- Email: [email protected]
Tyrosine phosphorylation of SHIP promotes its proteasomal degradation
Experimental Hematology 2010;38:392–402
Tyrosine phosphorylation of SHIP promotes its proteasomal degradation Jens Ruschmanna,b, Victor Hoa, Frann Antignanoa, Etsushi Kurodac, Vivian Lama, Mariko Ibarakia, Kim Snydera, Connie Kima, Richard A. Flavelld, Toshiaki Kawakamie, Laura Slyf, Ali G. Turhang, and Gerald Krystala a
The Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada; bFachbereich Biologie, Chemie, Pharmazie, Freie Universita¨tBerlin, Berlin, Germany; cDepartment of Immunology and Parasitology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu, Japan; dDepartment of Immunobiology, Yale University, School of Medicine and the Howard Hughes Medical Institute, New Haven, Conn., USA; eDivision of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, Calif., USA; fDepartment of Pediatrics, Division of Gastroenterology, BC Children’s Hospital and University of British Columbia, Vancouver, BC, Canada; gNSERM U935, Universite´ de Poitiers and Service d’He´matologie et d’Oncologie Biologique, CHU de Poitiers, Poitiers, France (Received 2 March 2010; revised 2 March 2010; accepted 8 March 2010)
Objective. The activity of the SH2-containing-phosphatidylinositol-50 -phosphatase (SHIP, also known as SHIP1), a critical hematopoietic-restricted negative regulator of the PI3 kinase (PI3K) pathway, is regulated in large part via its protein levels. We sought to determine the mechanism(s) involved in its downregulation by BCR-ABL and by interleukin (IL)-4. Materials and Methods. We used Ba/F3p210-tetOFF cells to study the downregulation of SHIP by BCR-ABL and bone marrowLderived macrophages to study SHIP’s downregulation by IL-4. Results. We show herein that BCR-ABL downregulates SHIP, but not SHIP2 or PTEN, and this can be blocked with the Src kinase inhibitor PP2, which inhibits the tyrosine phosphorylation of SHIP, or with the proteasomal inhibitor MG-132. We also show, using anti-SHIP immunoprecipitates, that c-Cbl and Cbl-b are associated with SHIP and that BCR-ABL induces SHIP’s polyubiquitination. This ubiquitination can be blocked with PP2, consistent with the tyrosine phosphorylation of SHIP acting as a signal for its ubiquitination. In bone marrowLderived macrophages, IL-4 also leads to the proteasomal degradation of SHIP but, unlike in Ba/F3p210-tetOFF cells, SHIP2 is also proteasomally degraded and the degradation of both inositol phosphatases can be prevented with PP2 or MG-132. Conclusion. Our results suggest that SHIP protein levels can be reduced via BCR-ABL and/or Src family member-induced tyrosine phosphorylation of SHIP because this triggers its polyubiquitination and degradation within the proteasome. Ó 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.
The 145-kDa hematopoietic-restricted SH2-containinginositol-50 -phosphatase (SHIP, also known as SHIP1), negatively regulates the PI3 kinase (PI3K) pathway by hydrolyzing the 50 -phosphate group from the critical PI3K-generated second messenger, PI-3,4,5-P3 [1]. In doing so, SHIP restrains both myelopoiesis and mature immune cell activation. Recent studies have revealed that SHIP’s activity can be regulated in large part by its protein levels [2,3]. For example, we have demonstrated that Toll-like receptors 4 and 9 trigger, via the MyD88-dependent pathway, the production of autocrineOffprint requests to: Gerald Krystal, Ph.D., The Terry Fox Laboratory, British Columbia Cancer Agency, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada; E-mail: [email protected]
acting transforming growth factorb. This cytokine, in turn, stimulates a 10-fold increase in SHIP protein levels and this increased SHIP is essential for preventing chronic bacterial or viral infections from triggering an overly enthusiastic host immune response, which can lead to septic shock and death [2,3]. On the other hand, studies looking at the effect of the chronic myelogenous leukemia oncogene, BCR-ABL, have shown that it causes a loss of SHIP protein and that this loss, via an increased PI3K pathway, gives a selective survival/proliferation advantage to late chronic myelogenous leukemia progenitors [4,5]. Moreover, very recent studies in our laboratory have shown that SHIP is downregulated during interleukin (IL)-4induced skewing of macrophages from an M1 ‘‘killer’’ to an M2 ‘‘healer’’ macrophage phenotype (L Sly
0301-472X/10 $–see front matter. Copyright Ó 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2010.03.010
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
et al, manuscript in preparation) and it is possible that this reduction in SHIP is important for this conversion. While the mechanism responsible for the upregulation of SHIP has been elucidated to some extent [2,3], very little is known about the mechanisms involved in the downregulation of SHIP and that is the focus of the current study.
Material and methods Reagents All reagents were purchased from Sigma-Aldrich (St Louis, MO, USA) unless otherwise stated. Tissue culture media, penicillin/ streptomycin, recombinant murine IL-3, recombinant murine macrophage colony-stimulating factor, and recombinant murine IL-4 were purchased from StemCell Technologies (Vancouver, BC, Canada). NP-40, PP2 (10 mM stock in dimethyl sulfoxide), and doxycycline (dox; 5 mg/mL in ethanol) were from Calbiochem (Darmstadt, Germany). MG-132 (carbobenzoxy-Leu-Leu-Leu-H) and E-64d were from Sigma-Aldrich. Antibodies (Abs) against c-Cbl (SC-170), Cbl-b (G-1), PTEN (SC-7974), or SHIP (P1C1) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Ab against glyceraldehyde-3-phosphate dehydrogenase was from Fitzgerald (Concord, MA, USA). Anti-SHIP2 antibody was kindly provided by Dr. Bayard Clarkson Memorial Sloan-Kettering Cancer Center, New York, NY, USA). A c-Abl Ab (Ab-3; Calbiochem) was used for the detection of BCR-ABL. Anti-mouse or anti-rabbit Abs conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Montreal, PQ, Canada) were used as secondary Abs for Western blot analysis. Anti-mouse IL-4 blocking Ab was purchased from R&D Systems (Burlington, Canada). The phospho-SHIP (Y1020) and SHIP (Cat#01506) Abs were from StemCell Technologies. Mice SHIPþ/þ (wild-type [WT]) F2 mice on a mixed C57Bl/6 x 129Sv background were used between 6 and 12 weeks of age. The mice were housed in the Animal Resource Centre of the British Columbia Cancer Research Centre under specific pathogen-free conditions and according to approved and ethical treatment of animal standards of the University of British Columbia. Animals were euthanized by CO2 asphyxiation. Femurs and tibia from mice deficient in Fyn and Lyn/Fyn were generously provided by Dr. J. Rivera (National Institutes of Health, Bethesda, MD, USA) and from mice deficient in c-Cbl from Dr. R. Hodes (National Institutes of Health). Femurs and tibia from mice deficient in Lyn, Hck, and Lyn/Hck were from T.K. and from mice deficient in signal transducers and activators of transcription (STAT) 6 from R.A.F. All these knockout mice were on a C57Bl/6 background and femurs and tibia obtained from WT C57Bl/6 littermates were sent and used as controls. Cell cultures Ba/F3p210-tetOFF cells were cultured in RPMI-1640 þ 10% fetal calf serum (FCS) and penicillin/streptomycin in a humidified 5% CO2 tissue culture incubator. Unless stated otherwise, Ba/F3p210-tetOFF cells were cultured in 5 ng/mL IL-3containing medium for at least 24 hours at 37 C before stimulation. For inhibitor studies, cells were resuspended at 1 105cells/mL in fresh 5 ng/mL IL-3containing medium and inhibitors added at the indicated concentrations. To
