect substrate for PKCα (Tatin et al., 2006), is not
75 diminished in this process and is not a likely mediator of the inhibition of raft internalization.
Figure 17: Proposed Working Model
76
Regulation of FR recycling has established implications in the receptor-mediated cellular uptake of reduced folate co-enzyme and also in FR-targeted drug delivery
(Leamon and Reddy, 2004). However, a variety of signaling processes occur through rafts. Various protein components of signaling complexes, including GPI-anchored proteins, receptors, non-receptor tyrosine kinases and GTP-binding proteins occur as constitutive residents of rafts or may translocate or become recruited to rafts upon activation by a signal (Simons and Toomre, 2000). Both GPI-anchored and non-GPI anchored membrane proteins as well as cytosolic proteins may be functional and/or structural components of such signaling complexes (Bilderback et al., 1999; Bruckner et al., 1999; Cheng et al., 1999; Couet et al., 1997; Deans et al., 1998; Doherty et al., 1993;
Field et al., 1997; Janes et al., 2000; Krauss and Altevogt, 1999; Langlet et al., 2000;
Mastick et al., 1995; Roy et al., 1999; Tansey et al., 2000; Wary et al., 1998). All of these processes can be mediated by non-caveolin rafts as evident from various cells that lack caveolin/caveolae. Certainly, the non-caveolin rafts enriched in FR also are likely associated with signaling molecules as previously reported (Miotti et al., 2000; Oh and
Schnitzer, 2001) and as confirmed in this study. Therefore, in the broader context, the population and residence time of GPI-protein rafts at the cell surface may be a key determinant of their signaling capacity and this may be regulated by endogenous signaling molecules and controlled by agents that specifically target this recycling pathway.
77
Internalization of rafts into endocytic compartments is an obvious means of
disassembling coalesced rafts (Simons and Toomre, 2000), but the consequence of GPI-
protein raft internalization on signaling function is yet to be thoroughly investigated.
More attention has been focused on signal transduction in relation to the internalization
of caveolae (Ikonen, 2001). For example, downregulation of PKCα activity after chronic
activation may occur by delivery of activated PKCα to endosomes via caveolae
(Prevostel et al., 2000). In contrast, internalization of the EGF receptor through caveolae
appears to result in MAP kinase activation in the early/sorting endocytic compartment
(Pol et al., 2000). It is conceivable that an external signal that increases the raft
population on the cell surface could further sensitize the cells to the same signal.
Conversely, the signal intensity could be down-regulated if the external signal
internalizes rafts. Obviously, a signaling molecule that alters raft recycling also could
cross-talk at an early stage with raft-mediated signaling pathways.
Whether the principles of regulation of FR raft recycling by phorbol ester
elucidated in this study may be extended to the recycling of caveolae or indeed lipid rafts
enriched in other GPI-anchored proteins remains to be tested. Such studies may have
implications in therapeutic interventions to target raft recycling such as the development
of immunomodulators and growth inhibitors or even drugs to disrupt the localization of a pathogenic protein such as the scrapie prion protein (Kaneko et al., 1997). The findings in this study also may be useful in investigating the mechanism and significance of the observation that in a variety of malignant cell lines, FR largely resided on the cell surface
78 at steady state so that phorbol ester did not produce such a marked change in its subcellular distribution (Kamen and Smith, 2004).
79 SUMMARY
The molecular and kinetic experiments in this study provide a detailed mechanism
by which FR recycling is regulated by PMA in MA104 cells. Some of the key players
identified include the Ca2+-dependent PKC isoform, PKCα and RACK1 which mediate
the PMA effect on FR recycling in these cells. Unlike RACK1, annexin II is required for
the internalization of FR rich rafts. Finally, the activation of Cdc42 is not diminished by
PMA treatment, and therefore is not a likely mediator of the inhibition of raft internalization. Whether this mechanism applies to FR-rich rafts only or whether it can be generalized to other GPI-anchored proteins that reside in rafts remains to be tested.
80 BIBLIOGRAPHY
Acs, P., Szallasi, Z., Kazanietz, M. G., and Blumberg, P. M. (1995). Differential
activation of PKC isozymes by 14-3-3 zeta protein. Biochem Biophys Res
Commun 216, 103-109.
Aiello, L. P., Clermont, A., Arora, V., Davis, M. D., Sheetz, M. J., and Bursell, S. E.
(2006). Inhibition of PKC beta by oral administration of ruboxistaurin is well
tolerated and ameliorates diabetes-induced retinal hemodynamic abnormalities in
patients. Invest Ophthalmol Vis Sci 47, 86-92.
Akimoto, K., Takahashi, R., Moriya, S., Nishioka, N., Takayanagi, J., Kimura, K., Fukui,
Y., Osada, S., Mizuno, K., Hirai, S., et al. (1996). EGF or PDGF receptors
activate atypical PKClambda through phosphatidylinositol 3-kinase. Embo J 15,
788-798.
Almholt, K., Arkhammar, P. O., Thastrup, O., and Tullin, S. (1999). Simultaneous
visualization of the translocation of protein kinase Calpha-green fluorescent
protein hybrids and intracellular calcium concentrations. Biochem J 337 ( Pt 2),
211-218.
Anderson, R. G. (1998). The caveolae membrane system. Annu Rev Biochem 67, 199-
225.
Anderson, R. G., Kamen, B. A., Rothberg, K. G., and Lacey, S. W. (1992). Potocytosis:
sequestration and transport of small molecules by caveolae. Science 255, 410-
411.
81 Balendran, A., Hare, G. R., Kieloch, A., Williams, M. R., and Alessi, D. R. (2000).
Further evidence that 3-phosphoinositide-dependent protein kinase-1 (PDK1) is
required for the stability and phosphorylation of protein kinase C (PKC) isoforms.
FEBS Lett 484, 217-223.
Bangs, J. D., Andrews, N. W., Hart, G. W., and Englund, P. T. (1986). Posttranslational
modification and intracellular transport of a trypanosome variant surface
glycoprotein. J Cell Biol 103, 255-263.
Bangs, J. D., Hereld, D., Krakow, J. L., Hart, G. W., and Englund, P. T. (1985). Rapid
processing of the carboxyl terminus of a trypanosome variant surface
glycoprotein. Proc Natl Acad Sci U S A 82, 3207-3211.
Bi, K., Tanaka, Y., Coudronniere, N., Sugie, K., Hong, S., van Stipdonk, M. J., and
Altman, A. (2001). Antigen-induced translocation of PKC-theta to membrane
rafts is required for T cell activation. Nat Immunol 2, 556-563.
Biener, Y., Feinstein, R., Mayak, M., Kaburagi, Y., Kadowaki, T., and Zick, Y. (1996).
Annexin II is a novel player in insulin signal transduction. Possible association
between annexin II phosphorylation and insulin receptor internalization. J Biol
Chem 271, 29489-29496.
Bilderback, T. R., Gazula, V. R., Lisanti, M. P., and Dobrowsky, R. T. (1999). Caveolin
interacts with Trk A and p75(NTR) and regulates neurotrophin signaling
pathways. J Biol Chem 274, 257-263.
82 Birn, H., Selhub, J., and Christensen, E. I. (1993). Internalization and intracellular
transport of folate-binding protein in rat kidney proximal tubule. Am J Physiol
264, C302-310.
