Deficient post-translational processing of Rap 1A in variant HL-60 cells.
Variant HL-60 cells resistant to differentiation induced by nitroprusside and cGMP analogs have normal guanylate cyclase and cGMP-dependent protein kinase (G-kinase) activity (J. Biol. Chem. 269, 32155-32161, 1994). We found decreased phosphorylation of a low molecular weight protein (pp23) in the variant cells and by co-migration on two-dimensional polyacrylamide gels, phosphopeptide mapping, immunoprecipitation and immunoblotting, we showed that pp23 was one of three post-translationally modified forms of Rap 1A expressed in HL-60 cells. Using an in vitro transcription/translation system, we studied each of the posttranslational processing steps of Rap 1A and we showed that pp23 represented fully processed Rap 1A. By immunoprecipitation, immunoblotting and 35S-methionine/cysteine incorporation, we showed that the variant cells were deficient in pp23, and thus in fully processed Rap 1A, but that these cells did express normal amounts of completely unprocessed Rap 1A and geranylgeranylated Rap 1A; the lack of Rap 1A processing beyond geranylgeranylation in the variant cells was not secondary to a change in Rap 1A's amino acid sequence. The variant cells had normal carboxyl methyltransferase activity suggesting they are deficient in proteolytic cleavage of Rap 1A. The deficient post-translational processing of Rap 1A had no effect on Rap 1A's subcellular distribution and we found no evidence for altered post-translational processing of H-Ras.
Scheele
J S
JS
Department of Biochemistry and Chemistry, University of California, San Diego, La Jolla 92093-0652, USA.
Pilz
R B
RB
von Lintig
F C
FC
Boss
G R
GR
D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D018922:HL-60 Cells / Q000737:chemistry* / Q000187:drug effects; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000032:analysis
D007202:Indicators and Reagents; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D009599:Nitroprusside; C016276:8-bromocyclic GMP; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569718:PRKG1 protein, human; D006152:Cyclic GMP