Mechanistic Analyses of kinase signaling complexes

Division Of Basic Sciences - Nci

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Abstract

Oncogenic kinase fusion proteins represent an important class of cancer drivers. Fibrolamellar hepatocellular carcinoma (FLHCC) is a rare liver cancer that predominantly affects adolescent and young adults with no history of liver diseases. It is driven by J-PKAca, which is a kinase fusion chimera of the J-domain of heat shock co-chaperone DNAJB1 with PKAca, the catalytic subunit of PKA, which has been used as a model system for the kinase family for the last 40 years. We determined the chimeric RIa2:J-PKAca2 complex, the first for chimeric PKA holoenzymes, and its wild-type counterpart RIa2:PKAca2 holoenzyme. Subsequent work has revealed mechanistic insights with respect to the RIa chimeric and wild-type holoenzyme conformations. Their biological relevance has been derived from analysis of these structures together with biochemical and biophysical data. We continue to study the impact of J-domain fusion of PKA complex structures and regulation. Using the structural knowledge we have gained, we are further developing inhibitor compounds directed against this fatal pediatric cancer driver J-PKAca for FLHCC. Additionally, our studies of J-PKAca could provide a model for exploring the pathways of oncogenic kinase fusion transformation in other cancers. The RAF kinases are key intermediates in the Ras signaling pathway, and they themselves are prominent drivers of human cancer. Elucidating the molecular mechanisms that regulate RAF signaling and identifying strategies to disrupt signal transmission in human disease states is a major scientific challenge. We determined cryo-EM structures of full-length BRAF complexes derived from mammalian cells: autoinhibited, monomeric BRAF:14-3-32:MEK and BRAF:14-3-32 complexes, and an inhibitor-bound, dimeric BRAF2:14-3-32 complex. These results, together with structure based mutational data, provide insights regarding how RAS binding facilitates the BRAF monomer to dimer transition. We continue to further elucidate the structural and regulatory differences between individual members of the RAF kinase family, with a long-term view of understanding the regulation of this key oncogenic pathway. A main focus in my group is the structure and regulation of the leucine-rich repeat kinase LRRK1 and LRRK2.. They are large multi-domain proteins containing two putative catalytic domains, a GTPase ROCO domain and a kinase domain, in addition to armadillo, ankyrin, leucin rich and WD40 domains. LRRK1 is slightly smaller than LRRK2 due to the lack of an N-terminal armadillo repeat domain. Despite similar domain organizations, LRRK1 and LRRK2 have distinct interactomes and distinct physiological functions. Mutations in LRRK2 that enhance kinase activity are a major genetic contributor to inherited Parkinson's disease (PD). Patients with the most common LRRK2 mutation can also have an overall increased risk of several cancers. Interestingly, LRRK1 has not been shown to associate with PD or cancer, but instead has an important role in bone biology. The current understanding of LRRK1 and LRRK2 will be greatly enhanced by revealing molecular mechanisms of their different functional states. The long-term goal of our studies is to gain a better understanding of how these large multi-domain kinases affect human health. Our ongoing studies are aimed at obtaining a comprehensive understanding of the inactive state of the LRRKs and their activation by revealing the structures and molecular mechanisms of full-length LRRK1 and LRRK2, both alone and in complex with regulatory proteins or substrates, such as the 14-3-3 proteins and the Rab small GTPases. Broadly, our goal is to gain a better understanding of how the LRRK proteins function in health and disease states, with an extended vision of developing therapeutic strategies to target this pathway.