Journal of Biological Chemistry
Volume 295, Issue 48, 27 November 2020, Pages 16239-16250
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Signal Transduction
CaMKK2 is inactivated by cAMP-PKA signaling and 14-3-3 adaptor proteins

https://doi.org/10.1074/jbc.RA120.013756Get rights and content
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The calcium-calmodulin–dependent protein kinase kinase-2 (CaMKK2) is a key regulator of cellular and whole-body energy metabolism. It is known to be activated by increases in intracellular Ca2+, but the mechanisms by which it is inactivated are less clear. CaMKK2 inhibition protects against prostate cancer, hepatocellular carcinoma, and metabolic derangements induced by a high-fat diet; therefore, elucidating the intracellular mechanisms that inactivate CaMKK2 has important therapeutic implications. Here we show that stimulation of cAMP-dependent protein kinase A (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. PKA-dependent phosphorylation of Ser495 directly impairs calcium-calmodulin activation, whereas phosphorylation of Ser100 and Ser511 mediate recruitment of 14-3-3 adaptor proteins that hold CaMKK2 in the inactivated state by preventing dephosphorylation of phospho-Ser495. We also report the crystal structure of 14-3-3ζ bound to a synthetic diphosphorylated peptide that reveals how the canonical (Ser511) and noncanonical (Ser100) 14-3-3 consensus sites on CaMKK2 cooperate to bind 14-3-3 proteins. Our findings provide detailed molecular insights into how cAMP-PKA signaling inactivates CaMKK2 and reveals a pathway to inhibit CaMKK2 with potential for treating human diseases.

Ca2+-calmodulin–dependent protein kinase kinase-2 (CaMKK2)
calmodulin (CaM)
cyclic AMP (cAMP)
protein kinase A (PKA)
inhibition mechanism
adaptor protein
14-3-3
Ca2+-calmodulin–dependent protein kinase (CaMK)
14-3-3 protein
Ca2+
calmodulin
CaMKK2
cAMP
PKA

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Author contributions—C. G. L., M. T. O., N. X. Y. L., T. A. D., J. S. O., B. E. K., and J. W. S. conceptualization; C. G. L., T. A. D., S. G., K. L., M. W. P., J. S. O., B. E. K., and J. W. S. supervision; C. G. L., B. E. K., and J. W. S. funding acquisition; C. G. L., M. T. O., K. R. N., L. M. M., U. D., A. H., N. X. Y. L., T. A. D., S. G., K. L., M. W. P., J. S. O., B. E. K., and J. W. S. investigation; C. G. L., M. T. O., K. R. N., L. M. M., U. D., A. H., N. X. Y. L., T. A. D., S. G., K. L., M. W. P., J. S. O., and B. E. K. methodology; C. G. L., M. T. O., B. E. K., and J. W. S. writing-original draft; C. G. L., B. E. K., and J. W. S. project administration; C. G. L., M. T. O., L. M. M., A. H., N. X. Y. L., T. A. D., S. G., K. L., M. W. P., J. S. O., B. E. K., and J. W. S. writing-review and editing; M. T. O. formal analysis.

Funding and additional information—This work was supported by National Health and Medical Research Council Grants 1138102 (to J. W. S.) and 1145265 (to J. S. O.) and the Jack Brockhoff Foundation Grant JBF-4206, 2016 (to C. G. L.). C. G. L. is a National Health and Medical Research Council Early Career Research Fellow and M. W. P. a National Health and Medical Research Council Senior Principal Research Fellow. B. E. K. was a National Health and Medical Research Council Fellow supported by the Australian Research Council (DP170101196). This project was supported in part by the Victorian Government's Operational Infrastructure Support Program. MS data were acquired at the Mass Spectrometry and Proteomics Facility, Bio21, University of Melbourne. We acknowledge the funding support of the Australian Cancer Research Foundation toward the ACRF Rational Drug Discovery Centre.

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Present address for Toby A. Dite: MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, United Kingdom.

Abbreviations—The abbreviations used are:

    CaM

    calmodulin

    CaMK

    Ca2+-calmodulin dependent protein kinase

    PKA

    protein kinase A

    AMPK

    AMP-activated protein kinase

    IBMX

    3-isobutyl-1-methylxanthine

    GLP

    glucagon-like peptide

    ANOVA

    analysis of variance

    SPR

    surface plasmon resonance.

These authors contributed equally to this work.