Inhibition of Ser/Thr Phosphatases Induces Capacitation-associated Signaling in the Presence of Src Kinase Inhibitors

Signaling events leading to mammalian sperm capacitation rely on activation/deactivation of proteins by phosphorylation. This cascade includes soluble adenylyl cyclase, an atypical bicarbonate-stimulated adenylyl cyclase, and is mediated by protein kinase A and the subsequent stimulation of protein tyrosine phosphorylation. Recently, it has been proposed that the capacitation-associated increase in tyrosine phosphorylation is governed by Src tyrosine kinase activity. This conclusion was based mostly on the observation that Src is present in sperm and that the Src kinase family inhibitor SU6656 blocked the capacitation-associated increase in tyrosine phosphorylation. Results in the present manuscript confirmed these observations and provided evidence that these inhibitors were also able to inhibit protein kinase A phosphorylation, sperm motility, and in vitro fertilization. However, the block of capacitation-associated parameters was overcome when sperm were incubated in the presence of Ser/Thr phosphatase inhibitors such as okadaic acid and calyculin-A at concentrations reported to affect only PP2A. Altogether, these data indicate that Src is not directly involved in the observed increase in tyrosine phosphorylation. More importantly, this work presents strong evidence that capacitation is regulated by two parallel pathways. One of them requiring activation of protein kinase A and the second one involving inactivation of Ser/Thr phosphatases. Signaling events leading to mammalian sperm capacitation rely on activation/deactivation of proteins by phosphorylation. This cascade includes soluble adenylyl cyclase, an atypical bicarbonate-stimulated adenylyl cyclase, and is mediated by protein kinase A and the subsequent stimulation of protein tyrosine phosphorylation. Recently, it has been proposed that the capacitation-associated increase in tyrosine phosphorylation is governed by Src tyrosine kinase activity. This conclusion was based mostly on the observation that Src is present in sperm and that the Src kinase family inhibitor SU6656 blocked the capacitation-associated increase in tyrosine phosphorylation. Results in the present manuscript confirmed these observations and provided evidence that these inhibitors were also able to inhibit protein kinase A phosphorylation, sperm motility, and in vitro fertilization. However, the block of capacitation-associated parameters was overcome when sperm were incubated in the presence of Ser/Thr phosphatase inhibitors such as okadaic acid and calyculin-A at concentrations reported to affect only PP2A. Altogether, these data indicate that Src is not directly involved in the observed increase in tyrosine phosphorylation. More importantly, this work presents strong evidence that capacitation is regulated by two parallel pathways. One of them requiring activation of protein kinase A and the second one involving inactivation of Ser/Thr phosphatases. IntroductionThe capacitation process is the major prerequisite for mammalian sperm to fertilize. This highly complex phenomenon occurs in the female reproductive tract, and renders the spermatozoa capable of binding and fusing with the oocyte (1.Yanagimachi R. Knobil E. Neill J. Mammalian Fertilization. Raven Press, New York1994: 189-317Google Scholar). The commonly accepted end point of capacitation is the time when sperm have obtained the ability to fertilize an egg. However, different physiological modifications of sperm have been correlated with the capacitated state. These include: cholesterol loss from the sperm plasma membrane, increased membrane fluidity, changes in intracellular ion concentrations (2.Salicioni A.M. Platt M.D. Wertheimer E.V. Arcelay E. Allaire A. Sosnik J. Visconti P.E. Soc. Reprod. Fertil. Suppl. 