The role of cyclin B1’s phosphate-binding pocket in ordered substrate phosphorylation

• Applicant: Professor Dr. Thomas Mayer
• Subject Area: Cell Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 532411725

The correct execution and timing of the various cell cycle phases represent important guarantees for the viability of any organism. Clocks for entry into and exit from the different cell cycle phases are cyclin-dependent kinases (Cdks) in complex with an activating cyclin subunit. While the central role of Cdks in cell cycle regulation is well known, we have only an incomplete understanding of how the multitude of Cdk/cyclin substrates are phosphorylated in the correct order, thus guaranteeing the correct sequence of the diverse cell cycle processes. Recently, a phosphate-binding pocket was identified in cyclin B1 that is highly conserved within B-type - but not A-type - cyclins. Via this positively charged pocket, cyclin B1 binds to a phosphorylated serine residue of separase, a regulator of Cdk1/cyclin B1. Based on these data, we postulate that the phosphate-binding pocket of cyclin B1 is important not only for interaction with the regulator separase but also for substrate binding. Accordingly, pre-phosphorylated substrates would bind to the phosphate-binding pocket of cyclin B1 and subsequently be phosphorylated at additional positions by Cdk1/cyclin B1. Such a mechanism could contribute significantly to the substrate specificity and timely substrate phosphorylations. Consistent with this hypothesis, we could already demonstrate that cells with a mutant phosphate-binding pocket in cyclin B1 exhibit mitotic defects. Using in vitro assays, we could exclude that the introduced mutations affect the kinase activity of Cdk1/cyclin B1 in a nonspecific manner. Mass spectrometry (MS) analyses identified subunits of the ubiquitin ligase APC/C, mitotic checkpoint proteins, and kinesins as proteins that interact with wild-type cyclin B1 but not with mutant cyclin B1. In this project, we will identify in these proteins both the phosphorylation sites that bind to the phosphate-binding pocket of cyclin B1 and the phosphorylation sites that are subsequently phosphorylated by Cdk1/cyclin B1. To this end, we will perform extensive MS-based phosphoprotein analyses. To investigate how these sequential phosphorylation events affect the function of the identified substrates, we plan to perform extensive studies in cells as well as in egg extract and in vitro. These studies will provide fundamental insights into how the phosphate-binding pocket in cyclin B1 contributes to both the specificity and temporally correct phosphorylation sequence of the multiple Cdk1/cyclin B1 substrates; two fundamental requirements for error-free cell cycle progression.

• DFG Programme: Research Grants