Substrate recognition by Ca2+/calmodulin-dependent protein kinase kinase: Role of the Arg-Pro-rich insert domain

Hiroshi Tokumitsu, Naomi Takahashi, Koh Eto, Shigetoshi Yano, Thomas R. Soderling, Masa Aki Muramatsu

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KK can also activate and phosphorylate protein kinase B (PKB). In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK α and β shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases. Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathione S-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site- directed mutagenesis of three conserved Arg in the RP-domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP-domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB. These results indicate that the RP- domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.

Original languageEnglish
Pages (from-to)15803-15810
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number22
DOIs
Publication statusPublished - May 28 1999
Externally publishedYes

Fingerprint

arginylproline
Calcium-Calmodulin-Dependent Protein Kinases
Protein Kinases
Phosphotransferases
Calcium-Calmodulin-Dependent Protein Kinase Type 4
Substrates
Proto-Oncogene Proteins c-akt
Calcium-Calmodulin-Dependent Protein Kinase Type 1
Chemical activation
Phosphorylation
Mutagenesis
Caenorhabditis elegans
Site-Directed Mutagenesis
Glutathione Transferase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Substrate recognition by Ca2+/calmodulin-dependent protein kinase kinase : Role of the Arg-Pro-rich insert domain. / Tokumitsu, Hiroshi; Takahashi, Naomi; Eto, Koh; Yano, Shigetoshi; Soderling, Thomas R.; Muramatsu, Masa Aki.

In: Journal of Biological Chemistry, Vol. 274, No. 22, 28.05.1999, p. 15803-15810.

Research output: Contribution to journalArticle

Tokumitsu, Hiroshi ; Takahashi, Naomi ; Eto, Koh ; Yano, Shigetoshi ; Soderling, Thomas R. ; Muramatsu, Masa Aki. / Substrate recognition by Ca2+/calmodulin-dependent protein kinase kinase : Role of the Arg-Pro-rich insert domain. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 22. pp. 15803-15810.
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