Multiple oligomerization domains of KANK1-PDGFRβ are required for JAK2-independent hematopoietic cell proliferation and signaling via STAT5 and ERK

Abstract

Background: KANK1-PDGFRB is a fusion gene generated by the t(5;9) translocation between KANK1 and the platelet-derived growth factor receptor beta gene PDGFRB. This hybrid was identified in a myeloproliferative neoplasm featuring severe thrombocythemia, in the absence of the JAK2 V617F mutation.

Design and methods: KANK1-PDGFRB was transduced into Ba/F3 cells and CD34(+) human progenitor cells to gain insights into the mechanisms whereby this fusion gene transforms cells.

Results: Although platelet-derived growth factor receptors are capable of activating JAK2, KANK1-PDGFRβ did not induce JAK2 phosphorylation in hematopoietic cells and a JAK inhibitor did not affect KANK1-PDGFRβ-induced cell growth. Like JAK2 V617F, KANK1-PDGFRβ constitutively activated STAT5 transcription factors, but this did not require JAK kinases. In addition KANK1-PDGFRβ induced the phosphorylation of phospholipase C-γ, ERK1 and ERK2, like wild-type PDGFRβ and TEL-PDGFRβ, another hybrid protein found in myeloid malignancies. We next tested various mutant forms of KANK1-PDGFRβ in Ba/F3 cells and human CD34(+) hematopoietic progenitors. The three coiled-coil domains located in the N-terminus of KANK1 were required for KANK1-PDGFRβ-induced cell growth and signaling via STAT5 and ERK. However, the coiled-coils were not essential for KANK1-PDGFRβ oligomerization, which could be mediated by another new oligomerization domain. KANK1-PDGFRβ formed homotrimeric complexes and heavier oligomers.

Conclusions: KANK1-PDGFRB is a unique example of a thrombocythemia-associated oncogene that does not signal via JAK2. The fusion protein is activated by multiple oligomerization domains, which are required for signaling and cell growth stimulation.

Figure 1.

JAK2 is not required for KPβ-induced cell proliferation or STAT activation. (A) Lysates of Ba/F3-KPβ or Ba/F3-JAK2-V617F cells that were cultured in the absence of IL3 were analyzed by western blot with anti-phospho-JAK2, anti-JAK2 and anti-β-actin antibodies. As a control, Ba/F3 cells transduced with the empty vector were stimulated with IL3 or left untreated (left lane). (B) The same cells were cultured in the presence of 25 nM imatinib or 0.5 μM Jak inhibitor I for 24 h. Cell proliferation was analyzed by measuring [³H]thymidine incorporation. Untreated cells were used as a reference. Total radioactivity incorporation in the DNA of Ba/F3 cells treated with IL3, Ba/F3-KPβ and Ba/F3-JAK2-V617F was 85543 ±2026 cpm, 59098 ±1577 cpm and 44899 ±4846 cpm, respectively. (C) STAT5 phosphorylation was monitored by flow cytometry using cells treated with 0.5 μM imatinib or 2 μM JAK inhibitor I for 4 h. (D, E) JAK2-deficient γ-2A cells were co-transfected with the erythropoietin receptor (EpoR), wild-type JAK2, JAK2-V617F, KPβ or the empty vector, as indicated. Cells were co-transfected with STAT5 and the luciferase reporter pGRR5-luc (D) or pLHRE-luc (E). Cells transfected with EpoR were stimulated by erythropoietin as indicated. STAT-dependent transcriptional activities were measured and normalized using the empty vector control as the reference. One representative experiment is shown with the standard deviation calculated from triplicate measurements.

Figure 2.

