Phosphatidylinositol 3-kinase p85{alpha} subunit-dependent interaction with BCR/ABL-related fusion tyrosine kinases: molecular mechanisms and biological consequences - PubMed (original) (raw)

Shu-Yue Ren et al. Mol Cell Biol. 2005 Sep.

Abstract

The p85alpha subunit of phosphatidylinositol 3-kinase (PI-3k) forms a complex with a protein network associated with oncogenic fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFbetaR, and NPM/ALK, resulting in constitutive activation of the p110 catalytic subunit of PI-3k. Introduction of point mutations in the N-terminal and C-terminal SH2 domain and SH3 domain of p85alpha, which disrupt their ability to bind phosphotyrosine and proline-rich motifs, respectively, abrogated their interaction with the BCR/ABL protein network. The p85alpha mutant protein (p85mut) bearing these mutations was unable to interact with BCR/ABL and other FTKs, while its binding to the p110alpha catalytic subunit of PI-3k was intact. In addition, binding of Shc, c-Cbl, and Gab2, but not Crk-L, to p85mut was abrogated. p85mut diminished BCR/ABL-dependent activation of PI-3k and Akt kinase, the downstream effector of PI-3k. This effect was associated with the inhibition of BCR/ABL-dependent growth of the hematopoietic cell line and murine bone marrow cells. Interestingly, the addition of interleukin-3 (IL-3) rescued BCR/ABL-transformed cells from the inhibitory effect of p85mut. SCID mice injected with BCR/ABL-positive hematopoietic cells expressing p85mut survived longer than the animals inoculated with BCR/ABL-transformed counterparts. In conclusion, we have identified the domains of p85alpha responsible for the interaction with the FTK protein network and transduction of leukemogenic signaling.

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Figures

FIG. 1.

FIG. 1.

Interaction of the p85α fragments with BCR/ABL. GST fusion proteins containing the indicated p85α fragments (A) were used for the pull-down assay along with the total cell lysates isolated from p210BCR/ABL-positive 32Dcl3 cells (B) or with p210BCR/ABL produced by IVTT (C). The reactions were resolved by SDS-PAGE and analyzed by Western blotting using anti-ABL (upper box) and anti-GST (lower box) primary antibodies to detect BCR/ABL and GST proteins, respectively.

FIG. 2.

FIG. 2.

Mutations in the p85α SH2 domains disrupt the interaction with p210BCR/ABL. GST fusion proteins containing the indicated p85α SH2 domains and their mutants (A) were used for the pull-down assay along with the total cell lysates isolated from p210BCR/ABL-positive 32Dcl3 cells (B). The reactions were resolved as described in the Fig. 1 legend.

FIG. 3.

FIG. 3.

Mutations in the p85α SH3 domain disrupt the interaction with p210BCR/ABL. GST fusion proteins containing the p85α SH3 domain and the indicated fragments or mutants (A) were used for the pull-down assay along with the total cell lysates isolated from p210BCR/ABL-positive 32Dcl3 cells (B). The reactions were resolved as described in the Fig. 1 legend.

FIG. 4.

FIG. 4.

Mutations in the p85α full-length protein affect the interaction with the p210BCR/ABL protein network. GST fusion proteins containing the p85α wild type (wt) or indicated mutants (A) were used for the pull-down assay along with the total cell lysates isolated from p210BCR/ABL-positive 32Dcl3 cells. (B) The reactions were resolved by SDS-PAGE and analyzed by Western blotting using anti-ABL (top), anti-p110α (middle), and anti-GST (bottom) primary antibodies to detect BCR/ABL, p110α, and GST proteins, respectively. (C) p210BCR/ABL and Flag-p85wt, Flag-p85mut, or empty plasmid were expressed in 293T cells and detected in total cell lysates (Lysate). The presence of BCR/ABL and Flag-p85 proteins in anti-Flag immunoprecipitates was also determined (IP:anti-Flag). (D) GST fusion proteins containing the p85α wild type (wt) or the W55F + T72I + R358L + R649L mutant (mut) were used for the pull-down assay along with the total cell lysates isolated from p210BCR/ABL-positive 32Dcl3 cells. The presence of Crk-L, c-Cbl, Gab2, and Shc in the reactions was determined by Western analysis. (E) GST fusion proteins containing p85wt or p85mut were used for the pull-down assay along with the total cell lysates isolated from BaF3 cells transformed by the indicated FTKs. The reactions were resolved by SDS-PAGE and analyzed by Western blotting using the kinase-specific antibody (upper boxes) and anti-GST antibody (bottom box) to detect the indicated FTKs and GST proteins.

FIG. 5.

FIG. 5.

cSH2 and SH3 domains of p85α interact with Gab2. GST fusion proteins containing p85α or the indicated p85α fragments were used for the pull-down assay along with the total cell lysates isolated from (A) p210BCR/ABL-positive 32Dcl3 cells, (B) 32Dcl3 cells expressing the p190BCR/ABL wild type (wt) or indicated mutants, and (C) GFP+ p210BCR/ABL-positive Gab2+/+ and Gab2−/− cells expressing similar levels of p210BCR/ABL kinase (Lysates). The reactions were examined by Western analysis to detect Gab2 (A, upper box), BCR/ABL (B, C, upper boxes), and GST (lower boxes) proteins.

FIG. 6.

FIG. 6.

The p85α mutant inhibits BCR/ABL-mediated leukemogenesis. (A) The p85α mutant inhibits PI-3k activation and phosphorylation of Akt in vivo. 32Dcl3 parental and BCR/ABL-positive counterparts were infected with p85mutFlag-IRES-GFP (p85mut) or empty IRES-GFP retroviral construct (e). PI-3k enzymatic activity was measured in the kinase assay in vitro using phosphatidylinositol as a substrate. Akt activation was assessed by Western analysis detecting the phosphorylated form of Akt; total Akt protein and actin were detected as loading controls. Anti-Flag antibody was used to confirm the expression of p85mut-Flag protein. (B, C) BCR/ABL-expressing 32Dcl3 cells were infected with p85αmutFlag-IRES-GFP (black bars) or empty IRES-GFP (gray bars) retroviral construct. The proliferation potential of GFP+ cells was analyzed by trypan exclusion in the presence of bovine serum albumin, FBS, or FBS plus IL-3 (B) or clonogenic assay (C). (D) Mouse mononuclear bone marrow cells were coinfected with pSRα-BCR/ABL and p85mutFlag-IRES-GFP retroviral construct or IRES-GFP empty construct. The clonogenic ability of GFP+ cells was evaluated in the absence of growth factors. Results in B, C, and D represent means ± standard deviations from three to four independent experiments (P value calculated by Student's t test). % of clonogenic cells represents the percentage of cells plated that formed colonies. (E) Survival of SCID mice injected with cells transfected with BCR/ABL and empty plasmid (BCR/ABL+E), or BCR/ABL and p85mut (BCR/ABL+p85mut). Survival of the animals was monitored weekly.

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