PKA and CaMKII mediate PI3K activation in bovine sperm by inhibition of the PKC/PP1 cascade

Abstract

To enable fertilization, spermatozoa must undergo several biochemical processes in the female reproductive tract, collectively called capacitation. These processes involve protein kinase A (PKA)-dependent protein tyrosine phosphorylation including phosphatidylinositol-3-kinase (PI3K). It is not known how PKA, a serine/threonine (S/T) kinase, mediates tyrosine phosphorylation of proteins. We recently showed that inhibition of S/T phosphatase 1 (PP1) causes a significant increase in phospho-PI3K. In this study, we propose a mechanism by which PKA and PP1 mediate an increase in PI3K tyrosine phosphorylation and implicate calmodulin-dependent kinase II (CaMKII) in this process. Inhibition of sperm PP1 or PKC, stimulated CaMKII phosphorylation/activation, and inhibition of PKC enhanced PP1 phosphorylation/inactivation. Inhibition of CaMKII, using KN-93, caused significant reduction in phospho-PP1, indicating its activation. Moreover, KN-93 prevented the dephosphorylation/inactivation of PKC. We therefore suggest that CaMKII inhibits PKC, leading to PP1 inhibition and the reciprocal auto-activation of CaMKII. Thus, CaMKII can regulate its own activation by inhibiting the PKC/PP1 cascade. Inhibition of Src family kinases (SFK) caused significant inhibition of CaMKII and PP1 phosphorylation, suggesting that SFK activity results in PP1 inhibition and CaMKII activation. Activation of sperm PKA by 8Br-cAMP revealed an increase in phosphoCaMKII, which was inhibited by PKA inhibitor. Tyrosine phosphorylation of PI3K was stimulated by 8Br-cAMP and by PKC or PP1 inhibition and was abrogated by CaMKII inhibition. Furthermore, phosphorylation/activation of the tyrosine kinase Pyk2 was enhanced by PP1 inhibition, and this activation is blocked by CaMKII inhibition. Thus, PKA activates Src, which inhibits PP1, leading to CaMKII and Pyk2 activation, resulting in PI3K tyrosine phosphorylation/activation.

How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions

“…In PC12 cells, Ca 2þ entry triggers both Pyk2 activation and cytonuclear accumulation, and the Ca 2þ -dependent Ser/Thr phosphatase calcineurin is required for these effects (Faure et al, 2007). The role of protein kinase A (PKA) and protein phosphatase 1(PP1) has also been suggested (Battistone et al, 2014;Park et al, 2000;Rotfeld et al, 2014) {Park, 2000Battistone, 2014 #808;Rotfeld, 2014 #807}. Pyk2 contains several putative (de)phosphorylation sites for these kinases and phosphatases, mainly located in the kinase-FAT linker (Oppermann et al, 2009) {Oppermann, 2009 Thus, Pyk2 appears to be able to detect multiple signals triggered by Ca 2þ .…”

Threonine-290 Regulates Nuclear Translocation of the Human Pregnane X Receptor through Its Phosphorylation/Dephosphorylation by Ca2+/Calmodulin-Dependent Protein Kinase II and Protein Phosphatase 1

“…PP1 is a key regulator of CaMKII, which functions as a serine/ threonine kinase by autophosphorylation at its threonine-286 through the inhibition of PP1, leading to the regulation of synaptic strength in forebrain postsynaptic density (Strack et al, 1997a;Blitzer et al, 1998;Baucum II et al, 2012) and mediates PI3K activation in bovine sperm capacitation (Rotfeld et al, 2014). Furthermore, the dephosphorylation of CaMKII in postsynaptic density was previously shown to be catalyzed by PP1 (Shields et al, 1985;Dosemeci and Reese, 1993;Strack et al, 1997b).…”

The role and importance of cofilin in human sperm capacitation and the acrosome reaction

“…It has also been shown that CaMKII negatively regulates SSH1L activity by phosphorylation/activation of LIMK1, which regulates the subcellular localization of SSH1L (Zhao et al 2012). In our recent study, we show that CaMKII activates Pyk2 leading to PI3K phosphorylation/activation in sperm (Rotfeld et al 2014), and it has been shown elsewhere that PI3K mediates the activation of SSH1L (Nishita et al 2004). Thus, it is possible that activation of CaMKII/ PI3K cascade during sperm capacitation together with the increase in [Ca 2+ ] i activates SSH1L which dephosphorylates p-cofilin towards the end of the capacitation process.…”