Increased expression of the Ras suppressor Rsu-1 enhances Erk-2 activation and inhibits Jun kinase activation.

Studies were undertaken to determine the effect of the Ras suppressor Rsu-1 on Ras signal transduction pathways in two different cell backgrounds. An expression vector containing the mouse rsu-1 cDNA under the control of a mouse mammary tumor virus promoter was introduced into NIH 3T3 cells and the pheochromocytoma cell line PC12. Cell lines developed in the NIH 3T3 background expressed p33rsu-1 at approximately twice the normal endogenous level. However, PC12 cell clones which expressed p33rsu-1 at an increased level in a regulatable fashion in response to dexamethasone were isolated. Analysis of proteins involved in regulation of Ras and responsive to Ras signal transduction revealed similar changes in the two cell backgrounds in the presence of elevated p33rsu-1. There was an increase in the level of SOS, the guanine nucleotide exchange factor, and an increase in the percentage of GTP-bound Ras. In addition, there was an increase in the amount of p120 Ras-specific GTPase-activating protein (GAP) and GAP-associated p190. However, a decrease in Ras GTPase-activating activity was detected in lysates of the Rsu-1 transfectants, and immunoprecipitated p120 GAP from the Rsu-1 transfectants showed less Ras GTPase-activating activity than GAP from control cells. Activation of Erk-2 kinase by growth factor and tetradecanyol phorbol acetate was greater in the Rsu-1 transfectants than in control cells. However, c-Jun amino-terminal kinase activity (Jun kinase) was not activatable by epidermal growth factor in Rsu-1 PC12 cell transfectants, in contrast to the PC12 vector control cell line. Transient expression of p33rsu-1 in Cos1 cells following cotransfection with either hemagglutinin-tagged Jun kinase or hemagglutinin-tagged Erk-2 revealed that Rsu-1 expression inhibited constitutive Jun kinase activity while enhancing Erk-2 activity. Detection of in vitro binding of Rsu-1 to Raf-1 suggested that in Rsu-1 transfectants, increased activation of the Raf-1 pathway occurred at the expense of activation of signal transduction leading to Jun kinase. These results indicate that inhibition of Jun kinase activation was sufficient to inhibit Ras transformation even in the presence of activated Erk-2.