Myosin phosphorylation by human cdc42-dependent S6/H4 kinase/gammaPAK from placenta and lymphoid cells.
The p21-activated kinase (PAK) family includes protein phosphotransferases regulated by the GTPases rho, rac, and cdc42. Sequence homology, activation mechanism, and substrate specificity suggest that the well-characterized human placenta S6/H4 kinase is a member of this family. In these studies, S6/H4 kinase purified to homogeneity from human placenta was activated in vitro by cdc42-GTP, or protease incubation and MgATP-dependent autophosphorylation. The cdc42-activated enzyme demonstrated an Mr 60,000, and shares sequence homology with the gammaPAK family. Antipeptide antibodies against one of the autophosphorylation site sequences recognized a single p60 protein in the purified placenta preparation or Jurkat cell extracts. An autophosphorylated Mr 40,000 protein, previously identified as the catalytic domain of the enzyme, was also detected by the antibody after protease activation. Crude PAK60 obtained from Mono Q chromatography of Jurkat cell extracts and purified placenta enzyme catalyzed phosphorylation of histone H4 and myelin basic protein as well as a variety of synthetic peptides previously identified as S6/H4 kinase substrates. In addition, Jurkat myosin II and the regulatory myosin light chain were phosphorylated by the Jurkat and placenta gammaPAK. Synthetic peptides were used to demonstrate that the site of light chain phosphorylation occurs at the serine which results in ATPase activation. The data suggest that human gammaPAK may regulate cell motility by a GTP-dependent and calcium-independent mechanism.
Ramos
E
E
Department of Biological Sciences, University of North Texas, Denton 76201, USA.
Wysolmerski
R B
RB
Masaracchia
R A
RA
D018797:Cell Cycle Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008214:Lymphocytes / Q000201:enzymology*; D018994:Myosin Light Chains / Q000378:metabolism*; D010766:Phosphorylation; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D019893:Ribosomal Protein S6 Kinases / Q000145:classification / Q000378:metabolism*; D013379:Substrate Specificity; D020764:cdc42 GTP-Binding Protein
D018797:Cell Cycle Proteins; D018994:Myosin Light Chains; D019893:Ribosomal Protein S6 Kinases; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein