Identification and characterization of novel components of a Ca2+/calmodulin-dependent protein kinase cascade in HeLa cells

FEBS Lett. 2003 Aug 28;550(1-3):57-63. doi: 10.1016/s0014-5793(03)00817-2.

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

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Benzimidazoles / pharmacology
  • Binding Sites
  • Calcium-Calmodulin-Dependent Protein Kinase Kinase
  • Calcium-Calmodulin-Dependent Protein Kinase Type 1
  • Calcium-Calmodulin-Dependent Protein Kinases / drug effects
  • Calcium-Calmodulin-Dependent Protein Kinases / genetics*
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Cloning, Molecular
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • HeLa Cells
  • Humans
  • Ionomycin / pharmacology
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Isoquinolines / pharmacology
  • Molecular Sequence Data
  • Naphthalimides
  • Phosphorylation
  • Point Mutation
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Signal Transduction / physiology*
  • Threonine / genetics
  • Threonine / metabolism

Substances

  • Benzimidazoles
  • Enzyme Inhibitors
  • Isoenzymes
  • Isoquinolines
  • Naphthalimides
  • Recombinant Proteins
  • STO 609
  • Threonine
  • Ionomycin
  • Protein-Serine-Threonine Kinases
  • CAMK1 protein, human
  • Calcium-Calmodulin-Dependent Protein Kinase Kinase
  • Calcium-Calmodulin-Dependent Protein Kinase Type 1
  • Calcium-Calmodulin-Dependent Protein Kinases