Regulation of Protein Phosphatase 1, Pp1 2, in Testis/Spermatozoa by Ppp1r11, Ppp1r7 and Ppp1r2

Home , Cellular compartment, PPP1CC, PPP1R11, PPP1R12A, PPP1R12B, PPP1R14A

REGULATION OF PROTEIN PHOSPHATASE 1, PP1γ2, IN TESTIS/SPERMATOZOA BY PPP1R11, PPP1R7 AND PPP1R2

A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Lina Cheng

May, 2008

Dissertation written by Lina Cheng B.S., M.D., Shanxi Medical University, 1998 M.S., Shanghai Medical University, 2001 Ph.D., Kent State University, 2008

Approved by

______,Dr. S. Vijayaraghavan

(Chair, Doctoral Dissertation Committee)

______,Dr. Douglas Kline

______,Dr. Jennifer L. Marcinkiewicz

______,Dr. Bansidhar Datta

______,Dr. Roger B. Gregory

Accepted by

______, Dr.Robert V. Dorman (Director, School of Biomedical Sciences)

______, Dr.John Starlvey (Dean, College of Arts and Sciences)

TABLE OF CONTENTS

LIST OF FIGURES………………………………………………………………………iv

ACKNOWLEDGEMENTS………………………………………………………….…..vii

AIMS………...... …..viii

INTRODUCTION...... ………….…...1

MATERIALS AND METHODS………...... 28

RESULTS...... 37

DISCUSSION……………...... 107

REFERENCES……………………………………………………………………...... 127

iii

LIST OF FIGURES

Fig. 1 The + and t homologs of the mouse t complex region ………………….………..25

Fig. 2 TCTEX5 antibody recognizes an I3-unrelated protein in testis/sperm…………...38

Fig. 3. Validation of the I3 antibody raised against the two peptides at N-terminus of I3

protein……………………………………………………………………………………39

Fig. 4. Northern blot analysis of Tctex5 mRNA expression in multiple mouse tissues…41

Fig. 5. I3 is a heat-stable protein present in nearly equal abundance in mouse sperm, testis

and a wide variety of somatic tissues…………………………………………………….42

Fig.6. GST-I3 binds Sds22 and PP1γ2 from testis or sperm extracts in vitro………...... 44

Fig. 7. Anti-PP1γ2 can co-precipitate Sds22 and I3, and anti-I3 can co-precipitate PP1γ2 and Sds22 from sperm (A) or testis (B)...... 46

Fig. 8. I3, Sds22 and PP1γ2 co-elute during chromatographic purification of testis

extracts...... 50

Fig. 9. I3, Sds22, and PP1γ2 from co-eluting column fractions are reciprocally co-

immunoprecipitated...... 54

Fig. 10. I3, Sds22, and PP1γ2 from co-eluting column fractions co-migrate by native

PAGE...... ,.55

Fig. 11. A protein molecular weight calibration curve for Superdex 200 column...... 56

Fig.12. Microsequencing of Coomassie-blue stained protein band containing Sds22/

PP1/I3...... 58

Fig 13. Evidence that PP1γ2, Sds22, and I3 are bound to each other in a male germ cell

complex that is larger than a trimer...... 62

Fig. 14. Actin co-precipitates with PP1γ2, Sds22, and/or I3 from testis...... 63

Fig. 15. Macromolecular complexes containing I3 and Sds22 are not PP1γ2 isoform-

specific...... 66

Fig. 16. Actin is also complexed with PP1β or PP1γ1 bound with I3 and Sds22...... 68

Fig. 17. PP1γ2 is inactive in Superose 6 fractions containing PP1γ2, Sds22, I3, and

actin...... 69

Fig. 18. Increasing steady state levels of I3, PP1γ2, and Sds22 in the testis parallel the temporal progression of spermiogenesis...... 71

Fig. 19. Cellular localization of PP1γ2, I3 and Sds22 in wild-type mouse testis

sections...... 72

Fig. 20. Both the steady state level and molecular weight of I3 are diminished in the

PP1γ-null testis, but its level and molecular weight increase in the PP1γ-null testis

producing low levels of PP1γ2 protein via transgene expression...... 76

Fig. 21. Difference of microcystin pulldown PP1γ2 and its binding proteins from sperm

and testis...... 78

Fig. 22. Amino acid sequence difference between t/t-I3 and wt-I3...... 80

Fig. 23. Validation of t/t-I3 antibody...... 82

Fig. 24. Comparison of the inhibitions of His-PP1γ2 by His-wt-I3 and His-t/t-I3...... 85

Fig. 25. Comparison of the binding of GST-wt-I3 and GST-t/t-I3 with PP1γ2 and Sds22 by GST pulldown assay...... 88

Fig. 26. Anti-PP1γ2 can co-precipitate t/t-I3 and Sds22, and anti-t/t-I3 can co-precipitate

PP1γ2 and Sds22 from t/t-testis protein extracts...... 89

Fig. 27. t/t-I3, Sds22 and PP1γ2 co-elute during chromatographic purification of t/t-testis

extracts...... 91

Fig. 28. t/t-I3, Sds22, and PP1γ2 from co-eluting column fractions co-migrate by native

PAGE...... 92

Fig. 29. In vitro phosphorylation study of GST-wt-I3 and GST-t/t-I3 using sperm extracts

as a source of kinase...... 94

Fig. 30. Characterization of I2 antibody and verification of the existence of inhibitor I2 in

testis...... 96

Fig. 31. Immunoprecipitation with I2 antibody from sperm or testis...... 98

Fig. 32. Northern blots of mouse tissue mRNAs...... 101

Fig. 33. PCR results of multiple tissue cDNAs...... 104

Fig. 34. Western blot of recombinant I2(17) protein...... 105

Fig. 35. Our affinity purified I2 antibody recognizes a 55kDa protein on western

blots...... 106

Table 1 Microsequencing Result...... 59

ACKNOWLEDGMENTS

I will express my deepest gratitude to my advisor, Dr. Srinivasan Vijayaraghavan,

for his constant support, guidance, encouragement and kindness. I would like to thank him for training and teaching me how to be a scientist. He helped me to achieve my goals during my research. He is an excellent professor, a fully devoted scientist and also a great

friend in life.

I thank our collaberator, Dr. Steven Pilder, and my committee members, Dr.

Douglas W. Kline, Dr. Jennifer L. Marcinkiewicz, and Dr. Bansidhar Datta. All your help

and kindness are greatly appreciated. Without your help, I could not have come this far.

I thank Kimbery Myers for her great help in the experiments and discussion, and

Dr. Michael Model for his help in the confocal microscopy. I thank the following experts

for their various help in my research, Dr.Edgar F. da Cruz e Silva, Dr.David Brautigan,

Dr. Mathieu Bollen, and Dr.Angus Nairn.

I thank all my friends for their kindness and their support. Thank them for

listening to me, encouraging me, and all the laughs and tears shared with me: Hongmei

Zhang, Kamil Gierszal, Qunying Zhu, Zhiping Jiang, Viorel Sandu, JJ Voelker, and

Karen Bonfiglio.

Finally I deeply thank my parents, my brothers and sister-in-laws, my lovely niece

and nephew, and all my other relatives. They strongly support me from every aspects of

my life. For my every achievement, I must share with them.

vii

AIMS

The phosphatase PP1γ2 is the only detectable PP1 isoform in mammalian spermatozoa. Its activity is inversely related to sperm motility suggesting that PP1γ2 has a fundamental, isoform-specific role(s) in mammalian sperm function. However, the biochemical nature of its essential role(s) in either sperm development or subsequent function during the fertilization process remains unclear. A number of PP1-interacting proteins in somatic cells have been detected via either biochemical or bioinformatic approaches, and considerable progress has been made in defining the functions of these proteins and how they regulate PP1 in those cells. In comparison, our understanding of the regulation of PP1γ2 in male germ cells is limited. Yeast two hybrid approaches have identified a number of PP1 interacting proteins in testis, yet because the specific locations of these interactions within testis are not presently known, the function of these proteins with respect to regulation of the PP1γ2 isoform remains unclear. In addition, as the haploid spermatid differentiates during spermiogenesis, there is progressively less transcriptional and translational activity, until the biosynthetic apparatus necessary to carry out these activities is finally eliminated in the terminally differentiated mature spermatozoon. This feature of sperm development precludes application to elongated spermatids and mature sperm of techniques (such as the yeast two hybrid approach) generally employed in somatic cells to study the role of the phosphatase and to identify its binding partners. However, by continuing to identify and characterize the relationships

viii

of PP1γ2 to its interacting partners in testis and spermatozoa by employing biochemical approaches, we hope to elucidate its function in developing and mature male gametes.

A potent heat-stable PP1 inhibitor, PPP1R11 (inhibitor 3 or I3), first discovered through yeast two-hybrid studies designed to characterize PP1 binding partners in human brain, has been shown to be involved in PP1 regulation in somatic cells. And, its mouse homologue, TCTEX5 (t-complex testis expressed gene 5), is implicatively involved in regulation of sperm motility based on the observation that a naturally occurring mutant isoform of TCTEX5 is involved in an abnormal sperm motility phenotype, “curlicue”.

So, this dissertation studies I3 in testis/sperm, particularly along with the mutant isoform of I3. In addition, there is no PPP1R1/inhibitor 1 (I1) activity present in sperm but there is PPP1R2/inhibitor 2-like (I2-like) activity in sperm, therefore the presence of I2 in sperm/testis will be examined in this dissertation as well.

This dissertation has the following three aims:

AIM 1: Characterize the protein phosphatase inhibitor PPP1R11/inhibitor 3

(I3) in testis and spermatozoa.

AIM 2: Conduct comparative studies of t/t-I3 versus wt-I3.

AIM 3: Characterize the protein phosphatase inhibitor PPP1R2/inhibitor 2

(I2) in testis and spermatozoa.

INTRODUCTION

1. Protein phosphatases

1a. Types of protein phosphatases:

Close to one third of all proteins in eukaryotes are regulated by reversible phosphorylation by kinases and phosphatases. Protein phosphatases and kinases are not present in equal numbers in cells. While there are just 30 human protein Ser/Thr phosphatase catalytic subunit genes, over 300 protein Ser/Thr kinase genes are in the human genome. Thus kinases outnumber phosphatases by 10 to 20-fold (1). Based on their catalytic subunit substrate specificities, catalytic mechanisms and amino acid sequences, protein phosphatase can be broadly classified into Serine/Threonine (Ser/Thr) protein phosphatase, Tyrosine (Tyr) protein phosphatase (PTP), and Histidine protein phosphatase (PHP).

Ser/Thr phosphatases catalyze the cleavage of phosphate from Serine and/or

Threonine residues in proteins. Ser/Thr phosphatases can be further sub-divided into the following two families, phosphoprotein phosphatase (PPP, including PP1, PP2A, and

PP2B), and metal-ion-dependent protein phosphatases (PPM, including PP2C). The PPP and PPM family members are metalloenzymes and dephosphorylate their substrates in a single reaction step. In spite of the fact that both PPP and PPM subfamily members utilize the same binuclear metal assisted catalytic mechanism, they have emerged through convergent evolution. PTPs, containing a highly conserved Cysteine residue essential for

1 2

the catalytic reaction, can also be further divided into transmembrane (receptor-like),

cytosolic (non-receptor), and dual-specificity phosphatases (DSP). PTPs are important in

cellular signaling because of its role in counterbalancing the effects of receptor and

cytosolic protein Tyr kinases and transmitting information from the plasma membrane.

Histidine (His) phosphorylation in eukaryotes is an emerging area of research in

comparison to the well studied Ser/Thr and Tyr phosphorylation mechanisms. A unique

protein phosphatase responsible for His dephosphorylation (PHP) has been identified

recently (2). Its primary structure which is different from Ser/Thr or Tyr phosphatases

and its insensitivity to known inhibitors of Ser/Thr or Tyr phosphatases put PHP into a

novel category of protein phosphatases. Interestingly, PHP is present in animals from

humans to nematodes but absent in bacteria (2).

1b. Evolution of protein phosphatases

The catalytic domains are highly conserved for each family of protein phosphatases.

Thirteen genes in human species and twelve genes in Saccharomyces cerevisiae encode

PPP family (3-5), and the catalytic subunits of PP1 and PP2 are highly conserved

throughout the evolution of multicellular eukaryotes and are among the most slowly

evolving proteins.

Structure based screening and function based cloning were used in the first cloning of the catalytic subunits of PP1 (PP1c). The structure based screening used oligonucleotide probes based on rabbit muscle PP1c peptide sequences encoded by PP1β cDNAs by

Berndt et al in 1987 (6)and PP1α cDNAs by Cohen (7) and Bai et al. in 1988 (8).

Function based cloning was used to map Dis2 (PP1α homologue in fission yeast) and the

genes dis2 m1 and dis2 m2 (the PP1α homologues in mice) by Ohkura et al. in 1988-

1989 from the study of cell cycle mutants (7). In 1989, the same S.pombe dis2 gene was

also reported by Booher and Beach (9). In 1989, Doonan and Morris found that BimG

gene from Emericella nidulans was PP1α homologue (10). In 1990, Sasaki et al. isolated

PP1γ1 and PP1γ2 from rats using dis2 m1 and dis2 m2 cDNA probes, and also cloned rat

PP1α and PP1β. Since the first cloning of the catalytic subunit of PP1, newer methods

and approaches have been used to identify isoforms of PP1 catalytic subunit in a wide

range of organisms., These methods include a combination of hybridization based

screening and PCR, EST sequencing, genome projects, and activity assay.

1c. The structure of PP1 catalytic subunit:

The catalytic subunit of PP1 is a polypeptide chain organized into ten alpha-helices

and fourteen beta sheets (11, 12). Three shallow grooves on the PP1 surface radiate from

the catalysis site, a hydrophobic groove, an acidic groove, and a C-terminal groove. The

three grooves provide potential binding sites for regulators and substrates (11, 12). The

basic residues of N-terminal to the phosphoserine of the PP1 binding partners can bind

the acidic residues in the acid groove of PP1; phosphoserine of PP1 binding partners can

bind to the active site of PP1, and C-terminal of the phosphoserine (or phosphothreonine) of PP1 binding partners can bind to the hydrophobic groove of PP1.

The catalytic site is located at the confluence of the three grooves. Mn2+ and Fe2+ situated at the catalytic site can bind to the phosphate moiety of the substrate. The position of the catalytic site at the base of a shallow cleft is consistent with the ability of

PP1 to dephosphorylate phospho-serine and phospho-threonine containing proteins (11).

The depth of the catalytic channel is equivalent to the length of phospho-serine and

phospho-threonine side-chains (11). Further specificity for phospho-serine/phospho-

threonine containing peptides and proteins may be provided by main-chain atoms of the

substrate interacting with the residues of the phosphatase (11). The molecular surface of

PP1 is relatively open and no peptide binding cleft is evident (11). This is consistent with

the ability of PP1 to dephosphorylate diverse substrates and suggests numerous modes of

peptide-protein recognition. PP1 also recognizes tertiary structural features of the

substrate (11).

Protein serine/threonine phosphatase of type-1 (PP1) regulates such diverse processes

as intermediate metabolism, glycogen metabolism, muscle contraction, cell cycle control,

cell division, mRNA splicing, transcription, cell motility, ion channel regulation,

neuronal activities, the organization of the cytoskeleton and apoptosis (13-17). All these

different processes are regulated by distinct PP1 holoenzymes containing the same

catalytic subunit (PP1c) but different regulatory subunits.

1d. Regulatory subunits of PP1

It has been established that PP1c is extremely well conserved during evolution (18).

The increasing diversity of phosphatase function from yeast to humans, and various

(sub)cellular functions of the same catalytic subunit are due to the fact that one catalytic

subunit is able to bind to one or more different regulatory subunits, forming the PP1

holoenzyme.

Evolutionary studies of the regulatory subunits of protein phosphatase 1 (17) revealed

that inhibitor 3 and Sds22 were the most ancient regulatory subunits because of their

widespread phylogenetic distribution. Inhibitor 2 and nuclear inhibitor of protein

phosphatase 1 (NIPP1) are also among the oldest inhibitors of PP1. Based on their

thermostable characteristics, inhibitor 1 and inhibitor 2 are the first PP1c interacting

proteins purified (19, 20), after which, large numbers of other PP1c interacting proteins

were found by various biochemical approaches, such as purification of the holoenzyme

complexes, affinity chromatography, co-immunoprecipitation, far-western analysis,

genetic interactions, yeast two hybrid and screening of expression libraries with labeled

PP1 (21).

Inhibitor 1 and inhibitor 2 have been widely used to identify PP1 activity (22). In

these studies, inhibitor 2 is preferable to inhibitor 1 because dephosphorylated inhibitor 2 is active while inhibitor 1 is only active after being phosphorylated by PKA. Inhibitor 2 is

part of the ATP-Mg-dependent protein phosphatase complex(15). Glycogen synthase

kinase 3 (GSK3) and ATP-Mg are required to (re-)activate the activity of the ATP-Mg-

dependent phosphatase. GSK3 can inactivate inhibitor 2 by transiently phosphorylating it in the presence of ATP and Mg, and this interaction aids the proper folding of newly synthesized PP1c and leads to the activation of protein phosphatase bound with inhibitor

2. These studies led to the suggestion that inhibitor 2 is a molecular chaperone of PP1

(23, 24). However, at higher concentration, inhibitor 2 acts as a negative regulator of

PP1c (25).

Up to 100 different binding proteins of PP1c, at least twenty in S. cerevisiae, have

been reported so far. Depending on their effect on PP1, the regulatory subunits can be

roughly divided into the following groups, targeting subunits and/or specific substrates,

and activity modulators:

1) Targeting subunits:

The concept of targeting subunits was first raised by Hubbard and Cohen in 1993, and further expanded by Faux and Scott in 1996. The targeting subunits direct PP1c to specific locations, like microtubules, mitochondria, membranes, myosin fibers, nucleus, centrosomes, ribosomes, glycogen particles or actin cytoskeleton, and control phosphatase specificity by tethering the enzyme activity towards the substrate proteins located at the given sub-cellular compartment.

Additionally, some targeting subunits play more roles than simply directing the

protein phosphatase to a specific location. For example, they can act as protein

phosphatase activity modulators and modify phosphatase specificity by activating the

enzyme activity against selected substrates. Some targeting subunits can also function as

signaling modules, also called ‘scaffolding’ proteins, whihc bring both protein kinases

and protein phosphatases in close proximity to their substrates, e.g. A-kinase-anchoring-

proteins (AKAPs) (26, 27), which include AKAP149, AKAP220, Yotiao, AKAP 350 and

CG-NAP/AKAP450, and MYPT1 (Myosin Phosphatase Targeting 1) (28-30).

