Abstract
The regulation of Rac1 by HACE1-mediated ubiquitination and proteasomal degradation is emerging as an essential element in the maintenance of cell homeostasis. However, how the E3 ubiquitin ligase activity of HACE1 is regulated remains undetermined. Using a proteomic approach, we identified serine 385 as a target of group-I PAK kinases downstream Rac1 activation by CNF1 toxin from pathogenic E. coli. Moreover, cell treatment with VEGF also promotes Ser-385 phosphorylation of HACE1. We have established in vitro that HACE1 is a direct target of PAK1 kinase activity. Mechanistically, we found that the phospho-mimetic mutant HACE1(S385E), as opposed to HACE1(S385A), displays a lower capacity to ubiquitinate Rac1 in cells. Concomitantly, phosphorylation of Ser-385 plays a pivotal role in controlling the oligomerization state of HACE1. Finally, Ser-385 phosphorylated form of HACE1 localizes in the cytosol away from its target Rac1. Together, our data point to a feedback inhibition of HACE1 ubiquitination activity on Rac1 by group-I PAK kinases.
Highlights
E3 ubiquitin ligases (E3s) are critical gatekeepers of cell homeostasis[1]
The GDP/GTP cycle is controlled by the following regulatory proteins: (i) Guanine nucleotide Exchange Factors (GEFs), which facilitate the exchange of GDP and GTP; (ii) GTPase activating Proteins (GAPs), which increase the intrinsic rate of GTP hydrolysis; and (iii) RhoGDIs, which sequester Rho proteins in the cytosol
Because PAK3 expression is restricted to the brain[5], we focused on PAK1, which is notably expressed in mammary glands, and on PAK2, which is ubiquitously expressed
Summary
E3 ubiquitin ligases (E3s) are critical gatekeepers of cell homeostasis[1]. While we have begun to appreciate their structure and the diversity of their targets, we fall short on knowledge about their general integration in cell signaling. One essential question in particular is to better understand the cross-talk between kinases and ubiquitin ligases[2] This is true for HACE1, which is a critical regulator of active Rac[1] flux for which we still lack identified regulators[3]. After endocytosis into the host cells, the toxin CNF1 translocates its catalytic domain into the cytosol, where it deamidates RhoA glutamine residue Q63 (Q61 in Rac[1] and Cdc42) into a glutamic acid[13,14] Because this residue is essential for the GTP hydrolysis, its deamidation by CNF1 impairs the GTPase activity of Rho proteins and locks them in a GTP-bound state. We show the essential roles of group-I PAK kinases on the phospho-regulation of HACE1 E3 ubiquitin ligase activity and its oligomerization state
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