Structure, function and regulation of a GEF (mCalDAG-I) and C. elegans Rap proteins

Abstract

I discovered that endogenous Rap1-GTP content and ERK activity were increased after stimulation of NIH3T3 and HEK293 cells with phorbol ester (TPA) and ionomycin (Ca2+ ionophore). Activation is blocked by PKC inhibitors or PKC depletion. CalDAG-I is a GEF with a high degree of substrate specificity for Rap1. The exchanger contains an N-terminal REM domain (unknown, but presumed structural function), a cdc25 region (promotes exchange of GTP for GDP) and several potential regulatory segments: two Ca2+ binding EF hands and a diacylglycerol (DAG) binding C1 region. Expression of a mouse CalDAG-I transgene in NIH3T3 and HEK293 cells fully restores Rap activation, and partially rescues ERK stimulation in the presence of PKC inhibitors. These results suggest that DAG/Ca2+ induced Rap/MAPK activation is mediated by two pathways: one is PKC dependent and another is controlled via CalDAG-I.;I discovered that mCalDAG-I is in an inactive signaling complex with B-Raf, Rap1 and F-actin. Signals that activate the Rap/MAPK pathway also rapidly and simultaneously (or earlier) disrupt the complex. DAG/Ca2+ or CAMP are second messengers required for activation. The GEF domain of mCalDAG-I is necessary and sufficient for association with B-Raf. The RA domain (a predicted classical Ras and Rap binding domain) of B-Raf is not essential for coupling with mCalDAG-I.;A potential MAPK docking site and binding motif are also evident in the mCalDAG-I sequence. A critical feature in MAPK phosphorylation sites (S*/T*-P) is also present at several sites in the linker region. All these observations indicated a possible relationship between CalDAG and MAPK. Co-immuno-precipitate experiments demonstrated that mCalDAG-I binds selectively to MAPK (ERK1, not ERK2) and p38 (an analog of ERK in a stress signaling cascade) in un-stimulated cells.;I established significant difference between Rap1ce and Rap2ce (60% identical sequences). Rap1ce can be activated by GTP binding in vitro and in vivo. In contrast, GTP exchange and activation are not detected in Rap2ce. GFP reporter assays utilizing Rap1ce and Rap2ce promoter enhancer DNA revealed that the two G-proteins are expressed in different cells in live worms. Expression of a Rap1ce transgene in WT C. elegans results in vulval structure abnormalities and egg laying defects. Depletion of Rap2ce expression in C. elegans via RNAi results lethality. Rap2ce null animals have been obtained and future experiments will link Rap2ce deficiency/rescue with specific physiological/developmental alterations. Similar approaches are being taken for Rap1ce and CalDAGce. (Abstract shortened by UMI.).