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Abstract
The Ca2+-calmodulin dependent protein kinase kinase-2 (CaMKK2) is a key regulator of neuronal function and whole-body energy metabolism. Elevated CaMKK2 activity is strongly associated with prostate and hepatic cancers, whereas reduced CaMKK2 activity has been linked to schizophrenia and bipolar disease in humans. Here we report the functional effects of nine rare-variant point mutations that were detected in large-scale human genetic studies and cancer tissues, all of which occur close to two regulatory phosphorylation sites and the catalytic site on human CaMKK2. Four mutations (G87R, R139W, R142W and E268K) cause a marked decrease in Ca2+-independent autonomous activity, however S137L and P138S mutants displayed increased autonomous and Ca2+-CaM stimulated activities. Furthermore, the G87R mutant is defective in Thr85-autophosphorylation dependent autonomous activity, whereas the A329T mutation rendered CaMKK2 virtually insensitive to Ca2+-CaM stimulation. The G87R and R139W mutants behave as dominant-negative inhibitors of CaMKK2 signaling in cells as they block phosphorylation of the downstream substrate AMP-activated protein kinase (AMPK) in response to ionomycin. Our study provides insight into functionally disruptive, rare-variant mutations in human CaMKK2, which have the potential to influence risk and burden of disease associated with aberrant CaMKK2 activity in human populations carrying these variants.
Highlights
The Ca2+-CaM dependence of calmodulin dependent protein kinase kinase-2 (CaMKK2) is unique among the CaM kinase subfamily, being contingent upon sequential phosphorylation of Ser[129], Ser[133] and Ser[137] located within a regulatory sequence N-terminal to the catalytic domain[5]
CaMKK2 autonomous activity was measured in the presence of 40 μM EGTA, which we determined by titration in order to chelate contaminating Ca2+ in the CaM preparation (Supplementary Fig. 1)
This is due to translation from Met[49], as a M49L mutation generated a single species corresponding to full length WT CaMKK2 that migrated with the upper band (Supplementary Fig. 2a)
Summary
The Ca2+-CaM dependence of CaMKK2 is unique among the CaM kinase subfamily, being contingent upon sequential phosphorylation of Ser[129], Ser[133] and Ser[137] located within a regulatory sequence N-terminal to the catalytic domain[5]. Nine rare-variant point mutations that occur in close proximity to regulatory phosphorylation sites and the catalytic site on human CaMKK2 have been detected in large-scale genetic studies[14,15], as well as in several cancer cell lines and tissues derived from cancer patients[16,17,18,19,20]. Rare-variant mutations are considered to be an important component in genetic susceptibility to common human diseases, and are more likely to be functionally disruptive and have larger effects on disease risk than common variants[21,22] This prompted us to analyze the functional effects of human CaMKK2 rare-variant mutations on enzyme activity and regulation by Ca2+-CaM and multisite phosphorylation
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