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Distinct structural mechanisms determine substrate affinity and kinase activity of protein kinase Cα

Lee, Sangbae and Devamani, Titu and Song, Hyun Deok and Sandhu, Manbir and Larsen, Adrien and Sommese, Ruth and Jain, Abhinandan and Vaidehi, Nagarajan and Sivaramakrishnan, Sivaraj (2017) Distinct structural mechanisms determine substrate affinity and kinase activity of protein kinase Cα. Journal of Biological Chemistry, 292 (39). pp. 16300-16309. ISSN 0021-9258. PMCID PMC5625059. https://resolver.caltech.edu/CaltechAUTHORS:20171009-115239146

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Abstract

Protein kinase Cα (PKCα) belongs to the family of AGC kinases that phosphorylate multiple peptide substrates. Although the consensus sequence motif has been identified and used to explain substrate specificity for PKCα, it does not inform the structural basis of substrate-binding and kinase activity for diverse substrates phosphorylated by this kinase. The transient, dynamic, and unstructured nature of this protein–protein interaction has limited structural mapping of kinase–substrate interfaces. Here, using multiscale MD simulation-based predictions and FRET sensor-based experiments, we investigated the conformational dynamics of the kinase–substrate interface. We found that the binding strength of the kinase–substrate interaction is primarily determined by long-range columbic interactions between basic (Arg/Lys) residues located N-terminally to the phosphorylated Ser/Thr residues in the substrate and by an acidic patch in the kinase catalytic domain. Kinase activity stemmed from conformational flexibility in the region C-terminal to the phosphorylated Ser/Thr residues. Flexibility of the substrate–kinase interaction enabled an Arg/Lys two to three amino acids C-terminal to the phosphorylated Ser/Thr to prime a catalytically active conformation, facilitating phosphoryl transfer to the substrate. The structural mechanisms determining substrate binding and catalytic activity formed the basis of diverse binding affinities and kinase activities of PKCα for 14 substrates with varying degrees of sequence conservation. Our findings provide insight into the dynamic properties of the kinase–substrate interaction that govern substrate binding and turnover. Moreover, this study establishes a modeling and experimental method to elucidate the structural dynamics underlying substrate selectivity among eukaryotic kinases.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1074/jbc.M117.804781DOIArticle
http://www.jbc.org/content/292/39/16300.shortPublisherArticle
http://www.jbc.org/content/292/39/16300/suppl/DC1PublisherSupporting Information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625059/PubMed CentralArticle
Additional Information:© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Received June 29, 2017. Revision received August 4, 2017. First Published on August 15, 2017. This work was supported in part by National Institutes of Health Director’s New Innovator Award 1DP2 CA186752-01 (to S. S.) and the Beckman Research Institute of the City of Hope (to N. V.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Author contributions—S. L., T. D., H. D. S., M. S., A. L., R. F. S., A. J., N. V., and S. S. planned, designed the experiments, and contributed equally for this work. S. L., T. D., and H. D. all contributed equally for the experiments and analyzing the results. S. L., T. D., H. D. S., N. V., and S. S. wrote the manuscript.
Funders:
Funding AgencyGrant Number
NIH1DP2 CA186752-01
City of HopeUNSPECIFIED
Issue or Number:39
PubMed Central ID:PMC5625059
Record Number:CaltechAUTHORS:20171009-115239146
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20171009-115239146
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:82212
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:09 Oct 2017 21:32
Last Modified:03 Oct 2019 18:52

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