393
stop the stimulations, cells were put on ice, counted, and washed once with ice-cold phosphate-buffered saline and lysed in 1 sodium dodecyl sulfate (SDS) sample buffer at 1 107cells/mL and boiled for 2 minutes at 95 C. Genomic DNA was sheared by passing samples 10 times through a 26-gauge needle. To generate bone marrowderived macrophages (BMM4s), bone marrow cells were aspirated from mouse femurs and tibia using ice-cold Iscove’s modified Dulbecco’s medium þ 10% FCS, pipetted through a 100-mm nylon cell strainer to break up cell clumps and cultured in bone marrow (BM) medium (Iscove’s modified Dulbecco’s medium, 10% FCS, 0.00125% monothioglycerol) in a 75-cm2 tissue culture flask for 18 hours for adherence depletion. Nonadherent cells (50 mL) were then cultured at 5 105 nucleated cells/mL in a 175-cm2 tissue culture flask in BMM4 medium (Iscove’s modified Dulbecco’s medium, 10% FCS, penicillin/streptomycin, 115 mM monothioglycerol, and 10 ng/mL recombinant murine macrophage colony-stimulating factor). The medium was replaced every 3 days. After 10 to 12 days, the medium was aspirated from the flasks and BMM4s lifted off the plates using Cell Dissociation Buffer. 1 mL 5 105c/mL BMM4s in BM medium was plated in a 12-well dish for 18 hours to allow cells to adhere. The medium was then replaced with fresh BMM4 medium (control) or BMM4 medium plus 10 ng/mL IL-4. If cells were treated with PP2 or MG-132, other samples were treated with an equal volume of DMSO as a solvent control. To lyse the cells, the medium was aspirated, washed once with ice-cold phosphate-buffered saline and 100 mL 1 SDS sample buffer added. Total cell lysates were transferred into fresh 1.7-mL reaction tubes. In one experimental protocol, cells were washed once with ice-cold phosphorylation solubilization buffer (PSB) [6] and lysed in 75 mL PSB lysis buffer (PSB þ2% TX100, 0.1% NP-40 5, 2 mg/mL leupeptin, 10 mg/mL aprotinin, 500 mM phenylmethylsulfonyl fluoride or they were washed with PSB þ 10 mM MG-132 and lysed in 75 mL PSB lysis buffer þ 10 mM MG-132. Lysed cells were then transferred into fresh 1.7-mL reaction tubes and 25 mL 4 SDS sample buffer added. Cell lysates were boiled for 2 minutes at 95 C and genomic DNA sheared.
Immunoprecipitations Anti-SHIP (clone P1C1) Ab was coupled to Protein G-agarosebeads by washing 40 mL Protein-G agarose beads three times in PSB þ 0.1% bovine serum albumin/0.1% TX100, resuspending in 90 mL of the same buffer and adding 2 mg Ab. The Protein-G agarose bead/Ab mix was then nutated at 4 C for 18 hours and the beads washed five times in the same buffer to remove unbound Ab and resuspended in 100 mL of the same buffer. Ba/F3p210tetOFF cells (5 106 cells, cultured as indicated) were washed once, resuspended in 200 mL PSB þ 0.1% bovine serum albumin and lysed with 200 mL PSB þ 0.1% bovine serum albumin/2% TX100/4 mg/mL leupeptin, 20 mg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride on a nutator at 4 C for 1 hour. The nuclei were then pelleted at 16,000g for 10 minutes at 4 C and the nuclei-free supernatant mixed with 100 mL prepared Ab-ProteinG agarose beads and nutated at 4 C overnight. The beads were washed five times with PSB þ 0.1% TX100, resuspended in 50 mL SDS sample buffer and heated at 95 C for 2 minutes. The supernatant was then subjected to Western blot analysis.
394
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
Western blot analysis SDS polyacrylamide gel electrophoresis was performed using a Protean II electrophoresis system. Cells were loaded as cell equivalents from total cell lysates. After separation by SDS polyacrylamide gel electrophoresis, proteins were transferred to polyvinylidine difluoride membranes using a Trans Blot Cell transfer system (Biorad) as per manufacturer’s instructions and probed as described previously [6].
Results BCR-ABL induces downregulation of SHIP but not SHIP2 or PTEN To confirm earlier reports that BCR-ABL reduces SHIP protein levels [4,5], we employed the Ba/F3p210tetOFF system depicted in Supplementary Figure E1A (online only, available at www.exphem.org). Using this cell system, Ba/F3p210-tetOFF cells were transferred from cytokine-free medium (since BCR-ABL expression enables these cells to grow in the absence of IL-3) into medium containing either IL-3 or IL-3 þ dox (to turn off BCR-ABL expression) and the expression of SHIP and the other two major lipid phosphatases, SHIP2 and PTEN, were monitored for up to 7 days by Western blot analysis. As shown in the top panel of Figure 1A, the addition of IL-3 þ dox dramatically reduced BCR-ABL protein levels within 24 hours and they remained low for the duration of the study. SHIP levels, in response, increased dramatically within 24 hours and plateaued at approximately fivefold the level observed in the presence of BCR-ABL for the 7 days of the study. On the other hand, the addition of IL-3 alone to these cells did not significantly increase SHIP levels. Cells from day 0 to day 3 (marked with an asterisk in Fig. 1A) were also subjected to intracellular staining with anti-SHIP Ab and confirmed that IL-3 alone did not change SHIP levels in the presence of BCR-ABL and that dox-induced reduction of BCR-ABL increased SHIP levels to the same extent in all cells (Supplementary Fig. E1B, online only, available at www.exphem.org). Interestingly, SHIP2 levels did not change upon the addition of either IL-3 þ dox or IL-3 alone (second panel of Fig. 1A). PTEN levels, on the other hand, were substantially increased (i.e., two- to threefold) when the cells were transferred into IL-3 þ dox. However, this increase was due to the addition of IL-3 rather than the loss of BCR-ABL because IL-3 alone also caused a similar increase (Fig. 1A). These results are consistent with BCR-ABL affecting SHIP but not SHIP2 or PTEN levels. Because IL-3 increased expression of PTEN, further experiments with Ba/F3p210-tetOFF cells were carried out in the presence of IL-3 to ensure that SHIP2 and PTEN levels remained constant throughout. To gain some insight into which pathways BCR-ABL used to reduce SHIP levels, Ba/F3p210-tetOFF cells were
grown in the absence of dox with different pathway inhibitors. As shown in Supplementary Figure E1C (online only, available at www.exphem.org), only PP2, a Src kinase family inhibitor, prevented the loss of SHIP protein in the presence of BCR-ABL. Because Src family members have been implicated as the kinases responsible for the tyrosine phosphorylation of SHIP [713], at least in some hematopoietic cells, this suggested that the tyrosine phosphorylation of SHIP might promote its own downregulation. Related to this, BCR-ABL’s tyrosine kinase activity has been reported to be required for reducing SHIP levels [5]. We therefore asked if BCR-ABL, directly or indirectly via a Src kinase, led to the tyrosine phosphorylation of SHIP and if SHIP’s tyrosine phosphorylation correlated with its reduced protein levels. As shown in the left lane of Figure 1B, SHIP was highly phosphorylated on its NPXY motifs (i.e., phosphorylated SHIP (pSHIP)/SHIP 5 3.1) in Ba/F3p210-tetOFF cells growing in IL-3 alone (so BCR-ABL is expressed). However, 24 hours after the addition of dox, when BCR-ABL levels were very low, we detected very low levels of pSHIP even though total SHIP levels were much higher (i.e., pSHIP/SHIP 5 