ABSTRACT
Increased intracellular adenosine 3':5'-monophosphate (cAMP) 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-6N,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 two-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, two proteins with a molecular weight of 43,000 were heavily phosphorylated. It is suggested that phosphorylation of cellular proteins by cAMP-dependent protein kinases might be involved in the cAMP-modulated biochemical changes in neuroblastoma cells.
Subject(s)
Neoplasm Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neuroblastoma/enzymology , Protein Kinases/metabolism , Animals , Bucladesine/pharmacology , Cell Line , Clone Cells , Cyclic AMP/pharmacology , Kinetics , Mice , Phosphorylation , Tubulin/metabolismABSTRACT
Proteins from mouse neuroblastoma cells treated with dibutyryl adenosine-3',5'-monophosphate (B2cAMP) were analyzed by high resolution, two-dimensional gel electrophoresis. Quantitative changes in proteins and charge modifications of proteins apparently induced B2cAMP were detected by isoelectric focusing. Some proteins appeared to be modified and one protein was increased 7- to 8-fold in cells treated with B2cAMP. Since neuroblastoma cells differentiate when treated with B2cAMP, understanding the protein changes induced by B2cAMP may help to understand cellular differentiation in neural tissue.