2007; 65: 245-259PubMed Google Scholar), hyperpolarization of the sperm plasma membrane (3.Hernández-González E.O. Sosnik J. Edwards J. Acevedo J.J. Mendoza-Lujambio I. López-González I. Demarco I. Wertheimer E. Darszon A. Visconti P.E. J. Biol. Chem. 2006; 281: 5623-5633Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), and increased protein tyrosine phosphorylation (4.Arcelay E. Salicioni A.M. Wertheimer E. Visconti P.E. Int. J. Dev. Biol. 2008; 52: 463-472Crossref PubMed Scopus (124) Google Scholar).Among ion fluxes that occur during capacitation, the transport of HCO3− into sperm promotes cAMP synthesis by the activation of an atypical soluble adenylyl cyclase (SACY) 2The abbreviations used are: SACYsoluble adenylyl cyclaseSFKfamily of protein tyrosine kinasePP2Aprotein phosphatase 2ABt2cAMPdibutyryl cyclic AMPCASACEROS computer-assisted semen analysisPKAprotein kinase ABSAbovine serum albuminIBMXisobutylmethylxanthinePVDFpolyvinylidene difluoridepYanti-phosphotyrosinePKIheat stable protein kinase A inhibitor. (5.Litvin T.N. Kamenetsky M. Zarifyan A. Buck J. Levin L.R. J. Biol. Chem. 2003; 278: 15922-15926Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) and subsequent PKA activation. cAMP-dependent phosphorylation of Ser/Thr residues is known to be a key regulator of tyrosine phosphorylation events linked to the process of capacitation. In mouse sperm exposed to HCO3−, cAMP rises to a maximum in <60 s, followed immediately by an increase in PKA-dependent phosphorylation (2.Salicioni A.M. Platt M.D. Wertheimer E.V. Arcelay E. Allaire A. Sosnik J. Visconti P.E. Soc. Reprod. Fertil. Suppl. 2007; 65: 245-259PubMed Google Scholar). However, tyrosine phosphorylation is only observed after incubations for at least 30 min in conditions conducive to capacitation (6.Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1129-1137Crossref PubMed Google Scholar). Despite the lack of temporal correlation of PKA-induced phosphorylation and the increase in tyrosine phosphorylation, it has been shown that PKA inhibition blocks the onset of tyrosine phosphorylation (7.Visconti P.E. Moore G.D. Bailey J.L. Leclerc P. Connors S.A. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1139-1150Crossref PubMed Google Scholar). The one or more tyrosine kinases responsible for the capacitation-associated increase in tyrosine phosphorylation have remained enigmatic. Recent proposals, however, have put forward the hypothesis that the Src family of protein tyrosine kinases (SFKs) mediates the increase in tyrosine phosphorylation in mouse and human sperm (8.Lawson C. Goupil S. Leclerc P. Biol. Reprod. 2008; 79: 657-666Crossref PubMed Scopus (50) Google Scholar, 9.Mitchell L.A. Nixon B. Baker M.A. Aitken R.J. Mol. Hum. Reprod. 2008; 14: 235-243Crossref PubMed Scopus (67) Google Scholar, 10.Varano G. Lombardi A. Cantini G. Forti G. Baldi E. Luconi M. Hum. Reprod. 2008; 23: 2652-2662Crossref PubMed Scopus (53) Google Scholar).The action of SFKs on mammalian cells has been extensively studied and demonstrated to be broadly pleiotropic, including effects on cell adhesion, proliferation, migration, differentiation, cell morphology, and survival (11.Pawson T. Warner N. Oncogene. 2007; 26: 1268-1275Crossref PubMed Scopus (95) Google Scholar). Several lines of evidence indicate that c-Src (the prototypic member of the SFKs) is activated through dephosphorylation of Tyr-527 (or the corresponding tyrosine in other members of the SFKs) (12.Zheng X.M. Resnick R.J. Shalloway D. EMBO J. 2000; 19: 964-978Crossref PubMed Scopus (206) Google Scholar). The currently accepted model proposes that c-Src tyrosine kinase (Csk) phosphorylates Tyr-527 allowing its intramolecular interaction with SH2 that keeps the protein in an inactive closed state. However, in its open state, Tyr-416 in the c-Src activation loop (or the corresponding tyrosine in other SFKs) can undergo autophosphorylation inducing kinase activity (13.Roskoski Jr., R. Biochem. Biophys. Res. Commun. 2005; 331: 1-14Crossref PubMed Scopus (449) Google Scholar).It has been shown that c-Src is present in mouse sperm and that SU6656, a c-Src kinase family inhibitor is able to block the capacitation-associated increase in tyrosine phosphorylation as well as the change in motility pattern, known as hyperactivation (14.Baker M.A. Hetherington L. Aitken R.J. J. Cell Sci. 2006; 119: 3182-3192Crossref PubMed Scopus (155) Google Scholar). This inhibitor is competitive with respect to ATP and is capable of affecting all SFKs, suggesting a role for these kinases in the capacitation process. In addition, more recent studies have shown that SFK inhibitors abrogate human and bovine sperm acrosome reactions (10.Varano G. Lombardi A. Cantini G. Forti G. Baldi E. Luconi M. Hum. Reprod. 