Phosphorylation of LIMK1 and COFILIN is regulated by PAK4 and Okadaic acid- sensitive protein phosphatases during mouse sperm capacitation.

ROMAROWSKI A; LUQUE GM; STIVAL C; UGO B; PUGA MOLINA LC; GILIO N; KRAPF D; BUFFONE MG

Seminario; The Batsheva de Rothschild Seminar on Challenges and Frontiers in Mammalian Reproduction.; 2016

Actin dynamics play a central role in controlling the process of exocytosis in somatic cells as well as in sperm from several mammalian species. We previously have shown the involvement of small GTPases of the Rho family in the signaling pathway that leads to actin polymerization during sperm capacitation through the activation of key regulatory proteins of the actin cytoskeleton, LIMK1 and COFILIN. The activity of both, LIMK1 and COFILIN is modulated by phosphorylation in Thr508 and Ser3, respectively. COFILIN, is phosphorylated during capacitation in a transiently manner and its function as a severing protein of actin filaments is modulated by this phosphorylation. In the present work we studied the role of PAK kinases and phosphatases in the regulation of LIMK1/COFILIN phosphorylation during capacitation. We observed that one of the widely known phosphatases of COFILIN in somatic cells, Slingshot homolog 1 (SSH1), is expressed in mouse sperm and localized in the acrosome and the principal piece of the flagellum. The dephosphorylation of SSH1 on Ser978 that results in the active form of SSH1, coincided with the time of incubation where COFILIN is dephosphorylated at Ser3. Okadaic acid- sensitive protein phosphatases were found to dephosphorylate SSH1 and their inhibition resulted in persistent levels of phosphorylated COFILIN. However, in presence of okadaic acid, the percentage of sperm that undergo acrosomal exocytosis remained unchanged. On the other hand, inhibition of PAK4 kinase by a specific inhibitor (PF -3758309) resulted in lower levels of pLIMK1 and a dramatic decrease in the percentage of sperm that undergo acrosomal exocytosis. In conclusion, we observed that PAK4 kinase and okadaic acid sensitive phosphatases participate in the regulation of LIMK1/COFILIN phosphorylation/dephosphorylation balance, which is essential for the dynamic changes of the actin cytoskeleton during capacitation and acrosomal exocytosis.