Archives of Andrology: Journal of Reproductive Systems, 53:169177, 2007 Copyright # Informa Healthcare USA, Inc.

ISSN: 0148-5016 print=1521-0375 online DOI: 10.1080/01485010701314032

Review and Hypothesis

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Role(s) of the Serine=Threonine Protein Phosphatase 1 on Mammalian Sperm Motility

Yibing Han and Christopher J. Haines Department of Obstetrics and Gynecology, Prince of Wales Hospital, Chinese University of Hong Kong, Hong Kong, SAR, China Huai L. Feng Center for Human Reproduction, North Shore University Hospital, NYU School of Medicine, New York, USA

Abbreviations: PPs: Protein serine=threonine phosphatases, PP1: Protein phosphatase 1, PIPs: PP1 interacting proteins, cAMP: Cyclic adenosine monophosphate, AKAP: A kinase anchor protein, CAMKII: Calmodulin kinase II, cdKs: Cyclin-dependent kinases, PKA: Protein kinase A, NLS: Nuclear localization signals, NTS: Nucleolar targeting signals, FSH: Follicular stimulating hormone, Spz1: Spermatogenic zip protein 1, GSK: Glycogen synthase kinase 3, DNA: Deoxyribonucleic acid, SARP: Several ankyrin repeat protein, ACE: Angiotensin-converting enzyme, Tesk: Testis-specific protein kinase, ADF: Actin-depolymerizing factor, MOs: Membranous organelles, ATP: Adenosin triphosphate Received 31 December 2006; accepted 26 February 2007. Address correspondence to Yibing Han, Ph.D., Department of Obstetrics and Gynecology, Prince of Wales Hospital, Chinese University of Hong Kong, Hong Kong, SAR, China. E-mail: ybhan@cuhk.edu.hk

Mammalian spermatozoa acquire the capacity for motility and fertilization during the transit through the epididymis under the control of different factors, such as cAMP, intracellular pH, intracellular calcium and phosphorylation of sperm proteins. As the acquisition of functional competence including gaining motility during epididymal transit occurs in the complete absence of contemporaneous gene transcription and translation on the part of the spermatozoa, it is widely accepted that post-translational modifications are the only means by which spermatozoa can acquire functionality. Serine-threonine protein phosphatase 1 (PP1) together with their testis= sperm-specific interacting proteins might be involved in this regulatory mechanism. PP1a, PP1b=d, PP1c1 and PP1c2 are all expressed in the testis whereas PP1c2 is the only isoform expressed on spermatozoa. I2, I3, sds22, 14-3-3 and hsp90 are associated with PP1c2 in spermatozoa located on the sperm head and tail. Activity of PP1c2 and the binding pattern to these regulatory proteins changes in spermatozoa recruited from the caput and those from the cauda part of the epididymis. In this review, we summarize the possible roles of PP1 on spermatozoa during spermatogenesis and flagellar motility control. We suggest that PP1 might take part in the inhibition of the sperm motility activation by interacting with AKAPs and CAMKII. A hypothesized signaling pathway of mammalian sperm motility activation and PP1s function has been proposed.
KEYWORDS motility activation, serine=threonine protein phosphatase, sperm, spermatogenesis

INTRODUCTION
Phosphorylation of proteins is a post-translational modification event that acts as one of the cells regulatory mechanisms to control various processes. Given that up to 30% of proteins undergone reversible phosphorylation in eukaryotic cells, it is estimated that protein phosphorylation may influence almost all stages of sperm development in the testis and the epididymis,
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from meiotic division to sperm maturation. In eukaryotic cells, one of the most common mechanisms for regulating protein activity is the addition and=or removal of phosphate groups from serine, threonine or tyrosine residues of protein moieties. Addition or removal of phosphate can induce allosteric modifications resulting in conformational changes in proteins leading either to their activation or inactivation. These processes are regulated by both protein kinases and protein phosphatases. In contrast to protein kinases that add a phosphate group to the hydroxyl group of serine, threonine or tyrosine residues, phosphatases remove it. In mammalian spermatozoa, the ability to actively swim is acquired during the transit through the epididymis under the control of different factors, such as cAMP, intracellular pH, intracellular calcium and phosphorylation of sperm proteins. As the acquisition of functional competence including gaining motility during epididymal transit occurs in the complete absence of contemporaneous gene transcription and translation on the part of the spermatozoa, it is widely accepted that post-translational modifications are the only means by which spermatozoa can become competent.

PP1 Isoforms and their Interacting Proteins (PIPs)

