Regulation of Ca2+/calmodulin-dependent protein kinase kinase β by cAMP signaling

Abstract

BACKGROUND: Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5'-AMP-activated protein kinase (AMPK), controlling Ca 2+-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca 2+ /CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear. METHODS: Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ. RESULTS: Our data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca 2+ /CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca 2+ /CaM-dependent enzyme. CONCLUSION: cAMP/PKA signaling may confer Ca 2+-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells. GENERAL SIGNIFICANCE: Our results suggest a novel cross-talk between cAMP/PKA and Ca 2+ /CaM/ CaMKKβ signaling through regulatory phosphorylation.

Development and Characterization of Novel Molecular Probes for Ca2+/Calmodulin-Dependent Protein Kinase Kinase, Derived from STO-609

“…Furthermore, we found that OA treatment induces additional phosphorylation at Thr144 (Figure 1B, Supplemental Figure S1). These results are consistent with previous reports demonstrating that human CaMKKβ is phosphorylated at Ser129, Ser133, and Ser137 by CDK5/GSK3 in transiently overexpressed COS-7 cells [20] and Ser511 in SH-SY5Y cells by DAPK [25] and Thr144 is maintained unphosphorylated state in unstimulated HeLa cells [21,22].…”

“…Moreover, feedback phosphorylation of Thr144 in the same region by activated AMPK converts CaMKKβ into a Ca 2+ /CaM-dependent enzyme [21], indicating that phosphorylation of the NRD suppresses the inhibitory effect of the region in the autoinhibitory mechanism. Recently, we have demonstrated that Thr144 in CaMKKβ is also rapidly phosphorylated by β-adrenergic stimulation through cAMP/PKA signaling; this has been confirmed by in vitro phosphorylation of Thr144 by purified PKA [22].…”

“…Recently, we identified Thr144 in the NRD of rat CaMKKβ by AMPK [21] and cAMP/PKA signaling in transiently expressed HeLa cells [22]. It has been shown that the Thr144 phosphorylation converts CaMKKβ into Ca 2+ /CaM-dependent enzyme [21,22]. These studies also indicated that the Thr144 phosphorylation was very weak without stimulation, suggesting that the Thr144 is maintained unphosphorylated by protein phosphatase(s) in resting cells.…”

“…Recombinant CaMKKβ (293 µg) was phosphorylated with purified PKA (1µg) at 30 o C for 240 min as described previously [22], followed by purification with CaM-sepharose column chromatography, resulting in approximately 160 µg of phosphorylated CaMKKβ. Phosphorylated CaMKKβ (80 µg/mL) was incubated without or with protein phosphatase 2A (4.6 µg/mL) or protein phosphatase 1 (5-50 µg/mL) in a solution containing 40 mM Tris-HCl pH 7.5, 100 mM KCl, 3 mM MgCl2, 1 mM DTT, and 0.1 mg/mL bovine serum albumin in the absence (for PP2A) or presence of 2 mM MnCl2 (for PP1) at 30 o C for the indicated time periods.…”

“…CaMKKβ has shown to be regulated by phosphorylation with multiple protein kinases in the NRD (residues 129-151, [19]) by GSK3 and CDK5 [20] and in the C-terminal region (at S511 in human CaMKKβ) by death-associated kinase (DAP kinase) [25] in transiently overexpressed cultured cells as well as in vitro. Recently, we identified Thr144 in the NRD of rat CaMKKβ by AMPK [21] and cAMP/PKA signaling in transiently expressed HeLa cells [22]. It has been shown that the Thr144 phosphorylation converts CaMKKβ into Ca 2+ /CaM-dependent enzyme [21,22].…”