0.2, a 15.5-fold drop) (right lane, Fig. 1B). Interestingly, a 24hour treatment with the Src kinase inhibitor PP2, which did not significantly affect BCR-ABL levels, resulted in total SHIP levels being higher than under normal culture conditions but lower than after dox treatment. Tyrosinephosphorylated SHIP levels, on the other hand, were lower than under normal culture conditions, but higher than in dox-treated cells (i.e., pSHIP/SHIP 51.1) (middle lane, Fig. 1B). Because PP2 has been reported to inhibit BCRABL as well as Src kinases [14], we cannot conclude from these studies that SHIP is tyrosine-phosphorylated by BCR-ABL directly or through the activation of a Src kinase. To determine how fast SHIP reaches its maximal levels after the addition of dox to Ba/F3p210-tetOFF cells, we monitored pSHIP and total and SHIP levels, as well as BCRABL protein levels, by Western analysis during a 24-hour period. As shown in Figure 1C, BCR-ABL levels started to drop steeply after 3 hours of dox treatment and were barely detectable by 24 hours. Tyrosine phosphorylated SHIP levels also decreased rapidly and showed a nice correlation with BCR-ABL levels. On the other hand, total SHIP levels only started to increase after 16 hours, reaching their maximum at 19 hours. Thus the loss of BCRABLinduced pSHIP precedes the increase in total SHIP protein levels. To gain some insight into how quickly SHIP levels drop in the presence of BCR-ABL, we preincubated Ba/F3p210-tetOFF cells growing in the absence of dox (i.e., high BCR-ABL levels) with PP2 (to maintain SHIP levels in the presence of BCR-ABL) and, after 24 hours, we washed out the PP2 and monitored total and pSHIP levels over time by Western analysis (Fig. 1D). As expected, BCR-ABL was expressed
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
395
Figure 1. BCR-ABL induces the downregulation of SH2-containing-phosphatidylinositol-50 -phosphatase (SHIP) but not SHIP2 or PTEN. (A) Ba/F3p210-tetOFF cells were incubated with interleukin (IL)-3 or IL-3 þ doxycycline (dox) for up to 7 days and Western analysis of total cell lysates (TCLs) was carried out using antibodies (Abs) to c-ABL, SHIP2, SHIP, PTEN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a loading control. (B) Ba/F3p210-tetOFF cells were treated for 24 hours 6 10 mM PP2 or 1 mg/mL dox and TCLs subjected to Western analysis using Abs to c-Abl, phosphorylated SHIP (pSHIP), SHIP, and GAPDH. (C) Ba/F3p210-tetOFF cells were incubated with 1 mg/mL dox for the indicated times and TCLs subjected to Western analysis as in (B). (D) Ba/F3p210-tetOFF cells were incubated with 10 mM PP2 for 24 hours. Cells were then washed twice to remove PP2 and TCLs taken at the indicated times for Western analysis as in (B). All blots shown are representative of at least two independent experiments.
396
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
Figure 2. BCR-ABL induces the polyubiquitination and proteasomal degradation of SH2-containing-phosphatidylinositol-50 -phosphatase (SHIP). (A) In the left panel, Ba/F3p210-tetOFF cells were treated 6 100 nM MG-132 for 24 hours and total cell lysates (TCLs) subjected to Western analysis for phosphorylated SHIP (pSHIP), SHIP, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the right panel, Ba/F3p210-tetOFF cells were treated or not with vehicle control (dimethyl sulfoxide), 100 nM MG-132, 1 mg/mL dox, 10 mM PP2, or 25 mM E64d for 24 hours and TCLs subjected to Western analysis for BCR-ABL, pSHIP, SHIP, SHIP2, PTEN, and GAPDH. (B) Ba/ F3p210-tetOFF cells were treated with 10 mM PP2, 100 nM MG-132, or 1 mg/mL doxycycline (dox) for 24 hours and SHIP immunoprecipitated with bead-bound anti-SHIP antibody (Ab). The immunoprecipitates were subjected to Western analysis with Abs to ubiquitin, BCR-ABL (left panel) and the ubiquitin blot reprobed for SHIP, using StemCell anti-SHIP #01506 (right panel). (C) Ba/F3p210-tetOFF cells were treated 6 10 mM PP2 or 1 mg/mL dox for 24 hours and TCLs subjected to Western analysis as indicated (left panel) or immunoprecipitated with bead-bound anti-SHIP Abs (right panel) and the immunoprecipitates subjected to Western analysis with antic-CBL, Cbl-b, and SHIP. All blots shown are representative of at least two independent experiments.
over the entire course of the experiment and any variation mirrored the variation in the glyceraldehyde-3-phosphate dehydrogenase loading control. After 24 hours with PP2 we found low levels of pSHIP and high total SHIP levels compared to control samples without PP2 treatment. After washing out the PP2, we observed a large increase in SHIP
phosphorylation within 30 minutes, but there was no significant drop in total SHIP levels until 3 hours, at which time the total and phosphorylated SHIP levels were back to control levels. This difference in timing suggested, once again, that the tyrosine phosphorylation of SHIP may act as a trigger for its downregulation.
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
BCR-ABL induces the polyubiquitination and proteasomal degradation of SHIP Because previous studies in our laboratory and others have shown that SHIP is a long-lived protein with a half-life O18 hours [5,6], its relatively short half-life of !3 hours in the presence of BCR-ABL (Fig. 1D) suggested that BCR-ABL may be inducing SHIP degradation rather than simply turning off SHIP gene transcription and/or translation. Because the proteasome is the main site of protein degradation in the cell and has been shown to play a critical role in BCR-ABLmediated pathogenesis [1521], we investigated the role of the proteasome in the BCRABLinduced reduction of SHIP levels. Specifically, we incubated Ba/F3p210-tetOFF cells, growing in IL-3 alone (so BCR-ABL is expressed), with the proteasome inhibitor MG-132 for 24 hours. As shown in the left panel of Figure 2A, both pSHIP and total SHIP levels increased dramatically with MG-132, suggesting that BCR-ABL downregulates SHIP by inducing its proteasomal degradation. However, because MG-132 has been reported, at high concentrations, to also inhibit calpains [22], we tested the calpain-specific inhibitor, E-64d, but found that even at 25 mM it did not increase SHIP or pSHIP levels (Fig. 2A, right panel). It is worthy of note in this figure, as in the left panel of Figure 2A, that in the presence of MG-132, the level of pSHIP increased significantly. In this same study, we examined the effect of MG-132, dox, and PP2 on the levels of SHIP2, PTEN and found that MG-132, dox, or PP2 did not increase SHIP2 or PTEN levels. To explore the putative proteasomal degradation of SHIP further, we immunoprecipitated SHIP from Ba/F3p210-tetOFF cells growing in IL-3 vs IL-3 plus either PP2, MG-132, or dox and carried out Western analysis using anti-ubiquitin Abs. As shown in the left panel of Figure 2B, SHIP was polyubiquitinated when BCR-ABL (IL-3 alone) or MG132 was present (to prevent proteasomal degradation of ubiquitinated SHIP), but was far less ubiquitinated when PP2 was added (consistent with the tyrosine phosphorylation of SHIP being a trigger for ubiquitination) or when dox was present (when BCR-ABL is turned off). An antiSHIP reprobe indicated the amount of SHIP in each lane (left panel of Fig. 2B) and showed that the higher molecular weight SHIP bands co-migrated with the ubiquitinated bands (right panel of Fig. 2B). Probing the same Western blot with antiBCR-ABL Abs demonstrated that SHIP was physically associated with BCR (left panel of Fig. 2B). This is consistent with, but does not prove that, BCR-ABL directly tyrosine-phosphorylates SHIP because Src family members have been reported to physically associate with BCR-ABL as well [2326]. However, attempts to show the presence of the Src family members Src, Lyn, Hck, or Fyn in reprobes of this or other similar Western blots were unsuccessful (data not shown). Because the ubiquitin ligase Cbl has been reported to interact with BCR-ABL [27], we then asked if it might
397
be involved in SHIP’s polyubiquitination. Before doing this, we first assessed the levels of Cbl, i.e., c-Cbl and Cbl-b, in total cell lysates from Ba/F3p210-tetOFF cells, treated with or without dox or PP2 for 24 hours. As shown in the left panel of Figure 2C, these two proteins were detectable under all three conditions and their levels were not affected by the presence or absence of BCR-ABL or PP2. We then immunoprecipitated SHIP (right panel of Fig. 2C) from Ba/F3p210-tetOFF cells treated with or without dox or PP2 for 24 hours and found that c-Cbl and Cbl-b were indeed present in SHIP immunoprecipitates. IL-4 directly downregulates SHIP and SHIP2 levels in BMM4s To explore the mechanism(s) involved in the downregulation of SHIP in normal primary cells, we examined IL-4induced M2 skewing of BMM4s because other studies in our laboratory demonstrated that IL-4 treatment of BMM4s downregulates SHIP levels while upregulating M2 markers, such as arginase 1 and Ym1 (Sly et al, manuscript in preparation). First, to determine if this IL-4induced reduction of SHIP occurred via IL-4 directly or through IL-4induced secretion and autocrine action of a secondary factor, we IL-4treated macrophage colony-stimulating factorderived BMM4s and examined SHIP levels during a 3-day period. As shown in Figure 3A, by 3 days, all the full-length SHIP was broken down into smaller fragments. We also found that SHIP2 was no longer detectable following 3 days of treatment with IL-4 (Fig. 3A). This was surprising because we did not observe a change in SHIP2 levels when BCR-ABL was expressed in Ba/F3p210-tetOFF cells. Thus, the mechanisms involved in reducing SHIP2 protein levels appear to vary with the cell type and/or stimulus involved. To determine if an IL4induced, autocrine-acting factor was involved in this reduction of SHIP and SHIP2, we added conditioned medium (CM) from BMM4s that had been incubated with IL-4 for 3 days to naı¨ve BMM4s in the presence or absence of IL4blocking Ab for 2 days. BMM4s treated with CM showed a reduction in SHIP and SHIP2 that was blocked by antiIL4. We therefore conclude that IL-4 directly induces the reduction of SHIP levels in BMM4s. Given that IL-4 was directly responsible for the downregulation of SHIP and SHIP2, and that IL-4 is known to mediate many of its effects through STAT6 [28], we also asked if the presence of STAT6 was required for IL-4induced reduction of SHIP levels and, as shown in Figure 3B, this was indeed the case. Interestingly, although SHIP levels were not reduced in IL4stimulated STAT6/ BMM4s, SHIP was still tyrosinephosphorylated, suggesting that STAT6 was required for SHIP degradation downstream of SHIP’s phosphorylation. IL-4-induced degradation of SHIP and SHIP2 is blocked with PP2 and MG-132 As shown in Figure 3A, SHIP levels decreased more slowly in IL-4treated BMM4s than in BCR-ABLexpressing
398
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
Figure 3. Interleukin (IL)-4 directly downregulates SH2-containing-phosphatidylinositol-50 -phosphatase (SHIP) and SHIP2 levels in bone marrowderived macrophages (BMM4s). (A) BMM4s in BMM4 medium were incubated with 10 ng/mL IL-4 for up to 3 days. As well, conditioned medium (CM) from day 3 IL-4treated cultures was diluted such that the IL-4 5 4 ng/mL, assuming no significant loss of IL-4 during the 3-day incubation, and incubated with day 3 BMM4s 6 aIL-4 (CM þ aIL-4) for 2 days and total cell lysates (TCLs) subjected to Western analysis with antibodies to SHIP2, SHIP, and glyceraldehyde3-phosphate dehydrogenase (GAPDH). (B) BMM4s derived from wild-type (WT) or signal transducers and activators of transcription (STAT)6/ mice were incubated in BMM4 medium without IL-4 (control) or with 10 ng/mL IL-4 for 2 or 3 days and TCLs subjected to Western analysis for phosphorylated SHIP (pSHIP), SHIP, and GAPDH. Blots shown are representative of at least two independent experiments. The arrows indicate full length-SHIP. The dashed lines on the blots indicate that while the lanes to the right and left of the dashed lines were from the same blot and the same exposure, they were not adjacent and so the blot was cut to juxtapose the lanes.
Ba/F3 cells, with the biggest drop in SHIP occurring between day 2 and day 3 (Fig. 3A). To determine if this reduction in SHIP protein was due to proteasomal degradation, we added MG-132 during the last 24 hours of IL-4 treatment (i.e., from day 2 to day 3). This was necessary because MG-132, even at 10 nM, was quite toxic and we could not expose the BMM4s to it for 3 days without causing significant cell death. We found the MG-132 inhibited the degradation of full-length SHIP that occurs between day 2 and 3, i.e., MG-132 prevented further reduction of SHIP protein levels compared to control samples (left panel of Fig. 4A), without any detectable loss in cell viability (data not shown). MG-132 also prevented the loss of SHIP2 protein, as shown in the right panel of Figure 4A. Because both IL-4 in BMM4s and BCR-ABL in Ba/ F3p210-tetOFF cells induced proteasomal SHIP degradation, we tested if PP2 was also able to prevent IL-4mediated SHIP degradation in BMM4s. Specifically, we incubated BMM4s with IL-4 for 2 days with or without PP2 and, as shown in Figure 4B, the presence of PP2 resulted in more full-length SHIP protein. Because there is no BCR-ABL present in BMM4s, it therefore seems likely that IL-4 induces SHIP degradation through Src kinases in these cells, while BCR-ABL may directly induce SHIP phosphorylation and subsequent degradation in Ba/F3p210-tetOFF cells. However, treatment of BMM4s deficient in different Src kinases (i.e., Lyn, Hck, Fyn, Fyn/Lyn, or Lyn/Hck) that have been shown to play a role in the tyrosine phosphorylation of SHIP [7,10,12,2931] could not prevent IL-4induced degradation of SHIP (Fig. 4C, left and right panels), indicating that Src kinases were either able to substitute for each other or that a PP2-sensitive non-Src kinase was inducing SHIP’s degradation in BMM4s.
SHIP in BMM4s is primed for proteasomal degradation Finally, given that we had shown in earlier studies that SHIP is rapidly degraded from the C-terminus if cells are lysed with nonionic detergents (e.g., NP40 or TX100) rather than with SDS sample buffer [6], we asked if SHIP was already primed for rapid degradation via the proteasome and all that was needed was a trigger, such as Src kinase-induced tyrosine phosphoryation, to induce this process. To test this, we treated BMM4s for 2 days with or without IL-4. The cells were then washed with phosphate-buffered saline and lysed with 2% TX100 þ 0.1% NP40 in the presence of protease inhibitors to prevent nonspecific protein degradation or washed and lysed in the same way in the presence of 10 mM MG-132 to inhibit proteasomal degradation. As shown in Figure 4D, after 2 minutes of lysis at 23 C, SHIP, but not glyceraldehyde-3-phosphate dehydrogenase, levels were greatly reduced in the absence of MG-132, regardless of whether they were treated or not with IL-4.
