Multisite autophosphorylation of p21-activated protein kinase gamma-PAK as a function of activation

J Biol Chem. 1999 Mar 19;274(12):8022-8. doi: 10.1074/jbc.274.12.8022.

Abstract

p21-activated protein kinase (PAK) is a family of serine/threonine kinases whose activity is stimulated by binding to small G-proteins such as Cdc42 and subsequent autophosphorylation. Focusing on the ubiquitous gamma-isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant gamma-PAK, while native gamma-PAK was isolated from rabbit reticulocytes. Two-dimensional gel electrophoresis of gamma-PAK followed by immunoblot analysis revealed a similar profile for native and recombinant gamma-PAK, both consisting of multiple protein spots. Following Cdc42-stimulated autophosphorylation, the two-dimensional profiles of native and recombinant gamma-PAK were characterized by a similar acidic shift, suggesting a common response to Cdc42. To understand the effect of differential phosphorylation on its activation status, gamma-PAK autophosphorylation was conducted in the presence or absence of activators such as Cdc42 and histone II-AS, followed by tryptic digestion and comparative two-dimensional phosphopeptide mapping. The major phosphopeptides were subjected to a combination of manual and automated amino acid sequencing. Overall, eight autophosphorylation sites were identified in Cdc42-activated gamma-PAK, six of which are in common with those previously reported in alpha-PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the gamma-form. Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with gamma-PAK activation.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Cycle Proteins / metabolism
  • GTP-Binding Proteins / metabolism
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Molecular Sequence Data
  • Peptide Mapping
  • Phosphopeptides / chemistry
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Rabbits
  • Sequence Alignment
  • cdc42 GTP-Binding Protein, Saccharomyces cerevisiae
  • p21-Activated Kinases

Substances

  • Cell Cycle Proteins
  • Phosphopeptides
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Protein-Serine-Threonine Kinases
  • p21-Activated Kinases
  • GTP-Binding Proteins
  • cdc42 GTP-Binding Protein, Saccharomyces cerevisiae