Characterization of Ca2+/calmodulin-dependent protein kinase I as a myosin II regulatory light chain kinase in vitro and in vivo.
Ca(2+)/calmodulin (CaM)-dependent protein kinase I (CaM-KI), which is a member of the multifunctional CaM-K family, is thought to be involved in various Ca(2+)-signalling pathways. In this report, we demonstrate that CaM-KI activated by an upstream kinase (CaM-K kinase), but not unactivated CaM-KI, phosphorylates myosin II regulatory light chain (MRLC) efficiently ( K (cat), 1.7 s(-1)) and stoichiometrically (approximately 0.8 mol of phosphate/mol) in a Ca(2+)/CaM-dependent manner in vitro. One-dimensional phosphopeptide mapping and mutational analysis of MRLC revealed that the activated CaM-KI monophosphorylates only Ser-19 in MRLC. Transient expression of the Ca(2+)/CaM-independent form of CaM-KI (CaM-KI(1-293)) in HeLa cells induced Ser-19 phosphorylation of myosin, II accompanied by reorganization of actin filaments in the peripheral region of the cells. CaM-KI-induced reorganization of actin filaments was suppressed by co-expression of non-phosphorylatable MRLC mutants (S19A and T18AS19A). Furthermore, a kinase-negative form of CaM-KI (CaM-KI(1-293,K49E)) significantly reduced reorganization of actin filaments, indicating a dominant negative effect. This is the first demonstration that the activation of the CaM-KI cascade induces myosin II phosphorylation, resulting in regulation of actin filament organization in mammalian cells.
Suizu
Futoshi
F
Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
Fukuta
Yasuaki
Y
Ueda
Kozue
K
Iwasaki
Takahiro
T
Tokumitsu
Hiroshi
H
Hosoya
Hiroshi
H
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism; D000818:Animals; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D018994:Myosin Light Chains / Q000235:genetics / Q000378:metabolism*; D024462:Myosin Type II / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism
D000199:Actins; D018994:Myosin Light Chains; D012694:Serine; C517596:CAMK1 protein, human; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D024462:Myosin Type II