Autophosphorylation-dependent degradation of Pak1, triggered by the Rho-family GTPase, Chp.
The Paks (p21-activated kinases) Pak1, Pak2 and Pak3 are among the most studied effectors of the Rho-family GTPases, Rac, Cdc42 (cell division cycle 42) and Chp (Cdc42 homologous protein). Pak kinases influence a variety of cellular functions, but the process of Pak down-regulation, following activation, is poorly understood. In the present study, we describe for the first time a negative-inhibitory loop generated by the small Rho-GTPases Cdc42 and Chp, resulting in Pak1 inhibition. Upon overexpression of Chp, we unexpectedly observed a T-cell migration phenotype consistent with Paks inhibition. In line with this observation, overexpression of either Chp or Cdc42 caused a marked reduction in the level of Pak1 protein in a number of different cell lines. Chp-induced degradation was accompanied by ubiquitination of Pak1, and was dependent on the proteasome. The susceptibility of Pak1 to Chp-induced degradation depended on its p21-binding domain, kinase activity and a number of Pak1 autophosphorylation sites, whereas the PIX- (Pak-interacting exchange factor) and Nck-binding sites were not required. Together, these results implicate Chp-induced kinase autophosphorylation in the degradation of Pak1. The N-terminal domain of Chp was found to be required for Chp-induced degradation, although not for Pak1 activation, suggesting that Chp provides a second function, distinct from kinase activation, to trigger Pak degradation. Collectively, our results demonstrate a novel mechanism of signal termination mediated by the Rho-family GTPases Chp and Cdc42, which results in ubiquitin-mediated degradation of one of their direct effectors, Pak1.
Weisz Hubsman
Monika
M
Department of Molecular Genetics, The Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, 1 Efron St. Bat-Galim, Haifa 31096, Israel.
Volinsky
Natalia
N
Manser
Edward
E
Yablonski
Deborah
D
Aronheim
Ami
A
D000818:Animals; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013601:T-Lymphocytes / Q000166:cytology / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism; D054462:p21-Activated Kinases; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D009363:Neoplasm Proteins; C465202:RHOV protein, human; D025801:Ubiquitin; C116367:PAK4 protein, human; C515903:PAK1 protein, human; C515907:PAK2 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D046988:Proteasome Endopeptidase Complex; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins