Structures and Mechanisms of Kinase Signaling Complexes

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Our research program investigates how key protein kinases function at the molecular level and how their dysregulation contributes to diseases such as cancer and Parkinson's disease. By determining high-resolution structures and studying their regulatory mechanisms, we aim to inform future therapeutic strategies. In FY2025, my lab continues to make advances in understanding the structural and mechanistic regulation of key kinase signaling complexes, including LRRK1 and LRRK2, RAF family kinases, and the oncogenic fusion protein J-PKACa. These efforts align with our broader objective to elucidate the molecular basis of kinase function and to translate structural insights into therapeutic strategies for diseases such as cancer and Parkinson's disease. Our main project focuses on the study of LRRK1 and LRRK2, two large and complex enzymes that play crucial roles in regulating fundamental cellular processes, most notably membrane trafficking. Mutations in LRRK1 have been linked to a rare bone disease, while mutations in LRRK2 are a major cause of inherited and sporadic Parkinson's disease. Certain hyperactive mutations in LRRK2 have also been associated with an increased overall risk of cancer in patients. Our research aims to understand the structure and function of these enzymes, including their inactive and active states, their regulation, and their interactions with other proteins such as 14-3-3 and Rab-family GTPases. Recently, we resolved the structure of the LRRK2:14-3-32 complex, revealing how 14-3-3 binding regulates LRRK2 kinase activity and how Parkinson's disease-associated mutations interfere with this regulation. Functional studies confirmed the inhibitory role of 14-3-3 on LRRK2 activity and helped us establish a robust in vitro system to study this mechanism. We also continue to work on structural studies of LRRK1. In our earlier work, we determined the first cryo-EM structure of full-length human LRRK1 in its inactive form, which revealed important regulatory features and differences between LRRK1 and LRRK2. We are now generating and analyzing active forms of LRRK1 to study its activation mechanism in greater detail. Another major project in the lab focuses on understanding RAF kinase signaling. RAF proteins are key intermediates in the RAS-RAF-MEK signaling pathway and are important drivers of many cancers. Our objective is to understand how RAS binding triggers the conformational changes required for RAF activation. Previously, we determined the cryo-EM structures of multiple full-length BRAF complexes, including the first autoinhibited form with a resolved RAS-binding domain. These structures revealed key transitions in the RAF activation process. In FY2025, we made additional progress in resolving the structure of autoinhibited full-length CRAF and obtained promising preliminary data on CRAF complexes, which sets the stage for future comparative analyses across RAF family members. The third project in my lab investigates the oncogenic fusion kinase J-PKACa, which drives fibrolamellar hepatocellular carcinoma, a rare type of liver cancer that mainly affects young adults with no prior history of liver disease. We aim to better understand how this fusion protein affects PKA signaling by comparing the structure and function of J-PKACa complexes with their wild-type counterparts. We have made significant progress in this area by determining the structures of both the chimeric RIa2:J-PKACa2 complex and the wild-type RIa2:PKACa2 complex. This marks the first structural analysis of a chimeric PKA holoenzyme. Our work continues to provide insight into how the fusion disrupts PKA signaling. In collaboration with the Molecular Targets Program at NCI, we identified and characterized natural product inhibitors targeting J-PKACa. These efforts led to the discovery of a new class of aplithianine analogues, for which we have filed patent applications. We are continuing to develop and optimize these compounds as potential therapeutics.