Biochimica et Biophysica Acta (BBA) - General Subjects
Regulation of Ca2+/calmodulin-dependent protein kinase kinase β by cAMP signaling
Graphical abstract
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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S. T. and S. O. contributed equally to this work.