Use of electroblotting to detect and analyze phosphotyrosine containing peptides separated by two-dimensional gel electrophoresis

Contor, Laura S.; Lamy, Françoise; Lecocq, Raymond

doi:10.1016/0003-2697(87)90069-8

Cited by 25 publications

(11 citation statements)

References 10 publications

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“…All 2-D gel samples were loaded on isoelectric focusing gels containing a mixture of pH 3.5-10, 5-7, and 6-8 Ampholines (LKB); the resolving gel for the second dimension contained 10% acrylamide. Proteins were electroblotted onto Immobilon (Millipore) and then alkali-treated with 1 M KOH at 56°C for 2 hr (10,24). RESULTS Comparison of pp42 and MAP Kinase by 2-D Gel Electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase.

Rossomando

Payne

Weber

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

391

185

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ABSTRACTpp42, a low-abundance 42-kDa protein, becomes transiently phosphorylated on tyrosine after stimulation of fibroblasts by a variety of mitogens, including epidermal growth factor, platelet-derived growth factor, phorbol 12-myristate 13-acetate, thrombin, and insulin-like growth factor II. The induction of pp42 phosphorylation on tyrosine by such diverse mitogenic agents suggests an important role for pp42 in the cascade of events necessary for cell transition from Go into the cell cycle. However, as with most proteins identified on the basis of their tyrosine phosphorylation, the function of pp42 in cellular regulation is unknown. In this manuscript we report evidence that suggests that pp42 is a serine/threonine-specific protein kinase. Stimulation of 3T3-L1 cells with insulin has been shown to activate a cytosolic serine/threonine kinase capable of phosphorylating microtubule-associated protein 2 (MAP-2) and ribosomal protein S6 kinase H. This cytosolic serine/threonine protein kinase, which itself is phosphorylated on tyrosine, has been termed "MAP kinase." We now report that pp42 phosphorylation and MAP kinase activation occur in fibroblasts in response to similar mitogens, that the two proteins comigrate on one-and two-dimensional polyacrylamide gels, and that the two proteins copurify chromatographically. The major peptides generated from purified MAP kinase by V8 protease digestion are present as a subset of the peptides in digests of pp42 excised from two-dimensional gels. Thus, the results suggest that MAP kinase is tyrosine-phosphorylated pp42.Many growth-factor receptors and oncogene products are tyrosine protein kinases. To understand the mechanisms of intracellular signaling used by these kinases, it will be important to identify and characterize their cellular substrate proteins. Although numerous tyrosine-phosphorylated proteins have been identified by gel electrophoresis in oncogenically transformed or growth factor-stimulated cells, in most cases the biological significance of these phosphorylations has not been determined. pp42 is among the most widely studied of these tyrosine-phosphorylated proteins; it was identified by gel electrophoresis in cells stimulated by any of a number of diverse mitogens [epidermal growth factor (EGF), plateletderived growth factor, insulin-like growth factor II, thrombin, or phorbol 12-myristate 13-acetate (PMA; also called TPA)](1-7) and also has been found in at least some oncogenically transformed cells (8-10). Because ofthe wide variety ofagents that stimulate this phosphorylation, pp42 is believed to be involved in some unknown step in intracellular signaling that is shared by all of these mitogens.Recently a novel serine/threonine protein kinase was identified in insulin-stimulated 3T3-L1 cells (11). This protein kinase has been shown to phosphorylate microtubuleassociated protein 2 (MAP-2) as well as ribosomal protein S6 kinase II in vitro (12). The kinase was also found to migrate on sodium dodecyl sulfate (SDS)/polyacrylamide gels with a molecular ...

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Section: Methodsmentioning

confidence: 99%

Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase.

Rossomando

Payne

Weber

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

391

185

View full text Add to dashboard Cite

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“…Protein Chemistry-Cleavage of the IGF-I receptor ␤-subunit with trypsin was performed essentially as described (Gibson, 1974;Cooper et al, 1983;Aebersold et al, 1987;Contor et al, 1987;Kamps and Sefton, 1989). Following transfer of proteins to nitrocellulose and visualization by autoradiography, the nitrocellulose region corresponding to the 95- The numbering and alignment of the tryptic peptides are based on the amino acid sequence of the two receptors as described in (Ullrich et al, 1986).…”

Section: Antibodies and Immunoprecipitations-␣-subunit Antibodies ␣Ir3mentioning

confidence: 99%

“…Finally, the tryptic peptides were resuspended in pH 1.9 chromatography buffer (see below) and separated by two-dimensional thin layer chromatography. Two-dimensional thin layer chromatography was performed essentially as described (Gibson 1974;Cooper et al, 1983;Aebersold et al, 1987;Contor et al, 1987;Kamps and Sefton, 1989). Chromatography plates (cellulose, without fluorescent indicator) 20 ϫ 20-cm square were purchased from Eastman Kodak Co. For separation in the first dimension, plates were electrophoresed for 1 h at 700 volts in pH 1.9 buffer (acetic acid, formic acid, butanol, and water).…”

Section: Antibodies and Immunoprecipitations-␣-subunit Antibodies ␣Ir3mentioning

confidence: 99%

Src Phosphorylates the Insulin-like Growth Factor Type I Receptor on the Autophosphorylation Sites

Peterson

Kulik

Jelı́nek

et al. 1996

Journal of Biological Chemistry

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“…for 2 h at 40°C, and its radioactivity was measured in a liquid scintillation counter after the addition of 12 Alkali treatment. After electrophoretic transfer of the proteins to nylon membranes, the sheets were treated with alkali in 1 N NaOH at 55°C for 2 h as previously described (8). The dried membranes were exposed to Kodak XAR5 film for a period calculated to reach a total of 1011 disintegrations (about 13 days).…”

mentioning

confidence: 99%

Differential protein phosphorylation in induction of thyroid cell proliferation by thyrotropin, epidermal growth factor, or phorbol ester.

Contor¹,

Lamy²,

Lecocq³

et al. 1988

Mol. Cell. Biol.

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Protein phosphorylation was studied in primary cultures of thyroid epithelial cells after the addition of different mitogens: thyrotropin (TSH) acting through cyclic AMP, epidermal growth factor (EGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA). EGF or TPA increased the phosphorylation of five common polypeptides. Among these, two 42-kilodalton proteins contained phosphotyrosine and phosphoserine with or without phosphothreonine. Their characteristics suggested that they are similar to the two 42-kilodalton target proteins for tyrosine protein phosphorylation demonstrated in fibroblasts in response to mitogens. No common phosphorylated proteins were detected in TSH-treated cells and in EGF- or TPA-treated cells. The differences in the protein phosphorylation patterns in response to TSH, EGF, and TPA suggested that the newly emerging cyclic AMP-mediated mitogenic pathway is distinct from the better known growth factor- and tumor promoter-induced pathways.

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Use of electroblotting to detect and analyze phosphotyrosine containing peptides separated by two-dimensional gel electrophoresis

Cited by 25 publications

References 10 publications

Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase.

Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase.

Src Phosphorylates the Insulin-like Growth Factor Type I Receptor on the Autophosphorylation Sites

Differential protein phosphorylation in induction of thyroid cell proliferation by thyrotropin, epidermal growth factor, or phorbol ester.

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