Discussion Considering the potential importance of BCR-ABLinduced and IL-4induced downregulation of SHIP, very little is known as yet about how they are regulated. Taken together, our data with Ba/F3p210tetOFF cells suggest a model of BCR-ABLinduced SHIP degradation, shown in Figure 5, in which BCR-ABL forms a complex with SHIP, c-Cbl, and Cbl-b and either tyrosine-phosphorylates SHIP directly or via a Src family member. The lack of highly specific Src family inhibitors and the fact that BCR-ABL has been shown to physically associate with Src family members [24,32] make this a difficult issue to resolve. Nonetheless, we propose that the tyrosine phosphorylation of SHIP triggers its
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
399
Figure 4. Interleukin (IL)-4induced degradation of SH2-containing-phosphatidylinositol-50 -phosphatase (SHIP) is blocked with PP2 and MG-132. (A) In the left panel, bone marrowderived macrophages (BMM4s) in BMM4 medium were incubated without IL-4 (control) or with 10 ng/mL IL-4 for the days indicated. For one sample, 10 nM MG-132 was added for 24 hours from day 2 to 3 and total cell lysates (TCLs) subjected to Western analysis for SHIP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Similar results were obtained when MG-132 was used at 0.1 mM and 1 mM (not shown). In the right panel, BMM4s in BMM4 medium were incubated without IL-4 (control) or with 10 ng/mL IL-4 for the days indicated. For one sample, 10 nM MG-132 was added for 24 hours from day 2 to 3 and TCLs subjected to Western analysis for SHIP2, SHIP, and GAPDH. (B) BMM4s in BMM4 medium were incubated without IL-4 (control) or with 10 ng/mL IL-4 for up to 2 days or with 10 ng/mL IL-4 þ 10 mM PP2 for 2 days. (C) BMM4s from either wild-type (WT), Lyn/, Hck/, or Lyn/Hck/ mice (left panel) or from WT, Fyn/, and Fyn/Lyn/ mice (right panel) were incubated in BMM4 medium without IL-4 (control) or with 10 ng/mL IL-4 6 10 mM PP2 for 2 days and phosphorylated SHIP (pSHIP) and SHIP levels determined by Western analysis. (D) BMM4s in BMM4 medium were incubated without IL-4 (control) or with 10 ng/mL IL-4 for 2 days. The cells were then either washed 1 with phosphate-buffered saline (PBS) and lysed with phosphorylation solubilization buffer (PSB) þ 2% TX100 þ 0.1% NP40 for 2 minutes at 23 C in the presence of protease inhibitors (-MG-132 lysis) or washed and lysed the same way but in the presence of 10 mM MG-132 (þMG-132 lysis). All blots shown are representative of at least two independent experiments. The arrows indicate full length SHIP. The dashed lines on the blots indicate that while the lanes to the left and right of the dashed lines were from the same blot and the same exposure, they were not adjacent and so the blot was cut to juxtapose the lanes.
400
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
Figure 5. Model of BCR-ABL- and interleukin (IL)-4induced proteasomal degradation of SH2-containing-phosphatidylinositol-50 -phosphatase (SHIP). BCR-ABL and other oncogenic tyrosine kinases (e.g., constitutively active c-kit), directly or indirectly via Src family kinases, tyrosine phosphorylate SHIP and this acts as a trigger for SHIP to be polyubiquitinated, possibly by c-Cbl and/or Cbl-b, and degraded in the proteasome. IL-4 treatment of bone marrowderived macrophages (BMM4s) also leads to the tyrosine phosphorylation of SHIP, via src family members, and its signal transducers and activators of transcription (STAT)6-dependent degradation in the proteasome. Dashed arrows indicate uncertain pathways.
polyubiquitination, via c-Cbl and/or Cbl-b, and subsequent proteasomal degradation. Our finding that, in the presence of BCR-ABL, SHIP’s tyrosine phosphorylation precedes its degradation and that PP2 reduces SHIP’s polyubiquitination and degradation lends support to this model. Interestingly, protein levels of SHIP2 and PTEN, two other lipid phosphatases that act as negative regulators of the PI3K pathway in hematopoietic cells, appear to be unaffected by the presence of BCR-ABL but PTEN levels are increased with IL-3. This increase in PTEN might serve as a negative feedback mechanism to limit IL-3induced PI3K activation and subsequent cell proliferation. The specific susceptibility of SHIP to BCR-ABLinduced degradation demonstrates that SHIP, SHIP2, and PTEN protein levels are regulated, at least in part, by different mechanisms in Ba/F3p210tetOFF cells. To understand how SHIP is downregulated by normal physiological regulators, we also investigated IL-4treated BMM4s and found that IL-4 induces the proteasomal degradation of SHIP directly and not through the secretion and autocrine action of a secondary factor (as observed in lipopolysaccharide (LPS)-induced upregulation of SHIP by secreted transforming growth factorb [2] and, as found
with BCR-ABLinduced degradation, it could be prevented with PP2 or MG-132. Moreover, we found in IL-4treated STAT6/ BMM4s that SHIP still becomes tyrosinephosphorylated but not degraded, suggesting that STAT6 is required either for polyubiquitination or a subsequent step prior to proteasomal degradation (Fig. 5). However, unlike in Ba/F3p210tetOFF cells, we found that treatment of BMM4s with IL-4 leads to reduced levels of SHIP2 as well, indicating that the regulation of SHIP2 varies with cell type and/or stimulus. Interestingly, in this regard, both SHIP and SHIP2 contain PEST domains, i.e., domains rich in prolines (P), glutamic acids (E), serines (S), and threonines (T) [33], and these domains have been shown to direct eukaryotic proteins for proteasomal degradation [34]. Our results further suggest that SHIP may be present in a complex in BMM4s that allows for rapid degradation through the proteasome, because the lysis of BMM4s with TX-100 and NP-40, instead of SDS sample buffer, results in breakdown of SHIP that is prevented when MG-132 is present in the lysis buffer. Such an effect was not seen for glyceraldehyde-3-phosphate dehydrogenase, suggesting that this was not due to nonspecific degradation and might
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
suggest that disruption of intracellular compartments by nonionic detergents might allow for Src family or other tyrosine kinases to phosphorylate SHIP, and perhaps other susceptible proteins, leading to proteasomal degradation. Because BCR-ABL was not present in our BMM4s, we hypothesize that the block in IL-4induced SHIP breakdown with PP2 is due to inhibition of a Src family kinase. However, as shown in Figure 4C, IL-4 treatment of BMM4s derived from mice lacking Lyn, Fyn, Hck, Fyn/ Lyn, or Lyn/Hck results in SHIP degradation to the same extent as in WT cells. Importantly, though, treatment with PP2 reduced the phosphorylation and prevented degradation. Thus we conclude that either other Src family members are able to substitute for the one(s) knocked out or PP2 inhibits a non-Src kinase that phosphorylates the critical tyrosine(s) involved in degradation. Relevant to the first possibility, c-Src has been shown to be upregulated in LPS-stimulated Lyn/Fgr/Hck triple knockout macrophages and compensates for the loss of these other Src family members in triggering nitric oxide and tumor necrosis factora secretion [35]. Moreover, a recent report showing that Lyn//Hck/ mice, which have a very similar phenotype to SHIP/ mice, can be rescued by expression of a membrane-bound form of SHIP suggests that the tyrosine phosphorylation of SHIP by Lyn plus Hck acts as a trigger for SHIP’s relocation [29]. This is supported by an earlier report showing that SHIP translocates to the actin cytoskeleton upon Src-mediated tyrosine phosphorylation in activated human platelets [36]. However, our current results show that tyrosine phosphorylation is also involved in SHIP degradation because this can be blocked by PP2. Thus, a dual role for SHIP’s tyrosine phosphorylation seems most likely. Related to this, the anti-pSHIP Abs we employed in this study were against one of the two NPXpY motifs and recent studies have shown that SHIP becomes tyrosine-phosphorylated following stimulation on other tyrosines as well [37], and it is conceivable that the two phosphorylated NPXY motifs, which are attracted to proteins with phosphotyrosine binding domains [38], are strictly involved in the translocation of SHIP to the plasma membrane to hydrolyze PI-3,4,5-P3, while some of these other tyrosines might be involved in triggering polyubiquitination and subsequent proteasomal degradation. If this is the case, it will be interesting to elucidate which tyrosinephosphorylation patterns induce SHIP’s translocation vs degradation. Acknowledgments We would like to thank Dr. R. Hodes (National Institutes of Health, Bethesda, MD, USA) for c-Cbl deficient bones and Christine Kelly for preparing the manuscript. This work was supported by the Terry Fox Foundation (Vancouver, BC, Canada) and the Canadian Cancer Society (Toronto, Ontario, Canada), with core support from the BC Cancer Foundation (Vancouver, BC, Canada) and the BC Cancer Agency (Vancouver, BC, Canada).
