title: Both normal and transforming PCPH proteins have guanosine diphosphatase activity but only the oncoprotein cooperates with Ras in activating extracellular signal-regulated kinase ERK1.
abstract: Previous reports from our laboratory described the activation of the PCPH gene 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 ribonucleotide 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 engineered to be GDPase deficient by targeted disruption of the single GDA1 allele did not complement their glycosylation-disabled phenotype, suggesting the existence of significant functional differences between the mammalian and yeast enzymes. Results from transient cotransfections into NIH3T3, COS-7, or 293T cells indicated that, in mammalian cells, both PCPH and mt-PCPH cause an overall down-regulation of the stimulatory effect of epidermal growth factor or the activated ras or raf oncogenes on the Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway. However, despite this overall negative regulatory role on Ras signaling, mt-PCPH, but not PCPH, cooperated with the Ras oncoprotein to produce a prolonged 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 determinant of the transforming activity of mt-PCPH.
authors.affiliation: Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 20007, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Recio
authors.forename: J G
authors.initials: JG
authors.lastname: Páez
authors.forename: B
authors.initials: B
authors.lastname: Maskeri
authors.forename: M
authors.initials: M
authors.lastname: Loveland
authors.forename: J A
authors.initials: JA
authors.lastname: Velasco
authors.forename: V
authors.initials: V
authors.lastname: Notario
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D006224:Cricetinae; D004926:Escherichia coli / Q000201:enzymology; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008647:Mesocricetus; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D014176:Protein Biosynthesis; D011755:Pyrophosphatases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000378:metabolism / Q000502:physiology*
chemical_list: D002457:Cell Extracts; D015513:Oncogene Proteins; C485537:Pcph protein, mouse; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C117634:ENTPD5 protein, human; D011755:Pyrophosphatases; C010357:guanosine-diphosphatase; D018631:ras Proteins