Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62

Wong, Gina; Müller, Oliver; Clark, Robin; Conroy, Leah; Moran, Michael F.; Polakis, Paul; McCormick, Frank

doi:10.1016/0092-8674(92)90455-l

Cited by 272 publications

(191 citation statements)

References 34 publications

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“…The recombinant p190 indeed showed GAP activity for the rho family members (Settleman et al, 1992b), suggesting that GAP-p190 complex may coordinate Ras and rho signaling pathways in cells in response to EGF. Cloning of a cDNA for GAP-associated p62 was also reported (Wong et al, 1992). Deduced amino acid sequence of this cDNA showed that it was related to heterogeneous nuclear ribonucleoprotein (hnRNP), speci®cally GRP33 and the puri®ed protein bound DNA and mRNA (Wong et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

“…Cloning of a cDNA for GAP-associated p62 was also reported (Wong et al, 1992). Deduced amino acid sequence of this cDNA showed that it was related to heterogeneous nuclear ribonucleoprotein (hnRNP), speci®cally GRP33 and the puri®ed protein bound DNA and mRNA (Wong et al, 1992). However, recent independent studies led to the conclusion that the previously cloned p62 cDNA is actually the gene encoding for Sam-68, a mitotic substrate of Src protein tyrosine kinase (Lock et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

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Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor

Tang

Feng

1997

Oncogene

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The SH3-SH3-SH3-SH2 adapter protein Nck links receptor tyrosine kinases, such as EGF and PDGF receptors, to downstream signaling pathways, among which p21 cdc42/rac -activated kinase cascade, Sos-activated Ras signaling and the human Wiskott-Aldrich Syndrome protein (WASp)-mediated actin cytoskeleton changes, have been implicated. In EGF stimulated cells, Nck coimmunoprecipitates with a number of phosphotyrosine proteins including the EGF receptor (Li et al., 1992 Mol. Cell. Biol. 12: 5824 ± 2833). To identify the phosphotyrosine protein(s) that directly interacts with Nck and to distinguish it from indirectly associated proteins, preexisting phosphoytrosine protein complexes in the cell lysate were dissociated by heat and SDS prior to the test for binding to Nck. We found that Nck does not directly bind to EGF receptor, instead it binds via its SH2 domain to a 62 kDa phosphotyrosine protein. We present evidence demonstrating that the Nck-bound p62 is related to the previously identi®ed GTPase-activating protein (GAP)-associated phosphotyrosine protein p62.(1) The Nck-bound and the GAP-bound p62 proteins comigrate with each other in SDS ± PAGE. (2) SH2 domains from Nck and GAP compete for binding to p62 in vitro. (3) Puri®ed GST-Nck-SH2 binds directly to the GAP-associated p62. Under these conditions, SH2 domains from PLCg, PI-3 kinase, SHC, and Grb2 did not bind p62. (4) Tryptic phosphopeptide maps of the Nck-and the GAP-associated p62 proteins are identical. However, Nck and GAP do not co-immunoprecipitate with each other and apparently bind to di erent pools of p62. This study suggests that the GAP-associated p62 acts as an SH2 domain docking protein and mediates the interaction between Nck and EGF receptor in response to EGF stimulation.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor

Tang

Feng

1997

Oncogene

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“…As a ®rst simple screening experiment we tested the electrophoretic mobility of plasmid DNA in an agarose gel in the presence of the puri®ed APC protein fragments ( Figure 5) (Wong et al, 1992). In this assay the DNA showed retardation only in presence of three APC fragments: APC(1340 ± 1901) that is located within the b-catenin binding region, and APC(2219 ± 2580) and APC(2581 ± 2843), the two carboxyterminal fragments.…”

Section: Recombinant Apc Protein Fragments Interact With Dnamentioning

confidence: 99%

“…APC precipitation with DNA-cellulose DNA binding of the APC protein present in the nuclear extracts was tested in a DNA-cellulose precipitation assay (Wong et al, 1992). 2.5 ± 5 mg cellulose carrying immobilized DNA (Pharmacia/Amersham/Biotec) in BI2 (50 mM Tris/ HCl (pH 7.6), 50 mM NaCl, 2.5 mM EDTA, 5% glycerol) was added to the nuclear lysate.…”

Section: Preparation Of Nuclear Extractsmentioning

confidence: 99%

“…Gel retardation assays were used for mapping the DNA binding domain, for determining the a nity of the APC protein fragments to DNA, for the binding site selection assay and for competition assays (Wong et al, 1992;Berman et al, 1987). For agarose gel shift experiments 1 mg plasmid DNA (pGEX4T3) was incubated with 100 ng of one of the bacterially expressed and puri®ed APC protein fragments for 30 min, electrophoresed in 0.5 ± 0.7% agarose and visualized by ethidium bromide staining.…”

Section: Electrophoretic Mobility Shift Assaysmentioning

confidence: 99%

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The APC protein binds to A/T rich DNA sequences

et al. 1999

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The tumor suppressor protein APC (Adenomatous Polyposis Coli) is localized in the cytosol and in the nucleus. In this study, we demonstrate that the nuclear APC protein level is high in cells in the basal crypt region of the normal colorectal epithelium. Strikingly, the APC protein staining resembles the staining pattern of a nuclear proliferation marker. As a ®rst step towards a possible role of the nuclear APC protein, we provide data showing the direct interaction of the nuclear APC protein with DNA. A nuclear APC isoform precipitates with matrix-immobilized DNA. Vice versa, the immunoprecipitation of APC from nuclear lysates results in coprecipitation of genomic DNA. Using recombinant APC fragments we mapped three DNA binding domains: one within the b-catenin binding and regulatory domain, and two in the carboxyterminal third of the APC protein. All these three domains contain clusters of repetitive S(T)PXX sequence motifs that were described to mediate the DNA interaction of many other DNA binding proteins. In analogy to S(T)PXX proteins, the APC protein binds preferentially to A/T rich DNA sequences rather than to a single DNA sequence motif.

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Novel RNA-binding motif: The KH module

Adinolfi¹,

Bagni

Morelli

et al. 1999

Biopolymers

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The KH motif has recently been identified in single or multiple copies in a number of RNA associated proteins. Here we review the current knowledge accumulated about the sequence, structure, and functions of the KH. The multidomain architecture of most of the KH‐containing proteins inspired an approach based on the production of peptides spanning the sequence of an isolated KH motif. Correct identification of the minimal length necessary for producing a folded peptide has had a number of important consequences for interpreting functional data. The presence of the KH motifs in fmr1, the protein responsible for the fragile X syndrome, and their possible role in the fmr1 functions are also discussed. © 1999 John Wiley & Sons, Inc. Biopoly 51: 153–164, 1999

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Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62

Cited by 272 publications

References 34 publications

Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor

Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor

The APC protein binds to A/T rich DNA sequences

Novel RNA-binding motif: The KH module

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