In certain oncogenic, transformed cell lines elevation of intracellular cyclic AMP (cAMP) results in the acquisition of normal cell growth and morphology. We have found that such """"""""reverse transformation"""""""" of Chinese hamster ovary (CHO) cells by cAMP or its analogs is accompanied by changes in the phosphorylation of specific proteins. This research is directed toward understanding how these phosphoproteins and the protein kinases which phosphorylate them function in the induction of the transformed cell phenotype and its reversal by cAMP. Two major changes are the dephosphorylation of pp20 which appears to be a myosin light chain, and the phosphorylation of pp55, a Mr = 55,000 soluble protein. Several cytoskeletal proteins including tubulin, and a Mr = 155,000 microtubule protein are phosphorylated. The unidentified cytoskeletal proteins will be characterized as components of microtubules, microfilament intermediate filaments, or adhesion plaques. The function of the phosphoproteins in reverse transformation will be examined by direct microinjection and the use of a permeable cell model which preserves fidelity of phosphorylation. We have found that a major change in cellular phosphotyrosine and phosphoserine accompanies morphological reversion, supporting the hypothesis that the activity of a transformation-specific tyrosine kinase (src-like enzyme) in CHO cells is reciprocally attenuated by cAMP-dependent protein kinases. The tyrosine-specific kinase will be purified and its interaction with cAMP-dependent kinases studied by in vitro phosphorylation. To test the concept that both tyrosine- and cAMPdependent kinases phosphorylate the same set of cytoskeletal proteins, the changes in p-tyrosine, p-serine, and p-threonine will be evaluated in individual proteins during reverse transformation. Our studies suggest that cAMP-dependent protein kinases are physically associated with the cytoskeleton, particularly microtubules and actomyosin bundles. We also have detected a tyrosine-specific protein kinase in isolated microtubules. The nature of the interaction of these enzymes with cytoskeletal structures will be explored by enzymological, immunofluorescent, and electron microscopic methods. The physiological effects of the kinases in mediating morphological transformation and reversion will be studied by microinjection and the use of a permeabilized cell model for in situ phosphorylation. (B)
| Lockwood, A H; Murphy, S K; Borislow, S et al. (1987) Cellular signal transduction and the reversal of malignancy. J Cell Biochem 33:237-55 |
