Structural analysis of normal and transforming mil(raf) proteins: effect of 5'-truncation on phosphorylation in vivo or in vitro.
The phosphorylation sites of the cellular proto-oncogene product p71/73c-mil(raf) from quail and from human cells were analyzed by two-dimensional peptide mapping and compared to the sites phosphorylated in proteins encoded by three transforming alleles of c-mil(raf). These alleles all were 5'-truncated resulting from either retroviral transduction (v-mil, v-raf) or promoter insertion mutagenesis (LTR-c-raf). The normal cellular proteins each were phosphorylated in vivo on three major sites, two of which were identical in the two protein species. MH2 p100gag-mil, murine sarcoma virus 3611 p75gag-raf, and LTR-c-raf p45-50 delta c-raf were phosphorylated in vivo on several sites. One site was shared between these transforming proteins and was also conserved in both avian and human p71/73c-mil(raf). All normal and transforming mil(raf) proteins were phosphorylated on serine in vivo while p100gag-mil and p75gag-raf occasionally also contained low levels of phosphothreonine. No specific phosphorylation of p71/73c-mil(raf) was detected in vitro under conditions that readily revealed presumed autophosphorylation of p100gag-mil, p75gag-raf, and p45-50 delta c-raf. However, the in vitro phosphorylated sites of these proteins were different to each other and to the sites phosphorylated in vivo. In contrast to the predominant threonine phosphorylation of the two viral proteins, only phosphoserine could be detected in p45-50 delta c-raf phosphorylated in vitro.
Patschinsky
T
T
Max-Planck-Institut für Molekulare Genetik, Otto-Warburg-Laboratorium, Berlin, Federal Republic of Germany.
Bister
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D002642:Chick Embryo; D005347:Fibroblasts / Q000378:metabolism; D007106:Immune Sera; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D027384:Oncogene Proteins v-raf; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D000090063:Proto-Oncogene Mas; D011784:Quail; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D007106:Immune Sera; C000715470:MAS1 protein, human; D010750:Phosphoproteins; D000090063:Proto-Oncogene Mas; D015682:Retroviridae Proteins, Oncogenic; D011505:Protein-Tyrosine Kinases; D027384:Oncogene Proteins v-raf