bromochloroacetic-acid and pervanadate

Rabbit antibodies may have favorable properties compared to mouse antibodies, including high affinities and better antigen recognition. We used a biochemical and reverse immunologic approach to generate and characterize rabbit anti-phospho-keratin and anti-keratin monoclonal antibodies (MAb). Human keratins 8 and 18 (K8/K18) were used as immunogens after isolation from cells pretreated with okadaic acid or pervanadate to promote Ser/Thr or Tyr hyperphosphorylation, respectively. Selected rabbit MAb were tested by immunofluorescence staining, immunoprecipitation, and 2-dimensional gels. Keratin phospho and non-phospho-mutants were used for detailed characterization of two unique antibodies. One antibody recognizes a K8 G61-containing epitope, an important epitope given that K8 G61C is a frequent mutation in human liver diseases. This antibody binds K8 that is not phosphorylated on S73, but its binding is ablated by G61 but not S73 mutation. The second antibody is bispecific in that it simultaneously recognizes two epitopes: one phospho (K8 pS431) conformation-independent and one non-phospho conformation-dependent, with both epitopes residing in the K8 tail domain. Therefore, a reverse immunologic and biochemical approach is a viable tool for generating versatile rabbit MAb for a variety of cell biologic applications including the potential identification of physiologic phosphorylation sites.

Topics: Animals; Antibodies, Bispecific; Antibodies, Monoclonal; Brain; Brain Chemistry; Cell Line; Colonic Neoplasms; Cricetinae; Enzyme Inhibitors; Epitopes; HT29 Cells; Humans; Immunoblotting; Immunohistochemistry; Keratin-18; Keratin-8; Keratins; Kidney; Liver; Liver Diseases; Mice; Mutation; Okadaic Acid; Phosphorylation; Rabbits; Serine; Stomach Neoplasms; Transfection; Vaccination; Vanadates

We used a proteomic approach to identify proteins that associate with keratins 8 or 18 (K8/K18) in a pervanadate-dependent manner. Pervanadate triggers Ran-K8/K18 binding and a gel-migration-shift of Ran from 25 to 27 kDa, which does not occur upon exposure to H2O2 or vanadate or if pervanadate is excluded during cell solubilization. Generation of 27-kDa Ran is not related to hyperphosphorylation, is heat-insensitive, but occurs upon conversion of Ran cysteines to cysteic acid. The pervanadate-mediated Ran cysteine --> cysteic acid oxidation and its related gel migration shift affects other proteins including actin. Mutation of the three Ran cysteines (Cys-85, -112, and -120) showed that Ran Cys-112 oxidation generates 27-kDa Ran and accounts for its keratin binding. Proteasome inhibition accentuates Ran-keratin binding after cell exposure to pervanadate. Therefore, cell-free exposure to pervanadate causes cysteine to cysteic acid oxidation of Ran and several other proteins and Ran-K8/K18 association. In cells, stabilization of oxidized Ran by proteasome inhibition promotes Ran-keratin interaction. Keratin sequestration of oxidized Ran may provide a back-up protective mechanism in some cases of oxidative injury.

Topics: Actins; Cell Line, Tumor; Cell Movement; Cell Survival; Cell-Free System; Cysteine; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Humans; Immunoprecipitation; Keratins; Mass Spectrometry; Models, Biological; Mutation; Oxygen; Phosphorylation; Proteasome Inhibitors; Protein Binding; Proteomics; ran GTP-Binding Protein; Tyrosine; Vanadates

Glandular epithelia express the keratin intermediate filament (IF) polypeptides 8, 18 and 19 (K8/18/19). These proteins undergo significant serine phosphorylation upon stimulation with growth factors and during mitosis, with subsequent modulation of their organization and interaction with associated proteins. Here we demonstrate reversible and dynamic tyrosine phosphorylation of K8 and K19, but not K18, upon exposure of intact mouse colon or cultured human cells to pervanadate. K8/19 tyrosine phosphorylation was confirmed by metabolic 32PO4-labeling followed by phosphoamino acid analysis, and by immunoblotting with anti-phosphotyrosine antibodies. Pervanadate treatment increases keratin solubility and also indirectly increases K8/18 serine phosphorylation at several known sites, some of which were previously shown to be associated with EGF stimulation, extracellular signal-regulated kinase (ERK), or p38 kinase activation. However, K8/19 tyrosine phosphorylation is independent of EGF signaling or ERK activation while inhibition of p38 kinase activity blocks pervanadate-induced K8/19 tyrosine phosphorylation. Our results demonstrate tyrosine phosphatase inhibitor-mediated in vivo tyrosine phosphorylation of K8/19, but not K18, and suggest that tyrosine phosphorylation may be a general modification of other IF proteins. K8/19 tyrosine phosphorylation involves a pathway that utilizes the p38 mitogen-activated protein kinase, but appears independent of EGF signaling or ERK kinase activation.

Topics: Animals; Binding Sites; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Colon; Enzyme Inhibitors; Epidermal Growth Factor; Humans; Keratins; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Serine; Signal Transduction; Solubility; Tyrosine; Vanadates