Tyrosine phosphorylated proteins in mammalian spermatozoa: molecular and functional aspects.
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During mammalian fertilization, spermatozoa must undergo capacitation and the acrosome reaction. These processes of sperm function are critically associated with various molecular events and one such process is protein tyrosine phosphorylation (PYP). This event is downstream of increases in intracellular Ca2+ and activities of HCO3- activated adenylate cyclase, cAMP-dependent-protein kinase-A and reactive oxygen species. Though, PYP is known to be mediated by tyrosine kinases and phosphatases, only a few of them have been identified and characterized in spermatozoa. Since most identified tyrosine kinases are soluble proteins from somatic cells, it is believed that distinct mechanisms could exist in spermatozoa for PYP. Such sperm-specific protein tyrosine kinases/ phosphatases still remain to be thoroughly characterized in most species, including hamsters. Nevertheless, a few tyrosine phosphorylated sperm proteins have been identified in hamsters and in other mammals as well. There is very limited information available on our understanding of the molecular and ultrastructural localization, as well as the characteristics of tyrosine phosphorylated proteins. Functionally, how sperm motility is regulated by PYP is also poorly understood. Knowledge of tyrosine phoshorylated proteins and how they regulate sperm function is of immense significance in our understanding of male (in)fertility and clinical management of fertility; especially, in the light of studies that implicate the hypo-tyrosine phosphorylated state of sperm proteins with asthenozoospermic condition in humans. This article provides a comprehensive review on PYP and its regulation by kinases and phosphatases.Keywords:
Capacitation
Acrosome reaction
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In the accompanying report (Visconti, P.E., Bailey, J.L., Moore, G.D., Pan, D., Olds-Clarke, P. and Kopf, G.S. (1995) Development, 121, 1129-1137) we demonstrated that the tyrosine phosphorylation of a subset of mouse sperm proteins of M(r) 40,000-120,000 was correlated with the capacitation state of the sperm. The mechanism by which protein tyrosine phosphorylation is regulated in sperm during this process is the subject of this report. Cauda epididymal sperm, when incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin do not display the capacitation-associated increases in protein tyrosine phosphorylation of this subset of proteins. This NaHCO3, CaCl2 or bovine serum albumin requirement for protein tyrosine phosphorylation can be completely overcome by the addition of biologically active, but not inactive, cAMP analogues. Addition of the active cAMP analogues to sperm incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin overcomes the inability of these media to support capacitation, as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. The effects of the cAMP analogues to enhance protein tyrosine phosphorylation and to promote capacitation appears to be at the level of the cAMP-dependent protein kinase (PKA), since two specific inhibitors of this enzyme (H-89 and Rp-cAMPS) block the capacitation-dependent increases in protein tyrosine phosphorylation in sperm incubated in media supporting capacitation. Capacitation, as assessed by the aforementioned endpoints, also appears to be inhibited by H-89 in a concentration-dependent manner. These results provide further evidence for the interrelationship between protein tyrosine phosphorylation and the appearance of the capacitated state in mouse sperm. They also demonstrate that both protein tyrosine phosphorylation and capacitation appear to be regulated by cAMP/PKA. Up-regulation of protein tyrosine phosphorylation by cAMP/PKA in sperm is, to our knowledge, the first demonstration of such an interrelationship between tyrosine kinase/phosphatase and PKA signaling pathways.
