Two point mutations in the transmembrane domain of P68gag-ros inactive its transforming activity and cause a delay in membrane association.
The transforming protein of the avian sarcoma virus UR2 is a 68-kDa transmembrane tyrosine protein kinase. We examined the relationship between membrane localization and transforming activity of P68 by changing Val-168-Val-169 in its hydrophobic domain into Asp-168-Glu-169. The resulting transmembrane (TM) mutant (P68TM) lost transforming activity toward chicken embryo fibroblasts (CEF). We found that the mutant protein was expressed and rapidly degraded into a smaller form which was still membrane associated and kinase active. The instability of the TM mutant protein is a phenomenon only manifested in CEF, because the same mutant protein was expressed with efficiency and stability similar to those of the wild-type protein in a transient expression system in COS cells. However, there are several differences between the wild-type and the TM mutant proteins in COS cells. The wild-type protein is more heavily phosphorylated and associated with membrane fractions in a cotranslational manner. It is enzymatically active when recovered from membrane fractions. The TM mutant protein is less phosphorylated, more labile toward protease degradation, and delayed in membrane association, with a lag period of 30 min or longer, and has little kinase activity when recovered from membrane fractions. Most of the kinase-active TM mutant protein was found in the cytosol fractions. Despite the delay, most of the TM protein in COS cells was found to be membrane associated, and its orientation on the cell surface was similar to that of the wild-type protein. It is probable that loss of the CEF-transforming activity of the TM mutant protein is due to its susceptibility to protease degradation resulting from improper membrane association of the newly synthesized product. The differences in the kinetics of membrane association and the distribution of kinase activity in COS cells might not be directly applicable in explaining the inability of the TM mutant to transform CEF but are intriguing as regards protein biosynthesis and translocation. The difference between CEF and COS cells implies that different factors or pathways are involved in the biosynthesis and processing of the TM mutant protein in these two cellular environments. Changes of P68TM in the kinetics of membrane association indicate that the transmembrane domain of ros, besides functioning as a membrane anchor, also plays a role in directing initial membrane association.
Jong
S M
SM
Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029-6574.
Wang
L H
LH
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002471:Cell Transformation, Neoplastic; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D015681:Fusion Proteins, gag-onc / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D015183:Restriction Mapping; D014162:Transfection
D004279:DNA, Viral; D015681:Fusion Proteins, gag-onc; D011505:Protein-Tyrosine Kinases