Rap2 is a member of the Ras family of GTPases and exhibits 60% identity to Rap1, but the function and regulation of Rap2 remain obscure. and inhibited Ras-dependent activation of Elk1 transcription factor, as did Rap1. The level MDV3100 supplier of GTP-Rap2 in rat 3Y1 fibroblasts was decreased by the expression of v-Src, and appearance of the GTPase-deficient Rap2 mutant inhibited MDV3100 supplier v-Src-dependent change of 3Y1 cells. Entirely, Rap2 is governed by an identical group of GEFs and Spaces as Rap1 and features as a slowly responding molecular switch in the Rap1 signaling cascade. The Ras family of G proteins consists of Ras (H-, N-, and K-), Rap1, Rap2, R-Ras, TC21, Ral, Rheb, and M-Ras (R-Ras3) (5). Compared to Ras, which has been extensively analyzed as a pivotal protein in cell growth and differentiation (4, 8), much less is known about the other Ras-family G proteins. Rap1, which shares its effector domain name with Ras, antagonizes Ras in many aspects. Overexpression of MDV3100 supplier Rap1 suppresses Ras-induced transformation of NIH 3T3 cells (30), Ras-induced c-activation (47), and Ras-dependent inhibition of muscarinic K+ channels (53). At least some of these effects appear Rabbit Polyclonal to COPZ1 to be due to the suppression of Ras-induced activation of Raf serine/threonine kinase and mitogenic ERK/mitogen-activated protein kinase (ERK/MAPK) (10, 19). In concordance with these findings, constitutive activation of Rap1 inhibits interleukin-2 (IL-2) gene production and causes T-cell anergy (7), and inhibition of Rap1 by insulin or lysophosphatidic acid stimulates Ras (40). However, Rap1 may activate the MAPK cascade in different milieus. Rap1 activates ERK/MAPK via the activation of B-Raf in neuronal cells (52, 54) and induces DNA synthesis and oncogenic transformation of Swiss 3T3 cells (1, 55). Rap1 circulates between GTP-bound active and GDP-bound inactive says. The activation is usually induced by guanine nucleotide exchange factors (GEFs); these include C3G, CalDAG-GEFI, and Epac (or cyclic AMP [cAMP]-GEF), which are activated by tyrosine kinases, Ca and diacylglycerol, and cAMP, respectively (12, 27, 28, 50). Thus, many signals converge at Rap1 via different GEFs. You will find four GTPase-activating proteins (GAPs) of Rap1: rap1Space, SPA-1, Space1IP48P, and tuberin (6). Little is known about the regulation of these GAPs, except for that of an isoform of rap1Space, rap1GAPII, which has recently been shown to bind to and transduce indication in the subunit of heterotrimeric Gi proteins (37). Understanding of Rap2, the amino acidity sequence which stocks 60% identification with Rap1, is bound. Rap2 is normally reported to localize generally in the endoplasmic reticulum (ER), whereas Rap1 localizes on the Golgi equipment (2, 3). Unlike Rap1, Rap2 cannot invert Ras-induced change of NIH 3T3 cells (23), no natural phenotype continues to be associated with Rap2 in the books. The legislation of Rap2 also continues to be unfamiliar. rap1Space stimulates Rap2 GTPase activity in vitro, albeit significantly more weakly than Rap1 (22). An attempt to purify a Space specific to Rap2 culminated in the isolation of rap1Space (22). Very recently, de Rooij et al. reported that a newly isolated GEF for Rap1, PDZ-GEF1, also activates Rap2 and that GTP-bound Rap2 makes up more than 50% of the Rap2 in A14 and COS1 cells (11). Rap2 shares most of the effector proteins with Ras and Rap1, except for a recently recognized RPIP8 (21, 38), suggesting that there is cross-talk among these three proteins. Here, MDV3100 supplier we display that GEFs and GAPs are shared between Rap1 and Rap2 and that, unlike the additional Ras family proteins, the GTP-bound active form makes up at least 50% of Rap2 in adherent cells due to a low level of sensitivity to GAPs. MATERIALS AND METHODS Plasmids. The cDNA fragment of Rap2A was amplified from a human being spleen cDNA library by PCR with primers 5-CCCTCGAGATGCGCGAGTACAAAGTGGTG-3 and 5-TTGCGGCCGCCTATTGTATGTTACATGCAGAACA-3. A constitutively active mutant of Rap2 was designed by analogy.