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A cell cycle ts mutant, tsJT16, is defective in p70 synthesis through protein kinase C-dependent and -independent
E Miyake1, T Takasuka, M Sakayama
1Department of Cellular and Molecular Biology, Hiroshima University School of Medicine, Japan.
Abstract:
tsJT16 is a G0/G1 ts mutant from the Fischer rat fibroblast line. It has a ts defect in a function operating early after growth stimulation with fetal bovine serum (FBS). A primarily induced gene product, p70, was not synthesized at 40 degrees C after stimulation with serum, while c-fos and c-myc mRNAs accumulated under the same condition. This paper reports that p70 was synthesized following stimulation of G0-arrested cells with platelet-derived growth factor, epidermal growth factor (EGF), and 12-0-tetradecanoylphorbol-13-acetate (TPA) at 34 degrees C, but not at 40 degrees C. However, it was synthesized at both temperatures after addition of A23187. In protein kinase C-deprived cells, peptide growth factors and A23187 induced p70 at 34 degrees C, whereas TPA did not. Fibroblast growth factor and insulin did not induce p70. Induction of c-fos and c-myc occurred at both temperatures after the stimulation with FBS, TPA or A23187. These results indicated that the defect in tsJT16 to induce p70 is likely to be located at the common downstream of protein kinase C-dependent and -independent pathways, but is independent from the pathway of calcium mobilization.
Insights
The tsJT16 mutant shows a temperature-sensitive defect in p70 protein synthesis, crucial for early cell growth. This defect affects pathways downstream of protein kinase C and calcium mobilization, impacting cell cycle regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The tsJT16 mutant, derived from Fischer rat fibroblasts, exhibits a temperature-sensitive (ts) defect.
- This defect impacts early events following growth stimulation, specifically the synthesis of a key protein, p70.
Purpose of the Study:
- To investigate the specific defect in tsJT16 cell cycle regulation.
- To identify the signaling pathways involved in p70 protein induction.
Main Methods:
- Utilized tsJT16 cells synchronized at G0/G1 phase.
- Stimulated cells with various growth factors (FBS, PDGF, EGF, FGF, insulin), TPA, and A23187 at permissive (34°C) and restrictive (40°C) temperatures.
- Analyzed p70 protein synthesis and mRNA accumulation of c-fos and c-myc.
- Performed experiments in protein kinase C-deprived cells.
Main Results:
- p70 protein was not synthesized at 40°C upon fetal bovine serum (FBS) stimulation, while c-fos and c-myc mRNA accumulated.
- Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and 12-O-tetradecanoylphorbol-13-acetate (TPA) induced p70 at 34°C but not at 40°C.
- A23187 induced p70 synthesis at both temperatures.
- In protein kinase C-deprived cells, peptide growth factors and A23187 induced p70 at 34°C, but TPA did not.
- Fibroblast growth factor (FGF) and insulin did not induce p70.
Conclusions:
- The tsJT16 defect in p70 induction is located downstream of both protein kinase C-dependent and -independent pathways.
- The defect is independent of calcium mobilization pathways.
- Cell cycle progression in tsJT16 cells is regulated by distinct signaling cascades affecting specific protein synthesis.