Multisite Autophosphorylation of p21-activated Protein Kinase γ-PAK as a Function of Activation*

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 γ-isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant γ-PAK, while native γ-PAK was isolated from rabbit reticulocytes. Two-dimensional gel electrophoresis of γ-PAK followed by immunoblot analysis revealed a similar profile for native and recombinant γ-PAK, both consisting of multiple protein spots. Following Cdc42-stimulated autophosphorylation, the two-dimensional profiles of native and recombinant γ-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, γ-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 γ-PAK, six of which are in common with those previously reported in α-PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the γ-form. Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with γ-PAK activation.

[1]  C. Walsh,et al.  PAK3 mutation in nonsyndromic X-linked mental retardation , 1998, Nature Genetics.

[2]  A. M. Reilly,et al.  A GTPase-independent Mechanism of p21-activated Kinase Activation , 1998, The Journal of Biological Chemistry.

[3]  J. Chant,et al.  Human Ste20 homologue hPAK1 links GTPases to the JNK MAP kinase pathway , 1996, Current Biology.

[4]  M. Siegmann,et al.  Active baculovirus recombinant p70s6k and p85s6k produced as a function of the infectious response. , 1993, The Journal of biological chemistry.

[5]  X. Q. Chen,et al.  Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes , 1997, Molecular and cellular biology.

[6]  G. Bokoch,et al.  Localization of p21-Activated Kinase 1 (PAK1) to Pinocytic Vesicles and Cortical Actin Structures in Stimulated Cells , 1997, The Journal of cell biology.

[7]  J. Chernoff,et al.  Identification of a Mouse p21Cdc42/Rac Activated Kinase (*) , 1995, The Journal of Biological Chemistry.

[8]  Jiahuai Han,et al.  Rho Family GTPases Regulate p38 Mitogen-activated Protein Kinase through the Downstream Mediator Pak1 (*) , 1995, The Journal of Biological Chemistry.

[9]  P. T. Tuazon,et al.  Autophosphorylation and protein kinase activity of p21-activated protein kinase gamma-PAK are differentially affected by magnesium and manganese. , 1998, Biochemistry.

[10]  T. Wong,et al.  A manual sequencing method for identification of phosphorylated amino acids in phosphopeptides. , 1991, Analytical biochemistry.

[11]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[12]  A. Roulston,et al.  Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[13]  L. Lim,et al.  A brain serine/threonine protein kinase activated by Cdc42 and Rac1 , 1994, Nature.

[14]  G. Bokoch,et al.  Membrane targeting of p21‐activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells , 1998, The EMBO journal.

[15]  J. Schlessinger,et al.  The adaptor protein Nck links receptor tyrosine kinases with the serine-threonine kinase Pak1 , 1996, The Journal of Biological Chemistry.

[16]  W. Bonner,et al.  Polyacrylamide gel electrophoresis of small peptides , 1984 .

[17]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[18]  P. T. Tuazon,et al.  Molecular Cloning and Sequencing of the Cytostatic G Protein-activated Protein Kinase PAK I (*) , 1996, The Journal of Biological Chemistry.

[19]  R. Cook,et al.  1,25-dihydroxyvitamin D3 modulates phosphorylation of serine 205 in the human vitamin D receptor: site-directed mutagenesis of this residue promotes alternative phosphorylation. , 1994, Biochemistry.

[20]  C. Hall,et al.  Molecular Cloning of a New Member of the p21-Cdc42/Rac-activated Kinase (PAK) Family (*) , 1995, The Journal of Biological Chemistry.

[21]  F. McCormick,et al.  A novel serine kinase activated by rac1/CDC42Hs‐dependent autophosphorylation is related to PAK65 and STE20. , 1995, The EMBO journal.

[22]  C. Monnig,et al.  Cleavage Arrest of Early Frog Embryos by the G Protein-activated Protein Kinase PAK I* , 1996, The Journal of Biological Chemistry.

[23]  G. Bokoch,et al.  Regulation of human leukocyte p21-activated kinases through G protein--coupled receptors. , 1995, Science.

[24]  S. Tahara,et al.  Differential activation of two protease-activated protein kinases from reticulocytes by a Ca2+-stimulated protease and identification of phosphorylated translational components. , 2005, European journal of biochemistry.

[25]  C. Kent,et al.  Identification of phosphorylation sites in rat liver CTP: phosphocholine cytidylyltransferase. , 1994, The Journal of biological chemistry.

[26]  P. Robinson,et al.  Okadaic acid interferes with phorbol-ester-mediated down-regulation of protein kinase C-alpha, C-delta and C-epsilon. , 1997, European journal of biochemistry.

[27]  C. Hall,et al.  Regulation of Phosphorylation Pathways by p21 GTPases , 1996 .

[28]  L. Heilmeyer,et al.  Sequence analysis of phosphoserine‐containing peptides , 1986, FEBS letters.

[29]  S. Tahara,et al.  Cyclic Nucleotide-independent protein kinases from rabbit reticulocytes. Identification and characterization of a protein kinase activated by proteolysis. , 1981, The Journal of biological chemistry.

[30]  G M Bokoch,et al.  Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. , 1997, Science.

[31]  H. Erdjument-Bromage,et al.  Methodical analysis of protein-nitrocellulose interactions to design a refined digestion protocol. , 1996, Analytical biochemistry.

[32]  S. Yang,et al.  Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family. , 1998, The Biochemical journal.

[33]  E. Alnemri,et al.  Cleavage and activation of p21-activated protein kinase gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity. , 1998, The Journal of biological chemistry.

[34]  M. Teo,et al.  Identification and Molecular Cloning of a p21cdc42/rac1-activated Serine/Threonine Kinase That Is Rapidly Activated by Thrombin in Platelets (*) , 1995, The Journal of Biological Chemistry.

[35]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[36]  C. Monnig,et al.  Determinants for substrate phosphorylation by p21-activated protein kinase (gamma-PAK). , 1997, Biochemistry.

[37]  A. Gatti,et al.  A two-dimensional peptide gel electrophoresis system for phosphopeptide mapping and amino acid sequencing. , 1999, Analytical biochemistry.

[38]  J. Chernoff,et al.  Emerging from the Pak: the p21-activated protein kinase family. , 1997, Trends in cell biology.