Expression of a mutant regulatory subunit of cAMP-dependent protein kinase in the Caco-2 human colonic carcinoma cell line.
Caco-2 human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of RI (regulatory subunit of the type 1 cAMP-dependent protein kinase), driven by the metallothionein 1 promoter. A stable transformant was isolated that expressed the mutant RI gene in a Zn(2+)-inducible manner. The consequences of the RI mutation on cAMP-dependent protein kinase activity, cell division, and regulation of chloride efflux were examined. When grown in the absence of ZnSO4, protein kinase activity in the transformant was stimulated 2.5-fold by cAMP and approached the levels of cAMP-dependent protein kinase activity seen in parental Caco-2 cells; when treated with ZnSO4, cAMP-dependent protein kinase activity in the transformant was inhibited by 60%. In the absence of ZnSO4 the transformant grew with the same doubling time and to the same saturation density as the untransformed parent. In the presence of ZnSO4 the transformant exhibited a cAMP-reversible inhibition of cell division, indicating that a functional cAMP-dependent protein kinase was required for the growth of these cells in culture. Induction of the mutant RI gene also abolished forskolin-stimulated chloride efflux from these cells, suggesting obligatory roles for cAMP and cAMP-dependent protein kinase in forskolin's actions on chloride channel activity. We anticipate that this transformant will be useful for further studies on the roles of cAMP and cAMP-dependent protein kinase in the regulation of intestinal epithelial cells, including regulation of cell proliferation and differentiation, and regulation of chloride channel activity by neurohormones and neurotransmitters.
Mihajlovic
V
V
Banting and Best Department of Medical Research, University of Toronto, Ont., Canada.
Krolczyk
A J
AJ
Auerbach
W
W
Buchwald
M
M
Schimmer
B P
BP
D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D002455:Cell Division / Q000502:physiology; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism; D003110:Colonic Neoplasms; D000242:Cyclic AMP / Q000502:physiology*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D047908:Intracellular Signaling Peptides and Proteins; D015640:Ion Channel Gating / Q000502:physiology; D008565:Membrane Proteins / Q000502:physiology*; D011494:Protein Kinases / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D015290:Second Messenger Systems / Q000502:physiology; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018118:Chloride Channels; D002712:Chlorides; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C015482:protein kinase modulator; D000242:Cyclic AMP; D011494:Protein Kinases