1-1) Targeting subunits that target PP1 to an organelle/specific molecules:

1-1-1) Targeting PP1 to glycogen particles, also called the glycogen-targeting subunits (G subunits): Some of G subunits in this category can also be substrates or activity modulators for PP1. For example, some G subunit can not only anchor PP1 to glycogen, but also bind the substrate glycogen synthase and increase the specific activity of the associated PP1 towards glycogen synthase (31). G subunits include

GM/RGL/PPP1R3A, GL, PPP1R5/PTG (Protein Targeting to Glycogen)/U5, and

PPP1R6/PPP1R3D. GM subunits, interacting indirectly with glycogen synthase through glycogen particles, can target PP1 to glycogen in skeletal muscle. Additionally, GM is a transmembrane protein of the sarcoplasmic reticulum, where it has been proposed to associate with phospholamban, which is another substrate of PP1 (32). GL subunits, interacting directly with glycogen synthase, can target PP1 to glycogen in liver. PPP1R5 and PPP1R6, which are widely distributed, can target PP1 to glycogen particles in various tissues (30).

1-1-2) Targeting PP1 to myofibril: These PP1 targeting subunits are also called M subunits (myosin-targeting subunits) which include MYPT1 (Myosin phosphatase- targeting subunit 1/PPP1R12A /Serine/threonine protein phosphatase PP1 smooth muscle regulatory subunit M110/130 kDa myosin-binding subunit of smooth muscle myosin phophatase (M130)), and MYPT2/PPP1R12B. MYPT1 is widely distributed in smooth muscle and non-muscle tissues while MYPT2 is mainly located in skeletal muscle, heart, brain and myofribrils (28-30). MYPT1 can not only bind both PP1c and myosin which is a PP1c substrate, but also enhance PP1c activity towards myosin and decrease its activity towards exogenous substrates. Thus MYPT1 can also act as a ‘substrate specifier’ of PP1

(28-30, 30, 33).

1-1-3) Targeting PP1 to nuclei: These targeting subunits include proteins such as -

NIPP1 (Nuclear Inhibitor of PP1/PPP1R8), involved in pre-mRNA splicing; PNUTS

(Phosphatase 1 Nuclear Targeting Subunit), involved in RNA processing; AKAP 149

(A-kinase Anchoring Protein 149), present in the nuclear envelope, involved in nuclear

envelope reassembly; and Sds22 (PPP1R7), widely distributed in somatic cell nucleus, is

involved in proteasome targeting and mitosis. More examples include PSF1

(Polypyrimidine tract-binding protein associated Splicing Factor), p99 (R111), Hox11

(homeodomain transcription factor) and HCF (Host Cell Factor).

1-1-4) Targeting PP1 to endoplasmic-ribosome: The molecule RIPP1 (Ribosomal

Inhibitor of PP1) located in the; ribosomal protein L5, widely distributed in ribosome,

GADD34 (Growth Arrest and DNA Damage protein/PPP1R15A), stress inducible and

widely distributed, are all proteins involved in regulating protein synthesis.

1-1-5) Targeting PP1 to plasma membrane/cytoskeleton centrosome/microtubule:

These targeting subunits include:

i. Neurabin I (PPP1R9A), involved in neurite outgrowth (34) and synapse morphology (35) and located in neuronal plasma membrane and actin cytoskeleton;

ii. Spinophilin (neurabin II/PPP1R9B), involved in glutamatergic synaptic transmission and dendritic morphology (36, 37) and located ubiquitously in plasma membrane and actin cytoskeleton;

iii. NF-L (neurofilament-L), involved in synaptic transmission and located in

neuronal plasma membrane and cytoskeleton, targeting PP1 to plasma membrane and actin cytoskeleton (38);

iv. AKAP220, involved in PKA and PP1 signaling pathway in brain and testis (27),

targeting PP1 to peroxisomes/cytoskeleton;

v. Yotiao (A-kinase anchoring protein), involved in synaptic transmission (as a

NMDA Neuronal receptor), targeting PP1 to microtubules (39),

vi. BH-protocadherin, involved in neuronal cell-cell interactions, targeting PP1 to neuronal membrane (40);

vii. Ryanodyne receptor, involved in calcium ion channel activity in skeletal and

cardiac muscle, targeting PP1 to plasma membrane (41);

viii. NKCC1 (Na-K-Cl Cotransporter), involved in transcellular chloride ion

transporting, targeting PP1 to plasma membrane (42);

ix. AKAP 350 (CG-NAP/AKAP450), widely distributed and involved in centrosomal

functions, targeting PP1 to cytoskeleton centrosome;

x. Tau, involved in microtubule stability, targeting PP1 to neuronal microtubules

(43);

xi. Nek2 (NIMA related protein kinase 2), widely distributed in cytoplasm and

involved in centrosome separation, targeting PP1 to cytoskeleton centrosome. And, Nek2

can also function as PP1 substrate, for example, in the complex of Nek2 bound with both

PP1 and C-Nap1 (Centrosomal Nek2-associated protein 1), C-Nap1 is the substrate for

both PP1 and Nek2 while Nek2 is also the substrate for PP1 (44).

1-1-6) Actin binding regulators: Some of the above PP1-binding proteins can also be categorized as actin-associated regulators of PP1. They include the neurabin family, the

MYPT family and the PHI (Phosphatase Holoenzyme Inhibitors) family. PP1-binding proteins in the neurabin family can associate integral membrane proteins with the subcortical actin cytoskeleton at cell-cell adhesion contacts, playing an important role in the formation of dendritic spines (45) and in morphogenesis (46). The MYPT family includes the myosin-phosphatase targeting subunits tethering PP1 to class-II myosin, leading to the dephosphorylation of the myosin regulatory light chain, thereby causing relaxation of the actomyosin fibers (47). The PHI family includes phosphatase holoenzyme inhibitors, i.e. PHI and the closely related CPI-17 (17-kDa PKC-potentiated inhibitory protein of PP1, also called protein phosphatase 1 regulatory (inhibitor) subunit

14A/PPP1R14A, which are the potent inhibitors for the MYPT-PP1 complexes (48).

1-2) Targeting subunits involved in apoptosis:

These subunits also act as specific substrates/regulatory subunits of phosphatase.

The proteins in this category are mainly PP1α binding partners and involved in apoptosis.

They can either target PP1 to the molecules involved in apoptosis, or can act as specific substrates for PP1 in the process of apoptosis, or can inhibit PP1 activity involved in apoptosis. For example,

Bad, located in rafts/cytosol/mitochondria which is a pro-apoptotic member of Bcl-2 family, can be dephosphorylated by PP1α that regulates interleukin-2 deprivation- induced apoptosis(49);

anti-apoptotic protein Bcl-2 (survival factor), a prototype of a family of survival

regulatory proteins and an integral membrane protein of the

cytoplasm/mitochondria/endoplasmic reticulum, and Bcl-xL and Bcl-w, two anti-

apoptotic members of Bcl-2 family, can target PP1α to Bad(50, 51);

p53BP2 (TP53BP2, p53-Binding Protein 2), widely distributed in cytosol and bound

to the tumor suppressor p53, binds to PP1 and results in dephosphorylation of p53(52);

Rb (Retinoblastoma protein), widely distributed in nucleus, interacts with PP1 catalytic subunits both as its substrate and also as a non-competitive inhibitor involved in

cell cycle progression(53);

2) Activity modulators/inhibitory subunits

2-1) These proteins, depending on their phosphorylation, can bind to the catalytic

subunit of PP1 and inhibit its catalytic activity. For example, inhibitor-1 (54) and its neuronal homologue, DARP-32 (Dopamine and cAMP-Regulated Phosphoprotein Mr

32000), can turn off PP1 activity due to the activity of the cAMP-dependent protein kinase (PKA) (55). Inhibitor 1 (I1 /PPP1R1)(56) is widely distributed in cytosol in various cell types, while DARPP-32 is mainly a cytosol protein in brain and kidney. In its non-phosphorylated form, inhibitor 2 (I2/PPP1R2) can inhibit PP1c. Inhibitor 3 (I3/HCG

V/TCTEX5)(57), widely distributed in both nucleus and cytosol, has been proposed to inhibit PP1 with its non-phosphorylated form. CPI-17, in its phosphorylated form, can inhibit PP1c in smooth muscle. PHI-2, widely distributed, can inhibit PP1 holoenzymes

(58). I-1 PP2A (PHAP1, Potent heat-stable protein phosphatase 2A inhibitor I1PP2A) and I-2 PP2A (SET, PHAPII, TAF1b), also widely distributed, can inhibit PP2A but can

stimulate PP1c by modifying PP1 substrate specificity (59). G-substrate (cGMP-

dependent protein kinase substrate) inhibits PP1c in brain (60). Grp78 (78-kDa Glucose-

Regulated Protein/BiP, immunoglobulin heavy chain-binding protein), stress inducible and widely distributed, is a molecular chaperone member of HSP-70 family and can bind to PP1γ2 in testis (61). Some activity modulators also function as a substrate for PP1c.

For example, the retinoblastoma protein interacts with PP1c both as a substrate and as a non-competitive inhibitor of PP1c (62).

2-2) Natural toxins can also function as protein phosphatase inhibitors (63-67), e.g.,

fatty acid polyether-like compounds, okadaic acid, calyculin A and tautomycin. Okadaic

acid accumulates in certain sponges and shellfish, and can cause diarrhetic poisoning.

Okadaic acid has a greater inhibitory potency for PP2A than for PP1. Calyculin A, an

octamethyl polyhydroxylated C28 fatty acid isolated from marine sponges, exhibits

similar cytotoxic and tumor promoting activities as okadaic acid. Tautomycin, a

polyketide toxin from a soil bacterium, has also been identified as a potent phosphatase

inhibitor. Another group of toxins produced by cyanobacteria include microcystins (a

cyclic heptapeptide toxin) (68) and nodularin (a pentapeptide toxin) that inhibits PP1 at

nanomolar concentrations (69). These toxins can cause both acute and chronic hazards to

human and animals.

3) Miscellaneous

These are proteins which can bind to PP1 - they are either enzymes themselves or are

protein not yet classified. For example, PP1bp80 was found to be PP1 binding protein by mixed peptide sequencing and database searching and was proposed to regulate

chaperones in skeletal muscle (70); MYPT3, widely distributed in microsomes and

cytosol, is a prenylatable myosin targeting subunit of protein phosphatase 1 (71, 71);

SARA (Smad Anchor for Receptor Activation), an anchor protein in TGF-beta signaling,

can bind to PP1 resulting in negative regulation of Dpp signaling in Drosophila

melanogaster(72); Aurora-B itself is a mitosis regulatory kinase and can bind to protein

phosphatase that acts as a negative regulator of kinase activation(73).

4) Protein phosphatase binding partners/subunits in yeast

The homologue of PP1 in yeast is Glc7. There are also homologues of PP1-binding

proteins in yeast. For example, REG1 (Islet of Langerhans regenerating protein) (74) and

REG2 (75) can bind to protein phosphatase and be involved in glucose metabolism in yeast. Similar to mammalian Nek2, the yeast protein REG1 is a substrate for the associated PP1c, and REG1 also transiently associates with the Snf1 protein kinase and with hexokinase II, thereby enabling dephosphorylation of these two proteins by PP1c

(76, 77); Gac1p/GAC1 are yeast homolog of mammalian GM subunit of PP1; GIP1,

GIP2 and GIP2h/YIL045w (Hypothetical protein) are also homologs of mammalian GM subunit of PP1 in yeast; SCD5 (also YFL023W and YAL014) is a suppressor of clathrin deficiency and a novel protein with a late secretory function in yeast; Yeast Sds22 is the homologue of mammalian Sds22 and required for the completion of mitosis, at least in part because of its involvement in the nuclear distribution of PP1; Ypi1 is a homologue of

inhibitor 3 in yeast (78); Glc8 is the homologue of inhibitor 2 in yeast(79).

1e. Binding of regulatory subunits and catalytic subunits:

1) PP1-binding motifs present in regulatory subunits:

Site directed mutagenesis and X-ray crystallographic studies were used to study the

structure of the PP1 holoenzyme formation (21), which revealed that PP1c establishes multiple contacts with each of its regulatory subunit, and that some interaction sites on

PP1c can be shared by different regulatory subunits. Most but not all of the regulatory

subunits contain the primary PP1-binding motif, RVxF, (R/K/H/N/S)x1(V/I/LX)x2 (F/W/

Y), where x1 may be absent or may be any residue except those with large hydrophobic

residues and x2 is any amino acid except those with large hydrophobic residues,

phosphoserine and probably aspartic acid (12, 80). In addition to RVxF sequence,

multiple PP1-binding sites have been mapped for regulatory subunits. These additional

binding sites usually comprise short protein fragments (4-20 residues). Although the

binding sites for PP1 can be distributed over large parts of the regulatory subunit, often

one or several binding sites are clustered around the RVxF sequence. One of these

additional sequences is F-X-X-R/K-X-R/K contributing to the cooperative association

between PP1c and its regulators.

RVxF motif can function as initial contact for PP1 which enables the regulatory

subunits to make additional contacts with the phosphatase in an ordered and cooperative

manner (14, 16). For example, four phosphatase-interaction sites, in addition to the RVxF

motif, have been identified for MYPT1 and inhibitor-2 (25, 28, 30). These additional

contacts, though weak, ensure a tight association of the regulatory subunits with PP1.

One consequence of this co-operation between interaction sites is that disruption of only

a single phosphatase-binding motif, such as the RVxF sequence, may be sufficient to

weaken or even destroy the interaction of the regulatory subunits with PP1. That is, the disruption of the RVxF-mediated interaction with PP1 might affect the activity and substrate specificity of the holoenzyme, even though the binding of the RVxF by itself to

PP1 does not cause significant conformational changes of the catalytic subunit nor does it have major effects on the activity of the phosphatase. For example, mutation of the hydrophobic (V/I/L) and/or aromatic (F/W/Y) residue in (R/K/H/N/S)x1(V/I/LX)x2 (F/W

/Y) motif on various regulatory subunits is sufficient to disrupt or weaken their interaction with the catalytic subunit of PP1 (25, 44, 77, 81).

The primary binding motif contained in some regulatory subunits is not RVxF, such as inhibitor 2 which binds to PP1c through its KLHY motif (the variant RVxF motif), while Sds22 binds PP1c by its leucine rich repeat sequences. Different regulatory subunits containing the same PP1c-binding motif will compete with each other and exhibit a mutually exclusive binding interaction (14, 17, 21).

In summary, binding of regulatory subunits to PP1c has some/all of the following features: 1) One primary binding motif which is short (4-6 residues) and degenerate sequence is generally used by each regulatory subunit to bind to PP1c; 2) Additional weak interaction sites ensure a tight association between most regulatory subunits and

PP1c and may decide the activity and substrate specificity of the holoenzyme; 3)

Regulatory subunits can share PP1 interaction sites but those with the same binding motif will compete with each other for PP1 binding (16).

2) PP1 sequences for regulatory subunit binding:

As mentioned earlier, the catalytic subunit of protein phosphatase is highly conserved,

especially its catalytic core sequence. For example, the amino acid residues 41-269 which

is the catalytic core sequence for mammalian PP1α’s is conserved in all PP1 isoforms

and exhibits a high similarity with the catalytic core sequence of PP2A and PP2B’s

catalytic subunits (11, 82). A truncated protein containing the catalytic core of PP1α

composed of 228 amino acid residues, compared with the entire 330 residues of PP1α,

has a broader substrate specificity but a dramatically reduced sensitivity to inhibitory toxins and protein regulators (54). So, the C- and N-terminus of the catalytic subunit of

PP1 appear to play a more important role in restricting PP1’s substrate specificity and are more important for regulatory subunit binding. Amino acid residues 270–296 at C- terminus of PP1 catalytic subunit contain at least two binding sites for regulatory subunits

(54, 54).

The three grooves on PP1 noted earlier, a hydrophobic groove, an acidic groove and

a C-terminal groove provide potential binding sites for regulators and/or substrates (11,

12). The major area of PP1 that interacts with regulatory subunits appears to be the

hydrophobic groove and neighboring negatively charged region (14). The hydrophobic

groove near the C-terminus of PP1 is for RVxF motif binding, and the groove mainly

interact with valine and phenylalanine residues in RVxF motif (11). This hydrophobic

groove is remote from the catalytic subunit (12). PP1 sequences necessary for RVxF-

binding (especially residues 287-293) are well conserved in all isoforms in all species.

The variant RVxF motif of inhibitor-2 (KLHY) can be accommodated in a similar way in

this channel, a site lying adjacent to the RVxF-binding groove (25, 83). The RVxF

binding pocket is surrounded by acidic residues that might provide additional binding sites for the basic residues often found N-terminal to the RVxF motif. On the other hand, various regulatory subunits that bind to PP1 do not seem to possess RVxF motifs, while they appear to interact with RVxF-binding hydrophobic groove on PP1.

The catalytic site and the β12/β13 loop are other surface areas of PP1 which can interact with regulatory subunits. The β12/β13 loop connecting β-sheet 2 with β-sheet 1 protrudes from the surface of PP1 at the edge of the tungstate-binding site (11, 12). The catalytic site and the β12/β13 loop are used in toxin inhibition of PP1. Toxins bound to the β12/β13 loop may inhibit PP1 activity by blocking substrate access to the catalytic site. Additionally, toxin binding may disrupt the loop conformation and induce a shift in

Tyr272, perturbing its potential interaction with metal ions (11, 12, 16, 21). The phosphorylated residues at or near the catalytic site are used for such regulatory subunits as phosphorylated inhibitor 1 (84), DARPP32 (85) and MYPT1 (28) to bind as pseudo- substrates for PP1. Additionally, the integrity of β12–β13 loop (residues 267–279 of the mammalian PP1α isoform), a common binding site for various regulatory subunits is needed for the inhibition of PP1 by some inhibitors (54). In addition to the catalytic site and the β12/β13 loop, the triangular region delineated by the α4/α5/α6-helices of PP1 have been identified as a major interaction site for Sds22 (17).

