A 32 kDa protein--whose phosphorylation correlates with oncogenic Ras-induced cell cycle arrest in activated Xenopus egg extracts--is identified as ribosomal protein S6.
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.
Pian
Jerry Pinghwa
JP
Graduate Center for Nutritional Sciences, University of Kentucky Medical Center, 800 Rose Street, Lexington, Kentucky 40536, USA.
Huang
Tun-Lan
TL
Tsai
Pei-Chi
PC
Shi
Jian-Peng
JP
Cu
Hong
H
Pan
Bin-Tao
BT
D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle / Q000502:physiology*; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010063:Ovum / Q000502:physiology*; D010766:Phosphorylation; D038601:Ribosomal Protein S6 / Q000032:analysis*; D014020:Tissue Extracts / Q000032:analysis*; D014981:Xenopus; D018631:ras Proteins / Q000378:metabolism*
D038601:Ribosomal Protein S6; D014020:Tissue Extracts; D018631:ras Proteins