Tyrosine phosphorylation, thiol status, and protein tyrosine phosphatase in rat epididymal spermatozoa.

Sperm thiol oxidation and the ability to undergo protein tyrosine phosphorylation are associated with the acquisition of sperm motility and fertilizing ability during passage of spermatozoa through the epididymis. Phosphotyrosine levels in various cells are controlled by tyrosine kinase versus phosphatase, with the latter known to be inhibited by oxidation. In the present paper we examine whether changes in thiol status during sperm maturation affect rat sperm protein phosphotyrosine levels and protein phosphotyrosine phosphatase (PTP) activity. Tyrosine phosphorylation, as demonstrated by immunoblotting (IB), was significantly increased in several sperm tail proteins during maturation in the epididymis. Sperm thiol oxidation with diamide enhanced tail protein phosphorylation; reduction of disulfides with dithiothreitol diminished phosphorylation. In the sperm head, a moderate increase in tyrosine phosphorylation was accompanied by altered localization of phosphotyrosine proteins during maturation. Blocking of thiols and PTP activity with N-ethylmaleimide led to increased tyrosine phosphorylation of protamine in caput sperm heads. Several PTP bands were identified by IB. In the caput spermatozoa, a prominent level of the 50 kDa band was present, whereas in the cauda spermatozoa a very low level of the 50 kDa band was found. PTP activity, measured by using p-nitrophenyl phosphate as a substrate, was significantly higher in the caput spermatozoa (high thiol content) than in the cauda spermatozoa (low thiol content). Our results show that PTP activity is correlated with sperm thiol status and suggest that tyrosine phosphorylation of sperm proteins during sperm maturation is promoted by thiol oxidation and diminished PTP.

Ca2+ promotes erythrocyte band 3 tyrosine phosphorylation via dissociation of phosphotyrosine phosphatase from band 3.

The anion-exchange band 3 protein is the main erythrocyte protein that is phosphorylated by protein tyrosine kinase (PTK). We have previously identified a band 3-associated phosphotyrosine phosphatase (PTP) that is normally highly active and prevents the accumulation of band 3 phosphotyrosine. Band 3 tyrosine phosphorylation can be induced by inhibition of PTP (vanadate, thiol oxidation), activation of PTK (hypertonic NaCl) or intracellular increased Ca(2+) (mechanism unknown). We now show that there is inhibition of dephosphorylation of band 3 in Ca(2+)/ionophore-treated erythrocytes and in membranes isolated from the treated cells. These membranes exhibit phosphatase activity upon the addition of exogenous substrate. Dephosphorylation of the endogenous substrate (band 3) can be activated in these membranes by the addition of Mg(2+). Thus the inability of PTP to dephosphorylate the band 3 phosphotyrosine is not due to inhibition of the enzyme itself. Ca(2+) rise in the erythrocyte causes dissociation of PTP from band 3, thus leaving the kinase unopposed. This is shown by a significant diminution in band 3/PTP co-precipitation. Addition of Mg(2+) to these membranes leads to reassociation of band 3 with PTP. The Ca(2+)-induced inhibition of band 3 dephosphorylation may be due to Ca(2+)-dependent alterations in membrane components and structure, affecting the interaction of band 3 with PTP. The Ca(2+)-induced tyrosine phosphorylation, involving an apparent PTP inhibition via dissociation from the substrate, may play a role in signal transduction pathways and in certain pathological disorders associated with increased cell Ca(2+).