title: Multisite phosphorylation of P-Rex1 by protein kinase C.
abstract: 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 S313, S319, S605 and S1169. We show that activation of protein kinase C (PKC) caused phosphorylation of S313, S319 and S1169. Activation of growth factor receptors induced phosphorylation of S1169 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 S313. Functional studies using cells with very low endogenous P-Rex1 expression, transfected with wild type P-Rex1 or a mutant form in which S313 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.
authors: Instituto de Biología Molecular y Celular del Cáncer, CSIC-Universidad de Salamanca, Spain.
authors: Juan Carlos
authors: JC
authors: Montero
authors: Instituto de Biología Molecular y Celular del Cáncer, CSIC-Universidad de Salamanca, Spain.
authors: Samuel
authors: S
authors: Seoane
authors: Instituto de Biología Molecular y Celular del Cáncer, CSIC-Universidad de Salamanca, Spain.
authors: Sara
authors: S
authors: García-Alonso
authors: Instituto de Biología Molecular y Celular del Cáncer, CSIC-Universidad de Salamanca, Spain.
authors: Atanasio
authors: A
authors: Pandiella
mesh_terms: D055785:Gene Knockdown Techniques; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D061986:MCF-7 Cells; D010766:Phosphorylation; D051745:Protein Kinase C-delta / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D020662:Guanine Nucleotide Exchange Factors; C453280:PREX1 protein, human; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate
keywords: GEFs; P-Rex1; PKC; Rac; breast cancer