1f. Regulation of PP1 activity

Reversible phosphorylation, allosteric regulation of the regulatory subunits, and targeting of the catalytic subunits are the biochemical mechanisms to regulate the holoenzyme activity. (1) Reversible phosphorylation of regulatory subunits: for the

regulatory subunits with RVxF motif, phosphorylation of Ser residue(s) within or close to the RVxF motif can disrupt the binding of RVxF to PP1c (31, 81, 86), resulting in an altered activity of the holoenzyme or a release of the catalytic subunit. By contrast, for other regulatory subunits, phosphorylation strengthens their interaction with PP1c by creating an additional binding site for PP1, e.g. inhibitor-1 and DARPP-32. (2) Allosteric regulation of regulators: For example, the allosteric binding of phosphorylase a to the C- terminal tail of the liver-type G subunit (GL) abolishes the activity of the associated PP1c towards glycogen synthase. (3) Targeting of PP1 holoenzymes to specific substrates or subcellular structures: The targeting of PP1 can sometimes be controlled independently of its subunit interactions. For example, the binding of MYPT1 to myosin is controlled by phosphorylation of MYPT1, which results in the binding or dissociation of PP1 with its specific substrate, myosin (87).

2. Roles of PP1 in Spermatozoa

The presence of serine/threonine phosphatase in testis, and thereby implication in mammalian spermatozoa, was first reported in 1975 (88). The protein phosphatase was proposed to be involved in rat spermatogenesis (89) due to its high level of expression and the postnatal developmental increase in its levels (90). More recently our laboratory showed PP1γ2 is the predominant isoform of Ser/Thr protein phosphatase in testis and the only detectable isoform of PP1 in spermatozoa (91). There are three PP1c gene encoding four isoforms of protein phosphatase catalytic subunit: PP1α, PP1β (also termed PP1δ),

PP1γ1 and PP1γ2 (92, 93). PP1γ1 and PP1γ2, differing only at their extreme C-termini,

are alternate splice products of the same gene. PP1α, PP1β and PP1γ1 are ubiquitously

expressed but PP1γ2 is weakly expressed in brain but predominant in testis (91). It is

notable that the genomes of species other than mammals, including birds, amphibians,

reptiles, and invertebrates, do not contain a PP1 orthologue resembling PP1γ2. Sperm from these species contain a protein phosphatase 1 catalytic subunit resembling mammalian PP1α, PP1β, or PP1γ1. Thus, PP1γ2 may be vital for sperm formation and function in mammals.

The intracellular regulators such as cAMP, calcium and pH have been shown to

mediate sperm motility by changing sperm protein phosphorylation (94). Since protein

phosphorylation is a balance between protein kinases and phosphatases, it was long suspected that protein phosphatases would be involved in regulation of sperm motility. A number of studies in our lab have shown that PP1γ2 plays a key role in sperm motility.

We confirmed the critical role of PP1γ2 in sperm motility by showing that inhibition of

PP1γ2 activity in caudal sperm is tightly linked to the onset of progressive motility, and to a significant increase in vigorous movement in already progressively motile sperm (91,

95, 96). A decline in PP1γ2 activity occurs during epididymal sperm maturation not due

to a decline in amounts of the enzyme but due to lowering of its catalytic activity. We

found that PP1 inhibitors, okadaic acid (OA) and calyculin A (CA) both initiate and

stimulate motility of epididymal spermatozoa. Other laboratories have shown that these

phosphatase inhibitors also promote hyperactivated sperm motility and acrosome reaction

(97, 98). The enzyme PP1γ2 is present in spermatozoa of a wide range of mammalian

species including humans and non-human primates (91, 95, 99). Other isoforms of PP1

are involved in regulating flagellar activity in rooster and Chlamidomonas (100, 101).

PP1γ2 is also implicated to play a key role in spermatogenesis and sperm morphogenesis. Our previous work has shown that in mouse testis, PP1γ2 is found to be

predominately located in the cytoplasm of secondary spermatocytes and round spermatids

as well as in elongating spermatids and testicular and epididymal spermatozoa, (102).

PP1γ gene knockout in mice provided evidence that in addition to its previously

documented role in motility, PP1γ2 may also play a key role in spermatogenesis and

sperm morphogenesis. Varmuza et al. showed (96) that, in PP1γ gene knockout mice,

spermiogenesis is arrested, the structure of seminiferous tubules is disrupted, and that

meiosis is disturbed with the presence of abnormal polyploid spermatids, resulting in

male infertility. An interaction between spermatogenic zip protein (Spz1), a transcription

factor, and PP1γ2 was identified and this protein was proposed to underlie the

biochemical mechanism for the involvement of PP1γ2 in the regulation of

spermatogenesis (102). Our studies showed that PP1γ2 is involved in sperm tail

morphogenesis (103). We found that sperm in Ppp1cc-null mice had malformed tails,

missing mitochondrial sheaths and disorganized outer dense fibers on the sperm tail while

sperm head showed a range of shapes, from round to oblong. Even though PP1α levels

appear to be upregulated, it does not compensate for the loss of PP1γ in testis unlike other

tissues and in female mice where there is no apparent phenotype due to the absence of

PP1γ isoforms. In summary, PP1γ2 has key roles in spermatogenesis, sperm morpho-

genesis and sperm motility in mammals.

3. PP1γ2 binding proteins in testis/sperm

To better understand the role of PP1γ2 in sperm motility/morphogenesis, various studies were carried out to identify the binding partners of PP1γ2 in testis/spermatozoa.

Sds22/PPP1R7:

Sds22 was originally identified in yeast as a positive regulator of protein phosphatase-

1, required for the mitotic metaphase/anaphase transition (104). This protein is a prototypic member of a protein family containing leucine-rich repeats (LRR). Both mammalian and yeast Sds22 contain 11-22 amino acid LRRs (104-106). Sds22 has been shown to enhance PP1 activity in yeast. However, based on a study in cultured mammalian cells, there was a partial inhibitory effect by a synthetic polypeptide corresponding to the Sds22 sixth LRR repeat on recombinant catalytic subunit of PP1

(107). The Sds22-PP1γ2 complex in spermatozoa was catalytically inactive (108) against the substrate phosphorylase a, a situation analogous to Sds22-bound yeast PP1 (109). The protein Sds22 is presently believed to be an inhibitor of PP1 in mammals. However, other components in the complex containing Sds22 and PP1γ2 were not identified.

14-3-3:

Protein 14-3-3 isoforms are highly conserved acidic proteins expressed in a variety of eukaryotic cells. 14-3-3 proteins are involved in various cellular processes, such as cell cycle progression, apoptosis, protein trafficking, cytoskeleton rearrangements, metabolism and transcriptional regulation of gene expression (110-112). The effect of 14-

3-3 binding depends on the nature of its ligand. Binding might activate or inhibit the enzyme activity or change the localization and phosphorylation status of proteins (113).

More than one hundred 14-3-3 binding partners have been identified in somatic cells through affinity chromatography coupled with proteomic analysis (114-117). Our lab first documented the expression of 14-3-3 in mature spermatozoa and showed that it is bound to a distinct pool of PP1γ2 in spermatozoa (118). It appears that PP1γ2 bound to 14-3-3 is phosphorylated. The physiological significance of this binding is still unknown. It is possible that 14-3-3 regulates PP1γ2 catalytic activity, phosphorylation or its interaction with other proteins. There is also evidence that, in addition to PP1γ2, at least three other

14-3-3 binding phosphoproteins exist. The identities of these proteins are still unknown

(118). Studies are underway to identify these other 14-3-3 binding phosphoproteins and the biological relevance of their binding to PP1γ2. Protein 14-3-3 is present in spermatozoa isolated from species as diverse as Xenopus, turkey, mouse, bull and man, suggesting an essential role for this protein in male gamete function.

Inhibitor 2 (I2)/PPP1R2:

Previous work from our lab was also focused on identifying heat-stable inhibitors of

PP1γ2. The first candidate protein examined was the ubiquitously expressed PKA- regulated inhibitor 1 (I1). It has been well established that I1 is inhibitory to PP1 when it is phosphorylated by PKA. The established role of PKA in sperm function made I1 a logical candidate for PP1γ2 regulation. Surprisingly, I1 activity was undetectable in bovine sperm extracts; however, substantial activity resembling inhibitor 2 (I2) was

present in heat-stable sperm extracts (95, 119). This activity was thought to be I2-like

because the inhibition of PP1 activity could be reversed by glycogen synthase kinase-3

(GSK-3). The investigation of I2 in spermatozoa is one of the aims in this dissertation.

4. Inhibitor 3 (I3)/PPP1R3

One of the main goals of this dissertation is to verify the presence of I3 in testis/sperm

and study its regulation of PP1γ2 activity in testis/sperm.

Wild-type (wt-) inhibitor 3

A potent heat-stable PP1 inhibitor was identified through yeast two-hybrid studies designed to identify PP1-binding proteins from human brain (57). This protein is

identical to the protein product of human hemochromatosis candidate gene HCG V,

orthologous to the mouse Ppp1r11, also called Tctex5. The gene, Ppp1r11, in mouse is

localized to the t complex, a naturally occurring polymorphism of the proximal third of

chromosome 17 and represented by a family of closely related t haplotypes that carry

similar mutant alleles of genes implicated in sperm function (120). The heavily mutated t-

allele of Ppp1r11 is thought to code for one of three tightly linked t haplotype proteins

whose expression in sperm from t/t males coincides with a flagellar waveform phenotype,

“curlicue”, which is strongly associated with the sterility of these males (121, 122, 122,

123). The protein product of HCG V/Tctex5 is a protein phosphatase 1 regulatory subunit

11 (PPP1R11, TCTEX5, inhibitor 3/I-3). Like protein phosphatase inhibitor 1 and

inhibitor 2, inhibitor 3 is hydrophilic, heat stable, behaves anomalously on SDS-PAGE

and is a specific PP1 inhibitor (57). Additionally, inhibitor 3 is extremely sensitive

to protease activity.

A naturally occurring mutant isoform of inhibitor 3

The wild type (+) and t haplotype (t) are the two types of the proximal one third region of mouse chromosome 17 existing in nature (124). A family member of the mouse t complex (the familial ancestor of modern-day t haplotypes) was first discovered about 75 years ago. All t haplotypes are closely-related, naturally occurring polymorphisms of the t complex region, exhibiting four inversions spanning the region relative to the wild-type homolog. By a nearly complete suppression of recombination, the integrity of a complete t haplotype, about 40 million base-pairs, is maintained along its 15-centimorgan (cM) length from the D17Leh48 locus to the H-2 complex from one generation to the next

((125, 126), resulting in a transmission ratio distortion (TRD), clearly against Mendel’s first law. At least five independent loci are involved in TRD demonstrated by genetic experiments (127, 128). Generally, only t haplotypes with a complete set of TRD loci are transmitted at high ratios, and only high-ratio t haplotypes survive for significant periods of time in a natural population (7). t haplotypes carry genes that either cause homozygous male sterility or embryonic lethal mutations (129, 130). As a result of TRD, numerous mutations have accumulated and become fixed in t haplotypes. With the precise mapping of mouse genome and manipulation of implicated genes for sperm motility, the mouse t haplotype affecting only male fertility has been used as a naturally-occurring model to study the regulation of sperm motility.

~40-MBP

+ FERTILE t STERILE t

In[17]1 CurlicueIn[17]2 In[17]3 In[17]4

Fig. 1. The + and t homologs of the mouse t complex region: (Top) The t complex is a 40-Mb pair region of mouse chromosome 17 proximal to the centromere (round ovals to the left). There are two forms of the t complex in wild mouse populations: the wild-type (+), and the naturally occurring mutant, the t haplotype (t). Blue and red arrows represent relative inversions (In[17]1-In[17]4) between the +- and t-regions; black arrowheads represent uninverted regions between inversions, allowing for production of rare recombinant homologs (partial t haplotypes) by +/t heterozygotes. (Bottom) +/+ and +/t males are fertile, but male mice carrying two t haplotypes (t/t) are sterile due to poor sperm motility observed as the “curlicue” phenotype (shown by scanning electron microscopy). “Curlicue” includes radically premature hyperactivation, sudden loss of vigorous progressive motility, and chronic negative curvature of the flagellum.

Spermatozoa from t/t males remain normal ultrastructure (131), but exhibit very poor motility, thus rendering them unable to reach the site of fertilization in vivo (121).

Evidence shows that these sperm do not activate progressive motility, but instead hyperactivate within minutes of their release from the caudae epididymides into complete

IVF (in vitro fertilization) medium. Subsequently and nearly as quickly, they exhibit chronic negative bending of the entire flagellum, and a significant loss of measurable flagellar beat frequency (121, 122). The chronic negative bend of the entire flagellum, including the principal piece, and loss of vigorous beat are reminiscent of a highly exaggerated form of calcium-induced flagellar arrest, where severe negative flagellar bending occurs normally in the midpiece (132, 133). The entirety of this complex phenotype is known as “curlicue” (121).

Until recently it had been difficult to isolate the genes responsible for t-associated motility aberrations, due to recombination suppression. However, several genetic “tricks” became available in the early 1990s allowing the circumvention of recombination suppression (134, 135), so that several candidate genes potentially responsible for the abnormal motility exhibited by sperm produced by t haplotype homozygous males were subsequently mapped to a high resolution in the t-complex In(17)4 region (136-139).

These included two categories of mutant genetic elements, those that produce abnormal sperm flagellar curvature primarily in the principal piece, and those that negatively impact the vigorous nature of a wild-type flagellar beat.

Three strong candidates for curvature abnormalities include Dnahc8, a gene coding for an axonemal outer arm γ dynein heavy chain exhibiting testis-restricted expression,

and confinement to the principal piece of the sperm flagellum, whose t-allele harbors 17

missense mutations (133, 140); Tsga2, a gene whose protein products may be molecular

adaptors in signaling complexes of the periaxonemal ODF and FS (139); and Btbd9, a

gene defined by its proximity to Dnahc8 and its mutant mRNA expression pattern in t/t

testis. A strong candidate for the loss of vigorous motility component of “curlicue” located in In(17)4 is Tctex5/Ppp1r11, one of the most ancient inhibitory subunits of PP1.

The t-alleles of Dnahc8, Tsga2, and Tctex5 also contain numerous nonsynonymous

mutations relative to wild-type orthologs, increasing the potential strength of each as a

candidate for “curlicue” worthy of further scrutiny. One of the goals of this dissertation is

to study wt-I3 and its mutant form t/t-I3.

MATERIALS AND METHODS

Protein Extract Preparation:

All tissues examined were homogenized in Buffer B (10 mM Tris, pH 7.2, 1 mM

EDTA, 1 mM EGTA, 10 mM benzamidine-HCl, 1 mM PMSF, 0.01 mM N-tosyl-L- phenylalanine chloromethyl ketone [TPCK], and 0.1% [V/V] β-mercaptoethanol). The homogenates were centrifuged at 16,000×g to remove insoluble material. To make heat- stable extracts, crude protein extracts were boiled for 10 min, chilled on ice for 1 min, and following centrifugation at 16,000×g for 15 min, the supernatants were collected.

Western Blot Analysis:

Protein samples were boiled in Laemmli sample buffer and separated by 12% SDS-

PAGE, then electrophoretically transferred to Immobilon-P, PVDF membranes

(Millipore Corp., Billerica, MA, USA). After blocking non-specific binding sites with 5% nonfat dry milk in Tween-Tris buffered saline (TTBS: 0.1% Tween-20 in 25 mM Tris-

HCl, pH 7.4, 150 mM NaCl), blots were incubated with primary antibody overnight at

4ºC. All primary antibodies were commercially prepared. Anti-PP1γ2 (Zymed

Laboratories, San Francisco, CA, USA) was raised against a peptide corresponding to the

unique carboxyl terminus of PP1γ2 (amino acid residues 315 to 337,

VGSGLNPSIQKASNYRNNTVLYE). Anti-Sds22 (Affinity Bio Reagents, Golden, CO,

USA) was raised against the 70th to 84th and the 347th to 360th amino acid residue,

ETINLDRDAEDVDLC and LPSVRQIDATYVRF. Anti-TCTEX5 is a custom antibody

28 29

raised against the sequence, EKPRAFGESSTESDE, of TCTEX5 (mouse homologue of

I3). Though the anti-TCTEX5 recognizes both wt-I3 and t/t-I3 proteins, it cross-reacts

strongly with an I3-unrelated protein on western blots, we then made the following two

antibodies against wt-I3 and t/t-I3, respectively. The rabbit polyclonal anti-wt-I3

(Affinity BioReagents, Golden, CO, USA) was raised against the synthetic peptide

containing the 1st to 16th and the 27th to 41st amino acid residue, MAETGAGISETVTETT

and EPENQSLTMKLRKRK. The rabbit polyclonal anti-t/t-I3 (Affinity BioReagents,

Golden, CO, USA) was raised against the synthetic peptide containing the 1st to 15h and the 22th to 35th amino acid residue, MAEAKAEMNETITET and QPENQKITIKLRKP.

Antibodies were affinity purified with the synthetic peptides conjugated to a sulfo-link column (Pierce, Rockford, IL, USA). Anti-actin was purchased from MP Biomedical,

OH, USA. Primary antibody dilution was 1:1000~1:4000. After washing, blots were incubated with anti-rabbit or anti-mouse secondary antibody (1:2000 dilution) conjugated

to horseradish peroxidase (GE Healthcare, Piscataway, NJ, USA) for 1 h at room

temperature. Blots were washed with TTBS twice for 15 min each and four times for 5 min each, and then developed with a homemade ECL chemiluminescence kit.

Expression of recombinant wt-I3 and t/t-I3 in the pGEX vector and pRSET vector:

The full length of mouse wt-I3 and t/t-I3 cDNA was inserted into the pGEX vector,

4T-2 (GE Healthcare, Piscataway, NJ, USA) and expressed it as a glutathione S-

transferase (GST) fusion protein in Escherichia coli. The recombinant protein was

subsequently purified on a Glutathione Sepharose 4B affinity column. wt-I3 or t/t-I3 gene

digested from the pGEX vector was ligated into pRSET A vector (Invitrogen, Carlsbad,

California, USA) at BamH1/EcoRI sites, expressed as a 6xHis-tagged recombinant protein according to manufacturer’s suggestion, and purified using Ni-NTA agarose from

QIAGEN (Valencia, CA, USA).

GST Pull-Down Assay:

Glutathione Sepharose 4B beads (GE Healthcare, Piscataway, NJ, USA) bound to

GST-wt-I3, GST-t/t-I3 or GST alone (as a negative control) were incubated with testis,

caudal or caput sperm extracts with rocking for 2 h at 4ºC. GST tag alone incubated with protein extracts and/or GST-I3 protein incubated with buffer were used as a negative control. After washing, the proteins were eluted with 20mM reduced glutathione, 50mM

Tris, pH 8.0. Western blot analysis was performed on the extracts used for pull-down assay and the eluted fractions to identify bound proteins.