Blagoev, B., Kratchmarova, I., Ong, S. E., Nielsen, M., Foster, L. J., and Mann, M.
(2003). A proteomics strategy to elucidate functional protein-protein interactions
applied to EGF signaling. Nat Biotechnol 21, 315-318.
Bohuslav, J., Horejsi, V., Hansmann, C., Stockl, J., Weidle, U. H., Majdic, O., Bartke, I.,
Knapp, W., and Stockinger, H. (1995). Urokinase plasminogen activator receptor,
beta 2-integrins, and Src-kinases within a single receptor complex of human
monocytes. J Exp Med 181, 1381-1390.
Brambilla, R., Zippel, R., Sturani, E., Morello, L., Peres, A., and Alberghina, L. (1991).
Characterization of the tyrosine phosphorylation of calpactin I (annexin II)
induced by platelet-derived growth factor. Biochem J 278 ( Pt 2), 447-452.
Brdickova, N., Brdicka, T., Andera, L., Spicka, J., Angelisova, P., Milgram, S. L., and
Horejsi, V. (2001). Interaction between two adapter proteins, PAG and EBP50: a
possible link between membrane rafts and actin cytoskeleton. FEBS Lett 507,
133-136.
Brichory, F. M., Misek, D. E., Yim, A. M., Krause, M. C., Giordano, T. J., Beer, D. G.,
and Hanash, S. M. (2001). An immune response manifested by the common
occurrence of annexins I and II autoantibodies and high circulating levels of IL-6
in lung cancer. Proc Natl Acad Sci U S A 98, 9824-9829.
83 Brown, D. A., and London, E. (2000). Structure and function of sphingolipid- and
cholesterol-rich membrane rafts. J Biol Chem 275, 17221-17224.
Brown, D. A., and Rose, J. K. (1992). Sorting of GPI-anchored proteins to glycolipid-
enriched membrane subdomains during transport to the apical cell surface. Cell
68, 533-544.
Bruckner, K., Pablo Labrador, J., Scheiffele, P., Herb, A., Seeburg, P. H., and Klein, R.
(1999). EphrinB ligands recruit GRIP family PDZ adaptor proteins into raft
membrane microdomains. Neuron 22, 511-524.
Brummelkamp, T. R., Bernards, R., and Agami, R. (2002). Stable suppression of
tumorigenicity by virus-mediated RNA interference. Cancer Cell 2, 243-247.
Bruns, R. R., and Palade, G. E. (1968). Studies on blood capillaries. I. General
organization of blood capillaries in muscle. J Cell Biol 37, 244-276.
Buist, M. R., Molthoff, C. F., Kenemans, P., and Meijer, C. J. (1995). Distribution of
OV-TL 3 and MOv18 in normal and malignant ovarian tissue. J Clin Pathol 48,
631-636.
Buys, S. S., Keogh, E. A., and Kaplan, J. (1984). Fusion of intracellular membrane pools
with cell surfaces of macrophages stimulated by phorbol esters and calcium
ionophores. Cell 38, 569-576.
Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982).
Direct activation of calcium-activated, phospholipid-dependent protein kinase by
tumor-promoting phorbol esters. J Biol Chem 257, 7847-7851.
84 Chamberlain, L. H. (2004). Detergents as tools for the purification and classification of
lipid rafts. FEBS Lett 559, 1-5.
Chang, W. J., Rothberg, K. G., Kamen, B. A., and Anderson, R. G. (1992). Lowering the
cholesterol content of MA104 cells inhibits receptor-mediated transport of folate.
J Cell Biol 118, 63-69.
Chasserot-Golaz, S., Vitale, N., Umbrecht-Jenck, E., Knight, D., Gerke, V., and Bader,
M. F. (2005). Annexin 2 promotes the formation of lipid microdomains required
for calcium-regulated exocytosis of dense-core vesicles. Mol Biol Cell 16, 1108-
1119.
Cheng, P. C., Dykstra, M. L., Mitchell, R. N., and Pierce, S. K. (1999). A role for lipid
rafts in B cell antigen receptor signaling and antigen targeting. J Exp Med 190,
1549-1560.
Chetcuti, A., Margan, S. H., Russell, P., Mann, S., Millar, D. S., Clark, S. J., Rogers, J.,
Handelsman, D. J., and Dong, Q. (2001). Loss of annexin II heavy and light
chains in prostate cancer and its precursors. Cancer Res 61, 6331-6334.
Choi, S. H., Hyman, T., and Blumberg, P. M. (2006). Differential effect of bryostatin 1
and phorbol 12-myristate 13-acetate on HOP-92 cell proliferation is mediated by
down-regulation of protein kinase Cdelta. Cancer Res 66, 7261-7269.
Chou, M. M., Hou, W., Johnson, J., Graham, L. K., Lee, M. H., Chen, C. S., Newton, A.
C., Schaffhausen, B. S., and Toker, A. (1998). Regulation of protein kinase C zeta
by PI 3-kinase and PDK-1. Curr Biol 8, 1069-1077.
85 Coghlan, M. P., Chou, M. M., and Carpenter, C. L. (2000). Atypical protein kinases
Clambda and -zeta associate with the GTP-binding protein Cdc42 and mediate
stress fiber loss. Mol Cell Biol 20, 2880-2889.
Couet, J., Sargiacomo, M., and Lisanti, M. P. (1997). Interaction of a receptor tyrosine
kinase, EGF-R, with caveolins. Caveolin binding negatively regulates tyrosine
and serine/threonine kinase activities. J Biol Chem 272, 30429-30438.
Cross, G. A. (1990). Glycolipid anchoring of plasma membrane proteins. Annu Rev Cell
Biol 6, 1-39.
Dai, J. G., and Murakami, K. (2003). Constitutively and autonomously active protein
kinase C associated with 14-3-3 zeta in the rodent brain. J Neurochem 84, 23-34.
Danielsen, E. M., and van Deurs, B. (1995). A transferrin-like GPI-linked iron-binding
protein in detergent-insoluble noncaveolar microdomains at the apical surface of
fetal intestinal epithelial cells. J Cell Biol 131, 939-950.
Deans, J. P., Robbins, S. M., Polyak, M. J., and Savage, J. A. (1998). Rapid redistribution
of CD20 to a low density detergent-insoluble membrane compartment. J Biol
Chem 273, 344-348.
Dempsey, E. C., Newton, A. C., Mochly-Rosen, D., Fields, A. P., Reyland, M. E., Insel,
P. A., and Messing, R. O. (2000). Protein kinase C isozymes and the regulation of
diverse cell responses. Am J Physiol Lung Cell Mol Physiol 279, L429-438.
Deora, A. B., Kreitzer, G., Jacovina, A. T., and Hajjar, K. A. (2004). An annexin 2
phosphorylation switch mediates p11-dependent translocation of annexin 2 to the
cell surface. J Biol Chem 279, 43411-43418.
86 Doherty, P., Singh, A., Rimon, G., Bolsover, S. R., and Walsh, F. S. (1993). Thy-1
antibody-triggered neurite outgrowth requires an influx of calcium into neurons
via N- and L-type calcium channels. J Cell Biol 122, 181-189.
Dong, L. Q., Zhang, R. B., Langlais, P., He, H., Clark, M., Zhu, L., and Liu, F. (1999).
