Abstract
The protein-tyrosine kinase (PTK) v-Fps induces protein kinase C (PKC)-dependent expression of the transformation-related 9E3 gene in chicken embryo fibroblasts (Spangler, R., Joseph, C., Qureshi, S.A., Berg, K., and Foster, D.A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7017-7021). We present evidence here that a GTP-binding protein (G-protein) is a component of this PKC-dependent signaling pathway. 1) A GTP analogue that stimulates G-protein-mediated signals induced 9E3 gene expression. 2) A GDP analogue that inhibits signaling through G-proteins inhibited expression of 9E3 and phosphorylation of a 67-kDa PKC substrate induced by v-Fps. The GDP analogue had no effect on phosphorylation of the PKC substrate or the expression of 9E3 induced by direct activation of PKC with phorbol ester. 3) Increased v-Fps PTK activity led to increased GTP binding to a 50-kDa protein. The molecular weight of this GTP-binding protein is consistent with the molecular weight of alpha-subunits of G-proteins of the heterotrimeric class. The data suggest that a G-protein functions upstream from PKC in a signaling pathway that connects v-Fps PTK activity to increased 9E3 gene expression.
Highlights
From the Institute for Biomolecular Structure and been restricted to signals initiated by membrane receptors of Function and the Department of Biological Sciences, the seven-transmembrane domain class (Bourne et al, 1991)
We previously demonstrated (Spangler et al, 1989) that theoncogenic PTK v-Fps uses a PKC-mediated signaling nals induced 9E3 gene expression.2)A GDP analogue pathway to induce the expression of the 9E3 gene, whose thatinhibits signaling throughG-proteinsinhibited expression of 9E3 and phosphorylation of a 67-kDa PKC substrate induced by v-Fps
We previously demonstrated that the PTv-KFps gene expression was theinduced either by shifting thecells to 34 "C
Summary
Theprotein-tyrosine kinase (PTK) v-Fps induces protein kinase C (PKC)-dependent expression of the transformation-related 9E3 gene in chicken embryo fibroblasts The. EXPERIMENTALPROCEDURES data suggest that a G-protein functions upstream fromMaterials-GTPyS, GDPPS, ATPyS, phorbol dibutyrate (PBt2), PKC in a signaling pathway that connectsv-Fps PTK saponin, and digitonin were obtained from Sigma. Permeabilization of CEF andRNA Analysis-Permeabilization to allow entry of guanine nucleotides was performed as described by Burch and Axelrod (1987).Cellswere incubated in serum-free medium containing saponin (80 pg/ml) for 3 min. PTK, protein-tyrosine kinase; CEF, chicken embryo fibroblasts; and subjected to SDS-polyacrylamide gel electrophoresis as previ-. 5’-O-(thiotriphosphate); GDPPS, guanosine 5’-0-(2-thiodiphos- Cross-linking of GTP to Cellular Proteins-CEF or tsNY225-inphate); ATP-& adenosine 5’-0-(thiotriphosphate); 8-N3GTP8,-azi- fected CEF were permeabilized for 3 min as described above for the doguanosine 5”triphosphate; SDS, sodium dodecyl sulfate; PIPES, analysis of phosphorylated proteins with permeabilization buffer.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
