Identification and characterization of novel components of a Ca2+/calmodulin-dependent protein kinase cascade in HeLa cells.
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.
Ishikawa
Yumi
Y
Department of Signal Transduction Sciences, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
Tokumitsu
Hiroshi
H
Inuzuka
Hiroyuki
H
Murata-Hori
Maki
M
Hosoya
Hiroshi
H
Kobayashi
Ryoji
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001562:Benzimidazoles / Q000494:pharmacology; D001665:Binding Sites; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D008969:Molecular Sequence Data; D053644:Naphthalimides; D010766:Phosphorylation; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology*; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D001562:Benzimidazoles; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D007546:Isoquinolines; D053644:Naphthalimides; D011994:Recombinant Proteins; C458525:STO 609; D013912:Threonine; D015759:Ionomycin; D017346:Protein Serine-Threonine Kinases; C517596:CAMK1 protein, human; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases