Mapping and identification of protein‐protein interactions by two‐dimensional far‐Western immunoblotting

Studies of protein‐protein interactions have proved to be a useful approach to link proteins of unknown function to known cellular processes. In this study we have combined several existing methods to attempt the comprehensive identification of substrates for poorly characterized human protein tyrosine phosphatases (PTPs). We took advantage of so‐called “substrate trapping” mutants, a procedure originally described by Flint et al. (Proc. Natl. Acad. Sci. USA 1997, 94, 1680—1685) to identify binding partners of cloned PTPs. This procedure was adapted to a proteome‐wide approach to probe for candidate substrates in cellular extracts that were separated by two‐dimensional (2‐D) gel electrophoresis and blotted onto membranes. Protein‐protein interactions were revealed by far‐Western immunoblotting and positive binding proteins were subsequently identified from silver‐stained gels using tandem mass spectrometry. With this method we were able to identify possible substrates for PTPs without using any radiolabeled cDNA or protein probes and showed that they corresponded to tyrosine phosphorylated proteins. We believe that this method could be generally applied to identify possible protein‐protein interactions.

[1]  S. Arkinstall,et al.  Identification of Tyrosine Phosphatases That Dephosphorylate the Insulin Receptor , 2000, The Journal of Biological Chemistry.

[2]  M. Bollen,et al.  Protein phosphorylation and protein phosphatases De Panne, Belgium, September 19–24, 1999 , 2000, The EMBO journal.

[3]  H. Idriss.  Do TNF-α-Insensitive Cancer Cells Escape α-Tubulin Nitrotyrosination? , 2000 .

[4]  P. Steinlein,et al.  Identification of Syntenin as a Protein of the Apical Early Endocytic Compartment in Madin-Darby Canine Kidney Cells* , 1999, The Journal of Biological Chemistry.

[5]  T. Haystead,et al.  Reg1p targets protein phosphatase 1 to dephosphorylate hexokinase II in Saccharomyces cerevisiae: characterizing the effects of a phosphatase subunit on the yeast proteome , 1999, The EMBO journal.

[6]  B. Kennedy,et al.  Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. , 1999, Science.

[7]  L. Huber,et al.  Subcellular fractionation, electromigration analysis and mapping of organelles. , 1999, Journal of chromatography. B, Biomedical sciences and applications.

[8]  R. V. Huijsduijnen.  Protein tyrosine phosphatases: counting the trees in the forest , 1998 .

[9]  D. Maysinger,et al.  From Vanadis to Atropos: vanadium compounds as pharmacological tools in cell death signalling. , 1998, Trends in pharmacological sciences.

[10]  G. Freiss,et al.  Extinction of insulin-like growth factor-I mitogenic signaling by antiestrogen-stimulated Fas-associated protein tyrosine phosphatase-1 in human breast cancer cells. , 1998, Molecular endocrinology.

[11]  T. Latifi,et al.  Structure-function analysis of Msx2-mediated transcriptional suppression. , 1997, Biochemistry.

[12]  D. Barford,et al.  Development of "substrate-trapping" mutants to identify physiological substrates of protein tyrosine phosphatases. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[13]  M. Kasuga,et al.  Overexpression of SAP-1, a transmembrane-type protein tyrosine phosphatase, in human colorectal cancers. , 1997, Biochemical and biophysical research communications.

[14]  B. Neel,et al.  From Form to Function: Signaling by Protein Tyrosine Phosphatases , 1996, Cell.

[15]  E. Fauman,et al.  Structure and function of theprotein tyrosine phosphatases , 1996 .

[16]  J. Olefsky,et al.  Protein Tyrosine Phosphatase 1B Interacts With the Activated Insulin Receptor , 1996, Diabetes.

[17]  D. Alexander,et al.  Protein tyrosine phosphatases in T-cell development, apoptosis and signalling. , 1996, Immunology today.

[18]  P. Leder,et al.  Formin binding proteins bear WWP/WW domains that bind proline‐rich peptides and functionally resemble SH3 domains. , 1996, The EMBO journal.

[19]  A. Shevchenko,et al.  Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry , 1996, Nature.

[20]  D. Barford,et al.  Protein tyrosine phosphatases take off , 1995, Nature Structural Biology.

[21]  S. Shenolikar.  Protein phosphatase regulation by endogenous inhibitors. , 1995, Seminars in cancer biology.

[22]  D. Barford,et al.  Structural basis for phosphotyrosine peptide recognition by protein tyrosine phosphatase 1B. , 1995, Science.

