Putative Roles of Tyrosine Kinase in Sperm Functions.Pierre Leclerc, Ph.D.

Abstract

An increase in protein tyrosine phosphorylation has been shown to occur during sperm capacitation. The effects of tyrosine kinase inhibitors strongly suggest the involvement of src-related kinase in this phenomenon. Our group has recently reported the presence and activity of SRC in human spermatozoa. This kinase is present both in the acrosomal region of the head and in the flagellum of ejaculated human spermatozoa. It is associated with the plasma membrane and its partial extraction with non-ionic detergents suggests that SRC is also associated with cytoskeletal elements. Its tyrosine kinase activity is dependent on the presence of Ca2+ and is modulated by cAMP. Our results show that SRC is active throughout sperm capacitation and might be involved in the increase in protein phosphotyrosine content related to different sperm functions. Although many SRC substrates, which are present in sperm, have been identified in different cell types, no direct SRC interacting protein has been identified in sperm. Two different approaches were used to identify putative SRC substrates in order to characterize its role in sperm functions. At first, sperm homogenates were incubated with active recombinant SRC and tyrosine phosphorylated proteins were identified by LC-MS/MS after 2D-gel electrophoresis. In the second assay, proteins extracted from sperm were subjected to 2D-electrophoresis, and a far western overlay was performed using a recombinant His-tagged SH2-SH3 domains from SRC. As for the first approach, proteins were identified by LC-MS/MS. The identified SRC substrates or interacting proteins will help to define the role of SRC mediated tyrosine phosphorylation in sperm functions.