Bicarbonate‐Induced phosphorylation of p270 protein in mouse sperm by cAMP‐Dependent protein kinase

Kaneto, Masako; Krisfalusi, Michelle; Eddy, Edward M.; O’Brien, Deborah A.; Miki, Kazuo

doi:10.1002/mrd.20839

Cited by 26 publications

(23 citation statements)

References 44 publications

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“…Kiyoshi Miki et al observed a 270 kDa band on a Western blot of post-capacitation spermatozoa lysates using anti-phospho-(Ser/Thr) PKA substrate antibody; however, they did not identify the protein (16). In the present study, we confirmed the presence of a 270 kDa protein band in post-capacitation spermatozoa lysates.…”

Section: +supporting

confidence: 76%

FSCB phosphorylation in mouse spermatozoa capacitation

Liu

Ni²,

Wang³

et al. 2011

BMB Reports

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It is generally accepted that spermatozoa capacitation is associated with protein kinase A-mediated tyrosine phosphorylation. In our previous study, we identified the fibrous sheath CABYR binding protein (FSCB), which was phosphorylated by PKA. However, the phosphorylation status of FSCB protein during spermatozoa capacitation should be further investigated. To this aim, in this study, we found that phosphorylation of this 270-kDa protein occurred as early as 1 min after mouse spermatozoa capacitation, which increased over time and remained stable after 60 min. Immunoprecipitation assays demonstrated that the tyrosine and Ser/Thr phosphorylation of FSCB occurred during spermatozoa capacitation. The extent of phosphorylation and was closely associated with the PKA activity and spermatozoa motility characteristics. FSCB phosphorylation could be induced by PKA agonist DB-cAMP, but was blocked by PKA antagonist H-89.Therefore, FSCB contributes to spermatozoa capacitation in a tyrosine-phosphorylated format, which may help in further elucidating the molecular mechanism of spermatozoa capacitation. [BMB reports 2011; 44(8): 541-546]

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Section: +supporting

confidence: 76%

FSCB phosphorylation in mouse spermatozoa capacitation

Liu

Ni²,

Wang³

et al. 2011

BMB Reports

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“…Specifically, bicarbonate stimulates motility and hyperactivation through the activation of ADCY10 and tyrosine phosphorylation of AKAP3 in human spermatozoa [47]. Moreover, bicarbonate/ADCY10-induced protein phosphorylation is likely important in the cAMP signaling cascades that lead to hyperactivation in mouse [48] and bull spermatozoa [49].Based on the data obtained in the present study, we would like to emphasize that functional ADCY10 ortholog proteins exist in the heads of boar ejaculated spermatozoa. This fact enables us to account for the effects of bicarbonate on the fertilization-related events in the boar sperm heads.…”

supporting

confidence: 53%

Evidence of the Existence of Adenylyl Cyclase 10 (ADCY10) Ortholog Proteins in the Heads and Connecting Pieces of Boar Spermatozoa

