THE ALTERNATIVE H-RAS PROTEIN P19 DISPLAYS PROPERTIES OF A NEGATIVE REGULATOR OF P21RAS

In previous work, we demonstrated that expression of mammalian H-ras g enes is controlled by alternative splicing. Mutational analyses indica ted that most H-ras premessenger RNA (pre-mRNA) is not processed into mRNA For the usual p21Ras protein, but is recognized instead for proce ssing through an alternative pathway that would produce mRNA For a hyp othetical p19Ras protein, which would be a truncated form of p21 with a different carboxyl-terminus. We have raised the possibility that p19 could be a negative regulator of p21, but only small amounts of matur e p19 mRNA could be detected and we had no evidence that the protein c ould be made. We now show by transient and stable transfection experim ents that p19 can be produced from complementary DNA (cDNA) expression vectors in human 293 cells, which express early adenoviral genes. How ever, p19 cDNA inhibited the Formation of drug-selected colonies of Ra t-1 cells and surviving colonies produced little p19. Colony formation and p19 expression were increased when the cDNA had mutations in the putative effector domain of p19 or when oncogenes believed to act down stream of p21Ras, such as adenoviral EIA, were included in the transfe ctions. These results indicate that p19 can act as an inhibitor of p21 . We also show that authentic p19 can be abundantly expressed in 293 c ells from H-ras gene constructs with mutations favoring the alternativ e splicing pathway, which suggests that regulated splicing could suppo rt overproduction of p19 under natural conditions. Our work indicates that mutations abolishing alternative H-ras splicing may not only cont ribute to oncogenesis by increasing the production of p21, but also by interfering with the production of an antiproliferative H-ras activit y.