Phosphorylation of Endogenous Proteins by Adenosine 3′:5′‐Monophosphate‐Dependent Protein Kinase in Mouse Neuroblastoma Cells

Abstract

Increased intracellular c[cyclic]AMP levels and activation of cAMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) in vivo were correlated in mouse neuroblastoma cells grown in the presence of 1 mM N6,O2''-dibutyryl 3'':5''-monophosphate (Bt2cAMP). The time course for activation showed that cAMP-dependent protein kinases were activated by 30 min. A heat-stable inhibitor protein inhibited a majority of activated cAMP-dependent protein kinase. Activation of cAMP-dependent protein kinase caused additional phosphorylation of proteins when compared with untreated control cells, as demonstrated by endogenous phosphorylation of proteins in vitro using [.gamma.-32P]ATP and analysis by 2-dimensional polyacrylamide gel electrophoresis. The phosphorylation data show selective phosphorylation of specific proteins by cAMP-independent and cAMP-dependent protein kinase. Among the proteins in the postmitochondrial supernatant fraction phosphorylated by cAMP-dependent protein kinases, 2 proteins with a MW of 43000 were heavily phosphorylated. Apparently, phosphorylation of cellular proteins by cAMP-dependent protein kinases might be involved in the cAMP-modulated biochemical changes in neuroblastoma cells.