简介

The Ras proteins are members of a large superfamily of Ras-related proteins that are key regulators of signal transduction pathways that control normal cell growth. Mutated Ras proteins are found in 30% of human cancers and promote uncontrolled cell growth, invasion, and metastasis. One major branch of the Ras superfamily consists of members of the Rho GTPases (e.g., RhoA, Rac1, Cdc42). Like Ras, Rho GTPases also serve as on-off switches to relay extracellular signal-mediated stimuli to cytoplasmic signaling pathways including those involved in cellular growth control. Distinct from Ras, however, Rho GTPase-mediated signaling pathways modulate cell morphology and actin cytoskeletal organization. Three RAS genes (HRAS, KRAS and NRAS) comprise the most frequently mutated oncogene family in cancer, with single point mutations predominately (99%) localized to codons 12, 13 and 61. A common feature of these point mutations is that they render Ras insensitive to down regulation by cellular factors called GTPase activating proteins (GAPs) that catalyze hydrolysis of GTP, resulting in constitutive, oncogenic signaling. As such, they have historically been considered oncogenic equivalents. However, recent observations suggest that amino acid and codon-specific Ras proteins show differences in their biochemical properties, ability to engage effectors, signaling and tumorigenic properties. Differences have also been observed in the response and resistance to specific anti-cancer therapies. These differences are likely to have important clinical and biological implications. Our lab is characterizing the molecular properties of amino acid-, codon- and isoform-specific Ras mutant proteins to better understand how these differences modulate Ras tumorigenicity. Another research focus of the lab lies in elucidating novel mechanisms by which post-translational modifications within the core Ras guanine nucleotide binding domain modulate its activity. Cell growth and differentiation are controlled by growth factor receptors coupled to the GTPase Ras. Oncogenic mutations disrupt GTPase activity, leading to persistent Ras signaling and cancer progression. Recent evidence indicates that monoubiquitylation of Ras leads to hyper-activation. Mutation of the primary site of monoubiquitylation impairs the ability of activated K-Ras (one of the three mammalian isoforms of Ras) to promote tumor growth. To determine the mechanism of human Ras activation, we chemically ubiquitylated the protein and analyzed its function by NMR, computational modeling and biochemical activity measurements. We established that monoubiquitylation has little effect on the binding of Ras to guanine nucleotide, GTP hydrolysis or exchange-factor activation but severely abrogates the response to GTPase-activating proteins in a site-specific manner. These findings reveal a new mechanism by which Ras can trigger persistent signaling in the absence of receptor activation or an oncogenic mutation. We are following up on these initial studies to better understand the molecular basis for how ubiquitin ligation regulates Ras conformational dynamics, GTP downregulation by GAP proteins and effector binding. We are also investigating the isoform dependence of ubiquitination and its role in Ras-mediated tumorigenesis, in collaboration with Atsuo Sasaki (U. of Cincinnati) and Channing Der (UNC-CH). In addition, we have initiated studies to determine whether other lysine-specific post-translational modifications modulate Ras function. Once activated, Ras can regulate numerous and complex pathways that control cellular growth. However, the activation state of Ras was previously thought to be regulated solely by protein modulatory factors. We are finding that the regulation is more complicated than previously envisioned. In addition to ubiquitination, reactive oxygen or nitrogen species can also regulate the activity of Ras and Rho GTPases. We are currently characterizing mechanisms of regulation by oxidative thiol modification. Current research projects in the Campbell laboratory include characterization of Ras and Rho GTPase post-translational modifications, including monoubiquitylation and redox regulation, using structural, biophysical and biochemical studies; characterization of small molecule Ras inhibitors; identification, characterization and structural elucidation of factors that act on Ras, Rap and Rho family GTPase proteins.