CaMKK2 is inactivated by cAMP-PKA signaling and 14-3-3 adaptor proteins

Christopher G Langendorf,Matthew T O'Brien,Kevin R.W Ngoei,Luke M Mcaloon,Urmi Dhagat,Ashfaqul Hoque,Naomi X.Y Ling,Toby A Dite,Sandra Galic,Kim Loh,Michael W Parker,Jonathan S Oakhill,Bruce E Kemp,John W Scott

doi:10.1074/jbc.ra120.013756

Abstract

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.

Highlights

The calcium ion (Ca21) is a dynamic second messenger that relays signals from ligand-activated receptors and voltagestimulated ion channels at the cell membrane to regulate a wide array of physiological functions [1]
COS7 cells expressing recombinant human Ca21CaM–dependent protein kinase kinase-2 (CaMKK2) were treated with forskolin and 3-isobutyl-1-methylxanthine (IBMX) to increase intracellular cAMP, after which we immunoprecipitated CaMKK2 and measured kinase activity over a range of CaM concentrations
We examined the effect of the glucagon-like peptide (GLP)-1 receptor agonist liraglutide on CaMKK2 activity in SHSY5Y neuroblastoma cells that endogenously express the GLP1 receptor [19]

Summary

Maximal activity

14-3-3 proteins exist functionally as dimers and contain two phosphopeptide-binding pockets that can interact with two sites simultaneously, often on the same target protein [23]. The pSer100Ser511 peptide (Fig. 4B) showed modest binding to 14-3-3 (KD 1.91 mM) relative to the Ser100-pSer511 peptide, which displayed a 5-fold increase in binding affinity (KD 0.35 mM) consistent with pSer511 conforming to the canonical 14-3-3 binding motif (Fig. 4C). Each dimer contained two phosphopeptide-binding sites, both of which showed strong electron density for the critical pSer100 and pSer511 residues (Fig. 5A). Coordination of the phosphoserines was conserved in both binding sites (mediated by Lys-4914-3-3, Arg-5614-3-3, Arg-12714-3-3, and Tyr-12814-3-3); the pSer511 end of the peptide bound in site 1 makes considerably more interactions through its main chain and side chain atoms compared with the pSer100 end in site 2 (Fig. 5, B and C). Only residues Leu and Leu101 on the pSer100 end make hydrogen bonds with 14-3-3 via main chain interactions with the side chains of Asn-22414-3-3 and Asn-17314-3-3, respectively

Discussion

Experimental procedures Cyclic AMP measurements

CaM overlay assay

Surface plasmon resonance binding assays