Capacitation
Acrosome reaction
Bovine serum albumin
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Protein tyrosine phosphatases (PTPs) constitute a large family of enzymes that can exert both positive and negative effects on signaling pathways. They play dominant roles in setting the levels of intracellular phosphorylation downstream of many receptors including receptor tyrosine kinases and G protein-coupled receptors. As observed with kinases, deregulation of PTP activity can also contribute to cancer. This review will examine a broad array of PTP family members that positively affect oncogenesis in human cancer tissues. We will describe the PTP family, their biological significance in oncology, and how recent progress is being made to more effectively target specific PTPs. Finally, we will discuss the therapeutic implications of targeting these oncogenic PTPs in cancer. Keywords: Cancer, Inhibitors, dual-specificity phosphatases (DSPs), Oncogene, Protein tyrosine phosphatases, Phosphorylation, Tumor suppressor, G protein-coupled receptors (GPCRs), Amplification, Gynecological Cancers
Receptor Protein-Tyrosine Kinases
Dual-specificity phosphatase
PTPN11
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Ejaculated spermatozoa must undergo 2 important preparatory changes namely capacitation and acrosome reaction to be able to fertilize an egg. Among the biochemical changes that take place during capacitation and acrosome reaction, protein tyrosine phosphorylation has been considered as an important event associated with different sperm functions. PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phophocholine), a potent signaling phospholipid, also regulates different sperm maturational processes. This study was undertaken to determine the effect of different concentrations of PAF as well as its incubation time on protein tyrosine phosphorylation during acrosome reaction of buffalo spermatozoa. Number of protein substrates were tyrosine phosphorylated and their degree of phosphorylation increased with increase in concentrations of PAF. However at higher concentrations of PAF (500 nM) a significant decrease in the extent of tyrosine phosphorylation was observed with respect to 20, 25 and 40 kDa proteins. It was also observed that with the increase in PAF incubation time the degree of protein tyrosine phosphorylation increased during acrosome reaction of buffalo spermatozoa. In conclusion, the effect of PAF on the extent of tyrosine phosphorylation of sperm proteins appeared to be dose related and time dependent and this compound can be utilized for the induction of acrosome reaction in buffalo spermatozoa.
Platelet-activating factor
Acrosome reaction
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Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH <7.8, mediated by calmodulin. To explore the mechanism of that inhibition, we incubated stallion sperm in media without added calcium, with calcium, or with calcium plus the calmodulin inhibitor W-7 (Ca/W-7 treatment). Treatment with inhibitors of calcium/calmodulin-dependent kinases, protein kinase A (PRKA), or Src family kinases suppressed the PY induced by the absence of added calcium, but not that induced by the Ca/W-7 treatment, indicating that PY in the absence of added calcium occurred via the canonical PRKA pathway, but that PY in the Ca/W-7 treatment did not. This suggested that when calmodulin was inhibited, calcium stimulated PY via a noncanonical pathway. Incubation with PF-431396, an inhibitor of focal adhesion kinases (FAKs), a family of calcium-induced protein tyrosine kinases, inhibited the PY induced both by the absence of added calcium and by the Ca/W-7 treatment. Western blotting demonstrated that both FAK family members, protein tyrosine kinases 2 and 2B, were phosphorylated in the absence of added calcium and in the Ca/W-7 treatment, but not in the presence of calcium without calmodulin inhibitors. Inhibition of FAK proteins inhibited PY in stallion sperm incubated under capacitating conditions (in the presence of calcium, bovine serum albumin, and bicarbonate at pH >7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways.
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During mammalian fertilization, spermatozoa must undergo capacitation and the acrosome reaction. These processes of sperm function are critically associated with various molecular events and one such process is protein tyrosine phosphorylation (PYP). This event is downstream of increases in intracellular Ca2+ and activities of HCO3- activated adenylate cyclase, cAMP-dependent-protein kinase-A and reactive oxygen species. Though, PYP is known to be mediated by tyrosine kinases and phosphatases, only a few of them have been identified and characterized in spermatozoa. Since most identified tyrosine kinases are soluble proteins from somatic cells, it is believed that distinct mechanisms could exist in spermatozoa for PYP. Such sperm-specific protein tyrosine kinases/ phosphatases still remain to be thoroughly characterized in most species, including hamsters. Nevertheless, a few tyrosine phosphorylated sperm proteins have been identified in hamsters and in other mammals as well. There is very limited information available on our understanding of the molecular and ultrastructural localization, as well as the characteristics of tyrosine phosphorylated proteins. Functionally, how sperm motility is regulated by PYP is also poorly understood. Knowledge of tyrosine phoshorylated proteins and how they regulate sperm function is of immense significance in our understanding of male (in)fertility and clinical management of fertility; especially, in the light of studies that implicate the hypo-tyrosine phosphorylated state of sperm proteins with asthenozoospermic condition in humans. This article provides a comprehensive review on PYP and its regulation by kinases and phosphatases.
