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

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46 Citations (Scopus)


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
Issue number22
Publication statusPublished - May 28 1999
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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