Immunoprecipitation:

Crude lysates or purified FPLC fractions of tissue protein extracts were incubated for

1 h at 4ºC with ~5μg of either anti-PP1γ2, anti-Sds22, anti-wt-I3, anti-t/t-I3, anti-actin antibody or diluted rabbit pre-immune serum as a negative control. Protein G-Sepharose

4 Fast Flow beads (GE Healthcare, Piscataway, NJ, USA) were washed once with distilled water and twice with TTBS. Each extract/antibody solution was incubated with the beads by rocking for 1 h at 4°C. After incubation, the beads were washed once with

TTBS and five times with Buffer B (10mM Tris-HCl, pH 7.0, 1mM EDTA, 1mM EGTA,

10mM Benzamidine-HCl, 1mM PMSF, 0.1mM N-tosyl-L-phenylalanine chloromethyl ketone [TPCK], 0.1% [V/V] b-mecaptoethanol). After washing, the beads were resuspended in 2xSDS reducing sample buffer (6%SDS, 25mM Tris-HCl pH 6.5, 50mM

DTT, 10% glycerol and Bromphenol blue), boiled for 10 min and centrifuged at

10,000×g for 10 min. The supernatant was separated by SDS-PAGE, followed by western blot analysis.

Column Chromatography:

All column procedures were conducted at room temperature following protocols

from the manufacturers (GE Healthcare, Piscataway, NJ, USA) on an AKTA FPLC

chromatographic system (GE Healthcare, Piscataway, NJ, USA). Tissue extracts were

first passed through two anion-exchange columns pre-equilibrated with 20mM Tris-HCl,

pH 8.0: a HiTrap DEAE FF (1ml) column and a Mono Q 5/50 GL (1ml) column, with

proteins eluting in a linear gradient of 0–1M NaCl in 20mM Tris-HCl, pH 8.0. Pooled

gradient fractions containing the co-eluted proteins, PP1γ2, Sds22 and I3, from the

MonoQ column were passed through the MonoQ column again and proteins were eluted

in a linear gradient of 250-550mM NaCl in 20mM Tris-HCl, pH 8.0. Subsequentially, the

fractions containing the co-eluted proteins were combined and passed through a HiTrap

Blue HP column pre-equilibrated with 50mM NaH2PO4, pH 7.0. Proteins were eluted in a

linear gradient of 0-1.5M NaCl in 50mM NaH2PO4, pH 7.0. The final step of purification

involved passing the co-eluting fractions through a size-exclusion column, a Superose 6

10/300 GL column pre-equilibrated with 20mM Tris-HCl, pH 8.0, 1mM EDTA and/or a

Superdex 200 10/300 GL column pre-equilibrated with 20mM sodium phosphate,

150mM NaCl, pH 7.0. The same approach was employed to isolate I3/PP1/Sds22-

containing fractions from mouse brain and PP1γ knockout mouse testis. Purified fractions were analyzed either by western blotting with anti-I3, anti-PP1γ2 and anti-Sds22, by

immunoprecipitation, or concentrated with Centricon-10 filters (Millipore Corp.,

Bedford, MA, USA) for native PAGE analysis.

To estimate the molecular weight of the protein fraction containing co-eluted

PP1/Sds22/I3, a molecular weight calibration curve for Superdex 200 column was

prepared from the elution profile of protein standards (Catalase [240-kDa], Aldolase

[158-kDa], Albumin [68-kDa] and Ovalbumin [43-kDa]) according to directions

provided in HMW and LMW calibration kits (GE Healthcare, Piscataway, NJ, USA).

Native PAGE:

Concentrated chromatography fractions containing all the three proteins (PP1γ2,

Sds22, and wt-I3/t/t-I3) were made 1X by the addition of 5X sample buffer (31% [V/V]

1M Tris-HCl pH 6.8, 5% [V/V] 1% bromophenol blue solution, 50% [V/V] glycerol) and separated by electrophoresis through a 10% Tris-HCl Ready Gel (Bio-Rad Laboratories,

Hercules, CA, USA) at ~20-25mA for about three hours followed by western blot

analysis with anti-wt-I3, anti-t/t-I3, anti-Sds22, anti-PP1γ2, or anti-E9 antibodies.

Phosphatase Assay:

Phosphatase activity was assayed using a 32P-labeled glycogen phosphorylase a

(Gibco-BRI) substrate, as previously reported (95). Briefly, purified fractions of caudal

sperm extracts by size-exclusion columns were preincubated in a buffer containing 1mM

Mn2+ in a total volume of 30μl at 30ºC for 5 min, or a serial dilution of 5 to 450nM of

His-wt-I3 or His-t/t-I3 was preincubated with 2ng His-PP1γ2 in a buffer containing 1mM

Mn2+ in a total volume of 30μl at 30ºC for 5 min, followed by addition of labeled

phosphorylase a to initiate the dephosphorylation reaction. At the end of a 10-minute

incubation, the reaction was terminated with 95μl of cold 20% trichloroacetic acid

(TCA), after which the reaction tubes were centrifuged for 5 min at 12,000×g at 4ºC.

32 PO4 counts released from phosphorylase a into the supernatants were quantitated. One

unit of enzyme activity is defined as the amount of enzyme that catalyzes the release of

32 1nmol of PO4 /min.

Immnohisochemistry of mouse testis:

Testes were fixed by immersion in 4% paraformaldehyde in PBS at 4°C for 40h.

The fixed testes were transferred to 75% Ethanol and dehydrated, permeabilized, and

embedded in paraffin using a Shandon Tissue Processor (Thermo Electron Corp.,

Waltham, MA, USA). Multiple 8μm-thick sections of the whole testis were attached to poly-L-lysine-coated slides, deparaffinized, and rehydrated using a standard procedure.

Antigen retrieval was performed using 1×Antigen Retrieval Citra Solution (BioGenex,

San Ramon, CA, USA). Sections immersed in Citra solution were microwaved for three

separate 3-min periods on high setting, with a cooling period of 1min between each

heating cycle. Slides were incubated for 1h at room temperature in a blocking solution

containing 10% normal goat serum in PBS, then incubated with primary antibodies for 2h

at room temperature or overnight at 4°C. After this, slides were washed three times with

PBS, and incubated with corresponding secondary antibody (1:250) conjugated to

indocarbocyanine (Cy3; Jackson Laboratories, West, Grove, PA) for 1h at room

temperature. The slides were washed five times with PBS, mounted with Vectashield

(Vector Laboratories, Burlingame, CA, USA) mounting media, and examined using a

Fluo View 500 Confocal Fluorescence Microscope (Olympus, Melville, NY, USA).

Control slides were processed in the same manner except that the primary antibody

incubation was omitted. Cell nuclei were stained with TO-PRO®-3 iodide (invitrogen,

Carlsbad, California) (1:500) for 20 mins.

In vitro phosphorylation:

0.5-5μg of either GST-t/t-I3 or GST-wt-I3 were incubated with sperm extracts for 30 min in the presence of 32P-ATP followed by quantification of label uptake by TCA

precipitation and separation by SDS-PAGE. Gels were dried, exposed to film, and

developed.

Microsequencing:

Microsequencing of the protein band of interest was conducted in the Proteomics lab at Cleveland Clinic, Cleveland, Ohio. Briefly, for the protein digestion, the bands were cut from the gel as closely as possible, with either a scalpel or a punch, and washed/destained in 50% ethanol, 5% acetic acid. The gel pieces were then reduced with

DTT and alkylated with iodoacetamide before digestion with trypsin overnight. The peptides that were formed were extracted from the polyacrylamide and the extract evaporated to <30 μL for LC-MS analysis. The LC-MS system was a ThermoFisher LTQ

ion trap mass spectrometer system. The HPLC column was a self-packed 8 cm x 75 μm

id Phenomenex Jupiter C18 reversed-phase capillary chromatography column. Two μL

volumes of the extract were injected and the peptides eluted from the column by an acetonitrile/0.05 M acetic acid gradient at a flow rate of 0.2 μL/min were introduced into the source of the mass spectrometer on-line. The microelectrospray ion source is operated

at 2.5 kV. The digest was analyzed using the data dependent multitask capability of the

instrument acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in successive instrument scans.

This mode of analysis produces approximately 2500 collisionally induced dissociation

(CID) spectra of ions ranging in abundance over several orders of magnitude. The data were analyzed by using all CID spectra collected in the experiment to search the NCBI databases, mouse and rat refseq databases, with the search program Mascot. Each identification is verified by manual inspection of several matching spectra. The interpretation process was aided by additional searches using the programs Sequest as needed. The results are summarized in Table 1.

Northern blot:

Total RNA was prepared from various sources using RNeasy Midi kit from

QIAGEN (Valencia, CA, USA). Mixed with RNA sample loading buffer (SIGMA, MI,

USA) in a ratio 2:1 to 5:1 followed by heated to 65℃ for 10mins and then chilled on ice,

RNA samples were electrophoresed on 1.3% formaldehyde-agarose gel, 125V for 3h, after which, the gel picture was taken with fluorescent ruler aside. The gel was then undergoing denaturation in 500ml of 0.05N NaOH for 20min followed by Neutralization in 500ml of 0.1M Tris-HCl (pH 7.5) and 0.15M NaCl for 2x15min, and then rinsed in

10xSSC for 20min. The gel was transferred to a Hybond-XL nitrocellulose membrane

(Amersham, USA) in ~3L 10 x SSC overnight. On the second day, the well positions were marked. Cross-link membrane (RNA-side up) on a piece of whatman paper in

Stratalinker using auto-crosslinking setting, or baked in an oven at 80℃ for 2h. Cut edge and label membrane with black sharpie. The membrane was washed briefly in 2xSSC

(100ml 10xSSC + 400ml DEPC water) for 30sec on a shaker. The membrane was prehybridized in 8ml Church buffer [13.4g Sodium Phosphate (7H2O) (0.5M), 50ml ddH2O, adjust pH to 7.2 with phosphoric acid, added with 200ul 500mM EDTA, then readjusted pH to 7.2, 7g SDS (7%), add ddH2O to 100ml] for at least 1 h in 65℃ rotisserie incubator, before hybridizing overnight with appropriate labeled fragments of cDNA (random primed) in 8ml fresh Church buffer in 65℃ rotisserie incubator overnight. Washes were performed 3-4 times for 5 min each at room temperature in

2xSSC/0.1%SDS, shaking at ~50RPM, followed by 5-10 min each at 65-68℃ in 0.1x

SSC/0.1%SDS. The washed blot was placed on bed of paper towels to blot off excess moisture wrapped in Saran Wrap. Expose to Kodak Biomak MS film in cassette with MS

Intensifying screen for overnight at -80℃, warm at 37℃ for 15min, develop film.

Microcystin-agarose chromatography:

Microcystin-agarose (MC-agarose) (Upstate Biotechnology, Lake Placid, NY) was washed twice with HB buffer supplemented with 5% BSA to prevent nonspecific binding. Protein extracts of sperm or testis were incubated with MC-agarose with shaking at 4℃ for 2 h. The pellet was washed five times with HB buffer. Proteins bound to MC- agarose were analyzed by western blot following boiling of the pellet with 6xSDS sample buffer.

RESULTS

A. Characterization of the protein phosphatase inhibitor PPP1R11/inhibitor 3 (I3) in testis and spermatozoa.

1. Generation and characterization of I3 antibodies.

To check the presence of I3 protein in testis and spermatozoa, we first made an antibody against the C terminus of I3 from mouse. This antibody, TCTEX5 antibody, while recognizing native and recombinant I3, also cross-reacted against a high molecular protein unrelated to I3 in sperm extracts (Fig.2). We then made a polyclonal antibody directed against two synthetic peptides at the N terminus of I3 (Materials and Methods).

This antibody after affinity purification specifically recognized recombinant I3 and endogenous I3 in cell extracts (Fig.3).

37 38

Fig. 2. TCTEX5 antibody recognizes an I3-unrelated protein in testis/sperm: Tissue extracts from bull caudal sperm, and mouse testis, lung, brain, spleen and stomach were analyzed by western blot probed with affinity purified TCTEX5 peptide antibody raised against amino acid residues 69-83rd of the TCTEX5 carboxyl terminus. The antibody, though recognizing I3 in all the tissues, cross-reacts an I3-unrelated protein at ~52kDa.

Fig. 3. Validation of the I3 antibody raised against the two peptides at N- terminus of I3 protein: Recombinant I3, sperm and testis protein extracts were separated by SDS-PAGE followed by western blot analysis with affinity purified rabbit polyclonal anti-I3. Size markers (left) were derived from β-Galactosidase (116- kDa), Phosphorylase b (97.4-kDa), Albumin (66-kDa), Glutamic dehydrogenase (55- kDa), Ovalbumin (45-kDa), Glyceraldehyde-3-phosphate dehydrogenase (36-kDa), Carbonic anhydrase (29-kDa), and Soybean trypsin inhibitor (20-kDa).

2. The I3 mRNA abundance is not indicative of its steady state protein level in testis.

A previous study documented the presence of I3 mRNA in testis, and showed that the

steady state level of I3 message is clearly higher in testis than in other tissues (141). In

collaboration with Dr. Steven Pilder (Temple University), we subsequently confirmed, by

Northern blot analysis of mRNAs isolated from testis and numerous other tissues, that I3

testicular mRNA was 100- to 1000-fold more abundant than it was in other tissues (Fig.

4). In order to determine if the steady state level of I3 protein in various tissues reflects

these mRNA results, an affinity-purified polyclonal antibody was prepared and

characterized as shown in Figure 3. Western blots of heat-treated protein extracts from testis, sperm and various somatic tissues, probed with the I3 antibody, established the existence of a single immunoreactive protein at 27-kDa (Fig. 5), presumably I3, at comparable steady state levels in all tissues, despite the documented difference between steady state levels of I3 testis and somatic tissue mRNA. These data suggested the possibility that an abundance of stable testis I3 mRNA may be required to counteract an inherent instability of I3 protein when transcriptional activity is reduced during spermiogenesis.

Fig. 4. Northern blot analysis of Tctex5 mRNA expression in multiple mouse tissues: A northern blot containing mRNAs from twelve mouse tissues (listed in top panel) was probed with a full-length Tctex5 cDNA. All tissues exhibited weak signals except muscle, which did not exhibit a signal, and testis, which exhibited a very strong signal (middle panel). The Tctex5 signal demonstrated a molecular size of ~500 bases on the blot. The lower panel shows the same blot probed with an β- actin fragment used as a mRNA loading control (141).

Fig. 5. I3 is a heat-stable protein present in nearly equal abundance in mouse sperm, testis and a wide variety of somatic tissues: heat-stable proteins from sperm, testis and somatic tissues were separated by SDS-PAGE followed by western blot analysis with the validated affinity purified I3 antibody.

3. I3 binds to testis-expressed PP1γ2 and Sds22 in vitro and in vivo.

I3 has been reported to co-localize with PP1γ1 in nucleoli and with PP1α in

centrosomes, and has been co-precipitated with PP1γ1 and PP1α but not PP1β in HEK cells (142). However, whether I3 can bind to PP1γ2 has not yet been determined. Our

previous studies have shown that PP1γ2 is highly expressed in testicular germ cells and is

the only isoform of PP1 detected in spermatozoa.

3.1 GST pulldown assay:

To determine if I3 is capable of binding to PP1γ2 in vitro, we made GST-tagged

recombinant I3 proteins in bacteria for pull-down assays (see Materials and Methods).

GST-I3 was immobilized on Glutathione Sepharose 4B beads and incubated with mouse

testis extracts. After extensive washing, the bound proteins released by reduced

glutathione were analyzed by western blotting. Blots were probed with an anti-PP1γ2

antibody. Figure 5 demonstrates that PP1γ2 from testis or sperm extracts was bound to

GST-I3 but not to GST alone. Because Sds22 is known to be a PP1γ2 binding protein in

testis/sperm, the same blot was also probed with an anti-Sds22 antibody. Interestingly,

Sds22 was also identified as a potential I3 and/or PP1γ2 binding protein in testis and

sperm extracts (Fig. 6).

Fig. 6. GST-I3 binds Sds22 and PP1γ2 from testis or sperm extracts in vitro: The recombinant GST-I3 protein, or control GST, was incubated with sperm or testis cell lysate in the presence of glutathione-Sepharose beads. GST-I3 incubated with buffer in the presence of glutathione-Sepharose beads was used as one of the negative controls. The eluted proteins, sperm and testis extracts are resolved on an SDS-PAGE gel and subjected to western blot probed with Sds22 and PP1γ2. “+” means the protein(s) was used in the GST pulldown assay, and “-” means otherwise.

3.2 Immunoprecipitation:

Immunoprecipitation was used to further explore whether endogenous I3 is bound

to PP1γ2 and/or Sds22. Antibodies to I3 or PP1γ2 were immobilized on Protein G-

Sepharose 4 beads. Following incubation of each antibody-bound bead slurry with testis or sperm extracts, the proteins immunoprecipitated by the antibodies were analyzed by

SDS-PAGE/western blotting. Blots were sequently probed with anti-Sds22, anti-I3, or anti-PP1γ2 antibodies. These studies showed that anti-I3 antibodies co-precipitated both

PP1γ2 and Sds22, whereas anti-PP1γ2 antibodies co-precipitated both I3 and Sds22 from sperm or testis extracts (Fig. 7A and B). Taken together with our GST-pull down results, these data strongly suggested that I3, Sds22, and PP1γ2 are part of a single complex or exist as multiple pairs of heterodimers in testis and sperm.

Fig. 7. Anti-PP1γ2 can co-precipitate Sds22 and I3, and anti-I3 can co-precipitate PP1γ2 and Sds22 from sperm (A) or testis (B): The protein extracts were incubated with anti-PP1γ2, anti-I3, and preimmune serum immobilized on Protein G-Sepharose-4 beads, respectively. The immunoprecipitates were subjected to SDS-PAGE and immunoblotting with the antibodies against the indicated proteins.

4. Column purification of the complex, PP1γ2/I3/Sds22, from in vivo.

4.1 Chromatographic fractionation of mouse testis/sperm protein extracts:

To further clarify the binding relationships of the three proteins in vivo, chromatographic fractionation of mouse testis or sperm protein extracts was performed to purify the complex(es) containing I3, PP1γ2 and Sds22 as described in Materials and

Methods. Column fractions were analyzed for I3, PP1γ2 and Sds22 immunoreactivities.

