Treatment of quiescent Swiss 3T3 fibroblasts with serum, or with the phosphatase inhibitors okadaic acid and vanadate, induced a 2-to 11-fold activation of the serine/ threonine RAC protein kinase (RAC-PK). Kinase activation was accompanied by decreased mobility of RAC-PK on SDS/ PAGE such that three electrophoretic species (a to c) of the kinase were detected by immunoblot analysis, indicative of differentially phosphorylated forms. Addition of vanadate to arrested cells increased the RAC-PK phosphorylation level 3-to 4-fold. Unstimulated RAC-PK was phosphorylated predominantly on serine, whereas the activated kinase was phosphorylated on both serine and threonine residues. Treatment of RAC-PK in vitro with protein phosphatase 2A led to kinase inactivation and an increase in electrophoretic mobility. Deletion of the N-terminal region containing the pleckstrin homology domain did not affect RAC-PK activation by okadaic acid, but it reduced vanadate-stimulated activity and also blocked the serum-induced activation. Deletion of the serine/ threonine rich C-terminal region impaired both RAC-PKa basal and vanadate-stimulated activity. Studies using a kinase-deficient mutant indicated that autophosphorylation is not involved in RAC-PKa activation. Stimulation of RAC-PK activity and electrophoretic mobility changes induced by serum were sensitive to wortmannin. Taken together the results suggest that RAC-PK is a component of a signaling pathway regulated by phosphatidylinositol (PI) 3-kinase, whose action is required for RAC-PK activation by phosphorylation.The RAC-PKs (for related to PKA and C protein kinases; also known as PKB/Akt) represent a subfamily of second messenger-regulated serine/threonine protein kinases (1). Two human genes have been identified, termed RACa and -Pf that are 90% homologous (2-4). Both genes appear to be widely expressed in human tissues, implying that they play an important role in cell signaling. Mouse RACa (c-akt) is the cellular homologue of the viral oncogene v-akt, generated by fusion of the Gag protein from the AKT8 retrovirus to the N terminus of mouse RAC-PKa, giving rise to a 105-kDa phosphoprotein that is myristilated at its N terminus (5, 6). The mouse protein is mainly cytosolic (90%), whereas the oncoprotein is apparently equally distributed between the plasma membrane, nucleus, and cytoplasm (6). Human RACf3 was found to be amplified in 10% of human ovarian carcinomas (4), suggesting the involvement of the RAC-PK subfamily members in regulation of cell growth. The Drosophila homologue (DRAC) shows 75% homology to the human isoforms and is ubiquitously expressed throughout the Drosophila life cycle (7,8).All characterized members of the RAC-PK subfamily have a similar domain structure: an N-terminal pleckstrin homologyThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.(PH) domain, a centrally located catalytic do...
A novel serine/threonine protein kinase (termed rac-PK) has recently been identified and cloned from cDNA libraries derived from the human cell lines MCF-7 and W138. A second form of this protein kinase, termed rac protein kinase ,, has been identified from cDNAs derived from the same cell lines.These two closely related forms show 90% homology, although the f form with a predicted Mr 60 200 has a carboxyl terminal extension of 40 amino acids in comparison to the a form. This extension has a high serine content with 11 serine residues in the last 30 amino acids. The jt form of the protein has been shown by both in vitro translation and bacterial expression to be -5000 Da larger than the a form. rac protein kinase ,8 is encoded by a 3.4-kb transcript and the a form is encoded by a 3.2-kb mRNA. Using gene-specific probes both transcripts were detected in all cell types analyzed, although levels of expression were different for the two forms. The catalytic domain of rac protein kinase ft shows a high degree of homology to both the protein kinase C and cyclic AMP-dependent protein kinase families, and hence rac protein kinases appear to represent a new subfamily of the second messenger serine/threonine protein kinases.
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