Immunological methods for the detection of phosphotyrosine-containing proteins in neural tissues

Ely, Constance M.; Parsons, Sarah J.; Parsons, J. Thomas

doi:10.1016/1058-6741(92)90026-t

Cited by 4 publications

(5 citation statements)

References 44 publications

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“…A bacterial trpE fusion protein containing the entire RasGAP molecule except the six NHE-terminal amino acids was expressed in Escherichia coli from a pATH1 plasmid (31), generated by insertion of the NaeI fragment of bovine RasGAP cDNA (68) into the SmaI site of the vector, trpE-RasGAP was purified from bacterial cell lysates by SDS-PAGE and elution from excised gel pieces in PBS (136 mM NaCI, 2.6 mM KC1, 10 mM Na2HPO4, 1.7 mM KI-I2PO 4, pH 7.2). Soluble, purified protein was emulsified with Freund's adjuvant and injected into New Zealand White rabbits according to the immunization protocol described by Ely et al (16). Antibodies were purified from immune sera first by protein A chromatography as described by Harlow and Lane (25) and Zola (73) and subsequently by GST-RasGAP-SH2-SH3-SH2 Sepharose affinity chromatography according to the procedure of Smith and Johnson (61).…”

Section: Polyclonal and Monoclonal Antibody Productionmentioning

confidence: 99%

c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation.

Chang

Gill

Settleman

et al. 1995

The Journal of Cell Biology

240

226

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Abstract. Analysis of C3H10T1/2 murine fibroblasts overexpressing wild type and dominant negative variants of c-Src has demonstrated a requirement for c-Src in EGF-induced mitogenesis. Correlating with the ability of c-Src variants to potentiate or inhibit EGFdependent DNA synthesis is the phosphotyrosine content of multiple cellular proteins, including pl90-RhoGAP, a protein thought to regulate growth factorinduced actin cytoskeleton remodeling by modulating the activity of the small GTP binding protein, Rho. Because the in vivo phosphotyrosine content of p190 varies with the level of active c-Src and not with EGF treatment, p190 is considered to be a preferred substrate of c-Src. To determine whether tyrosyl phosphorylation of p190 (by c-Src) could influence EGFdependent actin remodeling, we used conventional and confocal immunofluorescence microscopy to examine the intracellular distribution of p190, actin, and pl20RasGAP in EGF-stimulated or unstimulated 10T1/2 Neo control cells and cells that stably overexpress wild-type (K ÷) or kinase-defective (K-) c-Src. We found that in all cell lines, EGF induced a rapid and transient condensation of p190 and RasGAP into cytoplasmic, arclike structures. However, in K ÷ cells the rate of appearance and number of cells exhibiting arcs increased when compared with control cells. Conversely, K-cells exhibited delayed arc formation and a reduction in number of cells forming arcs. EGFinduced actin stress fiber disassembly and reassembly occurred with the same kinetics and frequency as did p190 and RasGAP rearrangements in all three cell lines. These results, together with the documented Rho-GAP activity intrinsic to p190 and the ability of Rho to modulate actin stress fiber formation, suggest that c-Src regulates EGF-dependent actin cytoskeleton reorganization through phosphorylation of p190.C -SRC is a ubiquitous, nonreceptor tyrosine kinase that localizes intracellularly to the cytoplasmic face of the plasma membrane and to poorly defined structures surrounding the nucleus (12, 35). Functionally, it is involved in a variety of biological processes which are associated with cytoskeletal reorganization, including malignant transformation (43), neuronal differentiation (28, 37), bone remodeling (62), and regulated secretion (17, 42). In addition, a requirement for c-Src and its family members in EGF-, PDGF-, and colony-stimulating factor I--dependent mitogenesis has been demonstrated by overexpression and microinjection studies (56,65,70). For example, stable overexpression of wild-type (wt I or K ÷) c-Src in C3H10T1/2 murine fibroblasts en-

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Section: Polyclonal and Monoclonal Antibody Productionmentioning

confidence: 99%

c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation.

Chang

Gill

Settleman

et al. 1995

The Journal of Cell Biology

240

226

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“…These protocols were performed as described in [7,10,15,16] with the following antibodies : c-Src-specific monoclonal antibodies (MAbs) GD11 and EB8 [17,18] ; MAb 2-17 (Quality Biotech, Camden, NJ, U.S.A.) ; c-Src-family rabbit polyclonal antibody Q0 [15,19] ; c-Src-specific rabbit polyclonal antibodies Q8 and Q9 [7] ; Syp\SHPTP2 (Src homology 2-containing phosphotyrosine phosphatase 2)-specific antibody (UBI, Lake Placid, NY, U.S.A.) ; AChRα-and AChRβ-specific MAbs (Transduction Laboratories, Lexington, KY, U.S.A.), AChR-specific MAb 35 [20] ; and the negative antibody controls p120 RasGAP-specific MAb 6F2 [21], anti-phosphotyrosine MAb 4G10 (UBI) and mouse γ-globulin (Jackson Immunoresearch Laboratory, West Grove, PA, U.S.A.). For immunoprecipitation, antigen-antibody complexes were captured on Protein A-Sepharose (Pharmacia).…”

Section: Immunoprecipitation and Immunoblottingmentioning

confidence: 99%

Phosphotyrosine phosphatase activity associated with c-Src in large multimeric complexes isolated from adrenal medullary chromaffin cells