2008; 23: 2652-2662Crossref PubMed Scopus (53) Google Scholar, 15.Etkovitz N. Tirosh Y. Chazan R. Jaldety Y. Daniel L. Rubinstein S. Breitbart H. Dev. Biol. 2009; 334: 447-457Crossref PubMed Scopus (61) Google Scholar). In the present work we have further characterized the involvement of SFKs in mouse sperm capacitation. Due to the importance of PKA in this process, we focused on the effect of SFK inhibitors on this kinase. Interestingly, our results indicate that, in addition to their abrogation of tyrosine phosphorylation, SFK inhibitors block PKA phosphorylation, sperm motility, and in vitro fertilization. Although these data suggest unspecific PKA inactivation by SFK inhibitors, in vitro activity assays show that this is not the case. Here, we provide evidence that Ser/Thr phosphatase inhibitors overcome the block by SFK inhibitors to all capacitation parameters, including in vitro fertilization. In addition, sperm from Src-null mice contained similar levels of capacitation-associated tyrosine phosphorylation as wild-type sperm. Taken together, these data have indicated that Src is not directly involved in the observed capacitation-associated changes in tyrosine phosphorylation, and further have strongly suggested that Ser/Thr protein phosphatase inactivation is necessary for sperm capacitation.DISCUSSIONIncreased tyrosine phosphorylation has been associated with the acquisition of sperm-fertilizing capacity in different species (6.Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1129-1137Crossref PubMed Google Scholar, 33.Krapf D. Visconti P.E. Arranz S.E. Cabada M.O. Dev. Biol. 2007; 306: 516-524Crossref PubMed Scopus (19) Google Scholar, 34.Vanichviriyakit R. Kruevaisayawan H. Weerachatyanukul W. Tawipreeda P. Withyachumnarnkul B. Pratoomchat B. Chavadej J. Sobhon P. Mol. Reprod. Dev. 2004; 69: 356-363Crossref PubMed Scopus (38) Google Scholar), and it is correlated with the onset of functional parameters such as hyperactivated motility and acrosomal responsiveness (35.Krapf D. O'Brien E.D. Cabada M.O. Visconti P.E. Arranz S.E. Biol. Reprod. 2009; 80: 311-319Crossref PubMed Scopus (12) Google Scholar, 36.Si Y. Okuno M. Biol. Reprod. 1999; 61: 240-246Crossref PubMed Scopus (154) Google Scholar, 37.Visconti P.E. Stewart-Savage J. Blasco A. Battaglia L. Miranda P. Kopf G.S. Tezón J.G. Biol. Reprod. 1999; 61: 76-84Crossref PubMed Scopus (183) Google Scholar, 38.Krapf D. Vidal M. Arranz S.E. Cabada M.O. Biol. Cell. 2006; 98: 403-413Crossref PubMed Scopus (13) Google Scholar). In mammalian sperm, capacitation is a HCO3−-dependent process (39.Visconti P.E. Westbrook V.A. Chertihin O. Demarco I. Sleight S. Diekman A.B. J. Reprod. Immunol. 2002; 53: 133-150Crossref PubMed Scopus (284) Google Scholar). A likely target for HCO3− is the regulation of cAMP metabolism through stimulation of a soluble adenylyl cyclase (SACY). Once activated, intracellular cAMP levels increase, promoting the activation of PKA through the release of its inhibitory subunits. As previously shown, inhibition of sperm PKA activity blocks the increase in tyrosine phosphorylation associated with capacitation. Recently, c-Src has been postulated to be the tyrosine kinase directly responsible for this increase in tyrosine phosphorylation. In addition, it has been proposed that c-Src is activated by a cAMP-mediated pathway via the inhibition of Csk, the tyrosine kinase responsible for the inactivation of c-Src in other cell types (14.Baker M.A. Hetherington L. Aitken R.J. J. Cell Sci. 2006; 119: 3182-3192Crossref PubMed Scopus (155) Google Scholar). Consistent with this model and with previous published results from different laboratories (8.Lawson C. Goupil S. Leclerc P. Biol. Reprod. 2008; 79: 657-666Crossref PubMed Scopus (50) Google Scholar, 10.Varano G. Lombardi A. Cantini G. Forti G. Baldi E. Luconi M. Hum. Reprod. 2008; 23: 2652-2662Crossref PubMed Scopus (53) Google Scholar, 14.Baker M.A. Hetherington L. Aitken R.J. J. Cell Sci. 2006; 119: 3182-3192Crossref PubMed Scopus (155) Google Scholar, 24.Baker M.A. Hetherington L. Curry B. Aitken R.J. Dev. Biol. 2009; 333: 57-66Crossref PubMed Scopus (44) Google Scholar), SU6656, an SFK inhibitor, blocked tyrosine phosphorylation in a concentration-dependent manner. Moreover, immunoprecipitated c-Src from capacitated sperm showed significantly higher activity than c-Src from non-capacitated sperm, suggesting that this kinase is activated during capacitation.The initial aim of the present work was to further investigate the role of c-Src tyrosine kinase in mouse sperm capacitation. For this purpose, the increase in tyrosine phosphorylation was analyzed in sperm from mice in which c-Src had been eliminated by homologous recombination (16.Soriano P. Montgomery C. Geske R. Bradley A. Cell. 1991; 64: 693-702Abstract Full Text PDF PubMed Scopus (1793) Google Scholar). Src-null mice have an osteopetrotic phenotype, are toothless, and normally succumb to various lesions (such as benign nasal polyps) by 15 weeks of age. Therefore, their reproductive phenotype has been difficult to analyze in detail. When the capacitation-associated increase in tyrosine phosphorylation was assessed in the Src-null sperm, no differences were found when compared with sperm from their wild-type littermates. These findings suggested that c-Src is not the enzyme that directly phosphorylates tyrosine residues as part of sperm capacitation, or, alternatively, that lack of c-Src can be compensated by other members of the SFKs.As mentioned, activation of tyrosine phosphorylation is downstream of a cAMP/PKA pathway. Analysis of PKA phosphorylation using anti-PKA substrate antibodies revealed that this phosphorylation was unaffected in cSrc KO sperm. However, PKA phosphorylation was blocked in the presence of SFK inhibitors suggesting that SU6656 and SKI606 may not be specific at the concentrations used. This possibility was ruled out by directly measuring PKA activity in vitro. At concentrations as high as 100 μm, the SFK inhibitors did not block PKA activity in vitro. Because SU6656 as well as SKI606 are competitive inhibitors of the SFK ATP binding site, the conclusion from these experiments is that the effect of these inhibitors on sperm is not due to unspecific inhibition of PKA activity. Another possibility was that SFK inhibitors could be blocking the capacitation pathway upstream of PKA activation. This possibility was also ruled out, because addition of cAMP agonists did not restore phosphorylation of PKA substrates or the increase in tyrosine phosphorylation in the presence of SFK inhibitors.In light of these data, we propose an alternative hypothesis in which SFK inhibitors act in a pathway parallel to PKA activation. The steady-state phosphorylation status of a protein is influenced by relative activities of both kinases and on has been shown that SFK members are able to the Ser/Thr phosphatase in its This phosphorylation results in the inactivation of J. PubMed Scopus Google Scholar, J. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. N. J. Biol. Chem. Full Text PDF PubMed Google Scholar, E. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, C. L. C. A.M. Biol. 2005; PubMed Scopus Google Scholar, L. L. A. G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). For has been associated with the of activity through phosphorylation J. J. J. 2009; PubMed Scopus Google Scholar). this into the effect of the SFK inhibitors on PKA and tyrosine phosphorylation in sperm could be by the hypothesis that Ser/Thr are from inhibition by tyrosine kinase Interestingly, both okadaic acid and two inhibitors of Ser/Thr the effect of the SFK inhibitors on PKA and tyrosine phosphorylation. However, PKA tyrosine phosphorylation was by okadaic acid when mouse sperm were incubated This of experiments that two parallel to be to the capacitation-associated phosphorylation of PKA substrates and the increase in tyrosine phosphorylation. One of the is the well HCO3−-dependent activation of cAMP synthesis by the second pathway on the inactivation of Ser/Thr Consistent with this okadaic acid was to increase phosphorylation by PKA or tyrosine phosphorylation when sperm were incubated with the PKA inhibitor further investigate the role of phosphatase inactivation in capacitation, the effect of SFK inhibitors and the of their action with okadaic acid was in motility and in vitro As because of their action on phosphorylation of PKA SFK inhibitors significantly blocked motility and fertilization. The SU6656 inhibition was overcome with the addition of okadaic acid that phosphatase inactivation an role in the regulation of these has been shown that inhibition of phosphatase activity promotes of sperm motility G.D. P. S. Biol. Reprod. PubMed Scopus Google Scholar, S. G.D. B. P. Biol. Reprod. PubMed Scopus Google Scholar), further a role of in the regulation of The concentrations of okadaic acid capable of the effect of SFK inhibitors the of the phosphatase in this which is more to okadaic acid than L. L. A. G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C. A. Biochem. J. PubMed Scopus Google Scholar, H. H. H. Y. N. S. D. