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

https://doi.org/10.1016/j.bbagen.2018.12.012Get rights and content

Highlights

  • cAMP/PKA signal enhances phosphorylation of CaMKKβ at Thr144 in HeLa cells.

  • PKA directly phosphorylates CaMKKβ at Thr144 in cultured cells and in vitro.

  • Phosphorylation at Thr144 by PKA converts CaMKKβ into Ca2+/CaM-dependent enzyme.

  • CaMKKβ is phosphorylated at Thr144 in mouse cerebellum.

Abstract

Background

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5’-AMP-activated protein kinase (AMPK), controlling Ca2+-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 Ca2+/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 Ca2+/CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme.

Conclusion

cAMP/PKA signaling may confer Ca2+-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 Ca2+/CaM/CaMKKβ signaling through regulatory phosphorylation.

Introduction

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) was originally identified as an activator of CaMKIα and CaMKIV by phosphorylating their activation loop Thr residue (Thr177 in CaMKIα and Thr196 in CaMKIV) [1,2]. In mammals, CaMKK is composed of two isoforms (α and β) and is expressed in lower eukaryotes, including Caenorhabditis elegans and Aspergillus nidulans [[3], [4], [5], [6], [7]]. Recently, accumulated evidence indicated that CaMKKβ activates 5’AMP-activated protein kinase (AMPK) through phosphorylation of Thr172 in AMPKα, resulting in various metabolic and pathophysiological responses including hepatic steatosis and cancer cell growth [[8], [9], [10], [11], [12], [13], [14], [15]]. CaMKK is a member of the CaMK family, which is regulated by intrasteric autoinhibition and activated by Ca2+/CaM-binding to the regulatory domain [16,17], however, does not belong to CAMK group according to the classification by Manning et al. [18]. In addition to Ca2+/CaM-binding, CaMKK is regulated by phosphorylation, including autophosphorylation [19] and trans-phosphorylation by multiple protein kinases. It has been demonstrated that CaMKKα is negatively regulated by phosphorylation with cAMP-dependent protein kinase (PKA), resulting in the recruitment of 14-3-3 proteins [[20], [21], [22]]. Unlike CaMKKα, which is strictly regulated by autoinhibitory mechanism [17], CaMKKβ contains an N-terminal regulatory domain (residues 129-151) that suppresses the autoinhibitory mechanism and, consequently, generates autonomous activity [23]. Despite the significant Ca2+/CaM-independent activity of CaMKKβ, the activation of CaMKKβ-mediated signaling, including the CaMKKβ/AMPK cascade, requires an increasing concentration of intracellular Ca2+ [[8], [9], [10]]. Recent studies demonstrated that the phosphorylation of Ser129, Ser133, and Ser137 in the N-terminal regulatory domain of human CaMKKβ by CDK5 and GSK3 reduced autonomous activity [24]. Moreover, feedback phosphorylation of Thr144 in the same region by activated AMPK converts CaMKKβ into a Ca2+/CaM-dependent kinase [25], indicating that phosphorylation of the N-terminal regulatory domain suppresses the inhibitory effect of the region in the autoinhibitory mechanism. This post-translational modification of the N-terminal regulatory domain of CaMKKβ may be essential for Ca2+-dependent activation of CaMK cascade although the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells remains unclear.

In this study, we investigated the intracellular signaling system controlling Thr144 phosphorylation of CaMKKβ in cultured cells. In addition, we identified the cAMP/PKA signaling, which contributes to the regulatory phosphorylation of CaMKKβ, maintaining CaMKKβ as a Ca2+/CaM-dependent enzyme that might be occurred in vivo.

Section snippets

cAMP/PKA signal enhances phosphorylation of CaMKKβ at Thr144 in HeLa cells

CaMKKβ is phosphorylated at Thr144 by activated AMPK, resulting in the conversion of the enzyme into a Ca2+/CaM-dependent kinase in vitro and in transfected cells [25]. To further examine the Thr144 phosphorylation in cultured cells, we stimulated HeLa cells expressing CaMKKβ [[8], [9], [10],26] with various agonists and performed immunoblot analysis to detect the phosphorylation of CaMKKβ at Thr144. Since the immunoreactivity of the antibody against phosphoThr144 of CaMKKβ is not sensitive

Discussion

Accumulated evidence indicates that the CaMKK-mediated phosphorylation cascade plays an important role in a wide variety of physiological responses, including neuronal and metabolic signaling as well as pathophysiological pathways, including cancer cell growth [30,31]. Conventional CaMK cascades (i.e., CaMKK/CaMKI and CaMKK/CaMKIV) are strictly regulated by the dual mode of Ca2+-signaling, including Ca2+/CaM-binding to downstream CaMKs (CaMKI and CaMKIV) and upstream CaMKKs for activation [1].

Materials

Recombinant rat CaMKKβ wild-type and Thr144Ala mutant were expressed in E. coli BL21 Star (DE3) cells and purified by CaM-sepharose and Q-sepharose chromatography [23,25]. GST-rat CaMKIα 1–293, Lys49Glu (GST-CaMKI 1–293, KE) was expressed in E. coli JM109 and purified as previously described [17]. Recombinant wild-type AMPK was expressed in E. coli BL21-CodonPlus (DE3) (Stratagene, La Jolla, CA, USA) using a tricistronic pγ1β1His-α1 plasmid (kindly provided by Dr. Dietbert Neumann, Swiss

Conflict of interest

The authors declare that they have no conflict of interest with the contents of this article.

Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research (C) (18K06113 to H.T.) from Japan Society for the Promotion of Science.

Author contributions

H. T. conceived and designed the study. S. T. and S. O. performed the experiments. N. H. performed mass spectrometry analysis to identify phosphorylation sites. T. S. and H. S. supervised experiments and contributed to drafting the manuscript. All authors contributed to the analysis and interpretation of the data. M. M. and N. K. supervised the experiments. H. T. wrote and prepared the final version of the manuscript.

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