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Time Course Analysis of Eight Autophosphorylation Sites on p21‐Activated Protein Kinase Pak2
Author(s) -
Jung JinHun,
White Jon,
Traugh Jolinda
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1050.10
Subject(s) - autophosphorylation , phosphorylation , serine , chemistry , kinase , microbiology and biotechnology , protein kinase a , biochemistry , biology
The p21‐activated protein kinase Pak2 is activated under cytostatic or apoptotic conditions. Binding of Cdc42(GTP) stimulates autophosphorylation of Pak2 up to 8 mol/mol, which is required for full activation. There are seven serine sites (S19, S20, S55, S141, S165, S192 and S197) in the regulatory domain, and one threonine (T402) in the activation loop. The time course of autophosphorylation of Cdc42‐activated Pak2 showed a gradual increase from 1 to 30 min. When autophosphorylated Pak2 was subjected to two‐dimensional tryptic phosphopeptide mapping and quantified, S197 was rapidly phosphorylated during first 10 min and constituted about 40% of the autophosphorylation, while the other sites were significantly less. To delineate the effect of each site in the molecular activation of Pak2, the sites were changed to alanine by site‐directed mutagenesis and the level of autophosphorylation was analyzed. In the presence of Cdc42, the four alanine mutants, S141A, S192A, S197A and T402A, showed a 50–60% reduction in autophosphorylation, while there was no effect on S19A/S20A, S55A and S165A, as compared to the level with WT Pak2. Thus, autophosphorylation of S141, S192, S197 and T402 are required to achieve maximum autophosphorylation, and phosphorylation of S197 contributes to promoting further autophosphorylation on other sites. Supported by NIH grant GM 26738 to J. A. T.