Phosphatidylinositol 3-kinase activation by IGF-I is independent of irs-1 in hl-60 cells

Daniel H. Schacher, Roger W. Vanhov, Sean Arkins, Gregory G. Freund, Keith W. Kelley

Research output: Contribution to journalArticle

Abstract

We have used the HL-60 cell line to study IGF-I signaling in myeloid progenitors. Using a radioligand displacement assay and Scatchard analysis, we determined that these cells have 2345 IGF-I receptors with a Kd of 0.719 nM. IP/Western blotting using an anti-phosphotyrosine (aPY) antibody following IGF-I stimulation of quiescent cells induced phosphorylation of a protein of approximately 105 kD which correlates with the expected size of the HL-60 IGF-IR. After IGF-I stimulation, phosphatidyl-inositol (PI) 3kinase activity precipitable with an aPY antibody was induced in a dosedependent manner (0.2 to 200 nM) with a maximal induction of 171-fold. Although PI 3-kinase is often activated after binding to 1RS-1, we used both RT-PCR and IP/western blotting with an IRS-1 specific antibody to determine that this protein is not expressed in HL-60 cells. A second molecule, IRS-2, has recently been implicated in PI 3-kinase activation. PI 3kinase activity co-immunoprecipitated with IRS-2 following IGF-I stimulation was increased 100-fold, indicating a role for this protein in IGF-I signaling. With the intracellular domain of the IGF-I receptor βsubunit derived from HL-60 cDNA as bait, we used the yeast two-hybrid system to screen an HL-60 cDNA library for other proteins which may interact with the phosphorylated receptor. Screening 8 x 106 transformants yielded 53 his+laccolonies. Initial analysis indicates that the p85 regulatory subunit of PI 3kinase directly interacts with the IGF-I receptor. This interaction, along with IRS-2, provides a potential mechanism for IRS-1 independent activation of PI 3-kinase in the HL-60 cells. (NIH Grant AG 026246).

Original languageEnglish (US)
Pages (from-to)A1323
JournalFASEB Journal
Volume10
Issue number6
StatePublished - Dec 1 1996

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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    Schacher, D. H., Vanhov, R. W., Arkins, S., Freund, G. G., & Kelley, K. W. (1996). Phosphatidylinositol 3-kinase activation by IGF-I is independent of irs-1 in hl-60 cells. FASEB Journal, 10(6), A1323.