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GTPase cycle diagram


The Johnson lab is primarily interested in mechanisms of small GTPase function in disease, with a particular emphasis on cancer. However, we invite collaboration and communication with any lab inside and outside the University of South Carolina community interested in protein chemistry and structural biology

Project 1: GTPase autophosphorylation
autoP cycle diagram

It has been known for ~50 years that active site mutations in GTPases can switch these enzymes from favoring GTP hydrolysis to favoring phosphoryl-transfer. However, the mechanism and biological function of this mutation-induced change in enzyme function is poorly understood. Surprisingly, several other small GTPases show potential for autophosphorylation, suggesting that this reaction has conserved yet unknown cellular functions. The goal of this project is to 1) understand the molecular mechanism of autophosphorylation and how it competes with GTP hydrolysis, 2) determine the extent of autophosphorylation as a conserved evolutionary function of wildtype small GTPases and its biological function(s), 3) determine how this post-translational modification influences GTPase interactions with other proteins, 4) specify the molecular interactions that allow auto-phosphorylated small GTPases to transform cells and participate in oncogenesis, and 5) explore autophosphorylation as a functional tool to study other members of the small GTPase superfamily.

Project 2: Mechanisms of GTPase nucleotide exchange

Activation of small GTPases require the exchange of GDP for GTP. For KRAS, HRAS, and NRAS (collectively RAS), enhancement of nucleotide exchange by guanine nucleotide exchange factors (GEFs) is well described structurally. The same is not true for the process of intrinsic nucleotide exchange, whereby exchange of GDP for GTP occurs without the aid of a partner GEF to bind the active site of RAS to catalyze nucleotide release. The reason why the processes of intrinsic nucleotide exchange has been largely ignored is because the exchange process occurs very slowly. However, many oncogenic and germline mutations of RAS proteins promote nucleotide exchange as a primary mechanism of activation. Therefore, a central question is whether 1) intrinsic nucleotide exchange in wildtype RAS and its mutants occur through a common change in protein conformation to allow release and binding of different nucleotides, or through a spectrum of conformational transitions? Further, 2) are these conformational transitions unique compared to GEF-mediated exchange, or do GEF and intrinsic nucleotide exchange mechanisms occur via a common conformational transition? While answering these questions are important for a better understanding of mutant RAS function, they’re also critical for the 3) design of chemical probes to investigate specific nucleotide-exchange promoting mutants of RAS and their role in pathogenesis.  

Project 3: Functional characterization of GTPase-RASSF interactions

The Ras-association domain family (RASSF) are a large group of putative tumor suppressors and scaffold proteins that are potentially regulated by small GTPases. All members of the RASSF family contain RalGDS/AF6 Ras association (RA) domains capable of binding the G-domain of small GTPases. A prominent example is the KRAS-RASSF5 complex, which has been shown by several groups to suppress cell growth in culture, and which appears to do so through the generation of several different ternary complexes. The molecular nature of these complexes, and their relevance for disease progression in vivo, remains to be elucidated. For instance, greater than half of all RASSF proteins form constitutive complexes with the evolutionarily conserved Hippo kinases MST1/2 which are critical for organ homeostasis, and this is true for RASSF5 as well, but how KRAS participates in MST1/2 function via RASSF5 is not understood. Therefore, the specific goals of this project are to 1) decipher the molecular interactions between KRAS, RASSF5, and MST1/2 proteins, 2) decode KRAS/RASSF5 interaction partners outside of MST1/2, 3) test the tumor suppressor functions of KRAS/RASSF in in vivo models of colon tumorigenesis. Further, besides KRAS and a few outstanding examples (e.g. GEM, RAP1), most other RASSF proteins lack an identified cognate GTPase. Thus, we will expand the knowledge of RASSF function by 4) identifying, validating, and characterizing at an atomic level other RASSF-GTPase pairs. In doing so, we will elucidate the basic functionality of RASSF-GTPase complexes at the atomic, molecular, and biological levels.

Project 4: Intra-cooperation between oncogenes and tumor suppressors of the MAPK signaling pathway. 

colon tumor

Oncogenic mutations of KRAS contribute to colorectal cancer progression through  activation of the RAF/MEK/ERK signaling pathway. Traditionally, co-occurring mutations in KRAS and other genes that regulate the MAPK signaling pathway have been viewed as  classic  examples of negative epistasis, whereby cancer cells select against the presence of multiple MAPK pathway activating mutations due to cellular toxicity. However, oncogenic mutations of KRAS are heterogenous, and KRAS allele heterogeneity in the context of genetic epistasis is understudied. In fact, some oncogenic KRAS alleles appear to favor co-mutations in genes of the MAPK-signaling pathway normally viewed as mutually exclusive. In this project, we seek to move the field beyond phenomenological descriptions of epistasis to 1) understanding the protein-protein interactions that define these relationships, 2) modelling of these interactions in vivo to understand their contribution to colorectal cancer, and lastly 3) testing their contributions to both primary and acquired drug resistance



Challenge the conventional. Create the exceptional. No Limits.