Binding of 14-3-3beta regulates the kinase activity and subcellular localization of testicular protein kinase 1.
Testicular protein kinase 1 (TESK1) is a serine/threonine kinase that phosphorylates cofilin and induces actin cytoskeletal reorganization. The kinase activity of TESK1 is stimulated by integrin-mediated signaling pathways, but the mechanism of regulation has remained unknown. By using the yeast two-hybrid system, we identified 14-3-3beta to be the binding protein of TESK1. Specific interaction between TESK1 and 14-3-3beta became evident in in vitro and in vivo co-precipitation assays. 14-3-3beta interacts with TESK1 through the C-terminal region of TESK1 and in a manner dependent on the phosphorylation of Ser-439 within an RXXSXP motif. Binding of 14-3-3beta inhibited the kinase activity of TESK1. During cell spreading on fibronectin, the TESK1/14-3-3beta interaction significantly decreased, in a time course that inversely correlated with increase in TESK1 kinase activity. Thus, the dissociation of 14-3-3beta from a TESK1/14-3-3beta complex is likely to be involved in the integrin-mediated TESK1 activation. In HeLa cells, TESK1, together with 14-3-3beta, 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 14-3-3beta plays important roles in regulating the kinase activity of TESK1 and localizing TESK1 to cell adhesion sites following integrin stimulation.
Toshima
J Y
JY
Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578, Japan.
Toshima
J
J
Watanabe
T
T
Mizuno
K
K
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D016023:Integrins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D005810:Multigene Family; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000378:metabolism*
D048948:14-3-3 Proteins; D016023:Integrins; D011993:Recombinant Fusion Proteins; C482982:YWHAB protein, human; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D005982:Glutathione Transferase; C097507:testis-specific protein kinase 1; D017346:Protein Serine-Threonine Kinases