Rabbit Anti-Human RAP2A Polyclonal antibody

The Rap family of small GTP-binding proteins is composed by four different members: Rap 1A, Rap 1B. Rap2A and Rap2B. In this work we report the identification and characterization of a fifth member of this family of small GTPases. This new protein is highly homologous to Rap2A and Rap2B, binds labeled GTP on nitrocellulose, and is recognized by a specific anti-Rap2 antibody. but not by an anti-Rap1 antibody. The protein has thus been named Rap2C. Binding of GTP to recombinant purified Rap2C was Mg2+-dependent. However. accurate comparison of the kinetics of nucleotide binding and release revealed that Rap2C bound GTP less efficiently and possessed slower rate of GDP release compared to the highly homologous Rap2B. Moreover, in the presence of Mg2+. the relative affinity of Rap2C for GTP was only about twofold higher than that for GDP, while, under the same conditions, Rap2B was able to bind GTP with about sevenfold higher affinity than GDP. When expressed in eukaryotic cells, Rap2C localized at the plasma membrane is dictated by the presence of a CAAX motif at the C-terminus. We found that Rap2C represented the predominant Rap2 protein expressed in circulating mononuclear leukocytes. but was not present in platelets. Importantly, Rap2C was found to be expressed in human megakaryocytes. suggesting that the protein may be down-regulated during platelets generation. This work demonstrates that Rap2C is a new member of the Rap2 subfamily of proteins. able to bind guanine nucleotides with peculiar properties, and differently expressed by various hematopoietic subsets. This new protein may therefore contribute to the still poorly clarified cellular events regulated by this Subfamily of GTP-binding proteins. (c) 2005 Elsevier SAS. All rights reserved.

BACKGROUND. Progression of prostate cancer to a fatal androgen-independent disease is associated with activation of MAP kinase, consistent with chronic stimulation of the Ras-signaling pathway. We have previously shown that Ras activation is sufficient to induce androgen-independent growth of prostate cancer cells. One mechanism of MAP kinase regulation is modulation of Ras signaling by other Ras family members, the Rap gene paralogs Rap1a/b and Rap2a/b. Here we ask if Rap proteins play a role in determining androgen sensitivity of human prostate cancer cells either alone or in the context of an activated Ras. METHODS. To evaluate the role of Rap proteins in androgen responsiveness we use Rap overexpression with or without mutated Ras co-transfection and Rap siRNA knockdown to evaluate androgen-dependent prostate-specific antigen (PSA) promoter reporter expression and cell growth in androgen-dependent LNCaP and independent C4-2 human prostate cancer cells. RESULTS. Rap1 is equally expressed between LNCaP and C4-2 cells and thus we focused on Rap2 which is minimally expressed in C4-2. Rap2a affects androgen-dependent PSA reporter expression in a dose-dependent manner in LNCaP and C4-2 cells. Low levels of Rap2a enhance PSA reporter expression, whereas higher concentrations inhibit expression. We show that Rap2a antagonizes the enhanced PSA reporter expression conferred by an active RasV12 gene in prostate cancer cells. siRNA knockdown data indicate that Rap2 has a greater effect on androgen-stimulated growth in LNCaP than in C4-2 cells. CONCLUSIONS. We show that Rap2 is involved in androgen-mediated transcriptional and growth responses of human prostate cancer cells.