Regulation of the Src Homology 2-containing Inositol 5-Phosphatase SHIP1 in HIP1/PDGFβR-transformed Cells

Djenann Saint-Dic,Samantha C Chang,Gregory S Taylor,Melissa M Provot,Theodora S Ross

doi:10.1074/jbc.m008336200

Djenann Saint-Dic, Samantha C Chang + Show 3 more

Open Access

PDF Available

https://doi.org/10.1074/jbc.m008336200

Copy DOI

Export

Save

Cite

Abstract
Highlights/Summary
Full-Text PDF
Similar Papers

Abstract

Listen

It has been shown previously that the Huntingtin interacting protein 1 gene (HIP1) was fused to the platelet-derived growth factor beta receptor gene (PDGFbetaR) in leukemic cells of a patient with chronic myelomonocytic leukemia. This resulted in the expression of the chimeric HIP1/PDGFbetaR protein, which oligomerizes, is constitutively tyrosine-phosphorylated, and transforms the Ba/F3 murine hematopoietic cell line to interleukin-3-independent growth. Tyrosine phosphorylation of a 130-kDa protein (p130) correlates with transformation by HIP1/PDGFbetaR and related transforming mutants. We report here that the p130 band is immunologically related to the 125-kDa isoform of the Src homology 2-containing inositol 5-phosphatase, SHIP1. We have found that SHIP1 associates and colocalizes with the HIP1/PDGFbetaR fusion protein and related transforming mutants. These mutants include a mutant that has eight Src homology 2-binding phosphotyrosines mutated to phenylalanine. In contrast, SHIP1 does not associate with H/P(KI), the kinase-dead form of HIP1/PDGFbetaR. We also report that phosphorylation of SHIP1 by HIP1/PDGFbetaR does not change its 5-phosphatase-specific activity. This suggests that phosphorylation and possible PDGFbetaR-mediated sequestration of SHIP1 from its substrates (PtdIns(3,4,5)P(3) and Ins(1,3,4,5)P(4)) might alter the levels of these inositol-containing signal transduction molecules, resulting in activation of downstream effectors of cellular proliferation and/or survival.

Highlights

Chronic myelomonocytic leukemia is a type of myelodysplastic syndrome characterized by dysplastic monocytosis, variable bone marrow fibrosis, and progression to acute leukemia
We provide evidence that a previously unidentified 130-kDa phosphorylated protein, from Ba/F3 cells transformed by plateletderived growth factor ␤ receptor gene (PDGF␤R) fusion proteins, appears highly related, if not identical, to the 125-kDa isoform of SHIP1
We show that Huntingtin interacting protein 1 gene (HIP1)/PDGF␤R and TEL/PDGF␤R physically associate with SHIP1, suggesting that SHIP1 phosphorylation may be catalyzed by the PDGF␤R tyrosine kinase moiety of these fusion proteins

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Culture—Ba/F3, 32D, and FL5.12 cells were grown in RPMI 1640 and 10% fetal calf serum with or without IL-3 (1 ng/ml). 293T cells were grown in Dulbecco’s modified Eagle’s medium and 10% fetal calf serum. Transient Expression in 293T Cells—Murine 293T cells were passaged 1:2 onto 15-cm plates in Dulbecco’s modified Eagle’s medium, 10% fetal calf serum, 10 units/ml penicillin/streptomycin. 48 h after plating, cells were transfected with 15 ␮g of DNA in 450 ␮l of Dulbecco’s modified Eagle’s medium and 90 ␮l of Superfect transfection reagent as described by the manufacturer (Qiagen). Antibody Production—The pGEX-3ЈHIP1 construct was used to express and purify recombinant protein as described by the supplier of the pGEX vector (Amersham Pharmacia Biotech). The 1,500 ϫ g supernatant was subsequently centrifuged for 30 min at 142,000 ϫ g This was designated the total membrane fraction. The membrane suspensions were incubated on ice for 30 min and centrifuged at 142,000 ϫ g for 30 min The supernatant from this was designated the peripheral membrane fraction.

RESULTS

DISCUSSION