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Identification of a potential physiological substrate for oncogenic Ras-activated protein kinases in activated
1Department of Surgery, University of Kentucky Chandler Medical Center, Lexington 40536, USA.
Abstract:
Activated Xenopus egg extracts are capable of undergoing cell-free cell cycling. Using these activated extracts, we previously showed that purified, bacterially expressed oncogenic human RasH protein arrests cell cycle progression. Because oncogenic Ras activates many serine/threonine protein kinases in Xenopus oocytes and egg extracts, it is possible that induction of cell cycle arrest involves the action of oncogenic Ras-activated kinases. Thus, the identification of the physiological substrates for oncogenic Ras-activated kinases is important for elucidating the molecular mechanism underlying oncogenic Ras-induced cell cycle arrest. We used 32P-orthophosphate as a label to identify the potential substrates. Our results demonstrated that the 32P-labeling of both a 32 and a 33 kDa protein were greatly enhanced by oncogenic Ras during the incubation of activated Xenopus egg extracts. The enhanced labeling correlated with the induced cell cycle arrest and was contributed by serine phosphorylation. Moreover, the 33 kDa protein was detected only in the presence of oncogenic Ras and was a serine-hyperphosphorylated form of the 32 kDa protein. Furthermore, new protein synthesis was not required for the enhanced labeling, consistent with the concept that the enhanced serine phosphorylation of the 32 kDa protein is by oncogenic Ras-activated protein kinases. In addition to serine phosphorylation, our results also suggested that an as yet unidentified modification of the 32 kDa protein might also be induced by oncogenic Ras. Our results suggest that the 32 kDa protein is a potential physiological substrate for oncogenic Ras-activated protein kinases.
Insights
Oncogenic Ras protein halts cell cycle progression in Xenopus egg extracts by activating kinases. Researchers identified a 32 kDa protein as a key substrate, crucial for understanding Ras-induced cell cycle arrest.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Activated Xenopus egg extracts enable cell-free cell cycling.
- Oncogenic human RasH protein arrests cell cycle progression in these extracts.
- Oncogenic Ras activates serine/threonine protein kinases, suggesting kinase action in cell cycle arrest.
Purpose of the Study:
- Identify physiological substrates for oncogenic Ras-activated kinases.
- Elucidate the molecular mechanism of Ras-induced cell cycle arrest.
Main Methods:
- Utilized 32P-orthophosphate labeling to identify potential protein substrates.
- Incubated activated Xenopus egg extracts with oncogenic Ras.
- Analyzed protein phosphorylation patterns.
Main Results:
- 32P-labeling of 32 kDa and 33 kDa proteins was significantly enhanced by oncogenic Ras.
- Enhanced labeling correlated with cell cycle arrest and involved serine phosphorylation.
- The 33 kDa protein is a serine-hyperphosphorylated form of the 32 kDa protein, requiring no new protein synthesis.
- Potential unidentified modification of the 32 kDa protein induced by oncogenic Ras.
Conclusions:
- The 32 kDa protein is a likely physiological substrate for oncogenic Ras-activated protein kinases.
- Understanding this substrate is key to unraveling the mechanism of Ras-induced cell cycle arrest.