The protein serine=threonine phosphatases (PPs) specifically hydrolyze serine=threonine phosphoesters, and are metalloproteins with extremely diverse and unrelated functions [Cohen 2002]. As a holoenzyme, the PPs have catalytic subunits and regulatory subunits. The catalytic subunits are divided into four major groups, including protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), protein phosphatase 2B (PP2B) and protein phosphatase 2C (PP2C). In mammals, there are four homologous isoforms of type 1 serine=threonine protein phosphatase (PP1a, PP1b=d, PP1c1 and PP1c2) [Bollen and Stalmans 1992]. These isoforms share > 89% identity and are encoded by three distinct genes, with PP1c1 and PP1c2 produced from the alternative splicing of the same primary transcript. The isoforms of PP1 vary in sequence at their extreme amino and carboxyl termini. Functions of PP1 include controlling metabolism, cell division, apoptosis and protein synthesis
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by dephosphorylation of key regulatory proteins [Cohen 2002; Bollen 2001; Ceulemans et al. 2002; Ceulemans and Bollen, 2004). All PP1s contain a Thr-Pro-Pro-Arg (TPPR) amino acid sequence segment at their carboxyl terminal, which is a consensus sequence for phosphorylation by cyclin-dependent kinases (Cdks). Phosphorylation of PP1 by Cdk1 and Cdk2 in somatic cells reduces the catalytic activity of this enzyme [Cohen 2002; Dohadwala et al. 1994; Kwon et al. 1997; Liu et al. 1999]. PP1s do not exist freely in the cell but are associated with a large variety of polypeptides that determine when and where PP1 acts. These PIPs (also called regulatory subunits) function as activity-modulators, targeting subunits and=or substrates. Hormones, growth factors and metabolites control the function of the PP1 holoenzymes mainly by modulating the interaction of the subunits. The available information suggests that these PIPs interact with PP1 via multiple, short-sequence motifs. The PIPs are structurally quite different, but almost all have a consensus binding motif (RK)-x0-1(VI)-{P}-(FW), where x denotes any residue and {P} any residue except Pro, and it is often called simply the RVxF-motif. The RVxF-motif binds to a hydrophobic channel near the C-terminus of PP1. The binding of the RVxF-motif not only has a PP1 anchoring function but also promotes the interaction of secondary, lower-affinity binding sites, often resulting in an altered activity and=or substrate specificity of PP1. The F-x-x-(RK)-x-(RK) motif represents a new consensus sequence for the recognition and binding of some Bcl-2 proteins to PP1 [Ayllon et al. 2002]. Currently, about 70 mammalian genes coding for more than 60 PIPs have been identified [GarciaGimeno et al. 2003]. Given there are approximately 10,000 phosphoproteins in mammals, many PIPs remain to be discovered. PIPs can be classified into mainly 8 categories of: glycogen metabolism, myofibriella, nuclear, endoplasmic-ribosomal, plasma membrane=cytoskeleton centrosome=microtubule, apoptosis and specific substrates and inhibitors. In addition to inhibitor-1 (I1) and inhibitor-2 (I2) representing two different ways of inhibiting PP1 phosphatase activity, there are also other protein phosphatase inhibitors without a clear mechanism [Garcia-Gimeno et al. 2003; Zhang et al. 1998;
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Hrabchak and Varmuza 2004; Mishra et al. 2003; Vijayaraghavan et al. 1996; Smith et al. 1996]. I1 activity is regulated by cAMP-dependent phosphorylation of a single threonine residue by protein kinase A (PKA) and by calcium=calmodulin-dependent dephosphorylation of the same residue [Shenolikar and Nairn 1991; Cohen 1989]. I2 binds to the catalytic subunit of PP1 to form an inactive cytoplasmic form of the enzyme (PP1I2) that can be converted to active PP1 by phosphorylation of the bound I2 by glycogen synthase kinase-3 [Bollen and Stalmans 1992; Cohen 1989; Hemmings et al. 1982]. In spermatozoa of mice, a novel PP1 inhibitor 3 (I3) containing both the RVxF-motif, nuclear localization signals (NLS) and nuclear targeting signals (NTS) has been discovered [Huang et al. 2005a; Han et al. 2007]. I1, I2 and I3 are heat-stable proteins all enriched in proline [Zhang et al. 1998]. It has been shown that PP1s nuclear translocation and nuclear retention depend on binding to RVxF-motif interactors [Lesage et al. 2004]. The PP1 nuclear interactors include PNUTS, Sds22, NIPP and SIPP1 that have both the RVxF-motif and NLS.

diffuse pool and in a few as-yet-unidentified foci [Twinkle-Mulcahy et al. 2006]. In addition to the nucleus, PP1 is also found within the axoneme. PP1c is anchored in the central pair apparatus of the axoneme in Chlamydomonas flagellar [Yang et al. 2000]. Ciliary and flagellar motility is controlled by phosphorylation [Brokaw 1987; Satir et al. 1993; Tash and Bracho 1994]. PP1 may be involved in the regulation of flagellar motility together with protein kinases [San Agustin and Witman 1994; Chaudhry et al. 1995]. Recently, PP1 was found to be involved in regulating the acquisition of motility [Huang et al. 2004; Huang et al. 2004b; Huang et al. 2005b].

Function and Expression of PP1/PIPs in the Testis, Epididymis and Spermatozoa

In the Testis
PP1a, PP1b and PP1c are all expressed in the testis [Tash and Bracho 1994]. Higher levels of PP1a in condensing spermatids and lower levels in other germ cell stages have been found. While PP1c1 is ubiquitously expressed, PP1c2 is conserved and expressed in the testis, spermatozoa and brain [Andreassen et al. 1998; Smith et al. 1996; Huang et al. 2002; Kitagawa et al. 1990]. PP1c2 is expressed in the nuclei of germ cells from the pachytene spermatocyte stage through the early spermatid stages, and in the spermatozoa head and flagella [Shima et al. 1993; Huang et al. 2004a; Huang et al. 2004b; Huang et al. 2005b]. The function of PP1c has been confirmed by PP1c= knock-out mice [Varmuza et al. 1999]. Males homozygous for a null mutation of PP1c gene are sterile, and display both germ cell and Sertoli cell defects. Histopathology from PP1c mutants indicates a more complex role for this protein in spermatogenesis. More than one defect is observed in mutant mice. The defects include elevated serum FSH [Oppedisano-Wells et al. 2002], increased DNA fragmentation in germ cells [Jurisicova et al. 1999] and increased aneuploidy in haploid gametes [Oppedisano-Wells et al. 2002]. Loss of spermatids begins at the round spermatid stage and increases in severity such that there is a marked reduction in elongating and condensing spermatids and an almost complete absence of mature sperm. Meiosis may be
Roles of Serine/Threonine Protein Phosphatase 1