The presence and abundance of all three proteins in DEAE, MonoQ, and Superose 6 column fractions is shown in Figure 8A, B, and C, respectively, providing further evidence that the three proteins may be part or all of a single complex in vivo.

4.2 Immunoprecipitation applied to the purified fractions containing I3,

PP1γ2 and Sds22:

To expand the assessment of whether the three co-eluting proteins were actually bound to one another, anti-I3, anti-PP1γ2, and anti-Sds22 antibodies were immobilized on Protein G-Sepharose 4 beads. Following incubation of each antibody-bound bead slurry with the purified fractions of testis extracts by a series of columns (DEAE,

MonoQ, Superose 6 and Superdex 200), the proteins immunoprecipitated by the antibodies were analyzed by SDS-PAGE/western blotting. Blots were probed with anti-

I3, anti-PP1γ2, or anti-Sds22 antibodies. In all cases, antibodies raised against each of the three proteins were able to reciprocally precipitate the other two proteins (Fig. 9).

4.3 Native gel electrophoresis of the column fractions containing I3, PP1γ2 and

Sds22:

The fractions containing the co-eluted proteins from the Superose 6 column were further analyzed by native PAGE/western blotting. Replicate blot strips were separately probed with anti-I3, anti-PP1γ2, or anti-Sds22 antibodies (Fig. 10). The results showed that all three proteins co-migrated on native PAGE, thus, offering further evidence that

I3, Sds22, and PP1γ2 are bound to one another in a single complex in testis and sperm.

5. Estimation of the molecular weight of the complex suggests that it consists of

more than three subunits.

While the studies for this dissertation were in progress, PP1α, I3, and Sds22 were

suggested to exist as a trimeric complex by overexpression of these proteins in cultured

mammalian cells (143). To test whether the testis/sperm complex containing PP1γ2, I3, and Sds22 was a trimer or existed as a larger multimer, the molecular weight was estimated of the purified complex by size exclusion chromatography through Superdex

200. The results showed that the three proteins were co-eluted at an elution volume of

13.06 ml, corresponding to a molecular weight of approximately 175 kDa estimated by the formula, y =-0.0871Ln(x)+1.3647, obtained from a calibration curve generated by proteins standards eluted from a Superdex 200 column (Fig. 11). Based on the primary sequences of the three proteins, the calculated molecular weight of a trimeric complex of

PPγ2, Sds22 and I3 should be ~96 kDa. This suggests that a complex of just the three

proteins either migrates anomalously through the sizing column or exists as part of a larger multimeric complex.

Fig. 8. I3, Sds22 and PP1γ2 co-elute during chromatographic purification of testis extracts: Western blot analysis of purified column fractions shows a consistent co- elution pattern of testicular PP1γ2/Sds22/I3 through a series of chromatographic media. A. Mouse testis protein extracts fractionated by DEAE chromatography shows PP1γ2, Sds22, and I3 co-eluting mainly in the A2, A3 and A4 fractions. B. A2, A3 and A4 fractions from the DEAE column were pooled and purified by MonoQ column fractionation. PP1γ2, Sds22, and I3 co-eluted in the B8-11 fractions. C. B8-B11 fractions from the MonoQ column were pooled and purified by Superose 6 column chromatography, from which PP1γ2, Sds22, and I3 remained co-eluted. In each instance 0.5 ml of co-eluting fractions were collected and concentrated, then assayed by SDS- PAGE/western blotting.

Fig. 8A.

Fig.8B.

Fig. 8C.

Fig. 9. I3, Sds22, and PP1γ2 from co-eluting column fractions are reciprocally co- immunoprecipitated: Superose 6 fractions containing co-eluted I3, PP1γ2, and Sds22 were incubated with anti- I3, anti-PP1γ2, anti-Sds22, and preimmune serum immobilized on Protein G-Sepharose 4 beads, respectively. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted for the expected co-precipitated proteins.

Fig. 10. I3, Sds22, and PP1γ2 from co-eluting column fractions co-migrate by native PAGE: Fraction of testis proteins purified by Superose 6 column chromatography containing I3, Sds22, and PP1γ2 was separated by native PAGE followed by western blot analysis. Triplicated blot strips probed with either anti-I3, anti-PP1γ2, or anti-Sds22 antibodies, respectively.

Fig. 11. A protein molecular weight calibration curve for Superdex 200 column: The curve was generated by the elution pattern of protein standards, Catalase (240kDa), Phosphorylase b (194kDa), Aldolase (158kDa), Albumin (68kDa), and Ovalbumin (43kDa) through Superdex 200 column. Kav=(Ve-Vo)/(Vt-Vo) (Ve is the elution volume of proteins by Superdex 200; Vo is void volume of Superdex 200, i.e. 8ml; Vt is the total elution volume of Superdex 200, i.e. 24ml).

6. Determination of other protein components of the complex containing PP1γ2/I3/

Sds22.

6.1 Microsequencing of the protein band containing PPγ2, Sds22 and I3 following native gel electrophoresis:

To determine if there are other protein components in addition to PPγ2, Sds22 and

I3 in the complex, non-denaturing gel electrophoresis of the Superdex 200 column fraction containing I3, PP1γ2, and Sds22 was performed in duplicate. One half of the gel was transferred to a PVDF membrane and used for a western blot probed with anti-I3, anti-Sds22, and anti-PP1γ2 antibodies, while the other half of the gel was stained with

Coomassie blue (Fig. 12). The band in the Coomassie blue-stained portion of the gel corresponding to the immunoreactive band in the western blot was excised from the gel and microsequenced following in-gel proteolysis. Interestingly, around 15 proteins were identified (Table 1). Of these, I3, Sds22, PP1γ1, PP1γ2, PP1α, PP1β, actin, nucleobindin, thioredoxin-like 2, and ubiquitin-activating enzyme E1 were highly represented.

Together, the elution profile of the complex on the size-exclusion column and the sequencing results following native gel electrophoresis revealed that I3, Sds22, and

PP1γ2 probably do not exist as a simple trimer.

band of interest

Fig. 12. Microsequencing of Coomassie-blue stained protein band containing Sds22/PP1/I3: Purified testis extracts were separated by native gel electrophoresis and the gel was stained with Coomassie blue. The band as indicated by the arrow in the Coomassie blue-stained portion of the gel corresponding to the immunoreactive band in the western blot was excised from the gel and microsequenced following in-gel proteolysis.

Table 1 Microsequencing Result

RefSeq# Protein Name Mascot Cover Score % 1 12963569 protein phosphatase-1 regulatory subunit 7 1122 68 2 6678483 ubiquitin-activating enzyme E1, Chr X 811 25 3 13994195 protein phosphatase 1, catalytic subunit, alpha 705 53 4 31980772 protein phosphatase 1, catalytic subunit, gamma 691 55 isoform 5 94374449 similar to Serine/threonine-protein phosphatase 544 32 PP1-beta, catalytic subunit 6 6679158 nucleobindin 1 409 32 7 31981269 thioredoxin-like 2 338 27 8 3385963 serine (or cysteine) proteinase inhibitor, clade A, 281 19 member 3K 9 6671509 actin, beta, cytoplasmic 266 23 10 66678079 serine (or cysteine) proteinase inhibitor, clade A, 231 21 member 1a 11 19072792 thioredoxin domain containing 4 169 19 12 7305563 t-complex protein 11 155 10 13 23943824 thioredoxin domain containing 2 (spermatozoa) 141 8 14 31980942 inositol (myo)-1(or 4)-monophosphatase 1 127 16 15 18390327 protein phosphatase 1, regulatory (inhibitor) 119 40 subunit 11 16 27501448 integrin beta 4 binding protein 89 17

6.2 Silver staining of the proteins immunoprecipitated by anti-I3 and anti-

Sds22 antibodies.

To further confirm that the complex(es) contained additional protein components,

the partially purified fraction from the Superdex 200 column was subjected to

immunoprecipitation with anti-I3 and anti-Sds22 antibodies followed by separation of the

immunoprecipitates by SDS-PAGE and subsequent silver staining of the resultant gel

(Fig. 13). Silver staining established the presence of other proteins in both immunoprecipitates that were not present in a negative control, and the protein profiles of

both anti-I3 and anti-Sds22 immunoprecipitates were remarkably similar.

6.3 Actin is the fourth member of PP1γ2/Sds22/I3 complex:

Microsequencing showed that actin was among the highly represented proteins in

the complex containing PP1 isoforms, I3, and Sds22. This was confirmed by

immunoprecipitation of the Superdex 200 fractions with anti-actin, anti-I3, anti-PP1, and

anti-Sds22 antibodies (Fig. 14). Western blots containing each immunoprecipitate were

probed with the anti-actin antibody as well as with anti-I3, anti-PP1γ2, and anti-Sds22

antibodies. Figure 14 showed that actin was co-precipitated with all three proteins in

testis, suggesting that PP1γ2, I3, Sds22, and actin constitute in entirety or parts of several

related multimeric protein complexes.

6.4. Nucleobindin, thioredoxin-like 2, and ubiquitin-activating enzyme E1 are

not part of the complex formed by PP1γ2/Sds22/I3:

Since nucleobindin, thioredoxin-like 2, and ubiquitin-activating enzyme E1 were

also highly represented in the microsequencing result (Table 1), we examined whether

they are also part of the PP1γ2/Sds22/I3 complex by immunoprecipitation with anti-actin, anti-I3, anti-PP1γ2, and anti-Sds22 antibodies. The immunoprecipitated proteins were

subjected to SDS-PAGE followed by immunoblotting with anti-nucleobindin, anti-

thioredoxin-like 2, and anti-ubiquitin-activating enzyme E1 antibodies, respectively. The

results showed that nucleobindin, thioredoxin-like 2, and ubiquitin-activating enzyme E1

failed to be co-precipitated by anti-actin, anti-I3, anti-PP1γ2, or anti-Sds22 antibodies

from the purified testis extracts, suggesting that they are not likely to be part of the

PP1γ2/Sds22/I3 complex. It was possible that these proteins happened to co-migrate with

the PP1γ2 complex in native PAGE.

Fig 13. Evidence that PP1γ2, Sds22, and I3 are bound to each other in a male germ cell complex that is larger than a trimer: Affinity-purified anti-I3 and anti-Sds22 antibodies, and preimmune serum were separately immobilized on Protein G-Sepharose 4 beads, and then incubated with a Superdex 200 purified fraction of testis proteins previously determined to contain I3, Sds22, and PP1γ2. The immunoprecipitate was separated by SDS-PAGE and the gel was silver-stained. Lane 2 shows silver-stained proteins immunoprecipitated by anti-I3 antibodies, lane 3, by anti-Sds22 antibodies, and lane 4, by preimmune serum. Based on the migration positions of expected immunoprecipitated proteins, I estimated the proteins numbered in the gel picture are as following: “1” is unknown, “2” to “7” include Sds22, actin, and PP1 isoforms, “8” is I3.

Fig. 14. Actin co-precipitates with PP1γ2, Sds22, and/or I3 from testis.: Protein extracts from mouse testis were immunoprecipitated with anti-actin, anti-PP1γ2, anti- Sds22, or anti-I3 antibodies. Following SDS-PAGE of the immunoprecipitates, the gels were analyzed by western blot with the same four antibodies. Results demonstrated that actin co-precipitated in immunoprecipitates of PP1γ2, I3, and/or Sds22, strongly suggesting that all four are present in a multimeric complex.

7. Is the PP1γ2/Sds22/I3 complex specific to PP1γ2 isoform?

7.1 Column purifications of testis from PP1γ knockout mice and mouse brain

extracts:

To answer the above question, protein extracts of brain from wild-type mice

(abundant in PP1γ1 with a minor amount of PP1γ2), and testis from PP1γ knockout mice

(the latter lacking both PP1γ1 and PP1γ2 but containing elevated levels of PP1α and

PP1β) were fractionated chromatographically on successive columns of DEAE, MonoQ, and Superose 6, and the collected fractions were evaluated by SDS-PAGE/western blotting with anti-I3, anti-Sds22, and either an anti-PP1γ1 antibody (for brain) or a monoclonoal antibody, E9, that recognizes all four PP1 isoforms (for PP1γ null testis).

The results demonstrated a co-elution pattern for Sds22, I3, and PP1γ1 in brain, or PP1α

and/or PP1β in PP1γ null testis (Figs. 15A & 15B, respectively), similar to that for Sds22,

I3, and PP1γ2 as described in Figure 8.

7.2 Immunoprecipitation and native gel electrophoresis of column fractions of brain or PP1γ knockout testis extracts.

Additionally, immunoprecipitation of brain extract fractions (in which PP1γ1, I3,

and Sds22 had co-eluted) with anti-PP1γ1 antibodies resulted in the co-precipitation of I3

and Sds22 (Fig. 15C). Finally, non-denaturing gel western blots containing I3, Sds22, and

PP1α/β co-eluting fractions from PP1γ null testis demonstrated that I3, Sds22, and

PP1α/β co-migrate (Fig. 15D). These data showed that all PP1 isoforms are capable of

forming complexes with I3 and Sds22, further demonstrating that PP1γ2 is not the only

PP1 isoform that can form a multimeric complex with I3 and Sds22. The

immunoprecipitated proteins from PP1γ null testis (Fig. 16A), and wild-type mouse brain

(Fig. 16B) with anti-I3, anti-PP1, and/or anti-Sds22 antibodies were subjected to SDS-

PAGE followed by immunoblotting with anti-actin. The results showed that actin is also

complexed with all isoforms of PP1 bound with I3 and Sds22.

8. PP1γ2 is catalytically inactive in a purified complex containing I3, and Sds22.

While Sds22 and I3 independently inhibit PP1, it was of interest to determine whether both proteins together affected the catalytic activity of the phosphatase. Because the testis contains all four PP1 isoforms as well as abundant PP2A co-eluting with these, I3, and

Sds22, I determined PP1γ2 activity in a complex also containing I3 and Sds22 purified by

size exclusion chromatography from spermatozoa, where PP1γ2 is the only PP1 isoform

detectable, and PP2A is not abundant in fractions containing PP1γ2, I3, and Sds22.

Phosphatase assays (see Materials and Methods) of fractions containing co-eluted I3,

PP1γ2, and Sds22 exhibited little if any serine-threonine phosphatase activity (Figs. 17A

& 17B).

Fig. 15. Macromolecular complexes containing I3 and Sds22 are not PP1γ2 isoform- specific: Western blot analyses of (A) wild-type mouse brain and (B) PP1γ-knockout testis protein extracts after fractionation by Superose 6 column chromatography. Sds22 and I3 co-eluted with PP1γ1 from brain in the B1-B4 fractions, while they co-eluted with PP1α/β in the B2-B3 fractions from PP1γ-knockout testis extracts. Western blot analysis of (C) co-precipitates from an I3-, Sds22-, and PP1γ1-containing MonoQ column fraction of wild-type brain using either an anti-PP1γ1 antibody or preimmune serum for immunoprecipitations; and western blot analysis of (D) a non-denaturing gel containing PP1γ-null testis protein extract from pooled and concentrated Superose 6 column fractions B2 and B3 in (B). Immunoblot probes included anti-Sds22, anti-I3, and a mouse monoclonal antibody (E9) which recognizes all isoforms of PP1. Pooled chromatography fractions of wild-type testis extracts served as positive controls.

A. Mouse brain fractionated by Superose 6

B. PP1γ knockout testis fractionated by Superose 6

C. D.

Fig. 16. Actin is also complexed with PP1β or PP1γ1 bound with I3 and Sds22: Protein extracts from (A) PP1γ-null testis, and (B) wild-type mouse brain were immunoprecipitated with anti-PP1β, or anti-PP1γ1, anti-Sds22, and/or anti-I3 antibodies. Following SDS-PAGE of the immunoprecipitates, the gels were analyzed by western blot with antibodies against the proteins as indicated. The secondary antibody for anti-actin is anti-mouse while the secondary antibody against the rest antibodies is anti-rabbit.

Fig. 17. PP1γ2 is inactive in Superose 6 fractions containing PP1γ2, Sds22, I3, and actin.: A. Protein phosphatase activity in 5-μl aliquots of Superose 6 fractions from caudal sperm protein extracts. B. SDS-PAGE/immunoblot analysis of the corresponding fractions probed with anti-Sds22, anti-PP1γ2, anti-I3, anti-PP2A, and anti-actin antibodies.

9. The mutual increasing abundance of I3, PP1γ2, and Sds22 in the testis from

adolescence to adulthood most closely reflects the progression of male germ cell

morphogenesis.

9.1 Developmental expressions of I3, PP1γ2, and Sds22 in wild-type testis:

We have previously shown that PP1γ2 levels increase in developing testis where its

expression levels parallel onset of spermatogenesis (103). To gain further insight into the

relationships between I3, Sds22, and PP1γ2 in developing male germ cells, the expression

and localization patterns of all the three proteins in testis were compared. Western blot

analysis showed that significant amounts of Sds22 were present at days 8 and 18 when

little PP1γ2 and I3 were detectable. However, the steady state levels of PP1γ2 and I3,

along with Sds22, rose to their highest levels by days 30 and 45. This pattern correlates

closely with a time course over which spermatids differentiate into mature testicular

spermatozoa (Fig. 18). Consistent with this data, immunohistochemical analysis of testis

sections indicated that Sds22 and PP1γ2 were most abundant in the cytoplasms of

spermatocytes, spermatids and spermatozoa, with PP1γ2 more predominant in spermatids

and Sds22 more pronounced in spermatocytes (Figs. 19A & 19B, respectively). I3 levels were highest in the cytoplasms of round and elongating spermatids and in spermatozoa

(Fig. 19C), resembling the PP1γ2 pattern somewhat more closely.

Fig. 18. Increasing steady state levels of I3, PP1γ2, and Sds22 in the testis parallel the temporal progression of spermiogenesis: In each experiment, postnatal testis levels of PP1γ2, Sds22, and I3 were monitored by assessing equal concentrations of protein (25μg) from extracts prepared from each age group. Proteins were separated by SDS- PAGE, and gels were assayed by western blotting with antibodies against PP1γ2, Sds22, and I3. Results indicating that postnatal expression of PP1γ2, Sds22, and I3 increased over the age range in which the initial round of spermiogenesis takes place, were visualized by chemiluminescence. A duplicate blot was probed with an anti-actin antibody to demonstrate equal protein loading.