[23]  John Calvin Reed,et al.  FAP-1: a protein tyrosine phosphatase that associates with Fas. , 1995, Science.

[24]  Charis Eng,et al.  Catalytic specificity of protein-tyrosine kinases is critical for selective signalling , 1995, Nature.

[25]  Hong Sun,et al.  The coordinated action of protein tyrosine phosphatases and kinases in cell signaling. , 1994, Trends in biochemical sciences.

[26]  M. Peter,et al.  Mapping of Ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[27]  J. Dixon,et al.  'Zip codes' direct intracellular protein tyrosine phosphatases to the correct cellular 'address'. , 1994, Trends in biochemical sciences.

[28]  D. Barford,et al.  Crystal structure of human protein tyrosine phosphatase 1B. , 1994, Science.

[29]  A. Kazlauskas.  Receptor tyrosine kinases and their targets. , 1994, Current opinion in genetics & development.

[30]  J. Inazawa,et al.  Molecular cloning of a human transmembrane-type protein tyrosine phosphatase and its expression in gastrointestinal cancers. , 1994, The Journal of biological chemistry.

[31]  W. Rutter,et al.  Interaction cloning: identification of a helix-loop-helix zipper protein that interacts with c-Fos. , 1992, Science.

[32]  D F Hochstrasser,et al.  Development of polyacrylamide gels that improve the separation of proteins and their detection by silver staining. , 1988, Analytical biochemistry.

[33]  D F Hochstrasser,et al.  Methods for increasing the resolution of two-dimensional protein electrophoresis. , 1988, Analytical biochemistry.

[34]  J. Otto.  Detection of vinculin-binding proteins with an 125I-vinculin gel overlay technique , 1983, The Journal of cell biology.

[35]  K. Weber,et al.  Calmodulin-binding proteins of the microfilaments present in isolated brush borders and microvilli of intestinal epithelial cells. , 1980, The Journal of biological chemistry.

[36]  C R Merril,et al.  Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[37]  J. den Hertog,et al.  Receptor protein-tyrosine phosphatase signalling in development. , 1999, The International journal of developmental biology.

[38]  S. Biffo,et al.  The identification of signaling molecules by the yeast two-hybrid system. , 1999, Methods in molecular biology.

[39]  Zhon-Yin Zhang,et al.  Protein–tyrosine phosphatases: Structure, mechanism, and inhibitor discovery , 1998, Biopolymers.

[40]  Z. Zhang,et al.  Protein-tyrosine phosphatases: biological function, structural characteristics, and mechanism of catalysis. , 1998, Critical reviews in biochemistry and molecular biology.

[41]  J. Dixon,et al.  Protein tyrosine phosphatases: mechanisms of catalysis and regulation. , 1998, Current opinion in chemical biology.

[42]  C. Pasquali,et al.  Preparative two‐dimensional gel electrophoresis of membrane proteins , 1997, Electrophoresis.

[43]  J. E. Celis,et al.  Cell Biology: A Laboratory Handbook , 1997 .

[44]  S. Elledge,et al.  Gene identification using the yeast two-hybrid system. , 1997, Methods in enzymology.

[45]  Z. Zhang,et al.  Structure, mechanism, and specificity of protein-tyrosine phosphatases. , 1997, Current topics in cellular regulation.

[46]  D. Hochstrasser,et al.  Improved and simplified in‐gel sample application using reswelling of dry immobilized pH gradients , 1997, Electrophoresis.

[47]  L. Huber,et al.  Subcellular fractionation of polarized epithelial cells and identification of organelle‐specific proteins by two‐dimensional gel electrophoresis , 1997, Electrophoresis.

[48]  N. Tonks.  Protein Tyrosine Phosphatases and the Control of Cellular Signaling Responses , 1996 .

[49]  J. Luban,et al.  The yeast two-hybrid system for studying protein-protein interactions. , 1995, Current opinion in biotechnology.

[50]  M. Peter,et al.  Mapping small GTP‐binding proteins on high‐resolution two‐dimensional gels by a combination of GTP binding and labeling with in situ periodateoxidized GTP , 1994, Electrophoresis.

[51]  D. Hochstrasser,et al.  A nonlinear wide‐range immobilized pH gradient for two‐dimensional electrophoresis and its definition in a relevant pH scale , 1993, Electrophoresis.

[52]  Amos Bairoch,et al.  Human liver protein map: A reference database established by microsequencing and gel comparison , 1992, Electrophoresis.

[53]  T. Rabilloud.  A comparison between low background silver diammine and silver nitrate protein stains , 1992, Electrophoresis.