Expression Localization and Possible Functions of PP1 Isoforms

PP1 is expressed in various cellular compartments but is most abundant in the nucleus. PP1a, PP1c and PP1b are closely related isoforms with distinct localization patterns. In somatic cells, PP1a, PP1c and PP1b are primarily located in the nucleus. PP1a is mainly attached to the nuclear matrix, while PP1c is predominantly found in the nucleoli. PP1b is present in the non-nucleolar chromatin fraction and the nucleoli [Andreassen et al. 1998; Twinkle-Mulcahy et al. 2001; Twinkle-Mulcahy et al. 2003]. Using the fluorescent fusion proteins, isoforms of PP1 have been delicately located in mammalian cells cultured in vitro. During interphase, PP1c was found in both cytoplasmic and nucleoplasmic pools, showing a prominent accumulation within nucleoli, targeting to kinetochores and chromatin. This implicates PP1c in multiple regulatory pathways, in agreement with previous studies linking its activity to the regulation of transcription, chromatin remodeling, chromosome condensation and segregation, cytokinesis, and reassembly of the nuclear envelope. PP1a is largely excluded from the nucleoli found mainly in a
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disrupted giving rise to polyploid spermatids. Interestingly, histones remain complexed with spermatid chromatin beyond when they are normally removed and replaced by protamines. Normal staging is disrupted in PP1 mutants. Some cell types are reduced in number, but none are absolutely missing [Varmuza et al. 1999]. A chimeric testis with wild type of Sertoli cells and PP1c= spermatids revealed intermediate phenotypes when compared with PP1c= . They did not sire pups derived from mutant germ cells, suggesting that expression of PP1c2 genes may be restricted to the spermatozoon [Oppedisano-Wells and Varmuza 2003]. Spermatogenic zip protein 1 (Spz1) has been identified as binding to the PP1c2 splice variant in the mouse testis [Hrabchak and Varmuza 2004]. Spz1 was a member of the basic helix-loop-helix family of transcription factors. Overexpression of spz1 and loss of PP1 c in the testis show similar phenotypes such as spermatogenic arrest and germ cell apoptosis [Hsu et al. 2004].

In the Epididymis and Spermatozoa

Human and primate sperm extracts contain PP activity that might be from PP1c2 or PP2A [Smith et al. 1996], whereas sea urchin [Swarup and Garbers 1992; Tash et al. 1988], caprine [Barua et al. 1985], canine and porcine [Tash et al. 1988] sperm extracts primarily contain PP2B (Calcineurin) activity. In bovine sperm extracts contain both PP2B and PP1 activity [Tash et al. 1988; Hrabchak and Varmuza 2004; Mishra et al. 2003; Huang et al. 2002; Huang et al. 2004a,b; Huang et al. 2005b; Varmuza et al. 1999; Oppedisano-Wells and Varmuza 2003; Tang and Hoskins 1975]. In human and primate spermatozoa, the heat-stable specific inhibitor of PP1 is neither I1 nor I2 [Smith et al. 1996]. The I2-like inhibitor is antagonized by the addition of Glucogen synthase kinase 3 (GSK-3) but the significance is not clear. A new PP1 inhibitor I3 has been identified in mouse spermatozoa [Han et al. 2007]. Sds22, 14-3-3 protein and hsp90 are potential regulators of PP1c2. PP1c2 may also regulate epididymal sperm motility [Hrabchak and Varmuza 2004; Mishra et al. 2003; Huang et al. 2004a,b; Huang et al. 2005b; Huang et al. 2002; Shima et al. 1993; Jurisicova et al. 1999]. Phosphorylation of PP1c2 increases exhibiting decreased activity during sperm maturation as
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motility of spermatozoa increase. Three pools of PP1c2 in caudal and caput epididymal spermatozoa are found. The caput pool includes the active form of PP1c2, phosphorylated and an active form of 14-3-3 binding PP1 c2 and the inactive form of hsp90 binding PP1 c2. The cauda pool includes the inactive form of sds22 binding PP1c2 [Mishra et al. 2003], phosphorylated and an active form of 14-3-3 binding PP1c2 and an inactive form of hsp90 binding PP1c2 [Huang et al. 2004a,b]. PP1c2 is inactivated by binding to sds22 and hsp90 while PP1c2 is activated and phosphorylated by binding to 14-3-3 protein [Huang et al. 2004a; Huang et al. 2002]. Sds22 is a mammalian homologue of yeast PP1 binding protein [Huang et al. 2002] belonging to a family of proteins that contain repeats of leucine-rich 22 amino acid segment. The 14-3-3 proteins belong to a family of abundant and widely expressed 2833 kDa acidic polypeptides that spontaneously self-assemble as dimmers. The 14-3-3 proteins bind to phosphoserine= threonine containing motifs in a sequencespecific manner [Yaffe and Elia 2001; Aitken et al. 1992; Tzivion and Avruch 2002]. The Hsp90 is a highly conserved ATP-dependent chaperone [Richter and Buchner 2001] and this protein is subject to tyrosine-phosphorylation during sperm capacitation in mice, rats and humans (Ecroyd et al. 2003). Cytosolic PP1 and GSK-3 activities appear to be inversely related to the motility of monkey epididymal sperm [Smith et al. 1996]. Higher concentration of GSK-3 and PP1 are present in immotile bovine caput epididymal sperm compated with motile cauda epididymal sperm, and may control motility [Vijayaraghavan et al. 1996; Miki et al. 2004; Somanath et al. 2004].

