A Chinese hamster fibroblast mutant defective in thrombin-induced signaling has a low level of phospholipase C-beta 1.
A mutant fibroblast, 2A4b, was isolated from the Chinese hamster lung cell line CCL39 by a previously described selection (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390) for cells deficient in thrombin-induced signaling. Although the antiporter activation by thrombin in 2A4b is only approximately 60% that in CCL39, the stimulation by serum is not significantly impaired, indicating that the defect in 2A4b lies upstream of the antiporter in the signaling pathway. The addition of thrombin to serum-starved 2A4b cells causes blunted responses both in production of inositol phosphates and in the cytosolic [Ca2+] transient, particularly when no Ca2+ is added to the external medium. The in vitro inositol phospholipid-specific phospholipase C (PLC) activity of 2A4b cytosol plus membrane extracts exceeds that in CCL39. However, immunoblots with antibodies to PLC isozymes show that although the levels of PLC-delta 1, PLC-gamma 1, and PLC-beta 3 are at least as great as those in CCL39, the amount of PLC-beta 1 in 2A4b is markedly deficient (< or = 10%). PLC-beta 1 is found primarily in the nucleus and in non-nuclear membranes of CCL39 and is proportionately low in these subcellular locations of 2A4b. Thrombin activation of phospholipases D and A2 is impaired in 2A4b. We postulate that the deficiency in PLC-beta 1 causes defective targeting of protein kinase C-alpha to specific membrane sites, which may be required for activation of these downstream phospholipases.
Fee
J A
JA
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Monsey
J D
JD
Handler
R J
RJ
Leonis
M A
MA
Mullaney
S R
SR
Hope
H M
HM
Silbert
D F
DF
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001692:Biological Transport; D002451:Cell Compartmentation; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D007527:Isoenzymes / Q000235:genetics*; D009154:Mutation; D010716:Phosphatidylinositols / Q000378:metabolism; D054799:Phospholipase C beta; D010741:Phospholipases A / Q000378:metabolism; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism*; D010738:Type C Phospholipases / Q000235:genetics*
D007527:Isoenzymes; D010716:Phosphatidylinositols; D017923:Sodium-Hydrogen Exchangers; D016718:Arachidonic Acid; D010741:Phospholipases A; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate