Specific protein phosphorylation during cyclic AMP-mediated morphological reversion of transformed cells

Treatment of transformed Chinese hamster ovary cells with dibutyryl cAMP or other agents that elevate cAMP results in the acquisition of growth and morphology characteristic of normal fibroblasts. The role of specific protein phosphorylation in this process of morphological reversion has been examine using metabolic labeling of Chinese hamster ovary (CHO) cells with 32P-orthophosphate in the presence or absence of N6O2'-dibutyryladenosine 3':5'-cyclic monophosphoric acid (Bt2cAMP). Analysis of labelled cultures by SDS gel electrophoresis and radioautography demonstrate dramatic changes in the phosphorylation of only 2 cellular proteins during reverse transformation. A 55,000 dalton protein (pp55) was phosphorylated and a 20,000 dalton protein (pp20) was dephosphorylated. The time course of these events was consistent with the kinetics of morphological reversion. The lower molecular weight species, pp20, was dephosphorylated within 15-30 minutes, prior to all morphological changes except membrane tranquilization. The higher molecular weight protein, pp55, was maximally phosphorylated over 1-2 hours following addition of Bt2cAMP, paralleling early stages in the establishment of fibroblastic form. The phosphorylated forms of pp20 and pp55 were both extracted from cellular cytoskeletons by 0.5% Triton X-100, but analysis of 35S-methionine-labelled cultures suggested that unphosphorylated pp 20 may be bound to the cytoskeleton. Since pp20 was found to comigrate with the 20,000 dalton myosin light chain, it is possible tht dephosphorylation of CHO cell myosin induced by cAMP may alter its interaction with actin microfilaments and modulate the assembly of stress fibers during morphological reversion.