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

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 PP1 gamma2, assessment for motility and co-immunoprecipitation of PP1gamma2 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 PP1 gamma2 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.