Purification and in vitro activities of p21-activated kinases

Abstract

Publisher Summary This chapter explains the purification and in vitro activities of p21-activated kinases (PAKs). The chapter describes various methods for generating recombinant, constitutively active PAKs for substrate phosphorylation studies and wild-type PAKs for kinase activation studies. The chapter discusses the regulation of PAK activity using dominant active mutants or in vitro GTP-labeled forms of Rac and Cdc42, which bind to the p21-binding (PBD) domain. The chapter also discusses the stimulation of PAK kinase activity with lipid compounds that leads to PAK autophosphorylation and substrate phosphorylation with similar kinetics as GTPase stimulation. Identification of PAKs and detection of the PAK activation state has been shown to be achieved in cells by in-gel kinase assays with specific substrates by 32p labeling and tryptic phospho peptide mapping or matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) analysis. The chapter concludes with an analysis of PAK activation and autophosphorylation by MALDI-TOF MS.