Identification of SRC as a key PKA-stimulated tyrosine kinase involved in the capacitation-associated hyperactivation of murine spermatozoa

Baker, Mark A.; Hetherington, Louise; Aitken, R. John

doi:10.1242/jcs.03055

Cited by 169 publications

(188 citation statements)

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“…It has been shown in other cell types that gelsolin can be phosphorylated by c-SRC on tyrosine 438 resulting in its release from actin filaments (19). In human sperm, SRC is involved in regulating sperm capacitation, calcium fluxes, tyrosine phosphorylation, and the AR (20,37,38). Previous studies indicated that SRC is not directly involved in tyrosine phosphorylation but rather inhibits protein phosphatase resulting in an increase of protein tyrosine phosphorylation (39).…”

Section: How Is Gelsolin Activity Inhibited During Spermmentioning

confidence: 99%

“…Moreover, it was shown that capacitation is regulated by two parallel pathways. The first one requires activation of protein kinase A leading to SRC activation, and the second one involves the inactivation of Ser/Thr phosphatases (37,40). SRC is also found in the sperm flagellum and head and is localized to membrane fraction (40).…”

Section: How Is Gelsolin Activity Inhibited During Spermmentioning

confidence: 99%

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Role and Regulation of Sperm Gelsolin Prior to Fertilization

Finkelstein

Etkovitz

Breitbart

2010

Journal of Biological Chemistry

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To acquire fertilization competence, spermatozoa should undergo several biochemical changes in the female reproductive tract, known as capacitation. The capacitated spermatozoon can interact with the egg zona pellucida resulting in the occurrence of the acrosome reaction, a process that allowed its penetration into the egg and fertilization. Sperm capacitation requires actin polymerization, whereas F-actin must disperse prior to the acrosome reaction. Here, we suggest that the actin-severing protein, gelsolin, is inactive during capacitation and is activated prior to the acrosome reaction. The release of bound gelsolin from phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by PBP10, a peptide containing the PIP 2 -binding domain of gelsolin, or by activation of phospholipase C, which hydrolyzes PIP 2 , caused rapid Ca 2؉ -dependent F-actin depolymerization as well as enhanced acrosome reaction. Using immunoprecipitation assays, we showed that the tyrosine kinase SRC and gelsolin coimmunoprecipitate, and activating SRC by adding 8-bromo-cAMP (8-Br-cAMP) enhanced the amount of gelsolin in this precipitate. Moreover, 8-Br-cAMP enhanced tyrosine phosphorylation of gelsolin and its binding to PIP 2(4,5) , both of which inactivated gelsolin, allowing actin polymerization during capacitation. This actin polymerization was blocked by inhibiting the Src family kinases, suggesting that gelsolin is activated under these conditions. These results are further supported by our finding that PBP10 was unable to cause complete F-actin breakdown in the presence of 8-BrcAMP or vanadate. In conclusion, inactivation of gelsolin during capacitation occurs by its binding to PIP 2 and tyrosine phosphorylation by SRC. The release of gelsolin from PIP 2 together with its dephosphorylation enables gelsolin activation, resulting in the acrosome reaction.

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Section: How Is Gelsolin Activity Inhibited During Spermmentioning

confidence: 99%

Section: How Is Gelsolin Activity Inhibited During Spermmentioning

confidence: 99%

Role and Regulation of Sperm Gelsolin Prior to Fertilization

Finkelstein

Etkovitz

Breitbart

2010

Journal of Biological Chemistry

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“…Baker et al [52] reported that PKA catalytic subunit can be immunodetected in the flagellar fraction of mouse spermatozoa, but barely in the head fraction. Our results in this study (Fig.…”

Section: Interaction Between Pka and Calyculin A-sensitive Protein Phmentioning

confidence: 99%

Calyculin A-Sensitive Protein Phosphatases are Involved in Maintenance of Progressive Movement in Mouse Spermatozoa In Vitro by Suppression of Autophosphorylation of Protein Kinase A

Goto

Harayama

2009

Journal of Reproduction and Development

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Abstract. Protein serine/threonine phosphorylation in mammalian sperm flagella has been considered to play important roles in regulation of motility. Protein phosphorylation state reflects balance of enzymatic activities between protein phosphatases and protein kinases [predominantly protein kinase A (PKA)]. The aims of this study were to disclose roles of protein phosphatases in the regulation of sperm motility and to provide evidence for suppression of PKA full activation by protein phosphatases in sperm flagella. Mouse epididymal spermatozoa were incubated with a cell-permeable protein phosphatase 1 (PP1)/protein phosphatase 2A (PP2A) inhibitor (calyculin A: 25-125 nM) at 37.5 C. After incubation, they were used for immunodetection of phosphorylated proteins, PKA and PP1γ2, assessment for motility and co-immunoprecipitation of PP1γ2 with PKA. Incubation with calyculin A enhanced the phosphorylation states of several proteins (>250 kDa, 170 kDa, 155 kDa, 140 kDa and 42 kDa for serine/threonine phosphorylation and 70 kDa for tyrosine phosphorylation) and PKA catalytic subunits [at the autophosphorylation residue (Thr-197) for its full enzymatic activation] in the flagella. Coincidently, this incubation induced changes of sperm flagellar movement from the progressive type to the hyperactivation-like type. Indirect immunofluorescence and co-immunoprecipitation showed that PKA was co-localized with PP1γ2 in the principal pieces of sperm flagella. These findings suggest that calyculin A-sensitive protein phosphatases (PP1/PP2A) suppress full activation of PKA as well as enhancement of the phosphorylation states of other flagellar proteins in sperm flagella in order to prevent precocious changes of flagellar movement from the progressive type to hyperactivation. Key words: Cyclic adenosine 3',5'-monophosphate (cAMP), Hyperactivation, Protein kinase, Protein phosphatase 1 (PP1), Protein phosphorylation (J. Reprod. Dev. 55: [327][328][329][330][331][332][333][334] 2009) ammalian spermatozoa are differentiated from spermatogonia in the testicular seminiferous tubules and then transferred through the duct of the epididymis. By arrival in the caudal epididymides, they have acquired the potentials to move in a forward direction, bind with the zona pellucida and fertilize oocytes [1], but they are temporally quieted in the epididymis by the influence of the acidic-base status of the luminal fluid [2,3] and interaction with membrane stabilizing factors [4,5]. After ejaculation into the female reproductive tract, the spermatozoa quickly initiate flagellar beating, which is regulated by the protein serine [Ser (S)]/threonine [Thr (T)] phosphorylation in the flagella [6]. Some of the phosphorylated proteins have previously been identified as dynein [6,7] and axokinin [8]. Subsequently, they gradually undergo a series of changes in their intracellular spaces as well as on their cell surfaces (capacitation), including dissociation of decapacitation factors (e.g., cholesterol and acrosomal stabilizing factors), elevation of th...

