Mechanistic Analyses of kinase signaling complexes

Division Of Basic Sciences - Nci

Investigators

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Abstract

The oncogenic kinase fusion J-PKAca is the primary driver for FLHCC. In the J-PKAca project, we ask the following questions: (1) What is the impact of the J-domain fusion on PKA holoenzyme structures and function? (2) Can the fusion oncoprotein be specifically targeted as a therapeutic approach for FLHCC? We were the first to determine the structure of J-PKAca in one of its most important physiological states, where it is associated in a holoenzyme complex, and further determined the structure of its wild-type counterpart. Our findings have demonstrated the J-domain fusion impact on PKA holoenzyme structure and regulation and are also highly informative to researchers for developing FLHCC therapeutics. We are continuing our studies on the impact of J-domain fusion on PKA holoenzymes and larger signaling complexes. Furthermore, we are also characterizing inhibitor compounds directed against J-PKAca using structural analysis. Our work has been and will continue to provide insights into the structure and function of the oncogenic J-PKACa chimera, with a potential to develop therapeutics against this fatal pediatric cancer. Mutations in the RAF kinases especially BRAF are a major contributor to human cancers. The major goal of the RAF kinase project is to reveal the structural and molecular mechanisms of the RASmediated RAF activation process. We want to address the following longstanding questions: (1) What are the structural and regulatory differences between autoinhibited BRAF and CRAF complexes? (2) How does RAS binding to the presignaling autoinhibited RAF monomer complex initiate the conformational changes required to form active RAF dimers? (3) Do BRAF homodimer complexes differ from BRAF/CRAF heterodimer complexes? To address these questions, reconstitution of physiological RAF complexes, multiple highresolution structures of these complexes in various states, and careful analysis of these structures are required. Our recent work on the BRAF complexes led to the determination of multiple new highresolution cryoEM structures of fulllength physiological BRAF complexes in both autoinhibited and active states. These results provide molecular basis for understanding the structural and biochemical changes of BRAF that occur upon RAS activation. We continue to decipher the molecular mechanism of the presignaling autoinhibited CRAF complex and the active BRAF/CRAF heterodimer complex. We are in a unique position, based on our combined structural and biochemical information, to advance the mechanistic understanding of RAF signaling in health and disease. Mutations in LRRK2 are a major genetic contributor to inherited PD. Patients with the most common LRRK2 mutation can also have an overall increased risk of several cancers. Given the limited highresolution structural and mechanistic information regarding the LRRKs, we propose to address the following essential questions: (1) What are the structures of fulllength LRRK proteins and how is LRRK2 misregulated by pathogenic mutations? (2) How is the structure and activity of LRRK2 affected by binding to upstream regulators? Our preliminary cryoEM data showed that both LRRK2 and LRRK1 are compact dimers, suggesting extensive inter and intramolecular interaction and potential regulatory relationships. Our work will provide a comprehensive understanding of the structure and function of LRRK proteins and has the potential to create new hypotheses for the development of LRRK2 driven disease therapeutics.