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Updated: Aug 22, 2026
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
A 32 kDa protein--whose phosphorylation correlates with oncogenic Ras-induced cell cycle arrest in activated Xenopus
Jerry Pinghwa Pian1, Tun-Lan Huang, Pei-Chi Tsai
1Graduate Center for Nutritional Sciences, University of Kentucky Medical Center, 800 Rose Street, Lexington, Kentucky 40536, USA.
Abstract:
Oncogenic Ras induces cell-cycle arrest in mammalian cells and in fertilized Xenopus eggs. How oncogenic Ras induces cell-cycle arrest remains unclear. We previously showed that oncogenic Ras induces cell-cycle arrest in activated Xenopus egg extracts (cycling extracts) and that the induced cell-cycle arrest correlates with hyperphosphorylation of a 32 kDa protein. However, the identity of the 32 kDa protein was not known. By using a sucrose density-gradient centrifugation, Triton X-100-acetic acid-urea (TAU)-gel electrophoresis, composite agarose-polyacrylamide gel electrophoresis (CAPAGE), SDS-PAGE, and partial tryptic peptide sequence analysis, the 32 kDa protein has now been identified as S6, a 40S subunit ribosomal protein. Hence, our results indicate that the oncogenic Ras-induced cell-cycle arrest is correlated with hyperphosphorylation of S6, suggesting that phosphorylation of S6 plays an important role in the induced cell-cycle arrest. It has been shown that conditional deletion of gene encoding S6 in mammalian cells prevents proliferation, demonstrating the importance of S6 in cell proliferation. The exact role S6 plays in cell proliferation is unclear. However, phosphorylation of S6 has been implicated in the regulation of protein synthesis. Thus, our results are consistent with the concept that oncogenic Ras induces S6 phosphorylation to influence protein synthesis, thereby contributing to the cell-cycle arrest. In addition, our results also demonstrate that composite agarose-polyacrylamide gel electrophoresis is suitable for the separation of large molecular complexes.
Insights
Oncogenic Ras causes cell-cycle arrest by hyperphosphorylating ribosomal protein S6. This phosphorylation impacts protein synthesis, highlighting S6
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Oncogenic Ras is known to induce cell-cycle arrest in various cell types.
- The precise mechanism by which oncogenic Ras triggers cell-cycle arrest remains incompletely understood.
- Previous studies indicated a correlation between Ras-induced cell-cycle arrest and hyperphosphorylation of a 32 kDa protein in Xenopus egg extracts.
Purpose of the Study:
- To identify the 32 kDa protein associated with oncogenic Ras-induced cell-cycle arrest.
- To elucidate the role of this protein's phosphorylation in the cell-cycle arrest mechanism.
- To investigate the potential involvement of protein synthesis regulation in Ras-mediated cell-cycle arrest.
Main Methods:
- Utilized sucrose density-gradient centrifugation, TAU-gel electrophoresis, CAPAGE, and SDS-PAGE for protein analysis.
- Performed partial tryptic peptide sequence analysis to determine protein identity.
- Employed activated Xenopus egg extracts (cycling extracts) for experimental models.
Main Results:
- Identified the 32 kDa protein as S6, a 40S subunit ribosomal protein.
- Demonstrated a strong correlation between oncogenic Ras-induced cell-cycle arrest and S6 hyperphosphorylation.
- Confirmed the suitability of composite agarose-polyacrylamide gel electrophoresis (CAPAGE) for separating large molecular complexes.
Conclusions:
- Oncogenic Ras-induced cell-cycle arrest is linked to the hyperphosphorylation of ribosomal protein S6.
- S6 phosphorylation likely plays a crucial role in mediating Ras-induced cell-cycle arrest.
- Results suggest Ras influences protein synthesis via S6 phosphorylation, contributing to cell-cycle arrest.
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