Induction of alkaline phosphatase in mouse L cells by overexpression of the catalytic subunit of cAMP-dependent protein kinase.
Mouse L929 cells were used to study the mechanism of cAMP induction of alkaline phosphatase (AP) activity. Following treatment with 200 microM 8-chlorophenylthio-cAMP (CPT-cAMP), alkaline phosphatase enzyme activity was observed to increase 80-fold after 24 h. The CPT-cAMP dose response of the alkaline phosphatase enzyme activity correlated well with the CPT-cAMP activation of cAMP-dependent protein kinase in L cells. A cDNA clone for the alkaline phosphatase was isolated and used to demonstrate a 10-fold increase in alkaline phosphatase mRNA levels after a 24-h treatment of L cells with CPT-cAMP. Increased mRNA levels were first detected 4-6 h, after CPT-cAMP treatment, and the level of alkaline phosphatase mRNA decreased rapidly after removal of CPT-cAMP. In vitro nuclear transcription studies showed that a 3-fold increase in alkaline phosphatase gene transcription was detectable 6 h after CPT treatment, and this increase was blocked by cycloheximide. In order to determine if the catalytic (C) subunit of cAMP-dependent protein kinase was able to mediate the induction of AP, L cells were transfected with expression vectors containing the metallothionein promoter and coding for the C alpha isoform of the catalytic subunit of cAMP-dependent protein kinase or for a catalytic subunit in which lysine 72 had been mutated to methionine (C alpha K72M). Zinc treatment of stably transfected cells expressing the wild-type C subunit showed an increase in protein kinase activity and an increase in AP activity. Zinc treatment of cells containing the mutant C subunit expression vector produced an increase in the amount of a protein which was recognized by C subunit antibodies on Western blots, but these cells showed no increase in protein kinase activity or in AP activity. We conclude that the C subunit is sufficient for transcriptional induction of the AP gene and that the phosphotransferase activity of the C subunit is required for this induction.
Brown
N A
NA
Department of Biochemistry, Oregon Health Sciences University, Portland 97201.
Stofko
R E
RE
Uhler
M D
MD
D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D004790:Enzyme Induction; D015870:Gene Expression; D005796:Genes / Q000187:drug effects; D007700:Kinetics; D007739:L Cells / Q000187:drug effects / Q000201:enzymology; D046911:Macromolecular Substances; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013431:Sulfates / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D015032:Zinc / Q000494:pharmacology; D019287:Zinc Sulfate
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D013431:Sulfates; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D019287:Zinc Sulfate; D008668:Metallothionein; D000242:Cyclic AMP; D011494:Protein Kinases; D000469:Alkaline Phosphatase; D015032:Zinc