Some Other Estimated PIPs from Database Expressed in the Testis and/or Spermatozoa
As mentioned above, two distinct docking consensus motifs (RK)-x0-1-(VI)-{P}-(FW) and F-x-x-(RK)-x(RK) have been identified in PIPs. The PIPs database at http://pp1signature.pasteur.fr/ includes all discovered proteins containing the two motifs. These include transcription factors, transport proteins as well as kinases. A putative transcription factor or DNA-associated protein called SARP (several ankyrin repeat protein) has been identified as a PIP. SARP has 3 splice
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variants, SARP1, SARP2 and SARP3. SARP1 and=or SARP2 are expressed at high levels in testis and spermatozoa, where they are shown to interact with both PP1c1 and PP1c2. SARP is highly abundant in the nucleus of mammalian cells, consistent with the putative nuclear localization signal at the N-terminus. The presence of a lucine zipper near the C-terminus of SARP1 and SARP2, and the binding of mammalian DNA to SARP2, suggests that SARP1 and SARP2 may be transcription factors or DNA-associated proteins that modulate gene expression [Browne et al. 2007]. Angiotensin-converting enzyme (ACE) is a zinccontaining dipeptidyl carboxypeptidase widely distributed in mammalian tissues and is thought to play a critical role in blood pressure regulation. Testis contain a unique androgen-dependent ACE isozyme, ACE-T, that is initially found in post-meiotic step 3 spermatids and then increases markedly during differentiation. ACE-T is strictly confined to the adluminal membrane face of elongating spermatids and localizing to the neck and midpiece region of released and ejaculated spermatozoa [Pauls et al. 2003]. Male mice homozygous for a disrupted ACE gene are almost infertile, despite showing normal mating behavior, testis histology and sperm parameters Reduced oviduct transport and zona pellucida binding of spermatozoa is observed [Krege et al. 1995; Hagaman et al. 1998]. It is estimated that the unique N-terminal of ACE-T bearing specific binding properties for an oviduct=ovum substrate may yield male sterility [Kessler et al. 2000]. Another putative PIP is testis-specific protein kinase (Tesk) 2 which is a member of the Tesk family with 48% amino acid identity with Tesk1. Tesk2, is predominantly expressed in the nucleus of Sertoli cells. It phosphorylates cofilin=ADF (actindepolymerizing factor) at Ser3 that induces actin reorganization. Since actin-depolymerizing and actin-severing activities of cofilin=ADF are abrogated by phosphorylation at Ser3, TESK2 seems to play an important role in actin filament dynamics by inhibiting cofilin=ADF activity [Toshima et al. 2001]. Fer-1, first discovered in Caenorhabditis elegans is another putative PIP that is mainly expressed in spermatocytes. It is prevalent when membranous organelles (MOs) fuse with the spermatid plasma membrane. Resembling some viral fusion proteins, fer-1 may play a direct role in MO-plasma membrane fusion [Achanzar and Ward 1997].
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Mechanisms of Spermatozoa Flagellar Motility Control

The force for flagellar movement is exerted through the sliding of outer-doublet microtubules. ATP is required to support coordinated movement of the central axoneme and surrounding flagellar structures [Mann and Lutwak-Mann 1981]. This is driven by dynein molecular motors [Inaba 2003] i.e., the dynein ATPase. Several studies have revealed that ciliary and flagellar motility is controlled by phosphorylation [Brokaw 1987; Satir et al. 1993; Tash and Brocho 1994; Chaudhry et al. 1995]. In vitro analysis has indicated that cAMP-dependent protein Skinase A, phosphatases PP1 and PP2A anchored in the axoneme are likely involved [Yang et al. 2000; an Agustin and Witman 1994; Chaudhry et al. 1995; Huang et al. 1982; Porter et al. 1992; Piperno et al. 1992; Piperno et al. 1994; Yoshimura and Shingyoji 1999]. Spermatozoa are stored in extratesticular ducts in an immotile state in many animals and their motility is activated on their release from the ducts. Activation of motility is inhibited by factors in the extratesticular plasma. Mammalian spermatozoa from the distal part of the epididymis show better motility activation than that from the proximal part of the epididymis as they acquire the capacity for motility and fertilization during epididymal transit. During this passage through the epididymis, changes are observed in the intracellular second messengerscAMP, pH, calcium and intra-sperm ATP content [Bedford and Hoskins 1990; Cooper 1986]. The relation of sperm motility activation and other morphological=physiological change(s) of sperm during spermiogenesis are still unclear. But, it is known that the potential for motility already exists in both immature testicular and epididymal sperm as evidenced by the ability of demembranated immature sperm to undergo motility activation [Mohri and Yanagimachi 1980; Yeung 1984]. Sperm motility in immature spermatozoa can be initiated by stimulating protein kinase activity or inhibiting protein phosphatase activity [Vijayaraghavan et al. 1996; Smith et al. 1996; Smith et al. 1999]. So, rather than acquiring the capacity for motility, spermatozoa in the epididymis might make themselves more sensitive to stimulating factors by gradually expelling inhibitors or by other unknown mechanisms. One possible signal is phosphorylation=dephosphorylation. Low protein kinase
Roles of Serine/Threonine Protein Phosphatase 1

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and high protein phosphatase activities most likely limit motility in immature spermatozoa. In a number of species, development of sperm motility in the epididymis is associated with increased intrasperm cAMP [Amann et al. 1982; Brandt and Hoskins 1980], associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity [Vijayaraghavan et al. 1996]. It is noted that the control factors of phosphatase to sperm motility might be confined to the axoneme [Habermacher and Sale 1996]. This is supported by the observation of the gain of motility after the addition of protein phosphatase 1 inhibitors to demembraned fowl spermatozoa [Ashizawa et al. 1994]. AKAPs [A Kinase Anchor Protein, cAMP-dependent protein kinase anchoring proteins, Miki and Eddy 1998] are the candidate target of PP1 in sperm flagella. AKAPs assemble multi-enzyme signaling complexes in proximity to substrate effector proteins, thus directing and amplifying membranegenerated signals. They form a transduceosome, an autonomous multivalent scaffold that assembles and integrates signals derived from multiple pathways. The AKAP family shares little overall primary sequence similarity excluding their functional domains including the targeting domain (as a scaffold and membrane anchor) and the amphipathic helical tethering domain (binding to regulatory subunits) that are highly conserved. In addition to binding to cAMP-dependent protein kinases such as protein kinase A, some AKAPs associate with protein kinase C and Ser=Thr phosphates. It has been shown that the amino terminus of AKAP121, a potential PIP, interacts with mitochondrial membranes and with tubulin. Tubulin, a heterodimer composed of two similar acidic isoforms (a and b) participates in the organization of eukaryotic microtubule networks. AKAP=PKA or AKAP=PP1 complexes anchored to spindles might regulate the dynamic assembly of microtubules by creating target sites of cAMP action [Cardone et al. 2002]. In contrast to AKAP121, the candidacy of AKAP4 as PIP is additionally experimentally supported. AKAP4 is the major fibrous sheath protein located in the principal piece of spermatozoa. It serves as a scaffold for proteins in signaling pathways involved in sperm maturation, motility, capacitation, hyperactivation and glycolysis. In the principal piece, the fibrous sheath replaces the
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mitochondria sheath. Outer dense fibers 3 and 8 are also substituted by the two longitudinal columns of the fibrous sheath. AKAP4 recruits PKA to the fibrous sheath and facilitates local phosphorylation to regulate flagellum function. Spermatozoa from AKAP4= mice lack motility and are infertile [Miki et al. 2002]. PP1 activity is decreased in the AKAP4= mice and might indicate the functional linkage between PP1 and AKAP4 [Huang et al. 2005b].

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A Hypothesis for the Signaling Pathways of Mammalian Spermatozoa Motility Activation

Both the cAMP and Ca2 signal transduction pathways are involved in activation of motility in immotile spermatozoa from the cauda epididymis in rat and mouse [Wade et al. 2003; Schulh et al. 2006]. A Ca2 dependent increase in cAMP initiates a signal transduction cascade for motility activation, which is independent of protein kinase A-mediated phosphorylation of flagellar proteins in immotile rat spermatozoa [Wade et al. 2003]. The concentration of cAMP increases with activation of motility in spermatozoa from the cauda epididymis of hamsters [Morton et al. 1974] and rats [Armstrong et al. 1994]. So, cAMP seems to be the first signal for sperm motility activation. cAMP-dependent protein kinase (PKA), the major downstream effector of cAMP signals in sperm, is then activated and through the AKAPs triggers protein phosphorylation that might be important for sperm motility [Si and Okuno 1995; Nolan et al. 2004]. Calmodulin Kinase II (CAMKII) is considered to be a ubiquitous protein mediating Cai2 signaling the activation of dynein ATPase in mammalian sperm [Hsu et al. 2004], and CAMKII-PP1 complex is proven to act together as a simple device in the Ca2 signal transduction in the synapses [Bradshaw et al. 2002]. CAMKII can be activated in a persistent manner by autophospholation at Thr286. When dephosphorylated at Thr286 by PP1, CaMKII is deactivated [Nomura et al. 2004]. Considering activation of the the sperm motility mechaninism and PP1s=PIPs function during spermatogenesis we propose the following twostep signaling pathway of PP1s in controlling spermatozoa motility as summarized in Figure 1. In the
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FIGURE 1 cAMP produced by adenylyl cyclase from ATP stimulates the activity of PKA. AKAPs then link the active PKA to both the microtubules of the spermatozoa flagella and the membrane of the mitochondria. PKA, now in the proximity of the flagella, then stimulates the microtubule local factors like dynein ATPase by creating target sites of cAMP and/or other unknown functions and makes the microtubules conditioned with the ability to respond to Ca2 calmodulin stimulation. AKAPs linked to the mitochondria membrane might stimulate production of more ATP. PP1 is involved in this pathway by interacting with AKAPs. We hypothesize that the binding of PP1 to the AKAPs might competitively inhibit their binding to PKA. Before binding to AKAPs, the microtubules are not sensitive to stimulating signals in the absence of key factors. In contrast, the Ca2 CamKII complex could initiate the motility of the conditioned microtubules by immediately activating dynein ATPase. Motility is maintained by the continuous consumption of ATP. PP1 could dephosphorylate and inactivate the CAMKII at Thr286. In both pathways, a decrease in the concentration of PP1 would favor the activation of sperm motility.

first step, motionless spermatozoa in the caput of the epididymis need to be conditioned through the AKAP signal transduction pathway which prepares the microtubules of the spermatozoa to a ready state for motility activation. In the second step, motility of the conditioned spermatozoa is then triggered by Ca2 -CAMKII signal transduction pathway with a functional dynein ATPase. PP1s are involved in the two signal transduction pathways by interacting with AKAPs and regulating activity of CAMKII, respectively.

REFERENCES
Achanzar, W. E. and Ward, S. (1997) A nematode gene required for sperm vesicle fusion. J. of Cell Sci 110:10731081. Aitken, A., Collinge, D. B., van Heusden, B. P., Isobe, T., Roseboom, P. H., Rosenfeld, G. and Soll, J. (1992) 14-3-3 proteins: A highly conserved, widespread family of eukaryotic proteins. Treads Biochem Sci 17: 498501. Amann, R. P., Hay, S. R. and Hammerstedt, R. H. (1982) Yield, characteristics, motility and cAMP content of sperm isolated from seven regions of ram epididymis. Biol Reprod 27:723733. Andreassen, P. R., Lacroix, F. B., Villa-Moruzzi, E. and Margolis, R. L. (1998) Differential subcellular localization of protein phosphatase-1 alpha, gamma1, and delta isoforms during both interphase and mitosis in mammalian cells. J Cell Biol 141:12071215. Armstrong, V. L., Clulow, J., Murdoch, R. N. and Jones, R. C. (1994) Intracellular signal transduction mechanisms of rat epididymal spermatozoa and their relationship to motility and metabolism. Mol Reprod Dev 38:7784. Ashizawa, K., Wishart, G. J., Tomonaga, H., Nishinakama, K. and Tsuzuki, Y. (1994) Presence of protein phosphatase type 1 and its involvement in temperature-dependent flagellar movement of fowl spermatozoa. FEBS Letters 350:130134.

ACKNOWLEDGMENT
This work was supported by a direct grant of the Chinese University of Hong Kong c001-2041219.
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Ayllon, C., Cayla, X., Garcia, A., Fleischer, A. and Rebollo, A. (2002) The antiapoptotic molecules Bcl-xL and Bcl-w target protein phosphatase 1 alpha to Bad. Eur J Immunol 32:18471855. Barua, M., Bhattacharyya, U. and Majumder, G. C. (1985) Occurrence of an ecto-phosphoprotein phosphatase in goat epididymal spermatozoa. Biochem Int 10:733741. Bedford, J. M. and Hoskins, D. D. (1990) The mammalian spermatozoa. In: Marshalls physiology of reproduction. Edinburgh: Churchill Livingstone, UK. Bollen, M. and Stalmans, W. (1992) The structure, role and regulation of type 1 protein phosphatase. Critical reviews in Biochemistry and molecular biology 27:227281. Bollen, M. (2001) Combinatorial control of protein phosphatase-1. Trends In Biochemical Sciences 26:426431. Bradshaw, J. M., Kubota, Y., Meyer, T. and Schulman, H. (2002) An ultrasensitive Ca2 =calmodulin-dependent protein kinase II-protein phosphatase 1 switch facilitates specificity in postsynaptic calcium signaling. Proc Natl Acad Sci USA 100:1051210517. Brandt, H. and Hoskins, D. D. (1980) A cAMP-dependent phosphorylated motility protein in bovine epididymal sperm. J Biol Chem 255:982987. Brokaw, C. J. (1987) Regulation of sperm flagellar motility by calcium and cAMP-dependent phosphorylation. J Cell Biochem 35:175184. Browne, G. J., Fardilha, M., Oxenham, S. K., Wu, W., Helps, N. R., da Cruz, E., Silva, O. A., Cohen, P. T. W., da Cruz, E. and Silva, E. F. (2007) SARP, a new alternatively spliced protein phosphatase 1 and DNA interacting protein. Biochem J 402:187196. Cardone, L., de Cristofaro, T., Affaitati, A., Garbi, C., Ginsberg, M. D., Saviano, M., Varrone, S., Rubin, C. S., Gottesman, M. E., Avvedimento, E. V. and Feliciello, A. (2002) A-Kinase anchor protein 84=121 are targeted to mitochondria and mitotic spindles by overlapping amino-terminal motifs. J of Mol Biol 320:663675. Ceulemans, H., Vulsteke, V., De Maeyer, M., Tatchell, K., Stalmans, W. and Bollen, M. (2002) Binding of the concave surface of the Sds22 superhelix to the alpha 4=alpha 5=alpha 6-triangle of protein phosphatase-1. J of Biological Chemistry 277:4733147337. Ceulemans, H. and Bollen, M. (2004) Functional diversity of protein phosphatase-1, a cellular economizer and reset button. Physiological Review 84:139. Chaudhry, P. S., Creagh, S., Yu, N. and Brokaw, C. J. (1995) Multiple protein kinase activities required for activation of sperm flagellar motility. Cell Motility and the Cytoskeleton 32:6579. Cohen, P. (1989) The structure and regulation of protein phosphatases. Annu Rev Biochem. 58:453508. Cohen, P. T. (2002) Protein phosphatase 1-targeted in many directions. J of Cell Science 15:241256. Cooper, T. G. (1986). The epididymis, sperm maturation and fertilization, Berlin, Germany: Heidelberg Springer-Verlag. Dohadwala, M., da Cruz E Silva, E. F., Hall, F. L., Williams, R. T., Carbonaro-Hall, D. A., Nairn, A. C., Greengard, P. and Berndt, N. (1994) Phosphorylation and inactivation of protein phosphatase 1 by cyclin-dependent kinases. Proc Natl Acad Sci USA 91:64086412. Ecroyd, H., Jones, R. C. and Aitken, R. J. (2003) Tyrosine phosphorylation of HSP-90 during mammalian sperm capacitation. Biol Reprod 69:18011807. Garcia-Gimeno, M. A., Munoz, I., Arino, J. and Sanz, P. (2003) Molecular characterization of Ypi1, a novel saccharomyces cerevisiae type 1 protein phosphatase inhibitor. J Biol Chem 278:4774447752. Habermacher, G. and Sale, W. S. (1996) Regulation of flagellar dynein by an axonemal type-1 phosphatase in Chlamydomonas. J Cell Sci 109:18991907. Hagaman, J. R., Moyer, U. S., Bachman, E. S., Sibony, M., Magyar, P. L., Welch, J. E., Smithies, O., Krege, J. H. and OBrien, D. A. (1998) Angiotensin-converting enzyme and male fertility. Proc Natl Acad Sci USA 95:25522557. Han, Y., Feng, H., Cheung, C. K., Lam, P. M., Wang, C. C. and Haines, C. J. (2007) Expression of a novel T-complex testis expressed 5 (Tctex5) in mouse testis, epididymis and spermatozoa. Molecular Reproduction and Development 74:11321140.

