Structual and function of kinase signaling complexes

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Project 1: Structure-function analyses of autoinhibited and active RAF kinases The family of Raf kinases (A-Raf, B-Raf and C-Raf) constitute core components of the RTK-RAS-RAF-MEK signaling cascade, which plays a major role in directing cell growth, differentiation and survival. Raf activity is frequently dysregulated in cancers. In quiescent cells, the wild type Raf kinase exists as an autoinhibited monomer in cytosol. Direct interaction with active GTP-bound Ras disrupts the autoinhibited state of Raf kinase and induces Raf dimer formation and activation. B-Raf and C-Raf are more active than the A-Raf in response to Ras activation and B-Raf/C-Raf heterodimers predominate in Ras-dependent signaling. Although Raf kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood due to the complexity of the activation process. The major goal of this project is to elucidate the molecular mechanisms of Raf activity regulation. We aim to define how B-Raf and C-Raf kinase monomers are autoinhibited and how co-factors in the autoinhibited complexes contribute to the autoinhibition and stabilization. we also aim to define the architectures of active B-Raf homodimers and B/C hetero dimers. We will further our understanding of the Raf activation process as well as study the structural and functional differences of the Raf isoforms as related to this process. To achieve this goal, we use an interdisciplinary approach that combines structural biology, biochemistry and cell biology techniques. Project 2: Structure-function analyses of Leucine Rich Repeat Kinase 2 (LRRK2) and its homologue LRRK1 LRRK2 is the leading genetic contributor to familial Parkinson's disease (PD) and currently one of the most promising therapeutic targets for drug design in PD. LRRK2 is a large multi-domain protein (2527 residues) containing two putative catalytic domains: a GTPase (ROC-COR) domain and a kinase domain as well as other domains. The PD pathological mutations clusters within the GTPase and kinase domains and increase kinase activities. LRRK2 kinase domain mutation carriers have an overall increased risk of cancer, especially for hormone-related cancer and breast cancer in women. However, little is known about the LRRK2 structure and even less is known about the regulation of its activity. LRRK2 and its singlet homologue LRRK1 belong to the same family as Raf kinases: Tyrosine Kinase Like family of protein kinases. LRRK1 is not shown be associated with PD and cancer. Little is known about the structure and function of LRRK1. The present understanding of LRRKs is severely handicapped by the lack of structural information, as only the ROC and WD40 domain structures of LRRK2 have been determined. There is no insight into the global relationship of the domains and the functional interactions of these domains. Many important mechanisms remain to be elucidated, including how the domains interact with and regulate each other, how the GTPase and kinase activities are regulated, and how these activities contribute to the overall functional output of LRRK2. The major goal to reveal the molecular mechanisms of LRRK function and pathology. We aim to advance the understanding of LRRK2 and LRRK1 structures, conformational states and regulation through interdisciplinary structural and biochemical approaches. This project will generate results that allow us to elucidate the structure and function of LRRKs and create new hypotheses for how to intervene with therapeutic strategies. Project 3: Structure and function analyses of kinase fusion protein DNAJB1-PKACA Fibrolamellar hepatocellular carcinoma (FLHCC) is a liver cancer that predominantly affects adolescents and young adults. The chimeric DNAJB1-PKACA protein is generated by a deletion of 400 kB between the first exon of the heat shock protein DNAJB1 and the first exon of the catalytic subunit of Protein Kinase PKA, PKACA, on one copy of chromosome 19 and has been recognized as the driver of FLHCC. Inhibition of the DNAJB1-PKACA chimeric tumor driver offers tremendous potential to treat FLHCC. We study the structure and dynamics of the DNAJB1-PKACA chimera and how it is regulated with the long-term goal of developing precision medicine strategies against this fatal pediatric cancer. In its inactive state in cells, wildtype PKA exists as a holoenzyme composed of two PKACA catalytic subunits and one regulatory subunit homodimer. There are four functionally nonredundant R-subunit isoforms. We aim to investigate the impact of the presence of the DNAJB1-domain fusion on PKACA holoenzyme formation and inhibition. In addition, we further our effort to design small molecules that selective block DNAJB1-PKACA activity or RIa2:DNAJB1-PKACA2 holoenzyme activation.