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

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

Rehovot

Congreso; The Batsheva de Rothschild Seminar on Challenges and Frontiers in Mammalian Reproduction. Weizmann Institute of Science.; 2016

Weizmann Institute of Science.

Actin dynamics play a central role in controlling the process of exocytosis in somatic cellsas well as in sperm from several mammalian species.!We previously have shown theinvolvement of small GTPases of the Rho family in the signaling pathway that leads toactin polymerization during sperm capacitation through the activation of key regulatoryproteins of the actin cytoskeleton, LIMK1 and COFILIN. The activity of both, LIMK1 andCOFILIN is modulated by phosphorylation in Thr508 and Ser3, respectively. COFILIN, isphosphorylated during capacitation in a transiently manner and its function as a severingprotein of actin filaments is modulated by this phosphorylation. In the present work westudied the role of PAK kinases and phosphatases in the regulation of LIMK1/COFILINphosphorylation during capacitation. We observed that one of the widely knownphosphatases of COFILIN in somatic cells, Slingshot homolog 1 (SSH1), is expressed inmouse sperm and localized in the acrosome and the principal piece of the flagellum. Thedephosphorylation of SSH1 on Ser978 that results in the active form of SSH1, coincidedwith the time of incubation where COFILIN is dephosphorylated at Ser3. Okadaic acidsensitiveprotein phosphatases were found to dephosphorylate SSH1 and their inhibitionresulted in persistent levels of phosphorylated COFILIN. However, in presence of okadaicacid, the percentage of sperm that undergo acrosomal exocytosis remained unchanged. Onthe other hand, inhibition of PAK4 kinase by a specific inhibitor (PF -3758309) resulted inlower levels of pLIMK1 and a dramatic decrease in the percentage of sperm that undergoacrosomal exocytosis. In conclusion, we observed that PAK4 kinase and okadaic acidsensitive phosphatases participate in the regulation of LIMK1/COFILINphosphorylation/dephosphorylation balance, which is essential for the dynamic changes ofthe actin cytoskeleton during capacitation and acrosomal exocytosis.