Induction of alkaline phosphatase in mouse L cells by overexpression of the catalytic subunit of cAMP-dependent protein kinase

Abstract

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.