Altered function of protein kinase C and cyclic adenosine monophosphate-dependent protein kinase in a cell line derived from a mouse lung papillary tumor.
Two serine/threonine protein kinases were compared in C10, a clone from the nontumorigenic NAL IA cell line derived from normal mouse lung epithelium, and PCC4, a cell line derived from a mouse lung adenoma. C10 cells are contact inhibited, whereas PCC4 cells are not. Upon treatment with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), the normally flattened C10 cells round up, while the normally bipolar, rounded PCC4 cells flatten out. Three proteins of 14,000, 20,000 and 116,000 molecular weight were phosphorylated in TPA-treated particulate fractions but not in untreated particulate fractions of PCC4 cells. In contrast, TPA caused a generalized increase in the phosphorylation of most membrane proteins in C10 cells. Cytosolic protein kinase C (PKC) specific activity was lower in PCC4 cells than in C10 cells, but particulate PKC activity was similar in the two cell lines. Both measurements of PKC activity and immunoblotting assays using anti-PKC antisera showed increased particulate PKC in TPA-treated C10 cells resulting from a quantitative translocation of PKC molecules from cytoplasm to plasma membrane. This PKC response to TPA was attenuated in PCC4 cells. While PCC4 particulate PKC activity was substantially increased after TPA treatment, PKC activity decreased only slightly in cytosolic fractions of TPA-treated PCC4 cells. Immunoblots of TPA-treated PCC4 cells showed a decline in cytosolic PKC content and increased particulate PKC concentration, but these changes were not of the same magnitude as the activity changes. This may represent an unmasking of latent PKC activity since particulate PKC activity in TPA-treated PCC4 cells was inhibited by staurosporine, a specific inhibitor of PKC when used at nanomolar concentrations. In addition, PCC4 cells had less mRNA coding for the R1 regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase (PKA) than C10 cells, as determined by Northern blotting using an R1 alpha cDNA probe. Consistent with this result, photolabeling with 8-azido-[32P]cAMP, a photoaffinity analog of cAMP, revealed that R1 from PCC4 cells incorporated less analogue than R1 from C10 cells. PKA-specific activity also was lower in PCC4 cells than in C10 cells. Thus, deficiencies in protein kinases which mediate the effects of diacylglycerol and cAMP second messengers were observed in neoplastic lung cells. This may dampen the responsiveness of PCC4 cells to extracellular signals that regulate cell growth and cell-cell interactions.
Nicks
K M
KM
Molecular and Environmental Toxicology Program, School of Pharmacy, University of Colorado, Boulder 80309.
Droms
K A
KA
Fossli
T
T
Smith
G J
GJ
Malkinson
A M
AM
D000818:Animals; D015153:Blotting, Western; D002291:Carcinoma, Papillary / Q000201:enzymology* / Q000473:pathology / Q000648:ultrastructure; D002455:Cell Division; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D007700:Kinetics; D008175:Lung Neoplasms / Q000201:enzymology* / Q000473:pathology / Q000648:ultrastructure; D051379:Mice; D008855:Microscopy, Electron, Scanning; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*
D009363:Neoplasm Proteins; D000242:Cyclic AMP; D011494:Protein Kinases; D011493:Protein Kinase C