Hemmings, B. A., Rensnik, T. J. and Cohen, P. (1982) Reconstitution of a Mg-ATP-dependent protein phosphatase and its activation through a phosphorylation mechanism. FEBD Letters 150:319324. Hrabchak, C. and Varmuza, S. (2004) Identification of the spermatogenic zip protein Spz1 as a putative protein phosphatase-1 (PP1) regulatory protein that specifically binds the PP1cgamm2 splice variant in mouse testis. J Biol Chem 279:3707937086. Hsu, S. H., Hsieh-Li, H. M. and Li, H. (2004) Dysfunctional spermatogenesis in transgenic mice overexpressing bHLH-Zip transcription factor, Spz1. Exp Cell Res 294:185198. Huang, B., Ramanis, Z. and Luck, D. J. (1982) Suppressor mutations in Chlamydomonas reveal a regulatory mechanism for Flagellar function. Cell 28:115124. Huang, H. S., Pozarowski, P., Gao, Y., Darzynkiewicz, Z. and Lee, E. Y. C. (2005a) Protein phosphatase-inhibitor-3 colocalized to the nucleoli and centrosomes with PP1c1 and PP1a, respectively. Arch Biochem and Biophy 443:3344. Huang, Z., Khatra, B., Bollen, M., Carr, D. W. and Vijayaraghavan, S. (2002) Sperm PP1gamma2 is regulated by a homologue of the yeast protein phosphatase binding protein sds22. Biol Reprod 67:19361942. Huang, Z., Myers, K., Khatra, B. and Vijayaraghavan, S. (2004a) Protein 14-3-3zetta binds to protein phosphatase PP1gamma2 in bovine epididymal spermatozoa. Biol Reprod 71:177184. Huang, Z. and Vijayaraghavan, S. (2004b) Increased phosphorylation of a distinct subcellular pool of protein phosphatase, PP1gamma2, during epididymal sperm maturation. Biol Reprod 70:439447. Huang, Z., Somanath, P. R., Chakrabarti, R., Eddy, E. M. and Vijayaraghavan, S. (2005b) Changes in intracellular distribution and activity of protein phosphatase PP1gamma2 and its regulating proteins in spermatozoa lacking AKAP4. Biol Reprod 72:384392. Inaba, K. (2003) Molecular architecture of the sperm flagella: Molecules for motility and signaling. Zoological Science 20:10431056. Jurisicova, A., Lopes, S., Meriano, J., Oppedisano, L., Casper, R. F. and Varmuza, S. (1999) DNA damage in round spermatids of mice with a targeted disruption of the PP1cgamma gene and in testicular biopsies of patients with non-obstructive azoospermia. Mol Human Reprod 5:323330. Kessler, S. P., Rowe, T. M., Gomos, J. B., Kessler, P. M. and Sen, G. C. (2000) Physiological non-equivalence of the two isoforms of antitension-converting enzyme. J Biol Chem 275:2625926264. Kitagawa, Y., Sasaki, K., Shima, H., Shibuya, M., Sugimura, T. and Nagao, M. (1990) Protein phosphatases possibly involved in rat spermatogenesis. Biochem Biophy Res Commun 171:230235. Krege, J. H., John, S. W., Langenbach, L. L., Hodgin, J. B., Hagaman, J. R., Vaqchman, E. S., Jennette, J. C., OBrien, D. A. and Smithies, O. (1995) Male-female differences in fertility and blood pressure in ACE-deficient mice. Nature 375:146148. Kwon, Y. G., Huang, H. B., Desdouits, F., Girault, J. A., Greengard, P. and Nairn, A. C. (1997) Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase. Proc Natl Acad Sci USA 94:35363541. Lesage, B., Beullens, M., Nuytten, M., Van Eynde, A., Keppens, S., Himpens, B. and Bollen, M. (2004) Interactor-mediated nuclear translocation and retention of protein phosphatase-1. J Biol Chem 279:5597855984. Liu, C. W., Wang, R. H., Rohadwala, M., Schonthal, A. H., Cilla-Moruzzi, E. and Berndt, N. (1999) Inhibitory phosphoryalation of PP1alpha catalytic subunit during the G(1)=S transition. Journal of Biological Chemistry 274:2947029475. Mann, T. and Lutwak-Mann, C. (1981). Male reproductive function and semen: Themes and trends in physiology, biochemistry and investigative andrology. Berlin, Germany: Springer-Verlag. Miki, K. and Eddy, E. M. (1998) Identification of tethering domains for protein kinase A type I alpha regulatory subunits on sperm fibrous sheath protein FSC1. J Biol Chem 273:3438434390. Miki, K., Willis, W. D., Brown, P. R., Goulding, E. H., Fulcher, K. D. and Eddy, E. M. (2002) Targeted disruption of the Akap4 gene causes

Syst Biol Reprod Med Downloaded from informahealthcare.com by Universitaets- und Landesbibliothek Duesseldorf on 11/17/13 For personal use only.

Y. Han et al.

176

defects in sperm flagellum and motility. Dev Biol 248: 331342. Miki, K., Qu, W., Goulding, E. H., Eillis, W. D., Bunch, D. O., Strader, L. F., Perreault, S. D., Eddy, E. M. and OBrien, D. A. (2004) Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility. Proc Natl Acad Sci USA 101:1650116506. Mishra, S., Somanath, P. R., Huang, Z. and Vijayaraghavan, S. (2003) Binding and inactivation of the germ cell-specific protein phosphatase PP1gamma2 by sds22 during epididymal sperm maturation. Biol Reprod 69:15721579. Mohri, H. and Yanagimachi, R. (1980) Characteristics of motor apparatus in testicular, epididymal and ejaculated spermatozoa. Exp Cell Res 127:191196. Morton, B., Harrigan-Lum, L., Albagli, L. and Joose, T. (1974) The activation of motility in quiescent hamster sperm from the epididymis by calcium and cyclic nucleotides. Biochem Biophys Res Commun 56:372379. Nolan, M. A., Babcock, D. F., Wennemuth, G., Brown, W., Burton, K. A. and McKnight, G. S. (2004) Sperm-specific protein kinase A catalytic subunit Calpha2 orchestrates cAMP signaling for male fertility. Proc Natl Acad Sci USA. 101:1348313488. Nomura, M., Yoshida, M. and Morisawa, M. (2004) Calmodulin=calmodulin-dependent protein kinase II mediates SAAD-induced motility activation of ascidian sperm. Cell Motil Cytoskeleton 59: 2837. Oppedisano-Wells, L., Haines, G., Hrabchak, C., Fimia, G., Elliott, R., Sassone-Corsi, P., Varmuza, S. (2002) The rate of aneuploidy is altered in spermatids from infertile mice. Human Reprod 17:710717. Oppedisano-Wells, L. and Varmuza, S. (2003) Protein phosphatase 1cgamma is required in germ cells in murine testis. Mol Reprod and Dev 65:157166. Pauls, K., Metzger, R., Steger, K., Knonisch, T., Danilov, S. and Franke, F. E. (2003) Isoforms of angiotension I-converting enzyme is the development and differentiation of human testis and epididymis. Andrologia 35:3243. Piperno, G., Mead, K. and Shestak, W. (1992) The inner dynein arms I2 interact with a dynein regulatory complex in Chlamydomonas flagella. J Cell Biol 118:14551463. Piperno, G., Mead, K., LeDizet, M. and Moscatelli, A. (1994) Mutations in the dynein regulatory complex alter the ATP-insensitive binding sites for inner arm dyneins in Chlamydomonas axonemes. J Cell Biol 125:11091117. Porter, M. E., Power, J. and Dutcher, S. K. (1992) Extragenic suppressors of paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that alter the inner dynein arms. J Cell Biol 118: 11631176. Richter, K. and Buchner, J. (2001) Hsp90: Chaperoning signal transduction. J Cell Physiol 1883:281290. San Agustin, J. T. and Witman, G. B. (1994) Role of cAMP in the reactivation of demembranated ram spermatozoa. Cell Motility and the Cytoskeleton 27:206218. Satir, P., Barkalow, K. and Hamasaki, T. (1993) The control of ciliary beat frequency. Trends in Cell Biol 3:409412. Schulh, S. M., Calson, A. E., McKnight, S., Conti, M., Hille, B. and Babcock, D. F. (2006) Signaling pathways for modulation of mouse sperm motility by adenosine and catecholamine agonists. Biol Reprod 74:492500. Shenolikar, C. and Nairn, A. C. (1991) Protein phosphatase: Recent progress. Adv Second Messenger Phosphoprotein Res 23:1121. Shima, H., Haneji, T., Hatano, Y., Kasugai, I., Sugimura, T. and Nagao, M. (1993) Protein phosphatase 1 gamma 2 is associated with nuclei of meiotic cells in rat testis. Biochem Biophy Res Commun 194:930937. Si, Y., and Okuno, M. (1995) Activation of mammalian sperm motility by regulation of microtubule sliding via cyclic adenosine 5-monophosphate-dependent phosphorylation. Biol Reprod 53:10811087.

Smith, G. D., Wolf, D. P., Trautman, K. C., Sila, E. F., Greengard, P. and Vijayaraghavan, S. (1996) Primate sperm contain protein phosphatase 1, a biochemical mediator of motility. Biol Reprod 54:719727. Smith, G. D., Wolf, D. P., Trautman, K. C. and Vijayaraghavan, S. (1999) Motility of macaque epididymal sperm: The role of protein phosphatase and glycogen synthase kinase-3 activities. J Androl 20:4753. Somanath, P. R., Jack, S. L. and Vijayaraghavan, S. (2004) Changes in sperm glycogen synthase kinase-3 serine phosphorylation and activity accompany motility initiation and stimulation. J Androl 25:605617. Swarup, D. and Garbers, D. L. (1992) Phosphoprotein phosphatase activity of sea urchin spermatozoa. Biol Reprod 26:953960. Tang, F. Y. and Hoskins, D. D. (1975) Phosphoprotein phosphatase of bovine epididymal sperm. Biochem Biophys Res Commun 62:328335. Tash, J. S. and Bracho, G. E. (1994) Regulation of sperm motility: Emerging evidence for a major role for protein phosphatase. J Androl 15:505509. Tash, J. S., Krinks, M., Patel, J., Means, R. L., Klee, C. B. and Means, A. R. (1988) Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm. J Cell Biol 106:16251633. Toshima, J., Toshima, J. Y., Takeuchi, K., Mori, R. and Mizuno, K. (2001) Cofilin phosphorylation and actin reorganization activities of testicular protein kinase 2 and its predominant expression in testicular Sertoli cells. J Biol Chem 276:3144931458. Trinkle-Mulcahy, L., Sleeman, J. E. and Lamond, A. I. (2001) Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells. J cell Sci 114:12071215. Trinkle-Mulcahy, L., Andrews, P. D., Wickramasinghe, S., Sleeman, J., Prescott, A., Lam, Y. W., Lyon, C., Swedlow, J. R. and Lamond, A. I. (2003) Time-lapse imaging reveals dynamic re-localization of PP1c throughout the mammalian cell cycle. Mol Biol Cell 14:107117. Trinkle-Mulcahy, L., Anderson, J., Lam, Y. W., Moorhead, G., Mann, M. and Lamonel, A. I. (2006) Repo-Man recruits PP1c to chromatin and is essential for cell viability. J Cell Biol 172:679692. Tzivion, G. and Avruch, J. (2002) 14-3-3 proteins: Active cofactors in cellular regulation by serine=threonine phosphorylation. J Biol Chem 277:30613064. Varmuza, S., Jurisicova, A., Okano, K., Hudson, J., Boekelheide, K. and Shipp, E. B. (1999) Spermiogenesis is impaired in mice bearing a targeted mutation in the protein phosphatase 1cgamma gene. Del Biol 205:98110. Vijayaraghavan, S., Stephens, D. T., Trautman, K., Smith, G. D., Khatra, B., da Cruz e Silva, E. F., Greengard, P. (1996) Sperm motility development in the epididymis is associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity. Biol Reprod 54:709718. Wade, M. A., Jones, R. C., Murdoch, R. N. and Aitken, R. J. (2003) Motility activation and second messenger signaling in spermatozoa from rat cauda epididymidis. Reprod 125:175183. Yaffe, M. B. and Elia, A. E. (2001) Phosphoserine=threonine-binding domains. Curr Opin Cell Biol 13:131138. Yang, P., Fox, L., Colbran, R. J. and Sale, W. S. (2000) Protein phosphatases PP1 and PP2A are located in distinct positions in the Chlamydomonas flagellar axoneme. J Cell Sci 113:91102. Yeung, C. H. (1984) Effects of cyclic AMP on the motility of mature and immature hamster epididymal spermatozoa studied by reactivation of the demembraned cells. Gamete Res 9:99114. Yoshimura, M. and Shingyoji, C. (1999) Effects of the central pair apparatus on microtubule sliding velocity in sea urchin sperm flagella. Cell Structure and Function 24:4354. Zhang, J., Zhang, L., Zhao, S. and Lee, E. Y. C. (1998) Identification and characterization of the human HCG V gene product as a novel inhibitor of protein phosphatase-1. Biochem 37:1672816734.

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