401
Conflict of Interest Disclosure G.K. is a founding member and Chief Scientific Advisor of Aquinox Pharmaceuticals Inc (Vancouver, BC, Canada), which is dedicated to identifying small molecule activators and inhibitors of SHIP.
References 1. Antignano F, Ruschmann J, Hamilton M, et al. The Src homology 2 containing inositol 5’ phosphatases. In: Bradsaw RA, Dennis EA, eds. Handbook of Cell Signalling. San Diego: Elsevier Inc; 2009. p. 1065–1084. 2. Sly LM, Rauh MJ, Kalesnikoff J, Song CH, Krystal G. LPS-induced upregulation of SHIP is essential for endotoxin tolerance. Immunity. 2004;21:227–239. 3. Sly LM, Hamilton MJ, Kuroda E, et al. SHIP prevents lipopolysaccharide from triggering an anti-viral response in mice. Blood. 2009;113:2945– 2954. 4. Sattler M, Salgia R, Shrikhande G, et al. The phosphatidylinositol polyphosphate 5-phosphatase SHIP and the protein tyrosine phosphatase SHP-2 form a complex in hematopoietic cells which can be regulated by BCR/ABL and growth factors. Oncogene. 1997;15:2379–2384. 5. Sattler M, Verma S, Byrne CH, et al. BCR/ABL directly inhibits expression of SHIP, an SH2-containing polyinositol-5-phosphatase involved in the regulation of hematopoiesis. Mol Cell Biol. 1999;19: 7473–7480. 6. Damen JE, Liu L, Ware MD, Ermolaeva M, Majerus PW, Krystal G. Multiple forms of SHIP are generated by C-terminal truncation. Blood. 1998;92:1199–1205. 7. Gardai S, Whitlock BB, Helgason C, et al. Activation of SHIP by NADPH oxidase-stimulated Lyn leads to enhanced apoptosis in neutrophils. J Biol Chem. 2002;277:5236–5246. 8. Malbec O, Attal JP, Fridman WH, Daeron M. Negative regulation of mast cell proliferation by FcgRIIB. Mol Immunol. 2002;38:1295–1299. 9. Hibbs ML, Harder KW, Armes J, et al. Sustained activation of Lyn tyrosine kinase in vivo leads to autoimmunity. J Exp Med. 2002; 196:1593–1604. 10. Baran CP, Tridandapani S, Helgason CD, Humphries RK, Krystal G, Marsh CB. The inositol 5’-phosphatase SHIP-1 and the Src kinase lyn negatively regulate macrophage colony-stimulating factor-induced Akt activity. J Biol Chem. 2003;278:38628–38636. 11. Harder KW, Quilici C, Naik E, et al. Perturbed myelo/erythropoiesis in Lyn-deficient mice is similar to that in mice lacking the inhibitory phosphatases SHP-1 and SHIP-1. Blood. 2004;104:3901–3910. 12. Hernandez-Hansen V, Smith AJ, Surviladze Z, et al. Dysregulated FcgRI signaling and altered Fyn and SHIP activities in Lyn-deficient mast cells. J Immunol. 2004;173:100–112. 13. Xiao W, Nishimoto H, Hong H, et al. Positive and negative regulation of mast cell activation by Lyn via the FcgRI. J Immunol. 2005;175: 6885–6892. 14. Tatton L, Morley GM, Chopra R, Khwaja A. The Src-selective kinase inhibitor PP1 also inhibits Kit and Bcr-Abl tyrosine kinases. J Biol Chem. 2003;278:4847–4853. 15. Jagani Z, Song K, Kutok JL, et al. Proteasome inhibition causes regression of leukemia and abrogates BCR-ABL-induced evasion of apoptosis in part through regulation of forkhead tumor suppressors. Cancer Res. 2009;69:6546–6555. 16. Crawford LJ, Windrum P, Magill L, et al. Proteasome proteolytic profile is linked to Bcr-Abl expression. Exp Hematol. 2009;37:357–366. 17. Magill L, Lynas J, Morris TC, Walker B, Irvine AE. Proteasome proteolytic activity in hematopoietic cells from patients with chronic myeloid leukemia and multiple myeloma. Haematologica. 2004;89:1428–1433. 18. Gatto S, Scappini B, Pham L, et al. The proteasome inhibitor PS-341 inhibits growth and induces apoptosis in Bcr/Abl-positive cell lines sensitive and resistant to imatinib mesylate. Haematologica. 2003; 88:853–863.
402
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
19. Tauchi T, Yoshimura A, Ohyashiki K. CIS1, a cytokine-inducible SH2 protein, suppresses BCR/ABL-mediated transformation. Involvement of the ubiquitin proteasome pathway. Exp Hematol. 2001;29: 356–361. 20. Dou QP, McGuire TF, Peng Y, An B. Proteasome inhibition leads to significant reduction of Bcr-Abl expression and subsequent induction of apoptosis in K562 human chronic myelogenous leukemia cells. J Pharmacol Exp Ther. 1999;289:781–790. 21. Dai Z, Quackenbush RC, Courtney KD, et al. Oncogenic Abl and Src tyrosine kinases elicit the ubiquitin-dependent degradation of target proteins through a Ras-independent pathway. Genes Dev. 1998;12: 1415–1424. 22. Kojima M, Oguro K, Sawabe K, et al. Rapid turnover of tryptophan hydroxylase is driven by proteasomes in RBL2H3 cells, a serotonin producing mast cell line. J Biochem. 2000;127:121–127. 23. Danhauser-Riedl S, Warmuth M, Druker BJ, Emmerich B, Hallek M. Activation of Src kinases p53/56 lyn and p59 hck by p210 bcr/abl in myeloid cells. Cancer Res. 1996;56:3589–3596. 24. Warmuth M, Bergmann M, Priess A, Hauslmann K, Emmerich B, Hallek M. The Src family kinase Hck interacts with Bcr-Abl by a kinaseindependent mechanism and phosphorylates the Grb2-binding site of Bcr. J Biol Chem. 1997;272:33260–33270. 25. Lionberger JM, Wilson MB, Smithgall TE. Transformation of myeloid leukemia cells to cytokine independence by Bcr-Abl is suppressed by kinase-defective Hck. J Biol Chem. 2000;275:18581–18585. 26. Wilson MB, Schreiner SJ, Choi HJ, Kamens J, Smithgall TE. Selective pyrrolo-pyrimidine inhibitors reveal a necessary role for Src family kinases in Bcr-Abl signal transduction and oncogenesis. Oncogene. 2002;21:8075–8088. 27. Brehme M, Hantschel O, Colinge J, et al. Charting the molecular network of the drug target Bcr-Abl. Proc Natl Acad Sci U S A. 2009;106:7414–7419. 28. Takeda K, Tanaka T, Shi W, et al. Essential role of Stat6 in IL-4 signalling. Nature. 1996;380:627–630.