Tate

Nakamura

Suzuki

et al. 2010

Journal of Reproduction and Development

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Abstract. The aim of this study is to provide evidence of the existence of the adenylyl cyclase 10 (ADCY10) ortholog proteins in boar spermatozoa. Experiments with RT-PCR techniques, nucleotide sequence analyses and Northern blot analyses revealed that boar testes exclusively express approximately 5.1-kbp RNA, the nucleotide sequence of which is highly similar to that of human ADCY10. Database analyses with CDART suggested that pig ADCY10 ortholog proteins conserve two catalytic domains of adenylyl cyclase. Western blot techniques and indirect immunofluorescence with a specific antiserum to pig recombinant ADCY10 ortholog proteins showed that 48-kDa and 70-kDa truncated forms of pig ADCY10 ortholog proteins are localized in the equatorial segments and connecting pieces of boar ejaculated spermatozoa. Finally, cell imaging techniques with fluo-3/AM indicated that incubation with sodium bicarbonate (an ADCY10 activator) can initiate the calcium influx in the boar sperm heads that is controlled via the cyclic AMP signaling cascades. These results are consistent with the suggestion that functional ADCY10 ortholog proteins exist in the heads of boar spermatozoa. This is the first direct evidence of the existence of ADCY10 proteins in the heads of mammalian spermatozoa. Key words: Bicarbonate, Calcium, Cyclic AMP, Pig, Sperm (J. Reprod. Dev. 56: [271][272][273][274][275][276][277][278] 2010) hile mammalian spermatozoa are transported through the epididymis, they gradually acquire potentials to move progressively and fertilize oocytes. This process is termed sperm maturation [1]. In the terminal region of the epididymis (caudal epididymides), stored spermatozoa are temporally quieted by the influence of the acid-base status of the luminal fluid [2,3] and interaction with membrane stabilizing factors [4,5]. Immediately after ejaculation, spermatozoa initiate flagellar beating in response to bicarbonate, which originates from the male accessory genital glands [6,7]. In the female reproductive tract, a relatively higher concentration of bicarbonate in the luminal fluid also promotes a series of sperm changes that are required for the expression of fertilizing ability, including phospholipid changes in the plasma membrane [8,9], changes in surface glycoconjugates [10], increases of phosphorylated proteins [11,12], increases of intracellular calcium [13,14], activation of phospholipase A2 [15] and polymerization and subsequent depolymerization of F-actin [16]. These molecular changes enable the spermatozoa to undergo the acrosome reaction in the heads and hyperactivation in the flagella.Extracellular bicarbonate enters spermatozoa through the plasma membrane by the actions of carbonic anhydrase [3,17,18], sodium-bicarbonate cotransporter [19] and bicarbonate/chloride exchanger [20,21]. The intracellular acceptor for bicarbonate in spermatozoa is an adenylyl cyclase 10 (ADCY10, formerly called soluble adenylyl cyclase) that is distinguished from the other isoforms by G protein-independent activation and lack of a membrane...

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“…However, there are only limited data regarding cAMP-dependent Ser/Thr phosphorylation in sperm proteins (chum salmon [42], boar [43,44], sea urchin [45], human [46] and mouse [47]). In this study, incubation with cBiMPS (0.1 mM) induced Ser/Thr phosphorylation of the sperm flagellar proteins, including the >250-kDa, 170-kDa, 155-kDa, 140-kDa and 42-kDa substrate proteins of AGC family kinases (Fig.…”

Section: Relationship Between Protein Phosphorylation and Calyculin Amentioning

confidence: 99%

“…1). One phosphorylated protein with a higher molecular mass than 250 kDa corresponds to the mouse sperm p270 protein that is quickly phosphorylated in response to bicarbonate and may be involved in the initial signaling events leading to activation and subsequent hyperactivation of flagellar movement [47]. Moreover, a major cBiMPS-dependent tyrosine-phosphorylated protein (estimated molecular mass: 70 kDa, Fig.…”

Section: Relationship Between Protein Phosphorylation and Calyculin Amentioning

confidence: 99%

“…Moreover, a major cBiMPS-dependent tyrosine-phosphorylated protein (estimated molecular mass: 70 kDa, Fig. 1) corresponds to the major 80-kDa bicarbonate-dependent tyrosinephosphorylated protein [47], and the difference in the molecular masses (70 vs. 80 kDa) may be due to the different protocols of SDS-PAGE and Western blotting. The increases in these phosphorylated proteins could be mimicked by incubation with calyculin A (125 nM, Fig.…”

Section: Relationship Between Protein Phosphorylation and Calyculin Amentioning

confidence: 99%

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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

J. Reprod. Dev.

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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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Bicarbonate‐Induced phosphorylation of p270 protein in mouse sperm by cAMP‐Dependent protein kinase

Cited by 26 publications

References 44 publications

FSCB phosphorylation in mouse spermatozoa capacitation

FSCB phosphorylation in mouse spermatozoa capacitation

Evidence of the Existence of Adenylyl Cyclase 10 (ADCY10) Ortholog Proteins in the Heads and Connecting Pieces of Boar Spermatozoa

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

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