Interactions between cyclic AMP‐ and phorbol ester‐dependent phosphorylation systems in S49 mouse lymphoma cells

Abstract

High‐resolution two‐dimensional gel electrophoresis of proteins labeled with either ³²P_i or [³⁵S]methionine was used to study interactions between cyclic AMP and tetradecanoyl phorbol acetate (TPA) at the level of intracellular protein phosphorylation. Cultured S49 mouse lymphoma cells were used as a model system, and mutant sublines lacking either the catalytic subunit of cyclic AMP‐dependent protein kinase or the guanyl nucleotide‐binding “N_s” factor of adenylate cyclase provided tools to probe mechanisms underlying the interactions observed. Three sets of phosphoproteins responded differently to TPA treatment of wild‐type and mutant cells: Phosphorylations shown previously ot be responsive to activation of intracellular cyclic AMP‐dependent protein kinase were stimulated by TPA in wild‐type cells but not in mutant cells, a subset of phosphorylations stimulated strongly by TPA in mutant cells was inhibited in wild‐type cells, and two novel phosphoprotein species appeared in response to TPA only in wild‐type cells. The latter two classes of TPA‐mediated responses specific to wild‐type cells could be evoked in adenylate cyclase‐deficient cells by treating concomitantly with TPA and either forskolin or an analog of cyclic AMP. Three conclusions are drawn from our results: (1) TPA stimulates adenylate cyclase in wild‐type cells causing increased phosphorylation of endogenous substrates by cyclic AMP‐dependent protein kinase, (2) activated cyclic AMP‐dependent protein kinase inhibits phosphorylation (or enhances dephosphorylation) of a specific subset of TPA‐dependent phosphoproteins, and (3) cyclic AMP‐dependent events facilitate TPA‐dependent phosphorylation of some substrate proteins.