Differentiation-specific increase of cAMP-dependent protein kinase in the 3T3-L1 cells.

The specific activity of CAMP-dependent protein kinase was studied during the differentiation of 3T3-Ll fibroblasts into adipocytes.8-Azido-adenosine 3':5'monophosphate, a photoaffinity-labeling analogue of CAMP, was used to quantitate the regulatory subunits of the type I and type 11 CAMP-dependent protein kinase in cell extracts.The catalytic subunit of CAMP-dependent protein kinase was studied by assaying for CAMPdependent histone kinase activity.A significant increase in CAMP-dependent protein kinase activity was observed during the adipose development of 3T3-Ll cells, promoted by 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin.This increase was attributed primarily to a 3-to 6-fold increase in the type I CAMP-dependent protein kinase, as demonstrated either by the incorporation of 8-azidoadenosine 3':5'-monophosphate into the 47,000-dalton regulatory subunit of the type I kinase or by the separation and analysis of the type I and type I1 kinases through DEAE-Sephacel column chromatography.The time course of increase in the type I CAMP-dependent protein kinase preceded increases both in malic enzyme and glutamine synthetase activities and was maximal at 5 days after initiation of the hormone treatment.The induction of CAMP-dependent protein kinase does not appear to involve an increase in intracellular CAMP.The addition of either 1 m~ dibutyryl CAMP or 0.5 m~ IBMX had little or no effect on the expression of CAMP-dependent protein kinase activity in the 3T3-Ll cells, although a marked increase in CAMP phosphodiesterase activity was observed in cells treated with dibutyryl CAMP.