DEFICIENT POSTTRANSLATIONAL PROCESSING OF RAP 1A IN VARIANT HL-60 CELLS

Variant HL-60 cells resistant to differentiation induced by nitropruss ide and cGMP analogs have normal guanylate cyclase and cGMP-dependent protein kinase (G-kinase) activity (J, Biol. Chem. 269, 32155-32161, 1 994). We found decreased phosphorylation of a low molecular weight pro tein (pp23) in the variant cells and by co-migration on two-dimensiona l polyacrylamide gels, phosphopeptide mapping, immunoprecipitation and immunoblotting, we showed that pp23 was one of three post-translation ally modified forms of Rap 1A expressed in HL-60 cells. Using an in vi tro transcription/ translation system, rye studied each of the posttra nslational processing steps of Rap 1A and we showed that pp23 represen ted fully processed Rap 1A. By immunoprecipitation, immunoblotting and S-35-methionine/cysteine incorporation, we showed that the variant ce lls were deficient in pp23, and thus in fully processed Rap 1A, but th at these cells did express normal amounts of completely unprocessed Ra p 1A and geranylgeranylated Rap 1A; the lack of Rap 1A processing beyo nd geranylgeranylation in the variant cells was not secondary to a cha nge in Rap 1A's amino acid sequence, The variant cells had normal carb oxyl methyltransferase activity suggesting they are deficient in prote olytic cleavage of Rap 1A, The deficient post-translational processing of Rap 1A had no effect on Rap 1A's subcellular distribution and we f ound no evidence for altered post-translational processing of H-Ras.