Identification and molecular cloning of a p21cdc42/rac1-activated serine/threonine kinase that is rapidly activated by thrombin in platelets
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1Glaxo-IMCB Group, Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge, Singapore.
Abstract:
The brain-enriched p21cdc42/rac1-activated serine/threonine kinase, p65PAK, was identified and purified on the basis of overlays with [gamma-32P]GTP-Cdc42 onto SDS-fractionated proteins (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L. (1994) Nature 367, 40-46). In this study, the ubiquitously expressed p21cdc42/rac1 binding protein with relative molecular weight of 62,000 was purified from rat testes and shown to contain peptides related to PAK. It has thus been designated as the gamma-PAK isoform (alpha- and beta-isoforms being brain enriched). Isolation of gamma-PAK cDNAs show that the kinase is highly conserved with alpha-PAK in both the p2 binding and kinase domains. The purified protein exhibited kinase activity that was activated by GTP-Cdc42 or GTP-Rac1 in vitro. In platelets, a p62 in situ renaturable kinase was recognized by antibodies raised against gamma-PAK. This thrombin-activated protein kinase appears to coprecipitate with another kinase of M(r) 86,000, suggesting that PAK may be part of a thrombin-responsive signaling complex.
Insights
Researchers identified gamma-PAK, a ubiquitously expressed kinase related to brain-enriched p65PAK. This protein kinase activity is activated by Cdc42 and Rac1, suggesting its role in cellular signaling pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- p65PAK, a brain-specific serine/threonine kinase, was previously identified.
- p21cdc42/rac1-activated kinases (PAKs) play crucial roles in cellular processes.
Purpose of the Study:
- To identify and characterize ubiquitously expressed PAK isoforms.
- To investigate the enzymatic activity and potential signaling roles of gamma-PAK.
Main Methods:
- Protein purification from rat testes.
- Peptide sequencing and cDNA isolation.
- In vitro kinase assays using GTP-Cdc42 and GTP-Rac1.
- Immunological detection in platelet lysates.
Main Results:
- A 62,000 molecular weight protein, designated gamma-PAK, was purified from rat testes.
- Gamma-PAK contains peptides related to PAK and exhibits conserved p21-binding and kinase domains.
- Purified gamma-PAK demonstrated kinase activity activated by GTP-Cdc42 and GTP-Rac1.
- A p62 kinase recognized by anti-gamma-PAK antibodies was found in platelets, suggesting involvement in thrombin signaling.
Conclusions:
- Gamma-PAK is a ubiquitously expressed isoform of the PAK family.
- Gamma-PAK possesses GTPase-dependent kinase activity.
- PAK kinases may function within thrombin-responsive signaling complexes in platelets.