Fig. 19. Cellular localization of PP1γ2, I3 and Sds22 in wild-type mouse testis sections: A. Sds22 expression is prominent in the cytoplasm of most germ cells in the testis (from spermatogonia, primary spermatocytes, secondary spermatocytes, and round spermatids to spermatozoa). B. PP1γ2 is prominently expressed in the cytoplasms of secondary spermatocytes, all spermatids, and mature testicular spermatozoa. C. I3 is prominently expressed in the cytoplasm of haploid germ cells (from round spermatids through spermatozoa). D. Immunohistochemistry performed without primary antibody (negative control). Blue fluorescence indicates TO-PRO3 staining of nuclei.

9.2 The expression level and cellular localizations of I3 rather than Sds22 seem highly linked to those of PP1γ2:

Immunohistochemical analysis of testis sections from PP1γ knockout and wild-type mice demonstrated that the I3 localization pattern was similar in both PP1γ knockout and

wild-type testis, although I3 immunoreactivity was significantly weaker in PP1γ null

testis (Fig. 20A), and this was confirmed by western blot analysis of testis protein extracts from wild-type and PP1γ knock out mice (Fig. 20B). Surprisingly, I3 in PP1γ

null testis migrated more quickly than I3 in wild-type testis on a denaturing

polyacrylamide gel (Fig. 20B), possibly indicating that I3 in the PP1γ null testis is devoid

of a wild-type post-translational modification(s). In contrast, Sds22 immunoreactivity and

size appeared to be comparable in wild-type and PP1γ knockout testis (Fig. 20B). Also of

interest were western blot results demonstrating that steady state levels of I3 increased,

though not to wild-type levels, and migrated on a denaturing gel with wild-type I3 when a

PP1γ2 transgene, driven by the testis specific PGK2 promoter, was expressed at ~30-40%

of wild-type levels in PP1γ null testis (Fig. 20B).

Fig. 20. Both the steady state level and molecular weight of I3 are diminished in the PP1γ-null testis, but its level and molecular weight increase in the PP1γ-null testis producing low levels of PP1γ2 protein via transgene expression.: A. Immunofluorescence of PP1γ-null (KO) and wild-type (WT) testis sections probed with anti-I3 antibody. Negative (-) control was performed without primary antibody. B. Western blot of PP1γ-null (lane 1), PP1γ-null/PP1γ2-rescued (lane 2), and wild-type (lane 3) testis protein extracts probed with anti-Sds22, anti-PP1γ2, anti-I3, and anti-actin antibodies. It is noteworthy that the size and steady state levels of Sds22 do not change.

10. The macromolecular complex formed by Sds22/PP1γ2/I3/actin could not bind to microcystin agarose.

Microcystin is an inhibitor of PP1 and microcystin affinity chromatography has been used to isolate PP1 binding proteins (144, 145). Previous work from our lab has demonstrated that Sds22-bound PP1γ2 could not bind to microcystin agarose (108), and at that time it was not known that a macromolecular complex composed of PP1/Sds22/I3 existed.

I examined whether the complex containing Sds22 complexed with PP1γ2/I3 could bind to microcystin, and whether there is a difference in the binding to microcystin by the components of the complex from testis versus from sperm. Mouse sperm or testis extracts were incubated with microcystin agarose that was pre-washed with homogenization buffer supplemented with 5 mg/ml BSA. The proteins bound to microcystin agarose, sperm and testis protein extracts used for microcystin agarose chromatography were analyzed by western blot analysis (Fig. 21) probed with anti-PP1γ2, anti-I3 and anti-

Sds22 antibodies. The results showed that Sds22 from either testis or sperm extracts failed to bind to microcystin agarose, consistent with the previous results (108), and I3 from testis rather than from sperm extracts were bound to microcystin agarose. The above results suggest that the complex containing Sds22/PP1γ2/I3 could not bind to microcystin agarose.

Fig. 21. Difference of microcystin pulldown of PP1γ2 and its binding proteins from sperm and testis: Sperm or testis protein extracts were incubated with microcystin agarose that was pre-washed with homogenization buffer supplemented with 5 mg/ml BSA. The microcystin bound fractions were prepared as outlined under Materials and Methods. The proteins bound to microcystin agarose, sperm and testis extracts were analyzed by western blot analysis probed with PP1γ2, Sds22 and I3 antibodies.

B. Comparative studies of t/t-I3 with wt-I3.

1. Comparison of the primary structures of t/t-I3 and wt-I3.

An alignment of the amino acid sequences (Fig. 22) showed that t/t-I3, composed of

126 amino acid residues, is 81% identical to wt-I3 which consists of 131 amino acid residues. Their sequence difference is mainly located at two extremes, N- and C-termini.

The middle part of the sequence is identical, containing a variant form of PP1-binding

motif, KKVEW, and the amino acid residues 54 to 83 of wt-I3 corresponding to the

amino acid residues 59 to 88 in t/t-I3, which are proposed to be necessary and sufficient

for PP1-binding. One of the biochemical features of wt-I3 is its highly hydrophilic nature.

- 43 out of 131 residues of wt-I3 are hydrophilic amino acids (8 Glycine, 2 Asparagine, 3

Glutamine, 11 Serine, 19 Threonine). Similarly, t/t-I3 contains 38 hydrophilic amino

acids out of 126 residues (7 Glycine, 3 Asparagine, 5 Glutamine, 7 Serine, 16 Threonine).

t: 1 MAEAKAEMNETITETTVTETT-----QPENQKITIKLRKPKPAKKVEWSSDTVDNEHMGR 55 MAE A ++ET+TETTVTETT +PENQ +T+KLRK KP KKVEWSSDTVDNEHMGR wt: 1 MAETGAGISETVTETTVTETTVTETTEPENQSLTMKLRKRKPEKKVEWSSDTVDNEHMGR 60 t: 56 RSSKCCCIYEKPRAFGESSTESDEDEEEGCGHTHCVWGHRKRRRPTTPGPTPTTPPQPPD115 RSSKCCCIYEKPRAFGESSTESDEDEEEGC H HCV GHRK RRPTTP PTPTTPPQPPD wt:61 RSSKCCCIYEKPRAFGESSTESDEDEEEGCSHKHCVRGHRKGRRPTTPAPTPTTPPQPPD120 t:116 PPQPPPGPMQH 126 P +PPPGPMQH Wt:121 PSKPPPGPMQH 131

Fig. 22. Amino acid sequence difference between t/t-I3 and wt-I3: t/t-I3, composed of 126 amino acid residues, is 81% identical to wt-I3 which consists of 131 amino acid residues. Their sequence difference is mainly located at two extremes, N- and C-termini. The middle part of the sequence is identical.

2. Generation and characterization of antibodies against t/t-I3.

The naturally occurring mutant isoform of I3 in the t-complex gene on mouse chromosome 17 is associated with sperm motility defects and male infertility. This provides an excellent model to examine the physiological function of I3 in testis and spermatozoa. To study the role of t/t-I3 protein in an abnormal sperm motility phenotype,

“curlicue”, we first made an antibody, TCTEX5 antibody, against a peptide sequence present in both wt-I3 and t/t-I3. While TCTEX5 antibody recognized both native and recombinant I3, it also cross-reacted against a high molecular protein unrelated to I3 in sperm extracts (Fig.2). We therefore made an affinity-purified polyclonal antibody directed against two synthetic peptides at the N terminus of t/t-I3 (Materials and

Methods). After affinity purification, this antibody specifically recognized recombinant

His-tagged-t/t-I3 and endogenous t/t-I3 in cell extracts (Fig. 23).

Fig. 23. Validation of t/t-I3 antibody: Rrecombinant His-I3 and t/t-testis protein extracts were separated by SDS-PAGE followed by western blot analysis with affinity purified rabbit polyclonal anti-t/t-I3.

3. t/t-I3 is also heat-stable and migrates anomalously on SDS-PAGE.

Since t/t-I3, similar to wt-I3, is highly hydrophilic, the question is whether t/t-I3 shares

some common biochemical features with wt-I3, such as anomalous migration on SDS-

PAGE and heat stability of the protein. To answer the question and to further study the

role of t/t-I3 played in the abnormal sperm motility phenotype, “curlicue”, western blot of

t/t-testis protein extracts probed with the affinity-purified polyclonal antibody against t/t-

I3 established the existence of a single immunoreactive protein at 25 kDa, presumably t/t-

I3, compared with its calculated molecular weight, 14.15 kDa. These data suggested t/t-I3

behaves similarly to wt-I3 with respect to heat stability and anomalous migration through

SDS-PAGE (Fig. 23).

4. Both wt- and t/t-I3 have similar potencies in inhibiting catalytic activity of PP1γ2.

It was shown in AIM 1 that PP1γ2 forms an inactive enzyme pool in sperm complexed

with its two inhibitory subunits, wt-I3 and Sds22. One of the questions that arose was

whether the ability of I3 to inhibit PP1γ2 is similar in both wt- and t/t-I3. To do this His- tagged recombinant wt- and t/t-I3 were generated for use in enzyme inhibition. His- tagged I3 instead of GST-tagged I3 was required for inhibition studies because it was noted that GST tag alone could inhibit PP1γ2 activity (data not shown). When a serial

dilution, from 5 to 450 nM, of either His-wt-I3 or His-t/t-I3 was incubated with 2ng of

His-PP1γ2 in the presence of Mn2+, the assay showed that both His-wt-I3 and His-t/t-I3

inhibit PP1γ2 with the same IC50 (20nM). Thus, in spite of their sequence differences, it

appears that, at least in vitro, both wt- and t/t-I3 inhibit PP1γ2 at comparable potencies

(Fig. 24).

Fig. 24. Comparison of the inhibitions of His-PP1γ2 by His-wt-I3 and His-t/t-I3: A serial dilution, from 5 to 450 nM, of either His-wt-I3 or His-t/t-I3 was incubated with 2ng of His-PP1γ2 in the presence of Mn2+, phosphorylase a used as a substrate. The assay showed that both His-wt-I3 and His-t/t-I3 inhibit PP1γ2 with the same IC50 (20nM).

5. The ability of t/t-I3 to form a complex with PP1γ2 and Sds22

That both recombinant wt- and t/t-I3 inhibit PP1γ2 in the same potential in vitro

implies that t/t-I3 may also have the similar ability as wt-I3 to bind to PP1γ2. As noted by the comparison of their amino acid sequences, PP1-binding motif and a protein fragment proposed to be necessary and sufficient for PP1-binding are intact in t/t-I3 compared with wt-I3. This suggests that t/t-I3 may have the similar ability as its wild type isoform to bind to PP1 and possibly also to another regulatory subunit of PP1, Sds22. In AIM 1 I

showed that testicular expressed wt-I3 binds to PP1γ2 and Sds22, forming a

macromolecular complex both in vitro and in vivo. The next question was whether t/t-I3

behaves in a similar fashion with respect to its ability to form a complex

5.1 GST pulldown assay:

First, the ability of wt-I3 or t/t-I3 binding to PP1γ2 was compared in vitro by the

GST-pull down assay. Figure 25 shows that PP1γ2 and Sds22 from either cauda or caput

sperm extracts can bind to either GST-wt-I3 or GST-t/t-I3 protein but not to GST alone.

The result is consistent with that of GST-wt-I3 pull down assay with testis extracts.

5.2 Immunoprecipitation:

Next, immunoprecipitation was used to further explore whether endogenous t/t-I3 is

bound to PP1γ2 and Sds22. Antibodies to t/t-I3 or PP1γ2 were immobilized on Protein G-

Sepharose 4 beads. Following incubation of each antibody-bound bead slurry with t/t

testis extracts, the proteins immunoprecipitated were analyzed by SDS-PAGE/western

blotting. Blots were subsequently probed with anti-t/t-I3, anti-PP1γ2, or anti-Sds22

antibodies. These studies established that anti-t/t-I3 antibodies co-precipitated both PP1γ2

and Sds22, and anti-PP1γ2 antibodies co-precipitated both t/t-I3 and Sds22 from mouse t/t testis extracts (Fig. 26). Taken together with the GST-pull down results, these data strongly suggested that t/t-I3, like wt-I3, can form a complex with PP1γ2 and Sds22 in vivo.

Fig. 25. Comparison of the binding of GST-wt-I3 and GST-t/t-I3 with PP1γ2 and Sds22 by GST pulldown assay: Cauda or caput sperm extracts were incubated with GST-wt-I3, GST-t/t-I3 protein or GST in the presence of glutathione-Sepharose beads. The eluted proteins and sperm extracts were resolved on a SDS-PAGE gel and subjected to western blot probed with Sds22 and PP1γ2.

1 2 3

Fig. 26. Anti-PP1γ2 can co-precipitate t/t-I3 and Sds22, and anti-t/t-I3 can co- precipitate PP1γ2 and Sds22 from t/t-testis protein extracts: The protein extracts were incubated with anti-PP1γ2, anti-t/t-I3, and preimmune serum immobilized on Protein G- Sepharose-4 beads, respectively. The immunoprecipitates were subjected to SDS-PAGE and immunoblotting with the antibodies against the expected precipitated proteins.

6. Column purification of the complex, t/t-I3/PP1γ2/Sds22, from testis protein

extracts.

6.1 Chromatographic purification:

To further clarify their binding relationships in vivo, chromatographic fractionation of

mouse t/t testis protein extracts, was performed to purify the complex(es) containing t/t-

I3, PP1γ2 and Sds22 as described in Materials and Methods. Column fractions were

analyzed for t/t-I3, PP1γ2 and Sds22 immunoreactivities. The presence and abundance of all these proteins in Superdex 200 column fractions, the last step of purification, is shown in Figure 27, providing further evidence that t/t-I3, like its wild type isoform, forms a

complex with PP1γ2 and Sds22 in vivo.

6.2 Native gel electrophoresis applied to the purified fractions of t/t-testis:

To examine the co-elution of the three proteins, t/t-I3, Sds22 and PP1γ2, through

various columns was due to they were actually bound to one another, the fractions

containing the co-eluted proteins from the final sizing column were further analyzed by

native PAGE/western blotting. Replicate blot strips were separately probed with anti-t/t-

I3, anti-PP1γ2, or anti-Sds22 antibodies (Fig. 28). The results showed that t/t-I3 co-

migrated with PP1γ2 and Sds22, similarly to previously reported spermatic and testicular

expressed wt-I3, on native PAGE, thus, offering conclusive evidence that t/t-I3, Sds22,

and PP1γ2 are bound to one another in a single complex in vivo.

Fig. 27. t/t-I3, Sds22 and PP1γ2 co-elute during chromatographic purification of t/t- testis extracts: Western blot analysis of purified column fractions shows a consistent co- elution pattern of testicular PP1γ2/Sds22/I3 through a series of chromatographic media. Here only shows the last step of purification by Superdex 200. 0.5 ml of co-eluting fractions were collected and concentrated, then assayed by SDS-PAGE/western blotting.

Fig. 28. t/t-I3, Sds22, and PP1γ2 from co-eluting column fractions co-migrate by native PAGE: The fractions containing the co-eluted proteins from the final sizing column were further analyzed by native PAGE/western blotting. Replicate blot strips were separately probed with anti-t/t-I3, anti-PP1γ2, or anti-Sds22 antibodies.

7. In vitro phosphorylation study of wt- and t/t-I3.

One of the known mechanisms to regulate PP1 holoenzyme is to change the binding

relationships between regulatory subunit(s) and the catalytic subunit by phosphorylation

or dephosphorylation of the regulatory subunit(s), for example, dephosphorylated

PPP1R2 (inhibitor 2) can bind and inhibit PP1, while PPP1R1 (inhibitor 1) and its

neuronal homologue, DARPP32, can only bind and inhibit PP1 after being

phosphorylated by PKA. So we turned our attention to examine the difference between

the potential phosphorylation sites in the two types of I3. The amino acid sequences of I3

show that there are four potential phosphorylation sites, Serine at the 9th, the 32nd, the

91st, and the 122nd, in wt-I3, replaced by Asparagine, Lysine, Glycine and Proline,

respectively, in t/t-I3, suggesting that the ability of t/t-I3 to be phosphorylated may be

different compared to that of wt-I3. To examine this, an in vitro phosphorylation study

was conducted with GST-wt-I3 and GST-t/t-I3 using sperm extracts as a source of kinase.

As shown in Figure 29, it was found that over 30min, GST-wt-I3 showed significantly

greater phosphorylation than GST-t/t-I3 when serial dilutions of 0.5-5μg of GST-t/t-I3 or

GST-wt-I3 were incubated with 2μg of sperm extracts for 30min in the presence of 32P-

ATP.

Fig. 29. In vitro phosphorylation study of GST-wt-I3 and GST-t/t-I3 using sperm extracts as a source of kinase: A serial dilution of 0.5-5μg of either GST-t/t-I3 or GST- wt-I3 was incubated with sperm extracts for 30 min in the presence of 32P-ATP followed by quantification of label uptake by TCA precipitation and separation by SDS-PAGE. Gels were dried, exposed to film, and developed.

C. Characterize the protein phosphatase inhibitor, PPP1R2/inhibitor 2 (I2) in testis

and spermatozoa.

1. Characterization of I2 antibody and verification of the existence of inhibitor I2 in

testis.

As noted in the introduction, a logical candidate for the regulation of PP1γ2 activity in

sperm is PPP1R1/inhibitor 1 (I1) (99). Surprisingly, however biochemical and

immunochemical approaches failed to detect I1 in spermatozoa. However sperm

contained what appeared to be I2-like activity (99). This conclusion was based on the

observation that the GSK-3 activatable PP1γ2 was present in sperm extracts and that a

heat stable I2-like entity was also present. However conclusive evidence for the presence of I2 was lacking. A reason for this was the lack of a reliable antibody to detect I2. We therefore generated antibodies against the 134~147 amino acid residues,

REKKRQFEMKRKLH, of I2 protein. The antibody was affinity purified and

characterized, and used for probing western blots of heat-treated protein extracts from

testis, brain and lung. The results showed the existence of a single immunoreactive

protein at 32kDa (Fig. 30), presumably I2, which is known to be a heat-stable protein.

This protein also anomalously migrates at 32kDa on 12% SDS-PAGE compared to its

calculated molecular weight, 23kDa.

Fig. 30. Characterization of I2 antibody and verification of the existence of I2 in testis: Western blot of heat stable proteins from mouse testis, brain and lung was probed with affinity purified polyclonal I2 antibody raised against the amino acid residues 134th~147th, EKKRQFEMKRKLH, of regular isoform of I2.

2. I2 from sperm and testis protein extracts is bound to PP1γ2 in vivo.

The protein I2 in somatic cells is well studied. It has been shown that I2 can bind to

PP1α, PP1β, and PP1γ1, but it is not known whether I2 can bind to PP1γ2.

Immunoprecipitation (IP) was used to explore whether endogenous I2 in testis and sperm is bound to PP1γ2. Following incubation of I2 antibody-bound Protein G-Sepharose 4 bead slurry with testis or sperm protein extracts, the proteins immunoprecipitated by I2 antibody were analyzed by SDS-PAGE and western blotting probed with anti-PP1γ2.

These results not only show that I2 binds to PP1γ2 in testis/sperm, but also further confirmed the presence of I2 in testis and sperm (Fig. 31).

Fig. 31. Immunoprecipitation with I2 antibody from sperm or testis: Sperm or testis protein extracts were incubated with anti-I2 or preimmune serum immobilized on Protein G-Sepharose-4 beads, respectively. The immunoprecipitates were subjected to SDS-PAGE and immunoblotting with anti-PP1γ2.

3. Evidence that a gene on mouse chromosome 17 encodes a novel protein similar to

I2.

A recent annotation of mouse chromosome 17 suggests the existence of an intronless

gene, located just outside of t-complex, which may ‘encode’ a protein similar to I2. This

intronless gene is referred as I2(17) in this dissertation compared to the regular I2 gene

on chromosome 16 referred as I2(16). I2(17) gene is an intronless gene containing an

extra N terminus sequence derived from 327bp which is absent in I2(16) coding

sequence. The rest of I2(17) gene, called C terminus in this dissertation, is 90% identical

to I2(16). To determine whether this gene can be transcribed in vivo, PCR with genomic

DNA was performed with primers at 5’- and 3’-flanking regions of mouse I2(17) gene.

The resulting PCR product was used as a probe for Northern blot analysis and used for in

vitro expression studies.

3.1. Northern blot:

Two probes used for Northern blots are the whole N-terminus (327bp) and C- terminus (621bp) of I2(17) generated from the above PCR fragment consisting of the whole I2(17) coding sequence. The Northern blot of total RNAs from mouse testis and multiple somatic tissues probed with 32P-labeled N terminus of I2(17) demonstrated a

radioactive band only in testis, indicating the presence of I2(17) RNA in testis (Fig. 32A).

While, the Northern blot of total RNAs from mouse testis probed with C terminus DNA

labeled with 32P (Fig. 32B) showed one extra band in testis in addition to the two

expected bands at 1.7kb and 2.7kb present in various tissues as reported (25). Consistent

with the result of multitissue Northern blot probed with I2(16) cDNA from Dr. Anna A.

100

DePaoli-Roach’s lab (25), the above Northern blot result probed with I2(17) C-terminus further suggested that the intronless gene is transcribed in vivo, probably highest in testis.

101

A B

Fig. 32. Northern blots of mouse tissue mRNAs: A. The Northern blot containing 10μg mRNAs from mouse testis, lung, kidney, liver and brain was probed with a 32P-labeled N terminus of I2(17) cDNA. All tissues exhibited no signals except testis, which exhibited a very strong signal. B. The Northern blot containing 10μg mRNAs from mouse testis was probed with a 32P-labeled C terminus of I2(17) cDNA. One extra radioactive band was present in testis in addition to the two expected bands at 1.7kb and 2.7kb present in various tissues

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3.2. RT-PCR:

To further confirm I2(17) gene is transcribed in vivo, PCR reactions were performed using commercially available multiple tissue cDNAs with primers designed to produce a

165bp PCR fragment which is unique to the mouse I2(17) gene (Fig. 33 A). The RT-PCR results indicated I2(17) gene is ubiquitously transcribed in various tissues (Fig. 33 B),

contradictory to the above Northern blot results which showed I2(17) RNA is specifically

present in testis. This inconsistency could be because that RT-PCR is far more sensitive

than Northern blot.

3.3. Expression studies in vitro:

To test whether the protein, I2(17), can be expressed in E.coli, PCR reactions were

performed using mouse genomic DNA as templates with the primers against the flanking

region of I2(17) gene as mentioned above. The resulting PCR fragment was inserted into

pRSET plasmids, expressed as a his-tagged protein in E.coli, and subsequently purified

with the Ni-NTA beads. The purified His-tagged recombinant protein was duplicately

separated by SDS-PAGE followed by immunoblotting with anti-his and anti-I2

antibodies, respectively. The western blot results demonstrated multiple protein bands

immunoreactive to anti-I2 antibody with a band of the highest immunoreactivity

positioning correspondingly to a single band immunoreactive to anti-His antibody

(Fig.34). This result suggests the I2(17) gene can be expressed as a protein in E.coli.

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4. A 55kDa protein from various tissues is recognized by I2 antibodies:

Various tissue proteins were separated by 12% SDS-PAGE followed by western blots

probed with rabbit peptide I2 antibodies. The results showed that one immunoreactive

protein at 55kDa strongly reacts with I2 antibody (Fig. 35), and the protein is not heat-

stable, while the more common I2 isoform is not consistently seen on western blots (Fig.

35), which may be due to the lability of I2. The 55kDa protein immunoreactive to I2

antibody migrates to the similar position to that of recombinant I2(17) protein on SDS-

PAGE, a position higher than that as expected by the calculated molecular weight of protein I2(17). This anomalous migration pattern is similar to that of protein I2(16).

Based on the shared biochemical features of the endogenous 55kDa protein from various

tissues and the recombinant I2(17) protein, their anomalous migration pattern and

migrating positions on SDS-PAGE, and their immunoreactivities to I2 antibodies, this

dissertation proposes that the endogenous 55kDa protein is the protein encoded by I2(17)

gene in vivo. In summary, the above results provided strong evidences that the intronless

gene outside of t-complex on mouse chromosomal 17 can be transcribed in vivo and

translated in vitro.

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Fig. 33. PCR results of multiple tissue cDNAs: A. Schematic of the sequence for I2(17) gene. Red solid rectangle depicts the full-length coding region of I2(17) gene, which consists of 327bp N terminus and 621bp C terminus labeled “regular I2” because it is highly identical to the coding sequence of mouse I2(16) gene. The two black bars underneath are 165bp PCR fragment unique to I2(17) gene produced from the PCR reaction performed with the left primer, 5’-TCAAACCATCACAGAGGCATAC-3’, and the right primer, 5’-TAGTCTCTTCCCTCCTAAATC-3’, indicated by pink arrows. B. PCR result of multiple tissue cDNAs with the above two primers. The result indicates that I2(17) gene is ubiquitously transcribed in various tissues.

105

Fig. 34. Western blot of recombinant I2(17) protein: Purified his- tagged I2(17) expressed in E. coli culture lyses transformed with I2(17)-pRSET construct was separated by SDS-PAGE followed by western blot probed with anti-his antibody (1:1000) (lane 1) and with anti-I2 antibody (1:2000) (lane 2). The black arrow indicates I2(17) protein.

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Fig. 35. Our affinity purified I2 antibody recognizes a 55kDa protein on western blots: Western blot of RIPA+ extracts from various tissues, and recombinant I2 protein used as a positive control, probed with our affinity purified peptide I2 antibody (1:1000) raised against amino acid residues 134~147th, EKKRQFEMKRKLH, of regular isoform of I2. A 55k Da protein strongly immunoreactive to our I2 antibody is ubiquitously present in various tissues.

DISSCUSSION

A. The protein PPP1R11/inhibitor 3 (I3) forms a macromolecular complex with

PP1γ2, Sds22 and actin.

Four protein phosphatase 1 isoforms are expressed in mammals. Of these, three

(PP1α, PP1β, and PP1γ1) are ubiquitous in somatic cells. Only PP1γ2, one of two alternatively spliced products of the Ppp1cc gene, is expressed at high levels in meiotically dividing and differentiating testicular germ cells. PP1γ2 is also the only detectable PP1 isoform in mammalian spermatozoa, where its inhibition in immotile or poorly motile caput sperm has been associated with the onset of vigorous progressive motility, and in already progressively motile cells, with a significant increase in vigorous movement (95). Interestingly, PP1γ2 is not found in any somatic tissue other than brain, where its expression is minor. It is also notable that a non-mammalian orthologue for this isoform does not exist. What differentiates PP1γ2 from other PP1 isoforms, which is possibly the reason for its uncommon cell- and tissue-type distribution in mammals, is its distinctive, almost completely conserved 21-amino acid C-terminal extension. However, the role of this unique molecular appendage in PP1γ2 function remains unknown, as it does not appear to provide PP1γ2 with additional catalytic activity (92, 146).

We previously established that aberrant spermatid development was the primary marker of infertility in PP1γ null male mice (103). In the same study, PP1γ1 was shown to localize predominantly in Sertoli (somatic) cells in the testis. Thus, we assumed that

107 108

the absence of PP1γ2 from the developing spermatids of PP1γ knockout mice was the

principal basis for the development of structurally abnormal male germ cell characters

and subsequent male sterility. Therefore, the study in AIM 1 was undertaken to test the

assumption that PP1γ2 plays a singular role in the development of mammalian male germ cells, and to elucidate the biochemical basis of its individuality.

We first set out to examine the presence of PPP1R11/TCTEX5/inhibitor 3 (I3) in testis/sperm, a highly conserved and potent PP1 inhibitor genetically linked to the male sterility phenotypes of impaired sperm tail development and poor sperm motility in t

complex mice (134, 135, 139, 147, 148), and the potential testicular interaction(s) of

PP1γ2 with I3, and with PPP1R7/Sds22, an evolutionarily ancient PP1 regulator bound to

PP1γ2 in mammalian caudal sperm (108). These prospective relationships were of

particular interest because in yeast and in cultured mammalian somatic cells, PP1

isoforms were known to form trimeric complexes with I3 and Sds22 in which the PP1

isoforms were held in a catalytically inactive state (143, 149).

Results of the present study have thus far indicated that in differentiating

spermatids, PP1γ2, I3, and Sds22 make up three parts of a tetrameric (or larger) protein

complex also containing actin and/or one or more of several other proteins. While this

study has not yet determined the state of PP1γ2 activity in this testicular complex, it was

established that PP1γ2 is rendered catalytically inactive in the spermatozoon equivalent

of this complex. Data in AIM 1 also suggest that PP1α and PP1β are fully capable of

forming similar complexes containing I3, Sds22, and actin, as they appear to do so in the

PP1γ null testis. However, their elevated expression and participation in these complexes

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in PP1γ null developing spermatids does not ameliorate the aberrations of spermatid development caused by the loss of the PP1γ isoforms (103). Therefore, the function of the unique PP1γ2 C-terminal extension could be to target PP1γ2 and its bound regulatory subunits to substrates in developing and mature male germ cells that PP1α and/or PP1β do not recognize. On the other hand, its function could be to bind to distinctive epitopes on otherwise common PP1 targets, such as their regulatory subunits that other PP1 isoforms never or rarely identify.

Data presented in this dissertation have not yet allowed discriminating between these two possibilities. Experiments in AIM 1 have established that the steady state level of I3 is dramatically diminished in PP1γ null testis, although the level of Sds22, another ancient regulatory subunit of PP1, remains equivalent to its level in wild-type testis. In addition, this study has demonstrated that de novo production of PP1γ2 (~30-40% of wild-type protein levels) in PP1γ null testis via PP1γ2 transgene expression corresponds to a significant increase in the level of I3, although not to wild-type levels. These data suggest, when evaluated together with our finding that I3 and PP1γ2 (as well as Sds22)

increase mutually in wild-type testis to their highest levels over the course of spermatid

morphogenesis, that one role for PP1γ2 in developing spermatids and mature

spermatozoa, both transcriptionally and translationally hypoactive/inactive cell types, is

to aid in the maintenance of I3 stability during and after the termination of biosynthetic

activities. The notion that I3 in developing male germ cells may be inherently unstable is

reinforced by the finding that I3 testicular mRNA is 100- to 1000-fold more abundant

than in somatic tissues, although the level of testicular I3 protein is no greater than its

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level in other tissues. Whether the mechanism by which PP1γ2 might stabilize I3 is one

involving direct binding or an indirect one routed through another substrate of PP1γ2 (or

a combination of both mechanisms), has not yet been determined.

It is interesting that in PP1γ null testis, I3 levels are not only significantly diminished, but I3 migrates more quickly on a denaturing gel than its counterpart from either wild-type testis or PP1γ null testis expressing a PP1γ2 transgene. This finding indicates a possible loss of one or more I3 post-translational modifications in PP1γ null

testis relative to wild-type testis, and suggests a model in which I3 is stabilized by post-

translational modifications. It may be that PP1γ2 specifically activates an enzyme by dephosphorylating it, which, in turn, would stabilize I3 in developing male germ cells by adding a post-translational modification to it. Alternatively, the role of PP1γ2 in stabilizing I3 might be a more direct one in which its unique C-terminus interacts with I3 in such a way that specific modifications of I3 residues which stabilize it are protected from an attack that would result in either loss or reversal of modification. Regardless of whether the role of PP1γ2 in stabilizing I3 is direct or indirect, it is likely that I3 stability

(and thus, activity) depends on maintaining specific modifications, without which it could be exposed to attack by tissue-specific proteases.

What type of enzyme(s) modifies and thus stabilizes I3 is currently under investigation.

Previous studies have demonstrated that O-GlcNAc transferase exists in an operative

complex with several PP1 isoforms in rat brain extract (150).

In addition, more recent studies have indicated that phosphorylation of CPI-

17/PPP1R14a, an endogenous inhibitor of PP1 in muscle, provides a mechanism for

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reciprocal coordination of kinase and phosphatase activities in which residue-specific

phosphorylation of CPI-17 increases its power to inhibit PP1 catalytic activity by >1000-

fold (151).

The role of Sds22 in the multimeric male germ cell complex is also not yet clear.

Recent studies indicate that while Sds22 can inhibit PP1 by itself in vitro, cell lysates that

were prepared under conditions that prevented the Sds22-induced inactivation of PP1

contained a catalytically inactive trimer of PP1, Sds22, and I3 in which PP1 was

sandwiched between Sds22 and I3, each binding PP1 at a different site (143). This

suggests that in some complexes containing catalytically inactive PP1, I3 is the major

inhibitor of PP1, while the main role of Sds22 might be to target PP1 to appropriate

substrates. However, whether the behavior of I3 and Sds22 in the larger multimers,

identified in the present study, is the same as in the trimeric complex, identified

previously in tissue culture protein lysates, has not yet been elucidated.

Perhaps Sds22 behaves in a similar fashion to MYPT1, which has the capacity to

target PP1 to the actin cytoskeleton in fibroblasts, where it then mediates changes in

cytoskeletal organization and cell motility under the influence of bound PP1 (152), or to

two neuronal-specific PP1 regulatory subunits (PPP1R9a/Neurabin and

PPP1R9b/Spinophilin) that target PP1γ1 to the actin cytoskeleton in different subsets of neurons (37, 153, 154). Interestingly, in the absence of PP1 in fibroblasts, expression of full-length MYPT1 appears to cause the radical disorganization of the actin cytoskeleton,

compellingly similar to what we have observed in PP1γ null developing spermatids, where the stable presence of Sds22 in the absence of PP1γ2 (and the sudden deficiency of

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I3) corresponds to disorganization of developing periaxonemal and sperm head structures

(103).

It is also interesting that at least one of the components of one or more of the

multimeric PP1γ2 conplex that we have identified in the mammalian testis and sperm is actin. In keeping with our previous observations that the fibrous sheath longitudinal columns of PP1γ null spermatids exhibit a subtle developmental abnormality (103), studies by Escalier and colleagues have demonstrated that a component(s) of fibrous sheath longitudinal columns co-localizes with the actin cytoskeleton throughout sperm tail morphogenesis and in the flagellum of mature sperm (155). Whereas the filamentous actin cytoskeleton of these cells might be targets of PP1 and its regulators, another target could be monomeric actin, especially in the mature sperm tail, where actin may be an inner arm dynein light chain, as it is in Chlamydomonas flagella (156). Thus, a multimer containing PP1γ2, I3, and Sds22, as well as monomeric actin, could be a complex

mediator of inner arm dynein function in mammalian sperm.

At present, we can only speculate about the ultrastructural location of the

PP1γ2/Sds22/I3/X complex (where X represents actin or another protein) in developing or mature male germ cells. In Chlamydomonas flagella, PP1 is primarily (but not exclusively) located in the C1 central singlet microtubule of the axoneme, where its activity, along with that of PP2A, presumably mediates dynein behavior by regulating the properties of mechano-chemical signals transduced through the radial spokes; however, nothing is yet known about how PP1 is anchored to the axoneme or what its substrates are (157). Interestingly, its primary location in Chlamydomonas may juxtapose PP1 to

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Radial Spoke Protein 1 (RSP1), part of the radial spoke head. In mouse, the orthologue of

RSP1 (RSPH1, formerly TSGA2) has several isoforms that are distributed not only in the sperm tail axoneme, but also in the two, nearly mammalian sperm-specific periaxonemal structures, the outer dense fibers and fibrous sheath, as well as at the cytosolic periphery around the circumference of the mitochondrial sheath (139). It is noteworthy that recent studies have suggested that mouse RSPH1 is a synergist of I3 activity, thus stimulating vigorous sperm motility (139, 148). Whether a similar synergy is a feature of outer dense fiber assembly and mitochondrial sheath differentiation (both adversely affected in PP1γ null testis) is not known. In either case, the biochemistry of this genetic synergism has not yet been resolved.

In our previous work, microcystin agarose affinity chromatography was intended as an independent verification of the immuno-affinity purification of the complex formed by

PP1γ2-Sds22 in sperm (108). However, it was found that PP1γ2 bound with Sds22 failed to bind to microcystin agarose. At that time, it was not known that a complex containing

PP1γ2, Sds22, I3 and actin existed in testis, sperm or any other tissue. Since work in this

dissertation has identified this complex, it would be interesting to know whether this

complex could bind to microcystin. Here we show that Sds22 from both testis and sperm

failed to bind to microcystin. However, I3 in sperm extracts did not bind to microcystin while I3 in testis extracts did. It may be noted that testis contain somatic cells, e.g. Leydig

and Sertoli cells. So I3 and actin bound to microcystin in testis may represent a distinct

pool of these two proteins which may be different from the complex of PP1γ2 with Sds22

and I3 in differentiating germ cells. The inability of Sds22 and I3 to bind to microcystin

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could also be simply due to the fact that the whole complex cannot bind to microcystin

rather than the possibility that microcystin displaced Sds22 and I3 from their complex

with PP1γ2.

