SCRAPE-LOADING OF SWISS 3T3 CELLS WITH RAS PROTEIN RAPIDLY ACTIVATES PROTEIN KINASE-C IN THE ABSENCE OF PHOSPHOINOSITIDE HYDROLYSIS

Abstract

Scrape-loading has been used to analyse the biochemical function of purified p21ras protein. We have shown that scrape-loading oncogenic p21ras into quiescent Swiss 3T3 cells causes morphological transformation of 90% of the cell population within 15h. Since large numbers of cells can be loaded with p21ras, early induced biochemical changes can be analysed. In this way we have shown that oncogenic p21ras causes rapid activation of protein kinase C five minutes after introduction of protein, but that ras protein fails to stimulate measurable inositol phosphate formation. It appears, therefore, that the stimulation of protein kinase C activity is due to a ras induced increase in diacylglycerol from a source other than inositol phospholipids. Efficient stimulation of DNA synthesis by oncogenic p21ras only occurs in the presence of insulin. This stimulation of DNA synthesis by ras is absolutely dependent on functional protein kinase C activity.