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Titolo:
Interaction of the insulin receptor beta-subunit with phosphatidylinositol3-kinase in bovine ROS
Autore:
Rajala, RVS; Anderson, RE;
Indirizzi:
Univ Oklahoma, Hlth Sci Ctr, Dept Ophthalmol, Oklahoma City, OK USA Univ Oklahoma Oklahoma City OK USA ept Ophthalmol, Oklahoma City, OK USA Univ Oklahoma, Hlth Sci Ctr, Dept Cell Biol, Oklahoma City, OK USA Univ Oklahoma Oklahoma City OK USA Dept Cell Biol, Oklahoma City, OK USA Univ Oklahoma, Hlth Sci Ctr, Dept Biochem & Mol Biol, Oklahoma City, OK 73190 USA Univ Oklahoma Oklahoma City OK USA 73190 iol, Oklahoma City, OK 73190 USA Dean A McGee Eye Inst, Oklahoma City, OK USA Dean A McGee Eye Inst Oklahoma City OK USA e Inst, Oklahoma City, OK USA
Titolo Testata:
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
fascicolo: 13, volume: 42, anno: 2001,
pagine: 3110 - 3117
SICI:
0146-0404(200112)42:13<3110:IOTIRB>2.0.ZU;2-7
Fonte:
ISI
Lingua:
ENG
Soggetto:
NUCLEOTIDE-GATED CHANNELS; OUTER SEGMENTS; GROWTH-FACTOR; TYROSINE KINASE; SIGNAL-TRANSDUCTION; HIGH-AFFINITY; I RECEPTORS; SH2 DOMAIN; PHOSPHORYLATION; ACTIVATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
49
Recensione:
Indirizzi per estratti:
Indirizzo: Rajala, RVS 608 Stanton L Young Blvd,Room 409, Oklahoma City, OK 73104 USA 608 Stanton L Young Blvd,Room 409 Oklahoma City OK USA 73104
Citazione:
R.V.S. Rajala e R.E. Anderson, "Interaction of the insulin receptor beta-subunit with phosphatidylinositol3-kinase in bovine ROS", INV OPHTH V, 42(13), 2001, pp. 3110-3117

Abstract

PURPOSE. To identify the tyrosine-phosphorylated protein(s) in bovine rod outer segments (ROS) that are associated with phosphatidylinositol 3-kinase(PI3K). METHODS. Glutathione-S-transferase (GST) fusion proteins containing two SH2 domains of the p85 regulatory subunit of PI3K-GST-p85 (N-SH2), GST-p85 (C-SH2), and respective SH2 mutants (N-SH2, R358A, and C-SH2, R649A)-were prepared and used to pull down tyrosine-phosphorylated proteins in bovine ROS. Protein identity was established by Western blot analysis. PI3K activity was determined in the pull-down mixtures and in immunoprecipitates by incubation with phosphatidylinositol-4,5-bisphosphate (PI-4,5-P-2) and [gamma P-32]adenosine triphosphate (ATP). RESULTS. The GST pull-down assays indicated the binding of a 97-kDa protein by GST-p85 (N-SH2) in tyrosine-phosphorylated (PY)-ROS that was not present in nonphosphorylated (N)-ROS. Binding was completely abolished when the Arg 358 in the N-SH2 domain was mutated to Ala. Increased binding of the p110 alpha catalytic subunit to GST-p85 (N-SH2) fusion protein was also observed in the presence of the 97-kDa phosphorylated protein. Biochemical evidence indicated that the 97-kDa protein was the beta -subunit of the insulin receptor beta -subunit (IR beta). Immunoprecipitates of PY-ROS and N-ROS with anti-PY antibodies, probed with anti-IR beta, indicated the presence of IR beta only in PY-ROS. Immunoprecipitates of PY-ROS and N-ROS with anti-IRbeta antibodies, probed with anti-p85 and anti-p110 alpha antibodies, indicated increased amounts of both p85 and p110 alpha in PY-ROS compared to N-ROS. Treatment of ROS with insulin, followed by immunoprecipitation with either anti-IR beta or anti-PY, resulted in increased PI3K activity. Expression and phosphorylation of the cytoplasmic tail of retina insulin receptor showed direct involvement with the p85 subunit of P13K in vitro. CONCLUSIONS. Tyrosine phosphorylation of the beta -subunit of the insulin receptor is involved in the regulation of P13K activity in ROS.

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Documento generato il 02/04/20 alle ore 12:35:31