In summary, the data in AIM 1 have led to the conclusion that in differentiating

spermatids, PP1γ2 most likely forms either a single multimeric complex or several

multimers with I3, Sds22, and at least one other protein. While we have confirmed that

one of these other proteins is actin, it is too early to tell whether PP1γ2 is targeted to

elements of the spermatid cytoskeleton. Even though the following three proteins,

ubiquitin ligase activating enzyme E1, nucleobindin, and thioredoxin 2-like protein, have

been examined and found to be not part of the complex, whether a multimeric complex forms between PP1γ2, Sds22, I3, and any of the additional members identified in Table 1 will be determined in future studies.

B. Biochemical properties of the naturally occurring mutant isoform of I3 .

In mouse, a gene located in t-complex on chromosome 17 encodes I3. t-complex is a region of 40 million base pairs (Mbp) proximal to the centromere of chromosome 17.

There are two types, wild type (wt) and t haplotype (t), of t-complex existing in nature. A family member of the mouse t complex (the familial ancestor of modern-day t haplotypes) was first discovered about 76 years ago. All t haplotypes are closely related, naturally occurring polymorphisms of the t complex region, exhibiting four inversions spanning the region relative to the wild-type homolog. By a nearly complete suppression of recombination, the integrity of a complete t haplotype, 30~40 million base-pairs, is

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maintained from one generation to the next, resulting in a transmission ratio distortion

(TRD), contrary to Mendel’s first law. The t haplotype carries genes that either cause

homozygous male sterility or embryonic lethal mutations. As a result of TRD, numerous

mutations have accumulated and become fixed in t haplotypes.

The mouse t haplotype affecting male fertility has been used as a naturally-occurring

model to study the regulation of sperm motility. It has been demonstrated that the coordinated activity of the protein products of three tightly-linked t complex genes in

sperm from t haplotype (t) homozygous males can lead to expression of an abnormal

flagellar waveform phenotype, “curlicue”, and so males with t/t sperm are sterile. The three candidate proteins are: DNAHC8, a principal piece-restricted, axonemal dynein heavy chain with a unique N-terminus; TSGA2, a testis/sperm-limited protein of

unknown biochemical/physiological function; and I3, an inhibitory subunit of

Serine/Threonine protein phosphatase 1.

Results in AIM 1 showed that wt-I3 is ubiquitously expressed in various tissues including testis and spermatozoa, and that its mRNA abundance is not indicative of its steady state protein level in testis. These studies using multiple approaches also provided strong evidence that wt-I3 binds to testicularly or spermatically expressed PP1γ2 and

Sds22 both in vitro and in vivo. We also showed for the first time that the macromolecular complex is not a trimer and that actin is also part of the complex.

Furthermore we showed that the expression of wt-I3, but not Sds22, is highly linked to the expression of PP1γ2 in wild type mice and in PP1γ null mice with a transgenic source of PP1γ2.

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To determine the role of t/t-I3 in the abnormal sperm motility phenotype, “curlicue”,

AIM 2 was focused on comparison of the properties of t/t-I3 with wt-I3 with respect to

their amino acid sequences, migration patterns on SDS-PAGE, inhibitory potential

against recombinant PP1γ2 activity in vitro, their ability to form a complex with PP1γ2 and Sds22 both in vitro and in vivo, and the ability of the proteins to be phosphorylated in vitro. The results in AIM 2 showed that t/t-I3, like its wild type isoform, is heat-stable and migrates anomalously on SDS-PAGE. It appears that the anomalous migration of I3 protein on SDS-PAGE is due to its high ratio of hydrophilic amino acid residues, 32.8% in wt-I3, similarly, 30.2% in t/t I3, which results in less SDS binding during electrophoresis, and so I3 migrates slower than it would based on its calculated molecular weight. It is perhaps not surprising that studies in AIM 2 found no significant difference between t/t-I3 and wt-I3 in terms of their binding to PP1γ2, their inhibitory potentials toward recombinant PP1γ2, and their ability to form a complex with PP1γ2 and Sds22, since the variant PP1-binding domain, KKVEW, and a peptide fragment demonstrated to be essential for PP1-binding (143) are unaltered in t/t-I3 relative to wt-I3. The study from

Bollen’s lab showed that PP1α is sandwiched between I3 and Sds22, and the formation of the sandwiched complex is highly specific to I3 since a NIPP-1 fragment containing only RVxF, PP1-binding motif, cannot displace I3 from the complex, and the 54th-83rd amino acid residues, are necessary and sufficient for PP1-binding.

The 120th-125th amino acid sequence, “ERRHRK”, at C-terminus was proposed to be

the nuclear localization signal (NLS) in Ypi1, a yeast homologue of I3. Ypi1 was

proposed to target Sds22 and PP1 into nuclei since the three can form a complex in both

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yeast and the cultured mammalian cells since even though Sds22 or PP1 lack traditional

NLS they are nevertheless found to be localized in nuclei of somatic cells. The

corresponding sequence, “ERRHRK”, of Ypi1 is “VRGHRK” (the 97th to the 101st amino acid) in wt-I3, and is “VWGHRK” (the 91st to the 96th amino acid) in t/t-I3. We propose

that these two variant sequences, “VRGHRK” and “VWGHRK”, may also be used as

NLS in wt- and t/t-I3 since a portion of I3 protein has been found to be in the nucleus of

germ cells in the testis.

One of the significant differences in the primary structure of t/t-I3 compared to wt-I3

is in the three potential phosphorylation sites, where Serine(s) are replaced by

Asparagine, Lysine, Glycine and Proline, respectively. Accordingly, the results of in vitro

phosphorylation studies showed that t/t-I3 is less phosphorylated in vitro than wt-I3 when

sperm extracts were used as a source of kinase. This indicates that differences in

phosphorylation may play a role in regulating conformation and/or activities of the two

types of I3 proteins in vivo. This phosphorylation difference may result in different

binding affinity and/or inhibitory potentials toward PP1γ2 in vivo, and thus affect the

formation and/or dissociation of the macromolecular complex with PP1γ2 and Sds22.

This may then be the biochemical basis for “curlicue” phenotype in mutant t-complex

mice. Indeed, many PP1 regulatory subunits are controlled by reversible phosphorylation,

and phosphorylation can either strengthen or weaken the protein-protein interactions. For

example, following an elevation of cAMP, the PPP1R1/inhibitor 1 (I-1) and its neuronal

homologue, DARPP-32 (Dopamine and cAMP-regulated Phosphoprotein), are inhibitory

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to PP1 after phosphorylation by PKA. Conversely, they lose their inhibitory potency

toward PP1 if dephosphorylated by calcineurin following elevation of calcium levels.

The phosphorylation of SAP155 (Spliceosome-associated protein 155/Splicing

factor 3B subunit 1) is required for its binding to NIPP1 (nuclear inhibitor of protein phosphatase 1) (158). Conversely, the transient phosphorylation of PPP1R2/inhibitor 2 by

GSK3 reduces the inhibition of PP1 by PPP1R2, resulting in a proper folding and so activation of PP1. The inhibition of PP1 by NIPP1 can be cancelled by PKA-mediated

phosphorylation of NIPP1. Similarly, the binding of PP1 catalytic subunit to GM or

neurabin I can be decreased by phosphorylation within or close to the RVxF motif. So,

our observation of the in vitro phosphorylation difference between wt- and t/t-I3 is potentially important for understanding the role of t/t-I3 in the “curlicue”, and so the regulation of PP1γ2 activity in maintaining normal sperm functions. At this point, it is not known how the phosphorylation and so the difference in phosphorylation affect the ability of I3 interact and inhibit PP1. We hypothesize that less phosphorylation of t/t-I3

results in t/t-I3 being less inhibitory to PP1 and/or that phosphorylation may stabilize I3.

We indeed have preliminary data suggesting that t/t-I3 is less stable than wt-I3. Also of

great interest is the nature of interaction of wt and t-type another t-complex gene product

TSGA2 since it is known that the presence of these two genes alone in the t-complex result in a “curlicue ’ phenotype in more that 70% of the spermatozoa. The identity of the kinase that may phosphorylate I3 and how this phosphorylation alters its biochemical properties are therefore obvious questions great interest for future research.

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C. PPP1R2/inhibitor 2 (I2) and a novel I2 isoform are present in testis and

spermatozoa.

The number of cellular functions in which PP1 is involved is quite extensive

although rigorous biochemical definitions of these roles have not been achieved in a

majority of cases, including spermatozoa. As noted earlier, PP1 is composed of a highly

conserved catalytic subunit (37kDa) and a targeting/regulatory subunit. The cellular

activity of PP1 is important when it is targeted. i.e., the specificity of PP1 is achieved by

targeting PP1 to a restricted microenvironment. Determination of the cellular functions of

PP1γ2 in sperm is challenging, as they will likely be dictated by the properties and

localization of the individual targeting/regulatory subunits. So, identification and

characterization of its targeting/regulatory subunits, and the elucidation of their

biochemical properties are essential to understand the role of sperm PP1γ2. The first two

aims in this dissertation involved studies with I3 and its naturally occurring mutant

isoform in testis/sperm. The third aim in this dissertation was focused on demonstrating

the presence of I2 in spermatozoa and examining if a hypothetical gene on mouse

chromosome 17 is transcribed in vivo and translated in vitro as a protein similar to I2.

The mechanism of I2 interaction with PP1 includes either inactivation or inhibition

of its catalytic acitivity (15, 159, 160). The protein I2 functions as an inhibitor of free

PP1, i.e., it causes an immediate inhibition of phosphatase activity of PP1. However, a slower time-dependent reaction takes place that results in the conversion of PP1 to a form that can be stimulated by the addition of Mn2+ and protein kinase GSK-3.

Phosphorylation of the inactive PP1-I2 heterodimer by GSK-3 at Thr72 allows access of

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Mn2+ to PP1 that results in transient PP1 activation. Activated PP1 permits an intramolecular auto-dephosphorylation of I2, during which PP1 is converted to an independent and fully active enzyme. Following this there is a slow conversion of PP1

back to the inactive form. Thus, the phosphorylation of this complex is unusual in that it

only results in a transient activation of PP1. Inhibition of PP1 by I2 can be reversed by

trypsin digestion, which destroys I2 and hydrolyzes the C-terminus of PP1, thereby

releasing the fully active catalytic core of PP1. However, when in an inactivated state,

PP1 activity cannot be activated by trypsin, rather PP1 is proteolysed by trypsin.

The first suggestion of the presence of I2 in spermatozoa came from the observation

that immotile caput sperm contains six-fold higher GSK-3 activity compared caudal

epididymal sperm (95). Inhibition of PP1 by the heat-stable sperm extract showed I2-like

activity. This inhibition could be reversed with purified glycogen synthase kinase-3

(GSK-3). Sperm extracts containing an inactive complex of PP1-heat stable protein could

also be activated by purified GSK-3 (95). GSK-3 is significantly less phosphorylated (i.e.

more active) in immotile caput compared to motile caudal epididymidal spermatozoa (95,

119). Furthermore, an increase or decrease in motility causes a corresponding increase or decrease in tyrosine and serine phosphorylation of GSK-3 (161). It is likely that one of

the consequences of inactive GSK-3 (e.g. in caudal spermatozoa) is to lower PP1γ2

activity because I2 is likely to be unphosphorylated and, hence, able to bind to PP1γ2.

Therefore, low GSK-3 and PP1γ2 activities might be prerequisites for the optimum

function of spermatozoa. The above results suggest a biochemical mechanism for the

development and regulation of sperm motility through the PP1/I2/GSK-3 system.

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The inhibitor I2, one of the ancient regulatory subunits of PP1 and widely used to

study PP1, is known to be present in somatic tissues. This study, for the first time,

demonstrated the presence of I2 in sperm using an affinity purified peptide antibody.

Even though the interaction of I2 with PP1 in somatic tissues has been extensively

studied, there have been few studies of the interaction of I2 with PP1γ2, a unique isoform

of PP1 in testis/sperm. The study of how I2 interacts with PP1γ2 in sperm will contribute to our basic understanding of these proteins and the possible roles of these proteins in sperm function.

This study, by using affinity purified peptide I2 antibodies, showed the presence of

I2 in testis/sperm and using immunoprecitation demonstrated that I2 is bound to PP1γ2.

Additionally, I2 antibodies from different preparations recognized an immunoreactive

protein present in various tissues which migrated at 55kDa in SDS-PAGE. Interestingly,

recent annotation of mouse chromosome 17 identified a gene located immediately outside

of t-complex which could potentially encode a protein similar to I2, referred as I2(17) in

this study to be distinguished from I2 encoded in mouse chromosome 16 referred as

I2(16). The protein I2(16) consists of 206 amino acid residues, while I2(17) consists of

315 amino acid residues. In addition to the C terminus which shares 96% amino acid

sequence identity with I2(16), I2(17) protein contains an extra N terminus composed of

109 amino acid residues. Intriguingly, the N-terminus also has a PP1 binding domain. It

has been previously argued that this 55kDa isoform may be a cross-reaction or an un-

dissociated complex of I2 and PP1 (162, 163). To test whether this intronless gene,

I2(17), can be transcribed and translated, Northern blot and RT-PCR were used.

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Examination of I2(17) gene demonstrates that of the 327bp encoding the N terminus of

I2(17), only the first 70bp from 5’ end is specific to mouse chromosome 17, and the rest

257bp of N-terminus is also present in the untranslated 5’-flanking region immediately upstream of the start codon of I2(16) gene. To ensure that the RT-PCR results are specific to the transcript from mouse chromosome 17, a downstream primer for PCR was designed against a region within the first 70bp from 5’ end of I2(17). As demonstrated in the Results section, the corresponding PCR product is ubiquitously present in multiple tissues, which suggested that I2(17) gene can indeed be transcribed in vivo.

A multi-tissue Northern blot probed with N-terminus of I2(17) gene demonstrated a single positive band only in testis, which indicated the presence of I2(17) RNA, while

Northern blot of testis RNA probed with C-terminus of I2(17) gene showed one extra band in testis in addition to the two expected bands at 1.7kb and 2.7kb in various tissues.

These Northern blot results with the C-terminus probe are consistent with that reported from Dr. Anna A. DePaoli-Roach’s lab (25). They showed that two transcripts, 1.7kb and

2.7kb, are present in all tissues including testis, but an extra third band is only present in testis. In contrast to the PCR data, the results of Northern blots suggested I2(17) is transcribed only in testis. This difference may be due to the differing sensitivities of the two methods.

To test whether I2(17) can be translated, the full length coding region of I2(17) gene was inserted into pRSET vector producing a His-tagged recombinant protein in

E.coli. The purified recombinant I2(17) protein was separated by SDS-PAGE followed by immunoblotting with anti-His and anti-I2 antibodies, respectively. Both antibodies

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recognized an immunoreactive band at about 55-57 kDa, while the calculated molecular weight of I2(17) is 35kDa. It has been known that I2(16) migrates anomalously on SDS-

PAGE at 32-34 kDa as compared to its calculated molecular weight of 23kDa. So the immunoreactive protein recognized by both anti-His and anti-I2 antibodies is likely to be the protein encoded by I2(17) gene. In summary, results from PCR of cDNAs, Northern blot, and in vitro expression study indicated that the hypothetical gene on mouse chromosome 17 is transcribed in vivo and quite likely also translated. Based on the above results, I hypothesize that the high molecular weight isoforms of I2, I2(17), may be converted to a lower molecular weight isoform of I2, I2(16), during sperm capacitation by its progressive degradation, thus promoting the onset and maintenance of sperm motility and hyperactivation, in part through inhibition of PP1γ2. For these biochemical and mechanistic studies a specific antibody is required which may be generated using the unique N-terminus region as an immunogen.

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In Summary, the major findings of this dissertation are as follows:

1. The protein phosphatase inhibitor PPP1R11/inhibitor 3 (I3) was found in

testis and spermatozoa.

2. The I3 mRNA abundance is not indicative of its steady state protein level in

testis.

3. I3 binds to testis/sperm-expressed PP1γ2 and Sds22 in vitro and in vivo as

shown by GST pulldown assay and immunoprecipitation.

4. Column purification of the complex, PP1γ2/I3/Sds22, in vivo combined with

immunoprecipitation and native gel electrophoresis of the purified fractions

confirmed I3, PP1γ2 and Sds22 forming a complex in vivo,

5. Microsequencing of the protein band containing PPγ2, Sds22 and I3 following

native gel electrophoresis was conducted to determine other protein components

of the complex.

6. Actin is the fourth member of PP1γ2/Sds22/I3 complex.

7. Nucleobindin, thioredoxin-like 2, and ubiquitin-activating enzyme E1 are not

part of the complex formed by PP1γ2/Sds22/I3 as shown by immunoprecipitation.

8. The formation of PP1γ2/Sds22/I3 complex is not specific to the PP1γ2 isoform

as shown by column purifications of testis from PP1γ knockout mice and mouse

brain extracts combined with immunoprecipitation and native gel electrophoresis

of column fractions.

9. Actin is also the fourth member of PP1γ1/Sds22/I3 and PP1β/Sds22/I3

complexes.

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10. PP1γ2 is catalytically inactive in a purified complex containing I3, and Sds22.

11. The mutual increasing abundance of I3, PP1γ2, and Sds22 in the testis from

puberty to adulthood most closely reflects the progression of male germ cell

morphogenesis.

12. The expression level and cellular localizations of I3 rather than Sds22 seem

highly linked to those of PP1γ2.

13. The macromolecular complex formed by Sds22/PP1γ2/I3/actin could not

bind to microcystin agarose.

14. t/t-I3 is also heat-stable and migrates anomalously on SDS-PAGE.

15. Both wt- and t/t-I3 have similar potencies in inhibiting catalytic activity of

PP1γ2.

16. t/t-I3 also can form a complex with PP1γ2 and Sds22 both in vitro and in vivo

as shown by GST pulldown assay and immunoprecipitation.

17. Column purification of the complex, t/t-I3/PP1γ2/Sds22, from testis protein

extracts combined with native PAGE further confirmed the existence of the

complex in vivo.

18. In vitro phosphorylation study of wt- and t/t-I3 found that GST-t/t-I3 showed

significantly greater phosphorylation than GST-wt-I3 with sperm extracts used as

a kinase source.

19. Verified the existence of inhibitor I2 in testis and sperm.

20. I2 from sperm and testis protein extracts is bound to PP1γ2 in vivo as shown

by immunoprecipitation.

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21. Evidence was provided that a gene on mouse chromosome 17 encodes a novel

protein similar to I2 were provided by the results of Northern blot, RT-PCR and

expression studies in vitro.

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