Substrates enhance autophosphorylation and activation of p21-activated protein kinase gamma-PAK in the absence of activation loop phosphorylation.
The p21-activated protein kinase gamma-PAK from rabbit, expressed in insect cells, is activated following binding of Cdc42(GTPgammaS). The rate of autophosphorylation is increased fivefold and the protein kinase activity 13-fold, as measured with the synthetic heptapeptide (AKRESAA). The mutant K278R, where the invariant lysine in the catalytic site is replaced by arginine, shows neither autophosphorylation nor activity. Replacement of the conserved threonine in the catalytic domain with alanine (T402A) reduces autophosphorylation and protein kinase activity to 1% that of the wild-type gamma-PAK, indicating autophosphorylation of Thr402 in the activation loop is essential for protein kinase activity. In contrast, certain protein substrates such as histone 2B, histone 4 and myelin basic protein, stimulate both autophosphorylation and protein kinase activity to levels similar to those observed with Cdc42(GTPgammaS). This substrate-level activation does not require autophosphorylation of Thr402 in the activation loop. As shown with T402A, the protein kinase activity with histone 4 is similar to that observed with recombinant wild-type gamma-PAK. Basic proteins or peptides which are not substrates of gamma-PAK, such as histone 1 and polylysine, do not stimulate autophosphorylation or activity. Other substrates such as the Rous sarcoma virus protein NC are phosphorylated by gamma-PAK following activation by Cdc42(GTPgammaS), but are not phosphorylated by T402A. The data suggest that some substrates can override the requirement for Cdc42(GTPgammaS), by activating gamma-PAK directly.
Jakobi
R
R
Department of Biochemistry, University of California, Riverside 92521, USA.
Huang
Z
Z
Walter
B N
BN
Tuazon
P T
PT
Traugh
J A
JA
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001358:Avian Sarcoma Viruses / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D006657:Histones / Q000378:metabolism; D006801:Humans; D007313:Insecta; D008239:Lysine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D004676:Myelin Basic Protein / Q000378:metabolism; D019590:Nucleocapsid Proteins / Q000378:metabolism; D010449:Peptide Mapping; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011107:Polylysine / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D054462:p21-Activated Kinases
D018076:DNA, Complementary; D006657:Histones; D004676:Myelin Basic Protein; D019590:Nucleocapsid Proteins; D010455:Peptides; D011994:Recombinant Proteins; D011107:Polylysine; D013912:Threonine; D001120:Arginine; C515907:PAK2 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D020764:cdc42 GTP-Binding Protein; D008239:Lysine