Cyclic AMP increasing agents rapidly stimulate vimentin phosphorylation in quiescent cultures of Swiss 3T3 cells

Abstract

The results presented here demonstrate that an elevation in the cellular levels of cyclic AMP (cAMP) increases the phosphorylation of an Mr = 58,000 cellular protein in quiescent cultures of Swiss 3T3 cells. The enhancement of 32Pi incorporation into the Mr 58,000 cellular protein was detected as early as 1 min and reached a maximum after 20 min of treatment. The role of cAMP in the phosphorylation of Mr = 58,000 protein is substantiated by the following lines of evidence: a) a variety of agents that cause cAMP accumulation in 3T3 cells, including cholera toxin, 5'-N-ethylcarboxamideadenosine (NECA), PGE1, and 3-isobutyl-1-methyl-xanthine (IBMX) increased the phosphorylation of the same Mr 58,000 cellular protein as demonstrated by peptide mapping; b) inhibitors of cyclic nucleotide phosphodiesterase potentiated the ability of low concentrations of the adenylate cyclase activators NECA, PGE1, and forskolin to increase Mr 58,000 phosphorylation; and c) permeable derivatives of cAMP such as 8BrcAMP were also effective and specific in promoting Mr 58,000 phosphorylation. Detergent extraction, immunoblotting, and immunoprecipitation identified the Mr = 58,000 phosphoprotein as vimentin, the main protein subunit of the intermediate filaments of mesenchymal cells including Swiss 3T3 cells. Studies with intact 3T3 cells revealed that an increase in the intracellular level of cAMP induced a marked redistribution and collapse of the intermediate filaments. These results raise the possibility that an intact intermediate filament network may restrict the reinitiation of DNA synthesis.