title: Differential proteomic analysis of lymphocytes treated with mycophenolic acid reveals caspase 3-induced cleavage of rho GDP dissociation inhibitor 2.
abstract: The antiproliferative immunosuppressive drug mycophenolic acid (MPA) is an uncompetitive inhibitor of inosine monophosphate dehydrogenase, a key enzyme in de novo synthesis of purine nucleotides. The latter are not only required for synthesis of DNA and RNA but also are essential for the regulation of numerous cellular signaling pathways modulated by guanine nucleotide binding proteins (G proteins). We undertook an analysis of the influence of MPA on protein expression in a T-lymphoblast cell line (CCRF-CEM), which displays concentration-dependent inhibition of proliferation by MPA to obtain insight into the influence of MPA on the cellular proteome. Cells were stimulated with phorbol myristate acetate/ionomycin and incubated in the presence or absence of MPA. Two-dimensional electrophoresis and densitometric imaging revealed 11 differentially expressed protein spots (P < 0.05) on MPA treatment, 6 with increased and 5 with decreased abundance. After in-gel tryptic digestion, proteins were identified by quadrupole time-of-flight mass spectrometry. Proteins displaying increased abundance after MPA treatment included splicing factor arginine/serine-rich 2, prostaglandin E synthase 3, peptidyl-prolyl cis-trans isomerase A, and deoxyuridine 5'-triphosphate nucleotidohydrolase. Endoplasmin, proliferating cell nuclear antigen, acidic leucine-rich nuclear phosphoprotein 32 family member A, and cofilin 1 showed decreased abundance after MPA treatment. Three separate spots (1 decreased and 2 increased abundance) were identified as Rho guanosine diphosphate dissociation inhibitor 2 (Rho GDI 2) proteins. Western blotting with a monoclonal antibody directed against the Rho GDI 2 site cleaved by caspase 3 demonstrated 1 spot with increased abundance to be the caspase 3-cleaved product of Rho GDI 2 lacking the first 19 amino acids. Rho GDI 2 plays a central regulatory role in the activation of Rho guanosine triphosphatases that function as molecular switches in cell signaling pathways affecting cell cytoskeletal dynamics and motility. Our data suggest that MPA can modulate Rho GDI 2 levels in T lymphocytes, thereby potentially disrupting cell signaling pathways important for T-cell function.
authors.affiliation: Department of Clinical Chemistry, University Medicine Göttingen, Robert-Koch-Strasse 40, Göttingen, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Heller
authors.forename: Abdul R
authors.initials: AR
authors.lastname: Asif
authors.forename: Darinka Todorova
authors.initials: DT
authors.lastname: Petrova
authors.forename: Yuliana
authors.initials: Y
authors.lastname: Doncheva
authors.forename: E
authors.initials: E
authors.lastname: Wieland
authors.forename: Michael
authors.initials: M
authors.lastname: Oellerich
authors.forename: Maria
authors.initials: M
authors.lastname: Shipkova
authors.forename: Victor William
authors.initials: VW
authors.lastname: Armstrong
mesh_terms: D053148:Caspase 3 / Q000378:metabolism*; D049109:Cell Proliferation; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism*; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008214:Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D009173:Mycophenolic Acid / Q000494:pharmacology*; D020543:Proteome / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
chemical_list: C569658:ARHGDIB protein, human; D020730:Guanine Nucleotide Dissociation Inhibitors; D007166:Immunosuppressive Agents; D020543:Proteome; D025521:Tumor Suppressor Proteins; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D053148:Caspase 3; D009173:Mycophenolic Acid