Autophosphorylation-dependent degradation of Pak1, triggered by the Rho-family GTPase, Chp

Abstract

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.

Pak3 inhibits local actin filament formation to regulate global cell polarity

“…Moreover, a recent biochemical analysis demonstrated that Pak interferes with the ability of an exchange factor beta-PIX to activate Rac (ten Klooster et al, 2006), suggesting a function for Pak as an upstream inhibitor of Rac function. Our analysis also fits with the observation that Paks can act downstream of Cdc42 to modulate Rac activation, without their being absolutely required for lamellipodial formation (Cau and Hall, 2005), with observations suggesting that Cdc42 inhibits Pak activity (Weisz Hubsman et al, 2007), and by the presence of both Pak and Cdc42 in a single complex that regulates polarity in yeast (Kozubowski et al, 2008). The effects of Paks on changes in the direction of cell migration, e.g., during neural growth cone turning (Ang et al, 2003;Hing et al, 1999;Newsome et al, 2000), may reflect a similar function for Paks in damping local actin polymerization to facilitate the necessary redistribution of actin filaments.…”

PAK1 Tyrosine Phosphorylation Is Required to Induce Epithelial–Mesenchymal Transition and Radioresistance in Lung Cancer Cells

“…Although a PAK1-specific E3 ligase has not been identified, Cdc42 homologous protein (Chp) is a Rho-family GTPase that mediates ubiquitinationdependent degradation of PAK1 (44). A previous report indicated that kinase activity of PAK1 is critical for PAK1 degradation (44). According to this study, Chp could mediate PAK1 degradation only when PAK1 was activated by autophosphorylation on several Ser/Thr residues including S57, S144, S149, S199/S203, and T423.…”

“…To understand how PAK1 stabilization is mediated by Tyr phosphorylation, it was important to determine which E3 ligase is responsible for PAK1 degradation. Although a PAK1-specific E3 ligase has not been identified, Cdc42 homologous protein (Chp) is a Rho-family GTPase that mediates ubiquitinationdependent degradation of PAK1 (44). A previous report indicated that kinase activity of PAK1 is critical for PAK1 degradation (44).…”

Regulation and functions of RhoU and RhoV

“…RhoV, as well as Cdc42, has been shown to stimulate the ubiquitination and proteasome-mediated degradation of PAK1 in Jurkat T-cells. 36 RhoV interaction with and activation of PAK1 appears to be required for its degradation, based on the effects of RhoV on a range of PAK1 mutants Furthermore, both N-terminal and C-terminal extension regions of RhoV were required to trigger PAK1 degradation, and the N-terminal region was required for PAK1 degradation but not its activation. This suggests a second unknown function for RhoV in PAK1 degradation.…”

“…This suggests a second unknown function for RhoV in PAK1 degradation. 36 In vivo studies RhoV has been described to be involved in developmental processes, although its function appears to differ from RhoU. For example, RhoU and RhoV have different functions in neural crest cells in Xenopus laevis.…”