Primary structure, tissue distribution, and expression of mouse phosphoinositide-
dependent protein kinase-1, a protein kinase that phosphorylates and activates
protein kinase Czeta. J Biol Chem 274, 8117-8122.
Drevot, P., Langlet, C., Guo, X. J., Bernard, A. M., Colard, O., Chauvin, J. P., Lasserre,
R., and He, H. T. (2002). TCR signal initiation machinery is pre-assembled and
activated in a subset of membrane rafts. Embo J 21, 1899-1908.
Economides, A. N., Ravetch, J. V., Yancopoulos, G. D., and Stahl, N. (1995). Designer
cytokines: targeting actions to cells of choice. Science 270, 1351-1353.
Edidin, M. (2003). The state of lipid rafts: from model membranes to cells. Annu Rev
Biophys Biomol Struct 32, 257-283.
Emoto, K., Yamada, Y., Sawada, H., Fujimoto, H., Ueno, M., Takayama, T., Kamada,
K., Naito, A., Hirao, S., and Nakajima, Y. (2001). Annexin II overexpression
correlates with stromal tenascin-C overexpression: a prognostic marker in
colorectal carcinoma. Cancer 92, 1419-1426.
Esposito, I., Penzel, R., Chaib-Harrireche, M., Barcena, U., Bergmann, F., Riedl, S.,
Kayed, H., Giese, N., Kleeff, J., Friess, H., and Schirmacher, P. (2006). Tenascin
C and annexin II expression in the process of pancreatic carcinogenesis. J Pathol
208, 673-685.
87 Fallon, R. J., and Schwartz, A. L. (1986). Regulation by phorbol esters of
asialoglycoprotein and transferrin receptor distribution and ligand affinity in a
hepatoma cell line. J Biol Chem 261, 15081-15089.
Fan, J., Vitols, K. S., and Huennekens, F. M. (1991). Biotin derivatives of methotrexate
and folate. Synthesis and utilization for affinity purification of two membrane-
associated folate transporters from L1210 cells. J Biol Chem 266, 14862-14865.
Ferguson, M. A., Duszenko, M., Lamont, G. S., Overath, P., and Cross, G. A. (1986).
Biosynthesis of Trypanosoma brucei variant surface glycoproteins. N-
glycosylation and addition of a phosphatidylinositol membrane anchor. J Biol
Chem 261, 356-362.
Ferguson, M. A., and Williams, A. F. (1988). Cell-surface anchoring of proteins via
glycosyl-phosphatidylinositol structures. Annu Rev Biochem 57, 285-320.
Field, K. A., Holowka, D., and Baird, B. (1997). Compartmentalized activation of the
high affinity immunoglobulin E receptor within membrane domains. J Biol Chem
272, 4276-4280.
Field, M. C., Menon, A. K., and Cross, G. A. (1991). A glycosylphosphatidylinositol
protein anchor from procyclic stage Trypanosoma brucei: lipid structure and
biosynthesis. Embo J 10, 2731-2739.
Fivaz, M., Vilbois, F., Thurnheer, S., Pasquali, C., Abrami, L., Bickel, P. E., Parton, R.
G., and van der Goot, F. G. (2002). Differential sorting and fate of endocytosed
GPI-anchored proteins. Embo J 21, 3989-4000.
88 Foster, L. J., De Hoog, C. L., and Mann, M. (2003). Unbiased quantitative proteomics of
lipid rafts reveals high specificity for signaling factors. Proc Natl Acad Sci U S A
100, 5813-5818.
Fra, A. M., Williamson, E., Simons, K., and Parton, R. G. (1994). Detergent-insoluble
glycolipid microdomains in lymphocytes in the absence of caveolae. J Biol Chem
269, 30745-30748.
Friedrichson, T., and Kurzchalia, T. V. (1998). Microdomains of GPI-anchored proteins
in living cells revealed by crosslinking. Nature 394, 802-805.
Fu, H., Subramanian, R. R., and Masters, S. C. (2000). 14-3-3 proteins: structure,
function, and regulation. Annu Rev Pharmacol Toxicol 40, 617-647.
Gerke, V., Creutz, C. E., and Moss, S. E. (2005). Annexins: linking Ca2+ signalling to
membrane dynamics. Nat Rev Mol Cell Biol 6, 449-461.
Gerke, V., and Moss, S. E. (2002). Annexins: from structure to function. Physiol Rev 82,
331-371.
Giovannucci, D. R., Groblewski, G. E., Sneyd, J., and Yule, D. I. (2000). Targeted
phosphorylation of inositol 1,4,5-trisphosphate receptors selectively inhibits
localized Ca2+ release and shapes oscillatory Ca2+ signals. J Biol Chem 275,
33704-33711.
Glenney, J. (1986). Two related but distinct forms of the Mr 36,000 tyrosine kinase
substrate (calpactin) that interact with phospholipid and actin in a Ca2+-
dependent manner. Proc Natl Acad Sci U S A 83, 4258-4262.
89 Glenney, J. R., Jr. (1985). Phosphorylation of p36 in vitro with pp60src. Regulation by
Ca2+ and phospholipid. FEBS Lett 192, 79-82.
Glenney, J. R., Jr., and Tack, B. F. (1985). Amino-terminal sequence of p36 and
associated p10: identification of the site of tyrosine phosphorylation and
homology with S-100. Proc Natl Acad Sci U S A 82, 7884-7888.
Gorodinsky, A., and Harris, D. A. (1995). Glycolipid-anchored proteins in neuroblastoma
cells form detergent-resistant complexes without caveolin. J Cell Biol 129, 619-
627.
Gould, K. L., Woodgett, J. R., Isacke, C. M., and Hunter, T. (1986). The protein-tyrosine
kinase substrate p36 is also a substrate for protein kinase C in vitro and in vivo.
Mol Cell Biol 6, 2738-2744.
Guo, M., Wu, M. H., Korompai, F., and Yuan, S. Y. (2003). Upregulation of PKC genes
and isozymes in cardiovascular tissues during early stages of experimental
diabetes. Physiol Genomics 12, 139-146.
Hayashi, A., Seki, N., Hattori, A., Kozuma, S., and Saito, T. (1999). PKCnu, a new
member of the protein kinase C family, composes a fourth subfamily with
PKCmu. Biochim Biophys Acta 1450, 99-106.
Heerklotz, H. (2002). Triton promotes domain formation in lipid raft mixtures. Biophys J
83, 2693-2701.
Heerklotz, H., Szadkowska, H., Anderson, T., and Seelig, J. (2003). The sensitivity of
lipid domains to small perturbations demonstrated by the effect of Triton. J Mol
Biol 329, 793-799.
90 Hjelle, J. T., Christensen, E. I., Carone, F. A., and Selhub, J. (1991). Cell fractionation
and electron microscope studies of kidney folate-binding protein. Am J Physiol
260, C338-346.
Hodges, R. R., Raddassi, I., Zoukhri, D., Toker, A., Kazlauskas, A., and Dartt, D. A.
(2004). Effect of overexpression of constitutively active PKCalpha on rat lacrimal
gland protein secretion. Invest Ophthalmol Vis Sci 45, 3974-3981.
Housey, G. M., Johnson, M. D., Hsiao, W. L., O'Brian, C. A., Murphy, J. P.,
Kirschmeier, P., and Weinstein, I. B. (1988). Overproduction of protein kinase C
causes disordered growth control in rat fibroblasts. Cell 52, 343-354.
Hug, H., and Sarre, T. F. (1993). Protein kinase C isoenzymes: divergence in signal
transduction? Biochem J 291 ( Pt 2), 329-343.
Ikonen, E. (2001). Roles of lipid rafts in membrane transport. Curr Opin Cell Biol 13,
470-477.
Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977). Studies on a cyclic
nucleotide-independent protein kinase and its proenzyme in mammalian tissues.
II. Proenzyme and its activation by calcium-dependent protease from rat brain. J
Biol Chem 252, 7610-7616.
Janes, P. W., Ley, S. C., Magee, A. I., and Kabouridis, P. S. (2000). The role of lipid rafts
in T cell antigen receptor (TCR) signalling. Semin Immunol 12, 23-34.
Jirousek, M. R., Gillig, J. R., Gonzalez, C. M., Heath, W. F., McDonald, J. H., 3rd, Neel,
D. A., Rito, C. J., Singh, U., Stramm, L. E., Melikian-Badalian, A., et al. (1996).
(S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-
91 1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-d
ione (LY333531) and related analogues: isozyme selective inhibitors of protein
kinase C beta. J Med Chem 39, 2664-2671.
Johannes, F. J., Prestle, J., Eis, S., Oberhagemann, P., and Pfizenmaier, K. (1994). PKCu
is a novel, atypical member of the protein kinase C family. J Biol Chem 269,
6140-6148.
Kamen, B. A., Johnson, C. A., Wang, M. T., and Anderson, R. G. (1989). Regulation of
the cytoplasmic accumulation of 5-methyltetrahydrofolate in MA104 cells is
independent of folate receptor regulation. J Clin Invest 84, 1379-1386.
Kamen, B. A., and Smith, A. K. (2004). A review of folate receptor alpha cycling and 5-
methyltetrahydrofolate accumulation with an emphasis on cell models in vitro.
Adv Drug Deliv Rev 56, 1085-1097.
Kamen, B. A., Wang, M. T., Streckfuss, A. J., Peryea, X., and Anderson, R. G. (1988).
Delivery of folates to the cytoplasm of MA104 cells is mediated by a surface
membrane receptor that recycles. J Biol Chem 263, 13602-13609.
Kaneko, K., Vey, M., Scott, M., Pilkuhn, S., Cohen, F. E., and Prusiner, S. B. (1997).
COOH-terminal sequence of the cellular prion protein directs subcellular
trafficking and controls conversion into the scrapie isoform. Proc Natl Acad Sci U
S A 94, 2333-2338.
Katagiri, Y. U., Kiyokawa, N., and Fujimoto, J. (2001). A role for lipid rafts in immune
cell signaling. Microbiol Immunol 45, 1-8.
92 Kenworthy, A. K., and Edidin, M. (1998). Distribution of a glycosylphosphatidylinositol-
anchored protein at the apical surface of MDCK cells examined at a resolution of
<100 A using imaging fluorescence resonance energy transfer. J Cell Biol 142,
69-84.
Kenworthy, A. K., Petranova, N., and Edidin, M. (2000). High-resolution FRET
microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma
membranes. Mol Biol Cell 11, 1645-1655.
Klausner, R. D., Harford, J., and van Renswoude, J. (1984). Rapid internalization of the
transferrin receptor in K562 cells is triggered by ligand binding or treatment with
a phorbol ester. Proc Natl Acad Sci U S A 81, 3005-3009.
Knopf, J. L., Lee, M. H., Sultzman, L. A., Kriz, R. W., Loomis, C. R., Hewick, R. M.,
and Bell, R. M. (1986). Cloning and expression of multiple protein kinase C
cDNAs. Cell 46, 491-502.
Krauss, K., and Altevogt, P. (1999). Integrin leukocyte function-associated antigen-1-
mediated cell binding can be activated by clustering of membrane rafts. J Biol
Chem 274, 36921-36927.
Lacey, S. W., Sanders, J. M., Rothberg, K. G., Anderson, R. G., and Kamen, B. A.
(1989). Complementary DNA for the folate binding protein correctly predicts
anchoring to the membrane by glycosyl-phosphatidylinositol. J Clin Invest 84,
715-720.
93 Lamaze, C., Dujeancourt, A., Baba, T., Lo, C. G., Benmerah, A., and Dautry-Varsat, A.
(2001). Interleukin 2 receptors and detergent-resistant membrane domains define
a clathrin-independent endocytic pathway. Mol Cell 7, 661-671.
Lambert, O., Gerke, V., Bader, M. F., Porte, F., and Brisson, A. (1997). Structural
analysis of junctions formed between lipid membranes and several annexins by
cryo-electron microscopy. J Mol Biol 272, 42-55.
Langlet, C., Bernard, A. M., Drevot, P., and He, H. T. (2000). Membrane rafts and
signaling by the multichain immune recognition receptors. Curr Opin Immunol
12, 250-255.
Le Good, J. A., Ziegler, W. H., Parekh, D. B., Alessi, D. R., Cohen, P., and Parker, P. J.
(1998). Protein kinase C isotypes controlled by phosphoinositide 3-kinase through
the protein kinase PDK1. Science 281, 2042-2045.
Leamon, C. P., and Reddy, J. A. (2004). Folate-targeted chemotherapy. Adv Drug Deliv
Rev 56, 1127-1141.
Lee, H. W., Smith, L., Pettit, G. R., and Smith, J. B. (1997). Bryostatin 1 and phorbol
ester down-modulate protein kinase C-alpha and -epsilon via the
ubiquitin/proteasome pathway in human fibroblasts. Mol Pharmacol 51, 439-447.
Lehnigk, U., Zimmermann, U., Woenckhaus, C., and Giebel, J. (2005). Localization of
annexins I, II, IV and VII in whole prostate sections from radical prostatectomy
patients. Histol Histopathol 20, 673-680.
94 Leontieva, O. V., and Black, J. D. (2004). Identification of two distinct pathways of
protein kinase Calpha down-regulation in intestinal epithelial cells. J Biol Chem
279, 5788-5801.
Lewis, C. M., Smith, A. K., and Kamen, B. A. (1998a). Receptor-mediated folate uptake
is positively regulated by disruption of the actin cytoskeleton. Cancer Res 58,
2952-2956.
Lewis, C. M., Smith, A. K., Nguyen, C., and Kamen, B. A. (1998b). PMA alters folate
receptor distribution in the plasma membrane and increases the rate of 5-
methyltetrahydrofolate delivery in mature MA104 cells. Biochim Biophys Acta
1401, 157-169.
Liu, J., Oh, P., Horner, T., Rogers, R. A., and Schnitzer, J. E. (1997a). Organized
endothelial cell surface signal transduction in caveolae distinct from
glycosylphosphatidylinositol-anchored protein microdomains. J Biol Chem 272,
7211-7222.
Liu, J., Rothermund, C. A., Ayala-Sanmartin, J., and Vishwanatha, J. K. (2003). Nuclear
annexin II negatively regulates growth of LNCaP cells and substitution of ser 11
and 25 to glu prevents nucleo-cytoplasmic shuttling of annexin II. BMC Biochem
4, 10.
Liu, P., Ying, Y., and Anderson, R. G. (1997b). Platelet-derived growth factor activates
mitogen-activated protein kinase in isolated caveolae. Proc Natl Acad Sci U S A
94, 13666-13670.
95 Madore, N., Smith, K. L., Graham, C. H., Jen, A., Brady, K., Hall, S., and Morris, R.
(1999). Functionally different GPI proteins are organized in different domains on
the neuronal surface. Embo J 18, 6917-6926.
Maizel, J. V., Jr., White, D. O., and Scharff, M. D. (1968). The polypeptides of
adenovirus. I. Evidence for multiple protein components in the virion and a
comparison of types 2, 7A, and 12. Virology 36, 115-125.
Mantovani, L. T., Miotti, S., Menard, S., Canevari, S., Raspagliesi, F., Bottini, C.,
Bottero, F., and Colnaghi, M. I. (1994). Folate binding protein distribution in
normal tissues and biological fluids from ovarian carcinoma patients as detected
by the monoclonal antibodies MOv18 and MOv19. Eur J Cancer 30A, 363-369.
Massague, J. (1996). Neurotrophic factors. Crossing receptor boundaries. Nature 382, 29-
30.
Mastick, C. C., Brady, M. J., and Saltiel, A. R. (1995). Insulin stimulates the tyrosine
phosphorylation of caveolin. J Cell Biol 129, 1523-1531.
Matsumura, M., Tanaka, N., Kuroki, T., Ichihashi, M., and Ohba, M. (2003). The eta
isoform of protein kinase C inhibits UV-induced activation of caspase-3 in normal
human keratinocytes. Biochem Biophys Res Commun 303, 350-356.
May, W. S., Jacobs, S., and Cuatrecasas, P. (1984). Association of phorbol ester-induced
hyperphosphorylation and reversible regulation of transferrin membrane receptors
in HL60 cells. Proc Natl Acad Sci U S A 81, 2016-2020.
Mayor, S., and Maxfield, F. R. (1995). Insolubility and redistribution of GPI-anchored
proteins at the cell surface after detergent treatment. Mol Biol Cell 6, 929-944.
96 Mayor, S., and Riezman, H. (2004). Sorting GPI-anchored proteins. Nat Rev Mol Cell
Biol 5, 110-120.
Mayor, S., Rothberg, K. G., and Maxfield, F. R. (1994). Sequestration of GPI-anchored
proteins in caveolae triggered by cross-linking. Science 264, 1948-1951.
Mayor, S., Sabharanjak, S., and Maxfield, F. R. (1998). Cholesterol-dependent retention
of GPI-anchored proteins in endosomes. Embo J 17, 4626-4638.
Mayor, S., Viola, A., Stan, R. V., and del Pozo, M. A. (2006). Flying kites on slippery
slopes at Keystone. Symposium on Lipid Rafts and Cell Function. EMBO Rep 7,
1089-1093.
McCahill, A., Warwicker, J., Bolger, G. B., Houslay, M. D., and Yarwood, S. J. (2002).
The RACK1 scaffold protein: a dynamic cog in cell response mechanisms. Mol
Pharmacol 62, 1261-1273.
McGraw, T. E., Dunn, K. W., and Maxfield, F. R. (1988). Phorbol ester treatment
increases the exocytic rate of the transferrin receptor recycling pathway
independent of serine-24 phosphorylation. J Cell Biol 106, 1061-1066.
Meller, N., Liu, Y. C., Collins, T. L., Bonnefoy-Berard, N., Baier, G., Isakov, N., and
Altman, A. (1996). Direct interaction between protein kinase C theta (PKC theta)
and 14-3-3 tau in T cells: 14-3-3 overexpression results in inhibition of PKC theta
translocation and function. Mol Cell Biol 16, 5782-5791.
Miotti, S., Bagnoli, M., Tomassetti, A., Colnaghi, M. I., and Canevari, S. (2000).
Interaction of folate receptor with signaling molecules lyn and G(alpha)(i-3) in
97 detergent-resistant complexes from the ovary carcinoma cell line IGROV1. J Cell
Sci 113 Pt 2, 349-357.
Moffett, S., Brown, D. A., and Linder, M. E. (2000). Lipid-dependent targeting of G
proteins into rafts. J Biol Chem 275, 2191-2198.
Mollinedo, F., and Gajate, C. (2006). Fas/CD95 death receptor and lipid rafts: new
targets for apoptosis-directed cancer therapy. Drug Resist Updat 9, 51-73.
Moran, P., Raab, H., Kohr, W. J., and Caras, I. W. (1991). Glycophospholipid membrane
anchor attachment. Molecular analysis of the cleavage/attachment site. J Biol
Chem 266, 1250-1257.
Nagai, T., Tanaka, M., Tsuneyoshi, Y., Matsushita, K., Sunahara, N., Matsuda, T.,
Yoshida, H., Komiya, S., Onda, M., and Matsuyama, T. (2006). In vitro and in
vivo efficacy of a recombinant immunotoxin against folate receptor beta on the
activation and proliferation of rheumatoid arthritis synovial cells. Arthritis Rheum
54, 3126-3134.
Nagayoshi, R., Nagai, T., Matsushita, K., Sato, K., Sunahara, N., Matsuda, T., Nakamura,
T., Komiya, S., Onda, M., and Matsuyama, T. (2005). Effectiveness of anti-folate
receptor beta antibody conjugated with truncated Pseudomonas exotoxin in the
targeting of rheumatoid arthritis synovial macrophages. Arthritis Rheum 52,
2666-2675.
Ng, T., Squire, A., Hansra, G., Bornancin, F., Prevostel, C., Hanby, A., Harris, W.,
Barnes, D., Schmidt, S., Mellor, H., et al. (1999). Imaging protein kinase Calpha
activation in cells. Science 283, 2085-2089.
98 Nichols, B. J., Kenworthy, A. K., Polishchuk, R. S., Lodge, R., Roberts, T. H.,
Hirschberg, K., Phair, R. D., and Lippincott-Schwartz, J. (2001). Rapid cycling of
lipid raft markers between the cell surface and Golgi complex. J Cell Biol 153,
529-541.
Nishizuka, Y. (1988). The molecular heterogeneity of protein kinase C and its
implications for cellular regulation. Nature 334, 661-665.
Nishizuka, Y. (1992). Intracellular signaling by hydrolysis of phospholipids and
activation of protein kinase C. Science 258, 607-614.
Nishizuka, Y. (1995). Protein kinase C and lipid signaling for sustained cellular
responses. Faseb J 9, 484-496.
Oh, P., and Schnitzer, J. E. (2001). Segregation of heterotrimeric G proteins in cell
surface microdomains. G(q) binds caveolin to concentrate in caveolae, whereas
G(i) and G(s) target lipid rafts by default. Mol Biol Cell 12, 685-698.
Ohmori, S., Shirai, Y., Sakai, N., Fujii, M., Konishi, H., Kikkawa, U., and Saito, N.
(1998). Three distinct mechanisms for translocation and activation of the delta
subspecies of protein kinase C. Mol Cell Biol 18, 5263-5271.
Oka, M., Hitomi, T., Okada, T., Nakamura Si, S., Nagai, H., Ohba, M., Kuroki, T.,
Kikkawa, U., and Ichihashi, M. (2002). Dual regulation of phospholipase D1 by
protein kinase C alpha in vivo. Biochem Biophys Res Commun 294, 1109-1113.
Ono, Y., Fujii, T., Igarashi, K., Kuno, T., Tanaka, C., Kikkawa, U., and Nishizuka, Y.
(1989). Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-
finger-like sequence. Proc Natl Acad Sci U S A 86, 4868-4871.
99 Ono, Y., Kikkawa, U., Ogita, K., Fujii, T., Kurokawa, T., Asaoka, Y., Sekiguchi, K., Ase,
K., Igarashi, K., and Nishizuka, Y. (1987). Expression and properties of two types
of protein kinase C: alternative splicing from a single gene. Science 236, 1116-
1120.
Oriente, F., Andreozzi, F., Romano, C., Perruolo, G., Perfetti, A., Fiory, F., Miele, C.,
Beguinot, F., and Formisano, P. (2005). Protein kinase C-alpha regulates insulin
action and degradation by interacting with insulin receptor substrate-1 and 14-3-3
epsilon. J Biol Chem 280, 40642-40649.
Ory, D. S., Neugeboren, B. A., and Mulligan, R. C. (1996). A stable human-derived
packaging cell line for production of high titer retrovirus/vesicular stomatitis virus
G pseudotypes. Proc Natl Acad Sci U S A 93, 11400-11406.
Parekh, D. B., Ziegler, W., and Parker, P. J. (2000). Multiple pathways control protein
kinase C phosphorylation. Embo J 19, 496-503.
Parker, E. M., Zaman, M. M., and Freedman, S. D. (2000). GP2, a GPI-anchored protein
in the apical plasma membrane of the pancreatic acinar cell, co-
immunoprecipitates with src kinases and caveolin. Pancreas 21, 219-225.
Perez, J. F., Ruiz, M. C., Chemello, M. E., and Michelangeli, F. (1999). Characterization
of a membrane calcium pathway induced by rotavirus infection in cultured cells. J
Virol 73, 2481-2490.
Pike, L. J. (2006). Rafts defined: a report on the Keystone Symposium on Lipid Rafts and
Cell Function. J Lipid Res 47, 1597-1598.
100 Pol, A., Lu, A., Pons, M., Peiro, S., and Enrich, C. (2000). Epidermal growth factor-
mediated caveolin recruitment to early endosomes and MAPK activation. Role of
cholesterol and actin cytoskeleton. J Biol Chem 275, 30566-30572.
Prevostel, C., Alice, V., Joubert, D., and Parker, P. J. (2000). Protein kinase C(alpha)
actively downregulates through caveolae-dependent traffic to an endosomal
compartment. J Cell Sci 113 ( Pt 14), 2575-2584.
Rajendran, L., and Simons, K. (2005). Lipid rafts and membrane dynamics. J Cell Sci
118, 1099-1102.
Ratnam, M., Marquardt, H., Duhring, J. L., and Freisheim, J. H. (1989). Homologous
membrane folate binding proteins in human placenta: cloning and sequence of a
cDNA. Biochemistry 28, 8249-8254.
Rebecchi, M. J., and Pentyala, S. N. (2000). Structure, function, and control of
phosphoinositide-specific phospholipase C. Physiol Rev 80, 1291-1335.
Reeves, S. A., Chavez-Kappel, C., Davis, R., Rosenblum, M., and Israel, M. A. (1992).
Developmental regulation of annexin II (Lipocortin 2) in human brain and
expression in high grade glioma. Cancer Res 52, 6871-6876.
Rety, S., Sopkova, J., Renouard, M., Osterloh, D., Gerke, V., Tabaries, S., Russo-Marie,
F., and Lewit-Bentley, A. (1999). The crystal structure of a complex of p11 with
the annexin II N-terminal peptide. Nat Struct Biol 6, 89-95.
Ritter, T. E., Fajardo, O., Matsue, H., Anderson, R. G., and Lacey, S. W. (1995). Folate
receptors targeted to clathrin-coated pits cannot regulate vitamin uptake. Proc
Natl Acad Sci U S A 92, 3824-3828.
101 Roberts, W. L., Myher, J. J., Kuksis, A., Low, M. G., and Rosenberry, T. L. (1988a).
Lipid analysis of the glycoinositol phospholipid membrane anchor of human
erythrocyte acetylcholinesterase. Palmitoylation of inositol results in resistance to
phosphatidylinositol-specific phospholipase C. J Biol Chem 263, 18766-18775.
Roberts, W. L., Santikarn, S., Reinhold, V. N., and Rosenberry, T. L. (1988b). Structural
characterization of the glycoinositol phospholipid membrane anchor of human
erythrocyte acetylcholinesterase by fast atom bombardment mass spectrometry. J
Biol Chem 263, 18776-18784.
Robinson, K., Jones, D., Patel, Y., Martin, H., Madrazo, J., Martin, S., Howell, S.,
Elmore, M., Finnen, M. J., and Aitken, A. (1994). Mechanism of inhibition of
protein kinase C by 14-3-3 isoforms. 14-3-3 isoforms do not have phospholipase
A2 activity. Biochem J 299 ( Pt 3), 853-861.
Rodgers, W., Crise, B., and Rose, J. K. (1994). Signals determining protein tyrosine
kinase and glycosyl-phosphatidylinositol-anchored protein targeting to a
glycolipid-enriched membrane fraction. Mol Cell Biol 14, 5384-5391.
Ron, D., Chen, C. H., Caldwell, J., Jamieson, L., Orr, E., and Mochly-Rosen, D. (1994).
Cloning of an intracellular receptor for protein kinase C: a homolog of the beta
subunit of G proteins. Proc Natl Acad Sci U S A 91, 839-843.
Ron, D., Jiang, Z., Yao, L., Vagts, A., Diamond, I., and Gordon, A. (1999). Coordinated
movement of RACK1 with activated betaIIPKC. J Biol Chem 274, 27039-27046.
Ron, D., and Kazanietz, M. G. (1999). New insights into the regulation of protein kinase
C and novel phorbol ester receptors. Faseb J 13, 1658-1676.
102 Ron, D., Luo, J., and Mochly-Rosen, D. (1995). C2 region-derived peptides inhibit
translocation and function of beta protein kinase C in vivo. J Biol Chem 270,
24180-24187.
Roseman, B. J., Bollen, A., Hsu, J., Lamborn, K., and Israel, M. A. (1994). Annexin II
marks astrocytic brain tumors of high histologic grade. Oncol Res 6, 561-567.
Rotenberg, S. A., and Sun, X. G. (1998). Photoinduced inactivation of protein kinase C
by dequalinium identifies the RACK-1-binding domain as a recognition site. J
Biol Chem 273, 2390-2395.
Rothberg, K. G., Heuser, J. E., Donzell, W. C., Ying, Y. S., Glenney, J. R., and
Anderson, R. G. (1992). Caveolin, a protein component of caveolae membrane
coats. Cell 68, 673-682.
Rothberg, K. G., Ying, Y. S., Kolhouse, J. F., Kamen, B. A., and Anderson, R. G. (1990).
The glycophospholipid-linked folate receptor internalizes folate without entering
the clathrin-coated pit endocytic pathway. J Cell Biol 110, 637-649.
Roy, S., Luetterforst, R., Harding, A., Apolloni, A., Etheridge, M., Stang, E., Rolls, B.,
Hancock, J. F., and Parton, R. G. (1999). Dominant-negative caveolin inhibits H-
Ras function by disrupting cholesterol-rich plasma membrane domains. Nat Cell
Biol 1, 98-105.
Rykx, A., De Kimpe, L., Mikhalap, S., Vantus, T., Seufferlein, T., Vandenheede, J. R.,
and Van Lint, J. (2003). Protein kinase D: a family affair. FEBS Lett 546, 81-86.
103 Ryves, W. J., Evans, A. T., Olivier, A. R., Parker, P. J., and Evans, F. J. (1991).
Activation of the PKC-isotypes alpha, beta 1, gamma, delta and epsilon by
phorbol esters of different biological activities. FEBS Lett 288, 5-9.
Sabharanjak, S., Sharma, P., Parton, R. G., and Mayor, S. (2002). GPI-anchored proteins
are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-
independent pinocytic pathway. Dev Cell 2, 411-423.
Sakai, N., Sasaki, K., Ikegaki, N., Shirai, Y., Ono, Y., and Saito, N. (1997). Direct
visualization of the translocation of the gamma-subspecies of protein kinase C in
living cells using fusion proteins with green fluorescent protein. J Cell Biol 139,
1465-1476.
Schechtman, D., and Mochly-Rosen, D. (2001). Adaptor proteins in protein kinase C-
mediated signal transduction. Oncogene 20, 6339-6347.
Schnitzer, J. E., McIntosh, D. P., Dvorak, A. M., Liu, J., and Oh, P. (1995). Separation of
caveolae from associated microdomains of GPI-anchored proteins. Science 269,
1435-1439.
Schuck, S., Honsho, M., Ekroos, K., Shevchenko, A., and Simons, K. (2003). Resistance
of cell membranes to different detergents. Proc Natl Acad Sci U S A 100, 5795-
5800.
Sharkey, N. A., Leach, K. L., and Blumberg, P. M. (1984). Competitive inhibition by
diacylglycerol of specific phorbol ester binding. Proc Natl Acad Sci U S A 81,
607-610.
104 Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., Rao, M., and
Mayor, S. (2004). Nanoscale organization of multiple GPI-anchored proteins in
living cell membranes. Cell 116, 577-589.
Shen, F., Ross, J. F., Wang, X., and Ratnam, M. (1994). Identification of a novel folate
receptor, a truncated receptor, and receptor type beta in hematopoietic cells:
cDNA cloning, expression, immunoreactivity, and tissue specificity.
Biochemistry 33, 1209-1215.
Shen, F., Wu, M., Ross, J. F., Miller, D., and Ratnam, M. (1995). Folate receptor type
gamma is primarily a secretory protein due to lack of an efficient signal for
glycosylphosphatidylinositol modification: protein characterization and cell type
specificity. Biochemistry 34, 5660-5665.
Shenoy-Scaria, A. M., Dietzen, D. J., Kwong, J., Link, D. C., and Lublin, D. M. (1994).
Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and
localization in caveolae. J Cell Biol 126, 353-363.
Shenoy-Scaria, A. M., Kwong, J., Fujita, T., Olszowy, M. W., Shaw, A. S., and Lublin,
D. M. (1992). Signal transduction through decay-accelerating factor. Interaction
of glycosyl-phosphatidylinositol anchor and protein tyrosine kinases p56lck and
p59fyn 1. J Immunol 149, 3535-3541.
Shirai, Y., Kashiwagi, K., Yagi, K., Sakai, N., and Saito, N. (1998). Distinct effects of
fatty acids on translocation of gamma- and epsilon-subspecies of protein kinase C.
J Cell Biol 143, 511-521.
105 Simons, K., and Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387, 569-
572.
Simons, K., and Toomre, D. (2000). Lipid rafts and signal transduction. Nat Rev Mol
Cell Biol 1, 31-39.
Simons, K., and van Meer, G. (1988). Lipid sorting in epithelial cells. Biochemistry 27,
6197-6202.
Smart, E. J., Foster, D. C., Ying, Y. S., Kamen, B. A., and Anderson, R. G. (1994).
Protein kinase C activators inhibit receptor-mediated potocytosis by preventing
internalization of caveolae. J Cell Biol 124, 307-313.
Smart, E. J., Ying, Y., Donzell, W. C., and Anderson, R. G. (1996). A role for caveolin in
transport of cholesterol from endoplasmic reticulum to plasma membrane. J Biol
Chem 271, 29427-29435.
Solomon, K. R., Kurt-Jones, E. A., Saladino, R. A., Stack, A. M., Dunn, I. F., Ferretti,
M., Golenbock, D., Fleisher, G. R., and Finberg, R. W. (1998). Heterotrimeric G
proteins physically associated with the lipopolysaccharide receptor CD14
modulate both in vivo and in vitro responses to lipopolysaccharide. J Clin Invest
102, 2019-2027.
Solomon, K. R., Rudd, C. E., and Finberg, R. W. (1996). The association between
glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein
alpha subunits in lymphocytes. Proc Natl Acad Sci U S A 93, 6053-6058.
106 Sprenger, R. R., Fontijn, R. D., van Marle, J., Pannekoek, H., and Horrevoets, A. J.
(2006). Spatial segregation of transport and signalling functions between human
endothelial caveolae and lipid raft proteomes. Biochem J 400, 401-410.
Stebbins, E. G., and Mochly-Rosen, D. (2001). Binding specificity for RACK1 resides in
the V5 region of beta II protein kinase C. J Biol Chem 276, 29644-29650.
Stefanova, I., Horejsi, V., Ansotegui, I. J., Knapp, W., and Stockinger, H. (1991). GPI-
anchored cell-surface molecules complexed to protein tyrosine kinases. Science
254, 1016-1019.
Stewart, J. M., Fleshner, N., Cole, H., and Sweet, J. (2006). Comparison of Annexin II,
p63 and AMACR immunoreactivity in prostatic tissue: A tissue microarray study.
J Clin Pathol.
Stewart, J. R., and O'Brian, C. A. (2005). Protein kinase C-{alpha} mediates epidermal
growth factor receptor transactivation in human prostate cancer cells. Mol Cancer
Ther 4, 726-732.
Subczynski, W. K., and Kusumi, A. (2003). Dynamics of raft molecules in the cell and
artificial membranes: approaches by pulse EPR spin labeling and single molecule
optical microscopy. Biochim Biophys Acta 1610, 231-243.
Takeuchi, Y., Yanagishita, M., and Hascall, V. C. (1992). Recycling of transferrin
receptors and heparan sulfate proteoglycans in a rat parathyroid cell line. J Biol
Chem 267, 14685-14690.
Tan, S. L., and Parker, P. J. (2003). Emerging and diverse roles of protein kinase C in
immune cell signalling. Biochem J 376, 545-552.
107 Tansey, M. G., Baloh, R. H., Milbrandt, J., and Johnson, E. M., Jr. (2000). GFRalpha-
mediated localization of RET to lipid rafts is required for effective downstream
signaling, differentiation, and neuronal survival. Neuron 25, 611-623.
Tatin, F., Varon, C., Genot, E., and Moreau, V. (2006). A signalling cascade involving
PKC, Src and Cdc42 regulates podosome assembly in cultured endothelial cells in
response to phorbol ester. J Cell Sci 119, 769-781.
Theti, D. S., Bavetsias, V., Skelton, L. A., Titley, J., Gibbs, D., Jansen, G., and Jackman,
A. L. (2003). Selective delivery of CB300638, a cyclopenta[g]quinazoline-based
thymidylate synthase inhibitor into human tumor cell lines overexpressing the
alpha-isoform of the folate receptor. Cancer Res 63, 3612-3618.
