Targeted neutralization of calmodulin in the nucleus blocks DNA synthesis and cell cycle progression.
Calmodulin (CaM) is a major intracellular calcium binding protein which has been implicated in the regulation of cell proliferation. Previous studies using chemically synthesized CaM antagonists and anti-sense RNA indicated that CaM is important for initiation of DNA synthesis and cell cycle progression. However, these methods reduce total intracellular CaM and globally interfering with all the CaM-dependent processes. In order to explore the function of nuclear CaM during the cell cycle, a CaM inhibitor peptide was targeted to the nucleus of intact mammalian cells. Cell progression through S-phase was assessed by incorporation of the thymidine analogue, BrdU. Cells were transfected for 48 h with either the CaM inhibitor peptide gene or the control plasmid prior to analysis. Approx. 70% of the control cells incorporated BrdU. In striking contrast, double immunofluorescent labeling demonstrated that none of the cells expressing the CaM inhibitor peptide entered S-phase. This result indicates that neutralization of nuclear CaM by targeted expression of a CaM inhibitor peptide blocks DNA synthesis and cell cycle progression.
University of Cincinnati, Department of Molecular and Cellular Physiology, OH 45267-0576, USA.
J
J
Wang
K M
KM
Moreira
B
B
Campos
M A
MA
Kaetzel
J R
JR
Dedman
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000502:physiology*; D002453:Cell Cycle / Q000502:physiology*; D002467:Cell Nucleus / Q000737:chemistry / Q000502:physiology*; D004247:DNA / Q000096:biosynthesis*; D015870:Gene Expression; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000235:genetics; D010455:Peptides / Q000494:pharmacology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014162:Transfection
D002147:Calmodulin; D010455:Peptides; D011993:Recombinant Fusion Proteins; D004247:DNA; D009219:Myosin-Light-Chain Kinase