29. Xiao W, Hong H, Kawakami Y, Lowell CA, Kawakami T. Regulation of myeloproliferation and M2 macrophage programming in mice by Lyn/Hck, SHIP, and Stat5. J Clin Invest. 2008;118:924–934. 30. Chari R, Kim S, Murugappan S, Sanjay A, Daniel JL, Kunapuli SP. Lyn, PKC-d, SHIP-1 interactions regulate GPVI-mediated platelet-dense granule secretion. Blood. 2009;114:3056–3063. 31. Xu R, Abramson J, Fridkin M, Pecht I. SH2 domain-containing inositol polyphosphate 5’-phosphatase is the main mediator of the inhibitory action of the mast cell function-associated antigen. J Immunol. 2001; 167:6394–6402. 32. Ptasznik A, Urbanowska E, Chinta S, et al. Crosstalk between BCR/ABL oncoprotein and CXCR4 signaling through a Src family kinase in human leukemia cells. J Exp Med. 2002;196:667–678. 33. Backers K, Blero D, Paternotte N, Zhang J, Erneux C. The termination of PI3K signalling by SHIP1 and SHIP2 inositol 5-phosphatases. Adv Enzyme Regul. 2003;43:15–28. 34. Schnupf P, Portnoy DA, Decatur AL. Phosphorylation, ubiquitination and degradation of listeriolysin O in mammalian cells: role of the PEST-like sequence. Cell Microbiol. 2006;8:353–364. 35. Leu TH, Charoenfuprasert S, Yen CK, Fan CW, Maa MC. Lipopolysaccharide-induced c-Src expression plays a role in nitric oxide and TNFalpha secretion in macrophages. Mol Immunol. 2006;43:308–316. 36. Giuriato S, Bodin S, Erneux C, et al. pp60c-src associates with the SH2-containing inositol-5-phosphatase SHIP1 and is involved in its tyrosine phosphorylation downstream of aIIbb3 integrin in human platelets. Biochem J. 2000;348:107–112. 37. Cao L, Yu K, Banh C, et al. Quantitative time-resolved phosphoproteomic analysis of mast cell signaling. J Immunol. 2007;179:5864– 5876. 38. DiNitto JP, Lambright DG. Membrane and juxtamembrane targeting by PH and PTB domains. Biochim Biophys Acta. 2006;1761:850–867. 39. Dugray A, Geay JF, Foudi A, et al. Rapid generation of a tetracyclineinducible BCR-ABL defective retrovirus using a single autoregulatory retroviral cassette. Leukemia. 2001;15:1658–1662.
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
402.e1
Supplementary Figure E1. (A) Ba/F3p210-tetOFF cells were derived from Ba/F3 cells as described in data from [39]. In the absence of doxycycline (dox) both BCR-ABL and the tTa (Tet repressor-VP16 fusion protein) get expressed. Increasing levels of tTa lead to higher activity of the minimal cytomegalovirus (CMV) (CMVmin) promoter, which, in turn, leads to increasing BCR-ABL and tTa levels. Upon addition of dox, tTa cannot bind to the CMVmin promoter and BCR-ABL as well as tTa levels decrease. (B) Ba/F3p210-tetOFF cells were transferred into medium containing IL-3 only (þIL-3) or IL-3 and dox (þIL-3/ dox) for up to 3 days and the cells intracellularly stained for SH2-containing-phosphatidylinositol-50 -phosphatase (SHIP). (C) Left panel, Ba/F3p210-tetOFF cells were incubated with IL-3 alone (þIL-3), IL-3 þ dox or IL-3 þ the indicated inhibitors for 24 hours and total cell lysates (TCLs) subjected to Western analysis. The blot shown is representative of two independent experiments. Right panel, list of inhibitors, their targets, and the final concentration used (based on studies showing these concentrations blocked the targeted pathways in Ba/F3 cells [unpublished]).
Tyrosine phosphorylation of SHIP promotes its proteasomal degradation Jens Ruschmanna,b, Victor Hoa, Frann Antignanoa, Etsushi Kurodac, Vivian Lama, Mariko Ibarakia, Kim Snydera, Connie Kima, Richard A. Flavelld, Toshiaki Kawakamie, Laura Slyf, Ali G. Turhang, and Gerald Krystala a
The Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada; bFachbereich Biologie, Chemie, Pharmazie, Freie Universita¨tBerlin, Berlin, Germany; cDepartment of Immunology and Parasitology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu, Japan; dDepartment of Immunobiology, Yale University, School of Medicine and the Howard Hughes Medical Institute, New Haven, Conn., USA; eDivision of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, Calif., USA; fDepartment of Pediatrics, Division of Gastroenterology, BC Children’s Hospital and University of British Columbia, Vancouver, BC, Canada; gNSERM U935, Universite´ de Poitiers and Service d’He´matologie et d’Oncologie Biologique, CHU de Poitiers, Poitiers, France (Received 2 March 2010; revised 2 March 2010; accepted 8 March 2010)
Objective. The activity of the SH2-containing-phosphatidylinositol-50 -phosphatase (SHIP, also known as SHIP1), a critical hematopoietic-restricted negative regulator of the PI3 kinase (PI3K) pathway, is regulated in large part via its protein levels. We sought to determine the mechanism(s) involved in its downregulation by BCR-ABL and by interleukin (IL)-4. Materials and Methods. We used Ba/F3p210-tetOFF cells to study the downregulation of SHIP by BCR-ABL and bone marrowLderived macrophages to study SHIP’s downregulation by IL-4. Results. We show herein that BCR-ABL downregulates SHIP, but not SHIP2 or PTEN, and this can be blocked with the Src kinase inhibitor PP2, which inhibits the tyrosine phosphorylation of SHIP, or with the proteasomal inhibitor MG-132. We also show, using anti-SHIP immunoprecipitates, that c-Cbl and Cbl-b are associated with SHIP and that BCR-ABL induces SHIP’s polyubiquitination. This ubiquitination can be blocked with PP2, consistent with the tyrosine phosphorylation of SHIP acting as a signal for its ubiquitination. In bone marrowLderived macrophages, IL-4 also leads to the proteasomal degradation of SHIP but, unlike in Ba/F3p210-tetOFF cells, SHIP2 is also proteasomally degraded and the degradation of both inositol phosphatases can be prevented with PP2 or MG-132. Conclusion. Our results suggest that SHIP protein levels can be reduced via BCR-ABL and/or Src family member-induced tyrosine phosphorylation of SHIP because this triggers its polyubiquitination and degradation within the proteasome. Ó 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.
