Asp (D) and MDV3100 purchase Glu (E) carboxyl groups

coordinate Ca2+; Gly and Ile contribute structural features; and “X” represents any amino acid (Gifford et al., 2007). Mutations that eliminate negative charge from positions 1 and 12 in EF hands substantially decrease or eliminate Ca2+ binding affinity. Therefore, Asp1 and Glu12 were replaced in both RGEF-1b EF hands (see Figure S1B). A quadruple mutant, named RGEF-1b(4A), was generated by substituting Asp432, Glu443, Asp461, and Glu472 with Ala. RGEF-1b-deficient animals were reconstituted with rgef-1::RGEF-1b-GFP and rgef-1::RGEF-1b(4A)-GFP transgenes. Expression of either WT or mutant GEF restored chemotaxis to odorants to near-WT levels ( Figure 8A). Thus, defective EF hand modules did not disrupt RGEF-1b-mediated chemotaxis. The data imply that an increase in DAG is sufficient to enable (C1 domain-mediated) RGEF-1b activation, production of LET-60-GTP and downstream signaling in neurons in vivo. Ca2+ binding by RGEF-1b EF hands is dispensable for chemotaxis. DAG-activated PKCs increase RasGRP3 catalytic activity by phosphorylating Thr133 (Zheng et al., 2005). Amino acid sequences

surrounding Thr133 in RasGRP3 and Ser135 in RGEF-1b are homologous. Moreover, RasGRP3 Thr133 and RGEF-1b Ser135 precede the catalytic domain by 18 amino acids and are embedded in a short linker region that couples REM to GEF domains in RasGRPs (Figures S1B and S1D). To determine if PMA elicits phosphorylation of Ser135 in situ, we prepared IgGs directed against an RGEF-1b peptide (amino

acids buy Idelalisib 128–143, Figure S1B) that contained phospho-Ser135. Cells expressing HA-RGEF-1b were incubated with PMA or vehicle and GTP exchanger was precipitated from cell extracts with anti-HA IgGs. Phosphorylation of RGEF-1b Ser135 out was minimal in unstimulated cells (Figure 8B, upper panel, lane 3). However, Ser135 phosphorylation increased substantially when endogenous PKCs were activated by PMA (Figure 8B, upper panel, lane 4). Specificity of the phosphopeptide-directed IgGs was verified by mutating Ser135 to Ala. RGEF-1bS135A was expressed and immunoprecipitated (Figure 8B, lanes 5 and 6, lower panel). No signals were detected when the blot was probed with phosphorylation-site selective IgGs (Figure 8B, upper panel, lanes 5 and 6). Thus, Ser135 in RGEF-1b is a target site for PMA-stimulated phosphorylation. The relevance of Ser135 phosphorylation to RGEF-1b-mediated chemotaxis was explored by reconstituting rgef-1−/− mutants with an rgef-1::RGEF-1bS135A-GFP transgene. RGEF-1bS135A-GFP rescued chemotaxis, yielding CI values for odorants that were similar to CIs obtained for WT C. elegans and null mutants expressing WT RGEF-1b-GFP ( Figure 8C). Thus, phosphorylation of Ser135 is not required for RGEF-1b-mediated activation of the LET-60-MPK-1 pathway in neurons that control odorant-induced chemotaxis. RasGRPs were discovered in studies on mammalian brain 12 years ago (Buday and Downward, 2008).