Generation of autonomous activity of Ca 2+/calmodulin-dependent protein kinase kinase β by autophosphorylation

Hiroshi Tokumitsu, Naoya Hatano, Tomohito Fujimoto, Saki Yurimoto, Ryoji Kobayashi

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Ca 2+/calmodulin-dependent protein kinase kinases (CaMKKs) phosphorylate and activate specific downstream protein kinases, including CaMKI, CaMKIV, and 5′-AMP-activated protein kinase, which mediates a variety of Ca 2+ signaling cascades. CaMKKs have been shown to undergo autophosphorylation, although their role in enzymatic regulation remains unclear. Here, we found that CaMKKα and β isoforms expressed in nonstimulated transfected COS-7 cells, as well as recombinant CaMKKs expressed in and purified from Escherichia coli, were phosphorylated at Thr residues. Introduction of a kinase-dead mutation completely impaired the Thr phosphorylation of these recombinant CaMKK isoforms. In addition, wild-type recombinant CaMKKs were unable to transphosphorylate the kinase-dead mutants, suggesting that CaMKK isoforms undergo Ca 2+/CaM-independent autophosphorylation in an intramolecular manner. Liquid chromatography-tandem mass spectrometry analysis identified Thr 482 in the autoinhibitory domain as one of the autophosphorylation sites in CaMKKβ, but phosphorylation of the equivalent Thr residue (Thr 446) in the α isoform was not observed. Unlike CaMKKα that has high Ca 2+/CaM-dependent activity, wild-type CaMKKβ displays enhanced autonomous activity (Ca 2+/CaM-independent activity, 71% of total activity). This activity was significantly reduced (to 37%) by substitution of Thr 482 with a nonphosphorylatable Ala, without significant changes in Ca 2+/CaM binding. In addition, a CaMKKα mutant containing the CaMKKβ regulatory domain was shown to be partially phosphorylated at Thr 446, resulting in a modest elevation of its autonomous activity. The combined results indicate that, in contrast to the α isoform, CaMKKβ exhibited increased autonomous activity, which was caused, at least in part, by autophosphorylation at Thr 482, resulting in partial disruption of the autoinhibitory mechanism.

Original languageEnglish
Pages (from-to)8193-8201
Number of pages9
JournalBiochemistry
Volume50
Issue number38
DOIs
Publication statusPublished - Sep 27 2011
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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