Biochemical and biological activity of phosphorylated and non-phosphorylated ras p21 mutants.
In contrast to all cellular ras oncogenes which carry a single activating mutation at codon 12, 13 or 61, all known retroviral ras oncogenes have two mutations at codons 12 and 59. To understand the role of the mutation at codon 59, we have constructed plasmids containing genes for Harvey ras: p21(Gly-12,Thr-59) and p21(Val-12,Thr-59). Escherichia coli expressed proteins and their respective phosphorylated (Pi) and non-phosphorylated (non-Pi) proteins were purified to 95% homogeneity by ion-exchange chromatography and gel filtration. GTPase, autophosphorylation and nucleotide exchange activities of the mutants were studied. When the mutants were microinjected into Xenopus oocytes, the non-phosphorylated forms of p21(Gly-12,Thr-59) and p21(Val-12,Thr-59) showed high activity. Surprisingly, their phosphorylated forms were inactive. These results suggest that threonine at position 59 endows the protein with transforming activity but that phosphorylation of the residue inhibits biological activity. A structural interpretation of the observation is presented.
Chung
H H
HH
Department of Chemistry, University of California, Berkeley.
Kim
R
R
Kim
S H
SH
D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D014981:Xenopus
D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D015689:Oncogene Protein p21(ras)