Multisite phosphorylation of P-Rex1 by protein kinase C

P-Rex proteins are guanine nucleotide exchange factors (GEFs) that act on the Rho/Rac family of GTP binding proteins. The activity of P-Rex proteins is regulated by several extracellular stimuli. In fact, activation of growth factor receptors has been reported to activate a phosphorylation/dephosphorylation cycle of P-Rex1. Such cycle includes dephosphorylation of serines 313 and 319 which negatively regulate the GEF activity of P-Rex1, together with phosphorylation of serines 605 and 1169 which favour P-Rex1 GEF activity. However, the kinases that regulate phosphorylation at these different regulatory sites are largely unknown. Here we have investigated the potential regulatory action of several kinases on the phosphorylation of P-Rex1 at S, S, S and S. We show that activation of protein kinase C (PKC) caused phosphorylation of S, S and S. Activation of growth factor receptors induced phosphorylation of S through a mechanism that was independent of PKC, indicating that distinct kinases and mechanisms control the phosphorylation of P-Rex1 at different regulatory serines. Genetic and biochemical studies confirmed that the PKC isoform PKCδ was able to directly phosphorylate P-Rex1 at S. Functional studies using cells with very low endogenous P-Rex1 expression, transfected with wild type P-Rex1 or a mutant form in which S was substituted by alanine, indicated that phosphorylation at that residue negatively regulated P-Rex1 exchange activity. We suggest that control of P-Rex1 activity depends on a highly dynamic interplay among distinct signalling routes and its multisite phosphorylation is controlled by the action of different kinases.Ministry of Economy and Competitiveness of Spain (BFU2009-07728 and BFU2012-39151), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD06/0020/0041 and RD12/0036/0003), and the Scientific Foundation of the Spanish Association Against Cancer (AECC). JCM is a recipient of a Miguel Servet fellowship program (CP12/03073). SGA is recipient of a predoctoral contract from the MINECO (BES-2013-065223). The Cancer Research Institute and the work carried out at our laboratory receive support from the European Community through the Regional Development Funding Program (FEDER).Peer Reviewe