Identification and molecular cloning of a p21cdc42/rac1-activated serine/threonine kinase that is rapidly activated by thrombin in platelets.
The brain-enriched p21cdc42/rac1-activated serine/threonine kinase, p65PAK, was identified and purified on the basis of overlays with [gamma-32P]GTP-Cdc42 onto SDS-fractionated proteins (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L. (1994) Nature 367, 40-46). In this study, the ubiquitously expressed p21cdc42/rac1 binding protein with relative molecular weight of 62,000 was purified from rat testes and shown to contain peptides related to PAK. It has thus been designated as the gamma-PAK isoform (alpha- and beta-isoforms being brain enriched). Isolation of gamma-PAK cDNAs show that the kinase is highly conserved with alpha-PAK in both the p2 binding and kinase domains. The purified protein exhibited kinase activity that was activated by GTP-Cdc42 or GTP-Rac1 in vitro. In platelets, a p62 in situ renaturable kinase was recognized by antibodies raised against gamma-PAK. This thrombin-activated protein kinase appears to coprecipitate with another kinase of M(r) 86,000, suggesting that PAK may be part of a thrombin-responsive signaling complex.
Teo
M
M
Glaxo-IMCB Group, Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge, Singapore.
Manser
E
E
Lim
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001921:Brain / Q000201:enzymology*; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013737:Testis / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D020764:cdc42 GTP-Binding Protein; D054462:p21-Activated Kinases
D018797:Cell Cycle Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D013917:Thrombin; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein