Abstract
In this report, we cloned a novel calmodulin-kinase (CaM-KIdelta) from HeLa cells and characterized its activation mechanism. CaM-KIdelta exhibits Ca(2+)/CaM-dependent activity that is enhanced (approximately 30-fold) in vitro by phosphorylation of its Thr180 by CaM-K kinase (CaM-KK)alpha, consistent with detection of CaM-KIdelta-activating activity in HeLa cells. We also identified a novel CaM-KKbeta isoform (CaM-KKbeta-3) in HeLa cells whose activity was highly Ca(2+)/CaM-independent. Transiently expressed CaM-KIdelta exhibited enhanced protein kinase activity in HeLa cells without ionomycin stimulation. This sustained activation of CaM-KIdelta was completely abolished by Thr180Ala mutation and inhibited by CaM-KK inhibitor, STO-609, indicating a functional CaM-KK/CaM-KIdelta cascade in HeLa cells.
MeSH terms
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Amino Acid Sequence
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Animals
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Base Sequence
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Benzimidazoles / pharmacology
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Binding Sites
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Calcium-Calmodulin-Dependent Protein Kinase Kinase
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Calcium-Calmodulin-Dependent Protein Kinase Type 1
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Calcium-Calmodulin-Dependent Protein Kinases / drug effects
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Calcium-Calmodulin-Dependent Protein Kinases / genetics*
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
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Cloning, Molecular
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Enzyme Activation
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Enzyme Inhibitors / pharmacology
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HeLa Cells
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Humans
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Ionomycin / pharmacology
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Isoenzymes / genetics
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Isoenzymes / metabolism
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Isoquinolines / pharmacology
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Molecular Sequence Data
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Naphthalimides
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Phosphorylation
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Point Mutation
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Sequence Homology, Amino Acid
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Signal Transduction / physiology*
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Threonine / genetics
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Threonine / metabolism
Substances
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Benzimidazoles
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Enzyme Inhibitors
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Isoenzymes
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Isoquinolines
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Naphthalimides
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Recombinant Proteins
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STO 609
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Threonine
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Ionomycin
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Protein Serine-Threonine Kinases
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CAMK1 protein, human
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Calcium-Calmodulin-Dependent Protein Kinase Kinase
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Calcium-Calmodulin-Dependent Protein Kinase Type 1
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Calcium-Calmodulin-Dependent Protein Kinases