Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1.
The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1alpha). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1alpha can act directly on phosphorylated Thr-402 in the activation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modification but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.
Wang
JinJun
J
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Wang
ZhiXin
Z
D000818:Animals; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D004789:Enzyme Activation; D007700:Kinetics; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera; D013379:Substrate Specificity; D054462:p21-Activated Kinases / Q000235:genetics / Q000378:metabolism*
D054462:p21-Activated Kinases; D054645:Protein Phosphatase 1; D053148:Caspase 3