Capacitation
Acrosome reaction
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Capacitation
Acrosome reaction
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Protein phosphorylation/dephosphorylation is considered a widespread mechanism of protein regulation. The covalent addition of a phosphate group to tyrosine residues in cellular proteins may occur as an appropriate response to a series of morphological and biochemical processes, with particular importance in complex functions such as growth, proliferation, differentiation, adhesion and motility. Protein tyrosine phosphorylation is regulated in the cell by the opposing activities of two classes of enzymes: protein tyrosine kinases, which phosphorylate specific tyrosine residues in protein using ATP as the phosphate source, and protein tyrosine phosphatases, which hydrolyze the phosphotyrosines yielding the restored amino acid residue and inorganic phosphate as products (Kolmodin & Aqvist, 2001; Mayer, 2008). The number of active protein phosphatases with the ability to dephosphorylate phosphotyrosine are very similar to the number of active tyrosine phosphatases. Both types of enzymes also display comparable tissue distribution patterns (Alonso et al., 2004). Abnormalities in tyrosine phosphorylation play a role in the pathogenesis of numerous inherited and acquired human diseases from cancer to immune deficiencies (Alonso et al., 2004)
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During the process of capacitation, spermatozoa go through a whole set of signaling cascade events in order to become fully competent at fertilizing the egg. An increase in sperm protein tyrosine phosphorylation has been described during this final maturational event in different animal species as well as in humans. Although the phosphotyrosine content of sperm protein is modulated by cAMP, Ca(2+), BSA, oxygen derivatives, and cholesterol, no protein tyrosine kinase (PTK) nor the phosphotyrosine protein phosphatase (PTPase) directly involved in the control of the phosphotyrosine content of sperm protein has been identified. Therefore, the goal of the present study was to identify the tyrosine kinases putatively responsible for the increases in sperm protein phosphotyrosine content. In the present study, we show that the src-related tyrosine kinase c-yes is present in the head of human spermatozoa in both membranes and Triton X-100-insoluble extracts. Our hypothesis was that c-yes is a tyrosine kinase responsible for at least some of the capacitation-induced increase in protein tyrosine phosphorylation. When spermatozoa were previously incubated in the presence of 3-isobutyl-1-methylxanthine or 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, treatments known to increase the phosphotyrosine content of human sperm proteins, an increase in the kinase activity of immunoprecipitated yes was measured using enolase as a substrate. These results suggest that cAMP activates while Ca(2+) inhibits human sperm c-yes kinase activity.
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Capacitation represents the final maturational steps that render mammalian sperm competent to fertilize, either in vivo or in vitro. Capacitation is defined as a series of events that enables sperm to bind the oocyte and undergo the acrosome reaction in response to the zona pellucida. Although the molecular mechanisms involved are not fully understood, sperm protein phosphorylation is associated with capacitation. The hypothesis of this study is that protein tyrosine phosphorylation and kinase activity mediate capacitation of porcine sperm. Fresh sperm were incubated in noncapacitating or capacitating media for various times. Proteins were extracted with SDS, subjected to SDS-PAGE, and immunoblotted with an antiphosphotyrosine antibody. An Mr 32 000 tyrosine-phosphorylated protein (designated as p32) appeared only when the sperm were incubated in capacitating medium and concomitant with capacitation as assessed by the ionophore-induced acrosome reaction. The p32 was soluble in Triton X-100. Fractionation of sperm proteins with Triton X-114 demonstrated that after capacitation, this tyrosine phosphoprotein is located in both the cytosol and the membrane. Enzyme renaturation of sperm proteins was conducted in gels with or without either poly glu:tyr (a tyrosine kinase substrate) or kemptide (a protein kinase A substrate). An Mr 32 000 enzyme with kinase behavior was observed in all gels but was preferentially phosphorylated on tyrosine, as assessed by phosphorimagery and by thin layer chromotography to identify the phosphoamino acids. Indirect immunolocalization showed that the phosphotyrosine residues redistribute to the acrosome during capacitation, which is an appropriate location for a protein involved in the acquisition of fertility.
Capacitation
Acrosome reaction
Phosphoprotein
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