Identification of a potential physiological substrate for oncogenic Ras‐activated protein kinases in activated Xenopus egg extracts: Correlation with oncogenic RAS‐induced cell cycle arrest

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 32 P‐orthophosphate as a label to identify the potential substrates. Our results demonstrated that the 32 P‐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. © 1996 Wiley‐Liss, Inc.