Abstract A17: Differential role of Ras isoforms in regulating PI3KC2β: Novel role for nucleotide-free H-Ras in cell signaling

Ras proteins, like all GTPases, cycle through an active GTP-bound state and an inactive GDP-bound state that arises through the intrinsic GTPase activity of the protein. Activation of Ras requires the activity of GEFs which promote the release of GDP leading to the subsequent binding of GTP due to the relatively higher levels of GTP in cells. Given the high affinity of nucleotide-free Ras (nf-Ras) for nucleotides in vitro, it has been proposed that nf-Ras is a transient intermediate in vivo. However, we have discovered a potentially new role for nf-H-Ras in the regulation of PI3KC2β (1). Previously, we discovered that the scaffold protein intersectin 1 (ITSN1) interacts with and activates H-Ras on endocytic vesicles (2). In addition, ITSN1 binds and activates PI3KC2β on a similar population of vesicles (3). PI3KC2β, like Class I PI3Ks, contains a Ras binding domain (RBD) although PI3KC2β does not interact with RasGTP or RasGDP (4). Our recent studies, however, indicate that nf-H-Ras binds the PI3KC2β RBD and inhibits PI3KC2β activity in vitro (1). Furthermore, the complex of nf-H-Ras with PI3KC2β RBD was not disrupted by addition of 1mM GTP or GDP suggesting that this complex may exist in the cellular milieu of high guanine nucleotides (1). Consistent with this in vitro data, PI3KC2β preferentially interacts with H-Ras dominant-negative (Ras17N)>H-Ras WT>activated H-Ras (Ras61L or 12V) in cells (1). To determine whether these results can be extended to other GTPases, we have examined the interaction of PI3KC2β with K-Ras and various Ras-related GTPases (see poster by RS Smith et. al.). Interestingly, PI3KC2β preferentially interacts with K-Ras12V>WT> dominant-negative (K-Ras15A). Furthermore, K-Ras localizes PI3KC2β to the plasma membrane whereas H-Ras localizes PI3KC2β to the perinuclear region. Thus, different Ras GTPases interact with PI3KC2β in distinct manners. We will present data on the contrasting role of K-Ras and H-Ras on regulation of PI3KC2β. Our findings suggest that PI3KC2β stabilizes nf-H-Ras and that interaction of H-Ras and PI3KC2β mutually inhibit one another. However, the role of K-Ras in regulation of PI3KC2β appears distinct. These findings have important implications for Ras-dependent tumorigenesis. References: 1. Wong, K. A., A. Russo, X. Wang, Y.-J. Chen, A. Lavie, and O. B. J. P. 2012. A new dimension to Ras function: a novel role for nucleotide-free Ras in Class II phosphatidylinositol 3-kinase beta (PI3K-C2β) regulation. PLoS ONE 7:e45360. 2. Mohney, R. P., M. Das, T. G. Bivona, R. Hanes, A. G. Adams, M. R. Philips, and J. P. O9Bryan. 2003. Intersectin activates Ras but stimulates transcription through an independent pathway involving JNK. J Biol Chem 278:47038-45. 3. Das, M., E. Scappini, N. P. Martin, K. A. Wong, S. Dunn, Y. J. Chen, S. L. Miller, J. Domin, and J. P. O9Bryan. 2007. Regulation of neuron survival through an intersectin-phosphoinositide 39-kinase C2beta-AKT pathway. Mol Cell Biol 27:7906-17. 4. Arcaro, A., S. Volinia, M. J. Zvelebil, R. Stein, S. J. Watton, M. J. Layton, I. Gout, K. Ahmadi, J. Downward, and M. D. Waterfield. 1998. Human phosphoinositide 3-kinase C2beta, the role of calcium and the C2 domain in enzyme activity. J Biol Chem 273:33082-90. Citation Format: Katy A. Wong, Russell Spencer Smith, Angela Russo, Arnon Lavie, John P. O9Bryan. Differential role of Ras isoforms in regulating PI3KC2β: Novel role for nucleotide-free H-Ras in cell signaling. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A17. doi: 10.1158/1557-3125.RASONC14-A17