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“…A variety of factors, including Ca 2ϩ , HCO 3 Ϫ , and H 2 O 2 stimulate soluble adenylyl cyclase leading to increased cytosolic levels of cAMP (4). This increase in cAMP then activates PKA, and the consequence is a significant increase in tyrosine phosphorylation of protein substrates localized in the flagellum such as AKAP3, AKAP4, CABYR, hsp-90, ODF2, and tubulin (3,(5)(6)(7)(8)(9)(10)(11)(12).…”

mentioning

confidence: 99%

FSCB, a Novel Protein Kinase A-phosphorylated Calcium-binding Protein, Is a CABYR-binding Partner Involved in Late Steps of Fibrous Sheath Biogenesis

Wang

Jha

et al. 2007

Journal of Biological Chemistry

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We report characterization of a novel testis-and sperm-specific protein, FSCB (fibrous sheath CABYR binding), that is expressed post-meiotically and localized in mouse sperm flagella. FSCB was identified as a binding partner of CABYR, a calcium-binding protein that is tyrosine-phosphorylated during capacitation. Orthologous genes of FSCB are present in other mammals, including rat and human, and conserved motifs in FSCB include PXXP, proline-rich and extensin-like regions. FSCB is phosphorylated by protein kinase A as shown by in vitro phosphorylation assay and also by determining phosphorylation sites in native FSCB from mouse sperm. Calcium overlay assay showed that FSCB is a calcium-binding protein from sperm. FSCB is a post meiotic protein first expressed at step 11 of mouse spermatogenesis in the elongating spermatids, and it subsequently incorporates into the flagellar principal piece of the sperm. Ultrastructurally, FSCB localized to a cortical layer of intermediate electron density at the surface of the ribs and longitudinal columns of the fibrous sheath. Due to its temporal appearance during spermiogenesis and location at the cortex of the fibrous sheath, FSCB is postulated to be involved in the later stages of fibrous sheath assembly.Hyperactivated motility along with changes in the sperm head that confer the capacity to fertilize an oocyte result from a series of time-dependent processes collectively referred to as capacitation (1, 2). Although the molecular mechanisms underlying capacitation, including hyperactivation, are incompletely understood, some fundamental processes related to capacitation have been elucidated.The presence of a protein source such as albumin, bicarbonate, and Ca 2ϩ and an energy substrate such as glucose, pyruvate, or lactate are essential to achieve in vitro capacitation (3). Capacitation is also marked by an increase in tyrosine phosphorylation through a unique signal transduction cascade involving a sperm-specific soluble adenylyl cyclase, protein kinase A (PKA), 3 and tyrosine kinase(s). A variety of factors, including Ca 2ϩ, HCO 3 Ϫ , and H 2 O 2 stimulate soluble adenylyl cyclase leading to increased cytosolic levels of cAMP (4). This increase in cAMP then activates PKA, and the consequence is a significant increase in tyrosine phosphorylation of protein substrates localized in the flagellum such as AKAP3, AKAP4, CABYR, hsp-90, ODF2, and tubulin (3,(5)(6)(7)(8)(9)(10)(11)(12).The fact that PKA is a key regulator of capacitation-associated changes such as protein phosphorylation and hyperactivation has been well established. Pharmacological stimulants that elevate intracellular cAMP, such as the phosphodiesterase inhibitors, caffeine, and pentoxifylline, enhance hyperactivated motility of sperm (13). cAMP agonists can accelerate protein tyrosine phosphorylation in sperm, whereas antagonists of PKA inhibit tyrosine phosphorylation and capacitation (14). Mice in which sperm-specific PKA ␣ is knocked out are infertile, owing to the complete absence of normal sperm movement...

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Identification of SRC as a key PKA-stimulated tyrosine kinase involved in the capacitation-associated hyperactivation of murine spermatozoa

Cited by 169 publications

References 58 publications

Role and Regulation of Sperm Gelsolin Prior to Fertilization

Role and Regulation of Sperm Gelsolin Prior to Fertilization

Calyculin A-Sensitive Protein Phosphatases are Involved in Maintenance of Progressive Movement in Mouse Spermatozoa In Vitro by Suppression of Autophosphorylation of Protein Kinase A

FSCB, a Novel Protein Kinase A-phosphorylated Calcium-binding Protein, Is a CABYR-binding Partner Involved in Late Steps of Fibrous Sheath Biogenesis

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