|Ph.D Student||Weisz-Hubshman Monika|
|Subject||Molecular Characterization of Chp Function|
|Department||Department of Medicine||Supervisor||Professor Ami Aronheim|
|Full Thesis text|
Rho GTPases form an intriguing subgroup of the Ras super-family. Till today 22 members are known that are further sub-grouped by homology, function and localization. Although the biochemical process used by the Rho GTPases to mediate signal transduction is rather simple, the exchange of GDP to GTP, they command a myriad of complex cellular processes. Rho GTPases hold key functions in processes like: actin cytoskeleton remodeling, MAPK pathways activation, cell movement, gene transcription, development, apoptosis, cellular proliferation, cell cycle progression and malignant transformation.
One of the most unique members of this protein family is Chp (Cdc42 homologue protein), that was identified in our lab. Over the years, we learned a lot about Chp domain structure, expression pattern and some of the signal transduction it mediates. Still its specific effector plethora and biological functions are unknown.
Chp was identified as Pak2 (p-21 activated kinase) interacting partner. This interaction is dependent on Chp C-terminal domain and its activity state. Chp also stimulates Pak group I kinase activity.
The p21-activated kinases (Pak) are among the most studied effectors of the Rho-family GTPases. Pak kinases influence a variety of cellular functions, but the process of Pak down-regulation, following activation, is poorly understood. Upon overexpression of Chp, we unexpectedly observed a T cell migration phenotype consistent with Pak inhibition. In line with this observation, overexpression of Chp or Cdc42 caused a marked reduction in the level of Pak1 in a number of different cell types. Rac induced Pak degradation to a much lesser extent. The regulation of Pak expression levels was found to be unique to group I Pak 1-3 and not for group II Paks 4-6. We focused our further analysis on Chp-Pak1 regulation. 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 (PBD), kinase activity, and a number of Pak1 autophosphorylation sites; whereas the PIX-binding site and NCK-binding site were not involved. Together, these results implicate Chp-induced kinase autophosphorylation in the degradation of Pak1. The N-terminal domain of Chp was required for Chp-induced degradation, though not for Pak1 activation, suggesting that Chp provides a second function, distinct from kinase activation to trigger Pak degradation. Collectively, our results suggest a novel mechanism for signal termination mediated by the Rho-family GTPases, Chp and Cdc42, which results in ubiquitin-mediated degradation of one of their direct effectors, Pak1.