Binding of 14-3-3 beta regulates the kinase activity and subcellular localization of testicular protein kinase 1

Testicular protein kinase 1 (TESK1) is a serine/threonine kinase that phosp horylates cofilin and induces actin cytoskeletal reorganization. The kinase activity of TESK1 is stimulated by integrin-mediated signaling pathways, b ut the mechanism of regulation has remained unknown. By using the yeast two -hybrid system, we identified 14-3-3 beta to be the binding protein of TESK 1. Specific interaction between TESK1 and 14-3-3 beta became evident in in vitro and in vivo co-precipitation assays. 14-3-3 beta interacts with TESK1 through the C-terminal region of TESK1 and in a manner dependent on the ph osphorylation of Ser-439 within an RXXSXP motif. Binding of 14-3-3 beta inh ibited the kinase activity of TESK1. During cell spreading on fibronectin, the TESK1/14-3-3 beta interaction significantly decreased, in a time course that inversely correlated with increase in TESK1 kinase activity. Thus, th e dissociation of 14-3-3 beta from a TESK1/14-3-3 beta complex is likely to be involved in the integrin-mediated TESK1 activation. In HeLa cells, TESK 1, together with 14-3-3 beta, accumulated at the cell periphery when cells were plated on fibronectin, whereas they were diffusely distributed in the cytoplasm in the case of non-stimulated cells. We propose that 14-3-3 beta plays important roles in regulating the kinase activity of TESK1 and locali zing TESK1 to cell adhesion sites following integrin stimulation.