[R01] Flipping the Switch in Cell Cycle Control: Dissecting the Molecular Mechanisms Governing Wee1 Kinase transition from Cdk Inhibitor to Substrate

Ente: National Cancer Institute

Scadenza: 2031-06-30

Importo max: 645.102 EUR

Paese: US

Descrizione

Abstract Cell cycle checkpoints are essential for maintaining genomic integrity, and their dysregulation is a hallmark of cancer. Among these, the G2-to-M transition is regulated by the kinase WEE1, which delays mitotic entry by phosphorylating and inactivating Cdk1. This modification prevents cell division under conditions of DNA damage or replication stress. Paradoxically, at the G2–M boundary, WEE1 is inactivated through Cyclin-Cdk–mediated phosphorylation to enable cell cycle progression. While WEE1’s opposing roles as kinase and substrate are well recognized, the molecular switch controlling this transition remains poorly understood. This proposal addresses a central, unresolved question in cell cycle biology: how does WEE1 shift between the function of being an enzyme to a substrate? We focus on the underexplored N-terminal intrinsically disordered region (N-IDR) of WEE1, which modulates its kinase activity. Prior structural studies have focused almost exclusively on the kinase domain, typically in inhibitor-bound states, leaving the regulatory N-IDR a mechanistic black box. However, emerging evidence indicates that the N-IDR is essential for full kinase activity and may serve as the critical interface mediating multisite phosphorylation and regulatory control. We propose a conceptual advance by reframing WEE1 as a phosphorylation-tunable molecular switch, in which IDR-mediated interdomain communication dictates whether WEE1 acts as an active kinase or CDK substrate. This hypothesis has remained unexplored due to technical limitations, but we are now uniquely positioned to address it using an integrated approach combining high-resolution NMR, proteomics, and live-cell imaging. Our preliminary data show distinct, time-resolved phosphorylation patterns induced by different Cyclin-Cdk complexes and modulated by docking proteins like CKS1, strongly supporting a hierarchical and temporally ordered regulatory mechanism. Computational approaches, ranging from AlphaFold to long-timescale MD simulations, fail to resolve phosphorylation-induced conformational changes or N-IDR–kinase domain interactions, underscoring the need for experimental insight. Our proposed studies will generate missing residue-level structural data and elucidate how multisite phosphorylation governs activation, inhibition, and degradation of WEE1. This work integrates the Arthanari lab’s strengths in structural biology with the Sicinski lab’s expertise in cell cycle regulation, supported by collaborators Davey and Pines. By resolving how the disordered N-IDR controls WEE1 function, this research fills a key gap in our understanding of cell cycle control. Our approach integrates atomic-resolution structural analysis, time-resolved proteomics, enzymology, and functional studies to elucidate how the N-IDR and its post- translational modifications orchestrate WEE1 regulation, revealing key principles of kinase signaling. Crucially, structural and enzymatic insights will be cross-v Istituzione: DANA-FARBER CANCER INST PI: Haribabu Arthanari Progetto: 1R01CA303520-01A1

Settori: National Cancer Institute

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