Both normal and transforming PCPH proteins have guanosine diphosphatase activity but only the oncoprotein cooperates with ras in activating extracellular signal-regulated kinase ERK1

Previous reports from our laboratory described the activation of the PCPH g ene into the PCPH oncogene (mt-PCPH reported previously as Cph) by a single point mutational deletion, As a consequence, the mt-PCPH oncoprotein is a truncated form of the normal PCPH protein. Although both proteins have ribo nucleotide diphosphate-binding activity, only mt-PCPH acted synergistically with a human H-Ras oncoprotein to transform murine NIH3T3 fibroblasts, We report here the expression of the PCPH and mt-PCPH proteins in Escherichia coli and the finding that the purified bacterial recombinant proteins have intrinsic guanosine diphosphatase (GDPase) activity. However, expression of the Syrian hamster PCPH and mt-PCPH proteins in haploid yeast strains engi neered to be GDPase deficient by targeted disruption of the single GDA1 all ele did not complement their glycosylation-disabled phenotype, suggesting t he existence of significant functional differences between the mammalian an d yeast enzymes. Results from transient cotransfections into NIH3T3, COS-7, or 293T cells indicated that, in mammalian cells, both PCPH and mt-PCPH ca use an overall down-regulation of the stimulatory effect of epidermal growt h factor or the activated ms or raf oncogenes on the Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathwa y. However, despite this overall negative regulatory role on Ras signaling, mt-PCPH, but not PCPH, cooperated with the Ras oncoprotein to produce a pr olonged stimulation of the phosphorylation of ERK1 but had no effect on the phosphorylation levels of ERK2, These results represent a clear difference between the mechanisms of action of PCPH and mt-PCPH and suggest that the ability to cause a sustained activation of ERK1 may be an important determi nant of the transforming activity of mt-PCPH.