Ca(2+)-dependent stimulation of retinoblastoma gene product phosphorylation and p34cdc2 kinase activation in serum-stimulated human fibroblasts.
In serum-deprived human fibroblasts IMR-90 and WI-38 cells, the addition of fetal calf serum or basic fibroblast growth factor stimulates DNA synthesis in an extracellular Ca(2+)-dependent manner; the effect of serum on [3H]thymidine incorporation into DNA is 4-16-fold greater at 2.0 mM CaCl2 as compared with that at 0.03 mM CaCl2. By contrast, in SV40 virus-transformed WI-38 (SV-WI-38) cells DNA synthesis is essentially independent of the extracellular calcium concentration ([Ca]out) and serum growth factors. To explore the role of Ca2+ in mitogenic signal transduction through G1 to S phase cell cycle progression, we studied and compared the effect of [Ca]out on phosphorylation of RB protein, the product of a tumor suppressor retinoblastoma gene. In IMR-90 and WI-38 cells, serum or basic fibroblast growth factor induces an increase in the amount of hyperphosphorylated forms of RB protein in a manner strictly dependent on [Ca]out. In sharp contrast, in SV-WI-38 cells, the extent of RB phosphorylation is little affected by [Ca]out or the presence or absence of serum growth factors. In addition, potent calmodulin antagonists W-7 and calmidazolium, but not an inactive analogue W-12 or W-5, strongly inhibit serum-induced increases in DNA synthesis and RB phosphorylation in IMR-90 and WI-38 cells, whereas in SV-WI-38 cells, the inhibitory effect is much more limited. Under the same treatment conditions, we measured histone H1 kinase activity associated with anti-p34cdc2 immunoprecipitate and found that the serum-induced increase in p34cdc2 kinase activity is strongly dependent on [Ca]out and is potently inhibited by the active calmodulin antagonists in IMR-90 and WI-38 cells, but not in SV-WI-38 cells. In IMR-90 cells that have been incubated with serum in 0.03 mM [Ca]out for 24 h, restoration of [Ca]out to 2.0 mM results in initiation of DNA synthesis after 13 h and concomitant increases in RB phosphorylation and p34cdc2 histone H1 kinase activity. These results suggest that in human fibroblasts, Ca2+/calmodulin regulates the signaling cascade leading to cdc2 kinase activation, RB protein phosphorylation, and DNA synthesis and that this Ca(2+)-dependent regulation is abrogated in SV40-transformed cells.
Takuwa
N
N
Department of Physiology, Faculty of Medicine, University of Tokyo, Japan.
Zhou
W
W
Kumada
M
M
Takuwa
Y
Y
D001769:Blood; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002460:Cell Line; D016895:Culture Media, Serum-Free; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D016161:Genes, Retinoblastoma; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008168:Lung; D008938:Mitosis / Q000187:drug effects; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000302:isolation & purification / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism*
D002147:Calmodulin; D016895:Culture Media, Serum-Free; D007093:Imidazoles; D016160:Retinoblastoma Protein; D013449:Sulfonamides; D016222:Fibroblast Growth Factor 2; C040184:W 12; C031938:calmidazolium; C017967:W 7; D004247:DNA; D016203:CDC2 Protein Kinase; D002118:Calcium; D013936:Thymidine