Dynamic interactions of c-fos protein in serum-stimulated 3T3 cells.
The c-fos gene, the cellular homologue of the transforming gene of the FBJ osteosarcoma virus, v-fos, is strongly induced in quiescent BALB/c 3T3 cells by growth factors and in other cell types by a wide variety of transmembrane signalling agents. c-fos is a member of a family of structurally related proteins which includes the fos-related antigens (fra). We have studied the dynamic state of the c-fos protein with an antibody prepared by immunizing rabbits with a plasmid-encoded fos fusion protein. In serum-stimulated BALB/c 3T3 cells, the antibody recognizes a nuclear antigen which resolves on SDS-PAGE as a 60-68-kD group of bands corresponding to c-fos, a doublet at 44-45-kD corresponding to the noncovalently associated p39 protein, as well as an approximately 50-kD band corresponding to a fra. We show that although c-fos protein synthesis is only transiently induced by serum, the c-fos protein persists within the cell after its synthesis has ceased, and it decays with a half-life of 2 hours. Significantly, newly synthesized p39 continues to appear in the immune-precipitated complex even at times when c-fos is no longer synthesized. These kinetics indicate that even following shutoff of c-fos protein synthesis, p39 is newly synthesized and can complex with c-fos protein or a fos-related antigen. During this time, c-fos also undergoes an extensive posttranslational modification. The modification is partially reversed by phosphatase treatment, which implicates protein phosphorylation. Together these results suggest that both interaction with p39 and phosphorylation may progressively modify the properties of c-fos and/or the fos-related antigens over a period of 4-8 hours following the shutoff of fos synthesis. We discuss the implications of the dynamic state of c-fos and fra protein interactions for the function of these proteins.
Vosatka
R J
RJ
Department of Biochemistry, Kaplan Cancer Center, New York, New York.
Hermanowski-Vosatka
A
A
Metz
R
R
Ziff
E B
EB
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002453:Cell Cycle; D002460:Cell Line; D005786:Gene Expression Regulation; D007158:Immunologic Techniques; D046911:Macromolecular Substances; D051379:Mice; D010750:Phosphoproteins / Q000502:physiology; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos; D011993:Recombinant Fusion Proteins / Q000276:immunology
D046911:Macromolecular Substances; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D011993:Recombinant Fusion Proteins