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PMID:10336483

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Citation

Tokumitsu, H, Takahashi, N, Eto, K, Yano, S, Soderling, TR and Muramatsu, M (1999) Substrate recognition by Ca2+/Calmodulin-dependent protein kinase kinase. Role of the arg-pro-rich insert domain. J. Biol. Chem. 274:15803-10

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 alpha and beta 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.

Links

PubMed

Keywords

Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Caenorhabditis elegans/enzymology; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Calcium-Calmodulin-Dependent Protein Kinases/chemistry; Calcium-Calmodulin-Dependent Protein Kinases/genetics; Calcium-Calmodulin-Dependent Protein Kinases/metabolism; Cloning, Molecular; Enzyme Activation; Helminth Proteins/chemistry; Helminth Proteins/genetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphorylation; Protein-Serine-Threonine Kinases/chemistry; Proto-Oncogene Proteins/metabolism; Proto-Oncogene Proteins c-akt; Recombinant Proteins/metabolism; Reverse Transcriptase Polymerase Chain Reaction; Sequence Deletion; Sequence Homology, Amino Acid

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

CAEEL:KKCC

enables

GO:0005516: calmodulin binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

CAEEL:KKCC

enables

GO:0004683: calmodulin-dependent protein kinase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

CAEEL:KKCC

involved_in

GO:0006468: protein phosphorylation

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

CAEEL:KKCC

involved_in

GO:0045860: positive regulation of protein kinase activity

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete


See also

References

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