Cellular concentrations of protein kinase A modulate prostaglandin and cAMP induction of alkaline phosphatase.
The relationship between the concentration of cAMP-dependent protein kinase (PKA) activity and the induction of alkaline phosphatase (AP) was examined in transfected L cell lines with altered PKA levels. C alpha 12 cells were generated by transfecting mouse L cells with an expression vector coding for the mouse C alpha catalytic subunit of PKA and were shown to contain 2.5-fold more PKA activity than L cells. RAB10 cells were generated by transfection with an expression vector for a mutant regulatory subunit and had 10-fold lower levels of PKA activity than L cells. AP induction by 8-chlorophenylthio-cAMP (CPT-cAMP) was found to be 2-fold greater in C alpha 12 cells than in L cells, while RAB10 cells lacked any induction of AP in response to CPT-cAMP. Northern blot and solution hybridization analyses of AP mRNA showed that induced AP mRNA levels were comparable in C alpha 12 and in L cells. Western blot analysis demonstrated that AP protein levels were greater in C alpha 12 cells and suggested that the increased AP protein level resulted from either increased stability of the AP protein or increased rate of translation of the AP mRNA. In contrast, Northern blot analysis of the RAB10 cells failed to detect AP mRNA after CPT-cAMP treatment and suggested that PKA is required for induction of AP mRNA. Stimulation of endogenous cAMP levels by treatment with prostaglandin E1 gave similar effects on AP activity as those seen with CPT-cAMP. These results indicate that cellular levels of PKA can determine the magnitude of cellular response to hormonal stimulation and also suggest that PKA can regulate AP gene expression at both the level of the AP mRNA and AP protein.
Uhler
M D
MD
Mental Health Research Institute, University of Michigan, Ann Arbor 48109.
Abou-Chebl
A
A
D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D004789:Enzyme Activation; D004790:Enzyme Induction; D015870:Gene Expression; D007739:L Cells; D051379:Mice; D010957:Plasmids; D011453:Prostaglandins / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013873:Thionucleotides / Q000737:chemistry; D014162:Transfection
D011453:Prostaglandins; D012333:RNA, Messenger; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D011494:Protein Kinases; D000469:Alkaline Phosphatase