CaMKK2 is inactivated by cAMP-PKA signaling and 14-3-3 adaptor proteins
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… from 10 μg of transfected cell lysate using 10 μl of anti-Flag M2 agarose beads (50% [v/v])
(Sigma-Aldrich), and then added to a 30 μl reaction containing assay buffer (50 mM Hepes.NaOH
[pH 7.4], 1 mM DTT, 0.02% [v/v] Brij-35), 200 μM CaMKKtide (Genscript), 200 μM [γ-32P … |
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The calcium-calmodulin (Ca-CaM) 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 Ca, 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 cyclic AMP (cAMP)-dependent protein kinase (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. PKA-dependent phosphorylation of Se... More
The calcium-calmodulin (Ca-CaM) 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 Ca, 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 cyclic AMP (cAMP)-dependent protein kinase (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. PKA-dependent phosphorylation of Ser495 directly impairs Ca-CaM 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 non-canonical (Ser100) 14-3-3 consensus sites on CaMKK2 co-operate 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.
14-3-3 protein, Ca2+, Ca2+/calmodulin-dependent protein kinase (CaMK), CaMKK2, PKA, cAMP, calmodulin, cyclic AMP (cAMP), inhibition mechanism, protein kinase A (PKA)