Hoek

Allen

Parsons

1997

Biochemical Journal

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Chromaffin cells, which secrete catecholamines in response to acetylcholine, express high levels of the Src-family tyrosine kinases. These kinases contain protein–protein interaction domains which bind signal transduction proteins that participate in a variety of cellular processes. To determine if signalling proteins bind c-Src in chromaffin cells, we examined c-Src immunocomplexes for co-precipitating proteins. We discovered a phosphotyrosine phosphatase (PTPase; EC 3.1.3.48) activity which associates with specific subcellular pools of c-Src in vivo and which preferentially binds the SH2 (Src homology 2) domain of c-Src in vitro. Known PTPases were not identified by blotting of c-Src immunocomplexes with a panel of anti-PTPase antibodies, suggesting that the PTPase may be a novel family member. The c-Src–PTPase complex is enriched in the plasma membrane fraction and exists in several large complexes, as revealed by gel-filtration analysis. This PTPase activity is altered rapidly following stimulation by secretagogues, decreasing within 30 s and returning to basal levels by 60 s of stimulation. Both the subcellular localization and rapid activity changes suggest that the c-Src-associated PTPase may function in early signalling events emanating from the nicotinic acetylcholine receptor. In support of this is the co-precipitation of a PTPase activity with the nicotinic acetylcholine receptor and co-chromatography of this receptor with one of the c-Src–PTPase complexes.

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“…All procedures were carried out as previously described (Oddie et al, 1989;Ely et al, 1992, Ely et al, A. In Vitro Kinase 1994) utilizing the following antibodies: Src-specific monoclonal antibody (Mab), GD11, whose epitope lies in the SH3 domain of c-Src (Parsons et al, 1984; Mab 2-17, specific for amino acid residues 2-17 of c-Src (Quality Biotech, Camden, NJ); rabbit anti-Fyn antiserum, raised against the peptide-containing residues 29-48 (Horak et al, 1989) (kindly provided by J. Bolen, DNAX Corp., Palo Alto, CA and R. Abraham of Mayo Clinic, Rochester, MN, or else obtained from Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-Lyn (residues 18-62) (Huang et al, 1991) (gifts of R. Abraham and J. Bolen); rabbit anti-Lck (residues 39-64) (Karnitz et al, 1992) (gift of R. Abraham); and rabbit anti-Fgr (residues 48-67), raised according to the methods outlined in Katamine et al (1988) (gifts of P. Leibson of Mayo Clinic, Rochester, MN and J.…”

Section: Antibodies Immunoprecipitations In Vitro Kinase Assays Anmentioning

confidence: 99%

“…In experiments depicted in Figures 1 and 3, one third of each immunoprecipitate was incubated in a kinase reaction mixture containing Y-[~~P]-ATP, and the phosphorylated products were analyzed by SDS-PAGE and autoradiography , while two thirds of each immunoprecipitate were subjected to Western immunoblotting, using QO as a developing antibody. Binding of QO to Src family members was detected with '251-protein A (Ely et al, 1992). In the experiment depicted in Figure 2, entire immunoprecipitates were subjected to immunoblotting , while in Figures 4 and 6, immunoprecitates were divided in half, and each portion was submitted either to an in vitro kinase assay or to a Western immunoblotting procedure.…”

Section: Antibodies Immunoprecipitations In Vitro Kinase Assays Anmentioning

confidence: 99%

Activation of Fyn tyrosine kinase upon secretagogue stimulation of bovine chromaffin cells

et al. 1996

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Adrenal medullary chromaffin cells derive from the neural crest during embryogenesis and differentiate into dedicated secretory cells that release catecholamines in response to acetylcholine in vivo or nicotinic agonists in vitro. Previous studies have indicated that tyrosine kinases participate in early secretagogue‐induced events in these cells and are required for exocytosis. Abundant levels of the cytoplasmic tyrosine kinases, c‐Src and c‐Yes, have been detected in chromaffin cells, thereby implicating them as kinases relevant to these events. However, c‐Src has been found to undergo a decrease in activity following secretagogue‐stimulation, and c‐Yes appears to exist in a constitutively low activity state, suggesting that other tyrosine kinases are involved. Furthermore, other members of the Src family of tyrosine kinases have been implicated as playing roles in secretion in a variety of cell types. Therefore, we sought to determine if other Src family members were present in chromaffin cells, and if so, to examine them for subcellular localization and changes in activity following treatment with nicotinic agonists. To this end, antibodies for Fyn, Lck, Lyn, and Fgr were assembled and used in immunoprecipitation, in vitro autokinase, and Western immunoblotting assays. Of these four kinases, only Fyn was found to be expressed at detectable levels. Differential centrifugation studies revealed that Fyn resides predominantly (>95%) in the crude plasma membrane fraction and undergoes nicotinic‐ and carbachol‐induced activation. This activation is reduced by the nicotinic antagonist, mecamylamine, is not elicited by muscarine, and is dependent upon the presence of extracellular Ca2+. These results suggest that Fyn is involved in signalling through the nicotinic receptor and may be one of the relevant kinases responsible for at least some of the tyrosine phosphorylations detected after stimulation. © 1996 Wiley‐Liss, Inc.

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Immunological methods for the detection of phosphotyrosine-containing proteins in neural tissues

Cited by 4 publications

References 44 publications

c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation.

c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation.

Phosphotyrosine phosphatase activity associated with c-Src in large multimeric complexes isolated from adrenal medullary chromaffin cells

Activation of Fyn tyrosine kinase upon secretagogue stimulation of bovine chromaffin cells

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