Understanding interplay between the Hsp70 chaperone code and Cyclin D1/Cln3

Hsp70 is vital molecular chaperone that folds a large proportion of the proteome. Research has primarily focused on how Hsp70 function specificity arises through regulation of a) expression of Hsp70, b) isoform differences in the Hsp70 protein family and c) the variety of co-chaperone proteins that bind to the Hsp70 molecule. Despite the identification of several phosphorylation sites on both yeast and mammalian Hsp70 through global proteomic screens, the biological function of most of these sites remains unknown. All organisms require correct coordination of the cell cycle to grow and proliferate. Uncontrolled cell cycle progression can result in either cell death or metastasis. Progression of the cell from G1 to G2 requires the activity of cyclin-dependent kinase (CDK) CDK4. The critical activity of CDK4 is regulated by binding to cyclin proteins such as Cyclin D1, which rises and falls in abundance periodically through the cell cycle. As such, altered Cyclin D1 activity (achieved through increased transcription or stability) is often correlated with tumorigenesis. Any strategy that lowers Cyclin D1 activity in cells may form the basis of novel anticancer therapies. Our studies have determined that phosphorylation of a single site on Hsp70 regulates chaperone function by altering both co-chaperone and client protein interactions. In particular, we have shown that increased Hsp70 T38 phosphorylation promotes Cyclin D1 destruction. In this proposal, we will investigate the role of other Hsp70 PTMs (the chaperone code) on Cyclin D1 levels in yeast and mammalian cells using the chaperone code array. We propose to use cross-linking mass spectrometry to fully characterize the Hsp70-Cyclin D1/Cln3 interaction, including binding surfaces and PTMs involved. The scope of this work has broad implications for a variety of diseases associated with both the cell cycle and molecular chaperone function, including many types of cancer and neurodegenerative illnesses. Project Number: 1R03CA289058-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Andrew Truman | Institution: UNIVERSITY OF NORTH CAROLINA CHARLOTTE, CHARLOTTE, NC | Award Amount: $150,784 | Activity Code: R03 | Study Section: Special Emphasis Panel[ZCA1 RTRB-Z (O1)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11033874