Ca(2+)-dependent stimulation of retinoblastoma gene product phosphorylation and p34cdc2 kinase activation in serum-stimulated human fibroblasts

• Published in: The Journal of biological chemistry 1993-01, Vol.268 (1), p.138-145
• Main Authors: Takuwa, N, Zhou, W, Kumada, M, Takuwa, Y
• Format: Article
• Language: English
• Subjects: Blood; Blotting, Western; Calcium - pharmacology; Calmodulin - antagonists & inhibitors; CDC2 Protein Kinase - metabolism; Cell Line; Culture Media, Serum-Free; DNA - biosynthesis; Enzyme Activation; Fibroblast Growth Factor 2 - pharmacology; Fibroblasts - cytology; Fibroblasts - drug effects; Fibroblasts - enzymology; Genes, Retinoblastoma; Humans; Imidazoles - pharmacology; Kinetics; Lung; Mitosis - drug effects; Phosphorylation; Retinoblastoma Protein - isolation & purification; Retinoblastoma Protein - metabolism; Sulfonamides - pharmacology; Thymidine - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)54125-9

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.