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, P. J. Biol. Chem. Full Text PDF PubMed Google Scholar, C. S. P. PubMed Scopus Google Scholar). is that the two Ser/Thr present in the sperm are a of S. R. Soc. Reprod. Fertil. Suppl. 2007; Google Scholar), and also present in other cell In sperm, the phosphorylation of in has been shown to be during and involved in phosphatase activity regulation S. Biol. Reprod. 2004; PubMed Scopus Google Scholar). This also to be in mouse sperm N. H. J. Reprod. Dev. 2009; PubMed Scopus Google Scholar). and N. H. J. Reprod. Dev. 2009; PubMed Scopus Google Scholar) in sperm protein L. S. S. S. 2009; PubMed Scopus Google Scholar), suggesting the of phosphatase with different The possibility that is during capacitation more In addition, has been shown to be capable of residues J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biochem. J. PubMed Scopus Google Scholar). This also be to involving tyrosine phosphorylation in the present presents evidence that c-Src is not directly involved in the increase in tyrosine phosphorylation that occurs during sperm capacitation in the This conclusion is based on results using sperm from Src-null as well as on the that Ser/Thr phosphatase inhibitors overcome the effect of SFK inhibitors used at concentrations in which no SFK activity be This observation also that at least one of the two sperm Ser/Thr be inactive in sperm to phosphorylation events to in sperm, c-Src could in this In this PKA activity and subsequent tyrosine phosphorylation observed in sperm from Src-null mice could be by activity of SFK we out the possibility that SU6656 and SKI606 at the concentrations tyrosine kinase different from This alternative hypothesis is with the high of SU6656 and with the inhibition observed with SKI606 at on the capacitation-associated increase in tyrosine phosphorylation. These concentrations are at least one of higher than the for c-Src in vitro. this is the we that this alternative kinase act upstream of Ser/Thr and be involved in their IntroductionThe capacitation process is the major prerequisite for mammalian sperm to fertilize. This highly complex phenomenon occurs in the female reproductive tract, and renders the spermatozoa capable of binding and fusing with the oocyte (1.Yanagimachi R. Knobil E. Neill J. Mammalian Fertilization. Raven Press, New York1994: 189-317Google Scholar). The commonly accepted end point of capacitation is the time when sperm have obtained the ability to fertilize an egg. However, different physiological modifications of sperm have been correlated with the capacitated state. These include: cholesterol loss from the sperm plasma membrane, increased membrane fluidity, changes in intracellular ion concentrations (2.Salicioni A.M. Platt M.D. Wertheimer E.V. Arcelay E. Allaire A. Sosnik J. Visconti P.E. Soc. Reprod. Fertil. Suppl. 2007; 65: 245-259PubMed Google Scholar), hyperpolarization of the sperm plasma membrane (3.Hernández-González E.O. Sosnik J. Edwards J. Acevedo J.J. Mendoza-Lujambio I. López-González I. Demarco I. Wertheimer E. Darszon A. Visconti P.E. J. Biol. Chem. 2006; 281: 5623-5633Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), and increased protein tyrosine phosphorylation (4.Arcelay E. Salicioni A.M. Wertheimer E. Visconti P.E. Int. J. Dev. Biol. 2008; 52: 463-472Crossref PubMed Scopus (124) Google Scholar).Among ion fluxes that occur during capacitation, the transport of HCO3− into sperm promotes cAMP synthesis by the activation of an atypical soluble adenylyl cyclase (SACY) 2The abbreviations used are: SACYsoluble adenylyl cyclaseSFKfamily of protein tyrosine kinasePP2Aprotein phosphatase 2ABt2cAMPdibutyryl cyclic AMPCASACEROS computer-assisted semen analysisPKAprotein kinase ABSAbovine serum albuminIBMXisobutylmethylxanthinePVDFpolyvinylidene difluoridepYanti-phosphotyrosinePKIheat stable protein kinase A inhibitor. (5.Litvin T.N. Kamenetsky M. Zarifyan A. Buck J. Levin L.R. J. Biol. Chem. 2003; 278: 15922-15926Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) and subsequent PKA activation. cAMP-dependent phosphorylation of Ser/Thr residues is known to be a key regulator of tyrosine phosphorylation events linked to the process of capacitation. In mouse sperm exposed to HCO3−, cAMP rises to a maximum in <60 s, followed immediately by an increase in PKA-dependent phosphorylation (2.Salicioni A.M. Platt M.D. Wertheimer E.V. Arcelay E. Allaire A. Sosnik J. Visconti P.E. Soc. Reprod. Fertil. Suppl. 2007; 65: 245-259PubMed Google Scholar). However, tyrosine phosphorylation is only observed after incubations for at least 30 min in conditions conducive to capacitation (6.Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1129-1137Crossref PubMed Google Scholar). Despite the lack of temporal correlation of PKA-induced phosphorylation and the increase in tyrosine phosphorylation, it has been shown that PKA inhibition blocks the onset of tyrosine phosphorylation (7.Visconti P.E. Moore G.D. Bailey J.L. Leclerc P. Connors S.A. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1139-1150Crossref PubMed Google Scholar). The one or more tyrosine kinases responsible for the capacitation-associated increase in tyrosine phosphorylation have remained enigmatic. Recent proposals, however, have put forward the hypothesis that the Src family of protein tyrosine kinases (SFKs) mediates the increase in tyrosine phosphorylation in mouse and human sperm (8.Lawson C. Goupil S. Leclerc P. Biol. Reprod. 2008; 79: 657-666Crossref PubMed Scopus (50) Google Scholar, 9.Mitchell L.A. Nixon B. Baker M.A. Aitken R.J. Mol. Hum. Reprod. 2008; 14: 235-243Crossref PubMed Scopus (67) Google Scholar, 10.Varano G. Lombardi A. Cantini G. Forti G. Baldi E. Luconi M. Hum. Reprod. 2008; 23: 2652-2662Crossref PubMed Scopus (53) Google Scholar).The action of SFKs on mammalian cells has been extensively studied and demonstrated to be broadly pleiotropic, including effects on cell adhesion, proliferation, migration, differentiation, cell morphology, and survival (11.Pawson T. Warner N. Oncogene. 2007; 26: 1268-1275Crossref PubMed Scopus (95) Google Scholar). Several lines of evidence indicate that c-Src (the prototypic member of the SFKs) is activated through dephosphorylation of Tyr-527 (or the corresponding tyrosine in other members of the SFKs) (12.Zheng X.M. Resnick R.J. Shalloway D. EMBO J. 2000; 19: 964-978Crossref PubMed Scopus (206) Google Scholar). The currently accepted model proposes that c-Src tyrosine kinase (Csk) phosphorylates Tyr-527 allowing its intramolecular interaction with SH2 that keeps the protein in an inactive closed state. However, in its open state, Tyr-416 in the c-Src activation loop (or the corresponding tyrosine in other SFKs) can undergo autophosphorylation inducing kinase activity (13.Roskoski Jr., R. Biochem. Biophys. Res. Commun. 2005; 331: 1-14Crossref PubMed Scopus (449) Google Scholar).It has been shown that c-Src is present in mouse sperm and that SU6656, a c-Src kinase family inhibitor is able to block the capacitation-associated increase in tyrosine phosphorylation as well as the change in motility pattern, known as hyperactivation (14.Baker M.A. Hetherington L. Aitken R.J. J. Cell Sci. 2006; 119: 3182-3192Crossref PubMed Scopus (155) Google Scholar). This inhibitor is competitive with respect to ATP and is capable of affecting all SFKs, suggesting a role for these kinases in the capacitation process. In addition, more recent studies have shown that SFK inhibitors abrogate human and bovine sperm acrosome reactions (10.Varano G. Lombardi A. Cantini G. Forti G. Baldi E. Luconi M. Hum. Reprod. 2008; 23: 2652-2662Crossref PubMed Scopus (53) Google Scholar, 15.Etkovitz N. Tirosh Y. Chazan R. Jaldety Y. Daniel L. Rubinstein S. Breitbart H. Dev. Biol. 2009; 334: 447-457Crossref PubMed Scopus (61) Google Scholar). In the present work we have further characterized the involvement of SFKs in mouse sperm capacitation. Due to the importance of PKA in this process, we focused on the effect of SFK inhibitors on this kinase. Interestingly, our results indicate that, in addition to their abrogation of tyrosine phosphorylation, SFK inhibitors block PKA phosphorylation, sperm motility, and in vitro fertilization. Although these data suggest unspecific PKA inactivation by SFK inhibitors, in vitro activity assays show that this is not the case. Here, we provide evidence that Ser/Thr phosphatase inhibitors overcome the block by SFK inhibitors to all capacitation parameters, including in vitro fertilization. In addition, sperm from Src-null mice contained similar levels of capacitation-associated tyrosine phosphorylation as wild-type sperm. Taken together, these data have indicated that Src is not directly involved in the observed capacitation-associated changes in tyrosine phosphorylation, and further have strongly suggested that Ser/Thr protein phosphatase inactivation is necessary for sperm capacitation.