Selective Recruitment of RSK Protein Kinases Through Non-Catalytic Docking Interactions

Loss-of-function mutations in ribosomal protein S6 kinase 2 (RSK2, encoded by the RPSKA3 gene) cause Coffin- Lowry syndrome, an X-linked dominant condition characterized by craniofacial abnormalities, short stature, and intellectual disability. The RSK2 N-terminal catalytic domain (NTD) shares a consensus phosphorylation site motif with numerous related kinases, including isoforms of AKT, SGK, and S6K. As such, it is difficult to rationalize how RSK2 targets a unique substrate pool, and the utility of this target motif to discover substrates of RSK2 and these other kinases is limited. Recently a cryptic allosteric pocket was identified in the RSK2 NTD that binds to a short linear sequence motif found in several of its interacting proteins. This RSK-interacting motif (RIM) consists of a core Val-Phe sequence, but the full motif has not yet been systematically defined. Furthermore, it is not known whether the analogous binding site in other related kinases might target distinct motifs. Here, we propose to comprehensively define RIM sequence space, screen the human proteome for functional RSK2-interacting sequences, and determine whether other kinases utilize the analogous binding pocket for protein-protein interactions. In preliminary studies, we have found the interaction between the RSK2 NTD and the RIM from its substrate protein SPRED2 is detectable by yeast two-hybrid (Y2H) analysis in a manner sensitive to mutation of a core residue. To comprehensively define the RIM, we will construct a Y2H double positional scanning library consisting of all pairwise combinations of residues at two positions in the context of the SPRED2 sequence. We will screen the library against RSK2 and a panel of related kinases. We will verify the importance of specific residues within the motif through quantitative measurements of kinase binding to synthetic peptides harboring targeted substitutions. Guided by these results, we will construct and screen a human proteomic Y2H library of candidate RIMs consisting of intrinsically disordered fragments of human proteins. RSK2-interacting sequences identified from the screen will be validated as direct binders of RSK2 in vitro. We will then investigate whether the full-length proteins harboring these sequences can act as RSK2 substrates or interactors in a manner dependent on the docking motif. These results will form the basis for subsequent in depth study of novel RSK2 substrates and their importance in normal physiology and in Coffin- Lowry syndrome. Our studies may furthermore reveal that a large number of related protein kinases utilize the corresponding interaction pocket in substrate docking, laying the groundwork for future studies to define their interaction networks and better understand how they function. Project Number: 1R21DE035244-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Dental and Craniofacial Research (NIDCR) | Principal Investigator: BENJAMIN TURK | Institution: YALE UNIVERSITY, NEW HAVEN, CT | Award Amount: $461,375 | Activity Code: R21 | Study Section: Cellular Signaling and Regulatory Systems Study Section[CSRS] View on NIH RePORTER: https://reporter.nih.gov/project-details/11214600