Signal transduction by KPβ, TEL-PDGFRβ and activated JAK2. Ba/F3 cells were transduced with KPβ, TEL-PDGFRβ (TPβ) or JAK2-V617F and cultured in the absence of IL3. (A) Cells were treated with 0.1 μM imatinib or 2 μM JAK inhibitor I for 4 h as indicated. As a control, Ba/F3 cells expressing the empty vector were cultured with or without IL3 for 4 h (left lanes). Cell lysates were immunoblotted with antibodies against phosphorylated or total PLC-γ, ERK1/2 or STAT5. (B) Cells were washed and seeded in microtiter plates in the presence of the indicated inhibitor for 24 h. As a control, Ba/F3 cells were cultured with IL3. Tritiated thymidine was added 4 h before harvest and radioactivity incorporated into DNA was quantified. The effect of both inhibitors was statistically significant in all cell lines (P<0.05).

Figure 3.

KPβ coiled coils play an important role in Ba/F3 proliferation and signaling. (A) A schematic representation of KPβ and mutants is shown. Ba/F3 cells were transduced with KPβ, one of the mutants, or the empty vector as control. CC: coiled-coil domain; KOD: KANK1 oligomerization domain; Ig5: Ig-like domain 5 of PDGFRβ; TM: transmembrane domain; Kinase: split kinase domain. (B) Cells were grown for 72 h in the absence of IL3 and proliferation was measured by [³H]thymidine incorporation. Ba/F3-KPβ cells were used as a reference. All cell lines proliferated to a similar extent in the presence of IL3 (data not shown). To quantify STAT5 and ERK1/2 phosphorylation, transduced cells were washed and cultured for 4 h without IL3. Cells were permeabilized, stained with antibodies directed against phospho-STAT5 or phospho–ERK1/2 and analyzed by flow cytometry. (C) Cell lysates were immunoprecipitated overnight with 3.3 μg of FLAG antibody at 4°C to capture KPβ or mutant proteins. Antibody complexes were collected by adding protein-A/G magnetic beads for 1 h at 4°C, washed extensively and analyzed by western blot with anti-PDGFR antibodies.

Figure 4.

Coiled coils are not required for KPβ oligomerization. Regions of KANK1 implicated in KPβ multimerization were identified by their ability to bind to K1-739, after transfection in HEK-293T cells. KPβ or mutants were immunoprecipitated with an anti-PDGFRβ antibody, which recognizes an epitope indicated by the arrow. Co-immunoprecipitated KPβ and K1-739 were visualized by western blot with anti-FLAG antibodies. Only m6 and m12 did not co-precipitate with K1-739. Non-relevant lanes were removed for clarity.

Figure 5.

The KANK1 part of KPβ is required for autophosphorylation and provides additional phosphorylated tyrosine residues. (A) Lysates of Ba/F3 cells expressing the indicated mutant were immunoprecipitated with anti-PDGFRβ antibodies and analyzed by western blot with anti-phosphotyrosine or anti-PDGFRβ antibodies. Alternatively, blots were analyzed using quantitative Odyssey technology, which indicated that normalized PDGFRβ phosphorylation (calculated as the phosphotyrosine to PDGFRβ signal ratio) was decreased by 64% in m1, 65% in m2 and 58% in m3 compared to KPβ. (B) HEK-293T cells were transfected with KPβ together with KANK1, K1-739 or an empty vector. Cells were treated for 4 h with the proteasome inhibitor MG132 (20 μM) to increase KANK1 expression level. KPβ was immunoprecipitated with anti-PDGFRβ antibodies and blotted sequentially with anti-phosphotyrosine and anti-FLAG antibodies. (C) Model for KPβ oligomerization, phosphorylation and signaling. See Figure 3 for abbreviations.

Figure 6.

KPβ stimulates the proliferation of CD34⁺ hematopoietic progenitor cells. CD34⁺ human hematopoietic cells were isolated from cord blood and transduced with lentiviral particles encoding the indicated KPβ construct or GFP. Cells were grown for 14 days in the absence of cytokines (A) or in the presence of a cytokine cocktail containing thrombopoietin, stem cell factor, FLT3-ligand and interleukin-6 (B). Viable cells were counted in the presence of trypan blue. One representative experiment is shown. At the end of the experiment, cells that had been cultured without cytokines were permeabilized and stained with anti-PDGFRβ (C) or anti-phospho-STAT5 (D) antibodies and analyzed by flow cytometry.