Thomas, P. M., and Samelson, L. E. (1992). The glycophosphatidylinositol-anchored
Thy-1 molecule interacts with the p60fyn protein tyrosine kinase in T cells. J Biol
Chem 267, 12317-12322.
Toker, A., and Newton, A. C. (2000). Cellular signaling: pivoting around PDK-1. Cell
103, 185-188.
Upla, P., Marjomaki, V., Kankaanpaa, P., Ivaska, J., Hyypia, T., Van Der Goot, F. G.,
and Heino, J. (2004). Clustering induces a lateral redistribution of alpha 2 beta 1
integrin from membrane rafts to caveolae and subsequent protein kinase C-
dependent internalization. Mol Biol Cell 15, 625-636.
Van Der Hoeven, P. C., Van Der Wal, J. C., Ruurs, P., and Van Blitterswijk, W. J.
(2000). Protein kinase C activation by acidic proteins including 14-3-3. Biochem
J 347 Pt 3, 781-785.
108 van Deurs, B., Roepstorff, K., Hommelgaard, A. M., and Sandvig, K. (2003). Caveolae:
anchored, multifunctional platforms in the lipid ocean. Trends Cell Biol 13, 92-
100.
Varma, R., and Mayor, S. (1998). GPI-anchored proteins are organized in submicron
domains at the cell surface. Nature 394, 798-801.
Veggian, R., Fasolato, S., Menard, S., Minucci, D., Pizzetti, P., Regazzoni, M.,
Tagliabue, E., and Colnaghi, M. I. (1989). Immunohistochemical reactivity of a
monoclonal antibody prepared against human ovarian carcinoma on normal and
pathological female genital tissues. Tumori 75, 510-513.
Vereb, G., Szollosi, J., Matko, J., Nagy, P., Farkas, T., Vigh, L., Matyus, L., Waldmann,
T. A., and Damjanovich, S. (2003). Dynamic, yet structured: The cell membrane
three decades after the Singer-Nicolson model. Proc Natl Acad Sci U S A 100,
8053-8058.
Wang, J., Gunning, W., Kelley, K. M., and Ratnam, M. (2002). Evidence for segregation
of heterologous GPI-anchored proteins into separate lipid rafts within the plasma
membrane. J Membr Biol 189, 35-43.
Wary, K. K., Mariotti, A., Zurzolo, C., and Giancotti, F. G. (1998). A requirement for
caveolin-1 and associated kinase Fyn in integrin signaling and anchorage-
dependent cell growth. Cell 94, 625-634.
Watts, C. (1985). Rapid endocytosis of the transferrin receptor in the absence of bound
transferrin. J Cell Biol 100, 633-637.
109 Way, K. J., Chou, E., and King, G. L. (2000). Identification of PKC-isoform-specific
biological actions using pharmacological approaches. Trends Pharmacol Sci 21,
181-187.
Wedegaertner, P. B., Wilson, P. T., and Bourne, H. R. (1995). Lipid modifications of
trimeric G proteins. J Biol Chem 270, 503-506.
Wu, M., Fan, J., Gunning, W., and Ratnam, M. (1997). Clustering of GPI-anchored folate
receptor independent of both cross-linking and association with caveolin. J
Membr Biol 159, 137-147.
Wu, M., Gunning, W., and Ratnam, M. (1999). Expression of folate receptor type alpha
in relation to cell type, malignancy, and differentiation in ovary, uterus, and
cervix. Cancer Epidemiol Biomarkers Prev 8, 775-782.
Yamada, E. (1955). The fine structure of the gall bladder epithelium of the mouse. J
Biophys Biochem Cytol 1, 445-458.
Yan, G., Luo, W., Lu, Z., Luo, X., Li, L., Liu, S., Liu, Y., Tang, M., Dong, Z., and Cao,
Y. (2006). Epstein-Barr virus latent membrane protein 1 mediates
phosphorylation and nuclear translocation of annexin A2 by activating PKC
pathway. Cell Signal.
Yan, W., and Ratnam, M. (1995). Preferred sites of glycosylphosphatidylinositol
modification in folate receptors and constraints in the primary structure of the
hydrophobic portion of the signal. Biochemistry 34, 14594-14600.
110 Yoshida, Y., Huang, F. L., Nakabayashi, H., and Huang, K. P. (1988). Tissue distribution
and developmental expression of protein kinase C isozymes. J Biol Chem 263,
9868-9873.
Zlatkine, P., Mehul, B., and Magee, A. I. (1997). Retargeting of cytosolic proteins to the
plasma membrane by the Lck protein tyrosine kinase dual acylation motif. J Cell
Sci 110 ( Pt 5), 673-679.
Zurzolo, C., van't Hof, W., van Meer, G., and Rodriguez-Boulan, E. (1994).
VIP21/caveolin, glycosphingolipid clusters and the sorting of
glycosylphosphatidylinositol-anchored proteins in epithelial cells. Embo J 13, 42-
53.
Zurzolo, C., van Meer, G., and Mayor, S. (2003). The order of rafts. Conference on
microdomains, lipid rafts and caveolae. EMBO Rep 4, 1117-1121.
111 ABSTRACT
FR quantitatively recycles, within minutes, between the cell surface and endocytic compartments via a Cdc42-regulated endocytic pathway. In this study, we investigated
the molecular mechanism by which FR recycling is regulated by PMA since its effects on
physiological processes are known to stimulate cell signaling through the second
messenger diacylglycerol. Phorbol-12-myristate-13-acetate causes a dose dependent increase in the number of receptor molecules residing on the cell surface by inhibiting
internalization. Moreover, the release of the intracellular calcium stores which is triggered by the ionophore cyclopiazonic acid results in a doubling of cell surface FR thereby simulating the effect of PMA. Since intracellular calcium is needed for externalization of FR, the PMA effect is probably mediated by a classical PKC isoforms.
Western blot of total MA104 cell lysates and quantitative real time RT-PCR show that
PKCα is the only classical PKC that can be detected in these cells. Overexpression of wild-type PKCα results in an increase in PMA-induced FR externalization relative to the control cells overexpressing GFP. Knockdown of PKCα using retrovirus or overexpression of a dominant negative PKCα mutant or a constitutively active PKCα which is targeted to the membrane but not specifically to rafts abrogate the PMA effect on FR recycling. These data further suggest that PKCα, targeted to specific membrane microdomains, is one of the key players in mediating the phorbol ester effect on FR recycling in MA104 cells. In order to identify other candidate proteins involved in FR recycling, we purified FR-rich rafts using an immobilized biotinylated folate probe.
112 Among the proteins identified, RACK1 mediates the PMA effect on FR recycling in
MA104 cells. The association of RACK1 with FR-rich rafts was further confirmed by electron microscopy in situ in MA104 cells. Though RACK1 has been reported to be present in the cytosol and in caveolae, no evidence for its association with rafts has been documented until now. One of the other proteins found to be associated with FR-rafts is the PKC substrate, annexin II. Unlike RACK1, annexin II is required for the internalization of FR rafts. Finally, the activation of Cdc42 is not diminished by PMA treatment, and therefore is not a likely mediator of the inhibition of raft internalization.
These detailed studies provide a comprehensive molecular picture of the effect of PMA on FR recycling in MA104.
113