The 145-kDa hematopoietic-restricted SH2-containinginositol-50 -phosphatase (SHIP, also known as SHIP1), negatively regulates the PI3 kinase (PI3K) pathway by hydrolyzing the 50 -phosphate group from the critical PI3K-generated second messenger, PI-3,4,5-P3 [1]. In doing so, SHIP restrains both myelopoiesis and mature immune cell activation. Recent studies have revealed that SHIP’s activity can be regulated in large part by its protein levels [2,3]. For example, we have demonstrated that Toll-like receptors 4 and 9 trigger, via the MyD88-dependent pathway, the production of autocrineOffprint requests to: Gerald Krystal, Ph.D., The Terry Fox Laboratory, British Columbia Cancer Agency, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada; E-mail: [email protected]
acting transforming growth factorb. This cytokine, in turn, stimulates a 10-fold increase in SHIP protein levels and this increased SHIP is essential for preventing chronic bacterial or viral infections from triggering an overly enthusiastic host immune response, which can lead to septic shock and death [2,3]. On the other hand, studies looking at the effect of the chronic myelogenous leukemia oncogene, BCR-ABL, have shown that it causes a loss of SHIP protein and that this loss, via an increased PI3K pathway, gives a selective survival/proliferation advantage to late chronic myelogenous leukemia progenitors [4,5]. Moreover, very recent studies in our laboratory have shown that SHIP is downregulated during interleukin (IL)-4induced skewing of macrophages from an M1 ‘‘killer’’ to an M2 ‘‘healer’’ macrophage phenotype (L Sly
0301-472X/10 $–see front matter. Copyright Ó 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2010.03.010
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
et al, manuscript in preparation) and it is possible that this reduction in SHIP is important for this conversion. While the mechanism responsible for the upregulation of SHIP has been elucidated to some extent [2,3], very little is known about the mechanisms involved in the downregulation of SHIP and that is the focus of the current study.
Material and methods Reagents All reagents were purchased from Sigma-Aldrich (St Louis, MO, USA) unless otherwise stated. Tissue culture media, penicillin/ streptomycin, recombinant murine IL-3, recombinant murine macrophage colony-stimulating factor, and recombinant murine IL-4 were purchased from StemCell Technologies (Vancouver, BC, Canada). NP-40, PP2 (10 mM stock in dimethyl sulfoxide), and doxycycline (dox; 5 mg/mL in ethanol) were from Calbiochem (Darmstadt, Germany). MG-132 (carbobenzoxy-Leu-Leu-Leu-H) and E-64d were from Sigma-Aldrich. Antibodies (Abs) against c-Cbl (SC-170), Cbl-b (G-1), PTEN (SC-7974), or SHIP (P1C1) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Ab against glyceraldehyde-3-phosphate dehydrogenase was from Fitzgerald (Concord, MA, USA). Anti-SHIP2 antibody was kindly provided by Dr. Bayard Clarkson Memorial Sloan-Kettering Cancer Center, New York, NY, USA). A c-Abl Ab (Ab-3; Calbiochem) was used for the detection of BCR-ABL. Anti-mouse or anti-rabbit Abs conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Montreal, PQ, Canada) were used as secondary Abs for Western blot analysis. Anti-mouse IL-4 blocking Ab was purchased from R&D Systems (Burlington, Canada). The phospho-SHIP (Y1020) and SHIP (Cat#01506) Abs were from StemCell Technologies. Mice SHIPþ/þ (wild-type [WT]) F2 mice on a mixed C57Bl/6 x 129Sv background were used between 6 and 12 weeks of age. The mice were housed in the Animal Resource Centre of the British Columbia Cancer Research Centre under specific pathogen-free conditions and according to approved and ethical treatment of animal standards of the University of British Columbia. Animals were euthanized by CO2 asphyxiation. Femurs and tibia from mice deficient in Fyn and Lyn/Fyn were generously provided by Dr. J. Rivera (National Institutes of Health, Bethesda, MD, USA) and from mice deficient in c-Cbl from Dr. R. Hodes (National Institutes of Health). Femurs and tibia from mice deficient in Lyn, Hck, and Lyn/Hck were from T.K. and from mice deficient in signal transducers and activators of transcription (STAT) 6 from R.A.F. All these knockout mice were on a C57Bl/6 background and femurs and tibia obtained from WT C57Bl/6 littermates were sent and used as controls. Cell cultures Ba/F3p210-tetOFF cells were cultured in RPMI-1640 þ 10% fetal calf serum (FCS) and penicillin/streptomycin in a humidified 5% CO2 tissue culture incubator. Unless stated otherwise, Ba/F3p210-tetOFF cells were cultured in 5 ng/mL IL-3containing medium for at least 24 hours at 37 C before stimulation. For inhibitor studies, cells were resuspended at 1 105cells/mL in fresh 5 ng/mL IL-3containing medium and inhibitors added at the indicated concentrations. To
393
stop the stimulations, cells were put on ice, counted, and washed once with ice-cold phosphate-buffered saline and lysed in 1 sodium dodecyl sulfate (SDS) sample buffer at 1 107cells/mL and boiled for 2 minutes at 95 C. Genomic DNA was sheared by passing samples 10 times through a 26-gauge needle. To generate bone marrowderived macrophages (BMM4s), bone marrow cells were aspirated from mouse femurs and tibia using ice-cold Iscove’s modified Dulbecco’s medium þ 10% FCS, pipetted through a 100-mm nylon cell strainer to break up cell clumps and cultured in bone marrow (BM) medium (Iscove’s modified Dulbecco’s medium, 10% FCS, 0.00125% monothioglycerol) in a 75-cm2 tissue culture flask for 18 hours for adherence depletion. Nonadherent cells (50 mL) were then cultured at 5 105 nucleated cells/mL in a 175-cm2 tissue culture flask in BMM4 medium (Iscove’s modified Dulbecco’s medium, 10% FCS, penicillin/streptomycin, 115 mM monothioglycerol, and 10 ng/mL recombinant murine macrophage colony-stimulating factor). The medium was replaced every 3 days. After 10 to 12 days, the medium was aspirated from the flasks and BMM4s lifted off the plates using Cell Dissociation Buffer. 1 mL 5 105c/mL BMM4s in BM medium was plated in a 12-well dish for 18 hours to allow cells to adhere. The medium was then replaced with fresh BMM4 medium (control) or BMM4 medium plus 10 ng/mL IL-4. If cells were treated with PP2 or MG-132, other samples were treated with an equal volume of DMSO as a solvent control. To lyse the cells, the medium was aspirated, washed once with ice-cold phosphate-buffered saline and 100 mL 1 SDS sample buffer added. Total cell lysates were transferred into fresh 1.7-mL reaction tubes. In one experimental protocol, cells were washed once with ice-cold phosphorylation solubilization buffer (PSB) [6] and lysed in 75 mL PSB lysis buffer (PSB þ2% TX100, 0.1% NP-40 5, 2 mg/mL leupeptin, 10 mg/mL aprotinin, 500 mM phenylmethylsulfonyl fluoride or they were washed with PSB þ 10 mM MG-132 and lysed in 75 mL PSB lysis buffer þ 10 mM MG-132. Lysed cells were then transferred into fresh 1.7-mL reaction tubes and 25 mL 4 SDS sample buffer added. Cell lysates were boiled for 2 minutes at 95 C and genomic DNA sheared.
Immunoprecipitations Anti-SHIP (clone P1C1) Ab was coupled to Protein G-agarosebeads by washing 40 mL Protein-G agarose beads three times in PSB þ 0.1% bovine serum albumin/0.1% TX100, resuspending in 90 mL of the same buffer and adding 2 mg Ab. The Protein-G agarose bead/Ab mix was then nutated at 4 C for 18 hours and the beads washed five times in the same buffer to remove unbound Ab and resuspended in 100 mL of the same buffer. Ba/F3p210tetOFF cells (5 106 cells, cultured as indicated) were washed once, resuspended in 200 mL PSB þ 0.1% bovine serum albumin and lysed with 200 mL PSB þ 0.1% bovine serum albumin/2% TX100/4 mg/mL leupeptin, 20 mg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride on a nutator at 4 C for 1 hour. The nuclei were then pelleted at 16,000g for 10 minutes at 4 C and the nuclei-free supernatant mixed with 100 mL prepared Ab-ProteinG agarose beads and nutated at 4 C overnight. The beads were washed five times with PSB þ 0.1% TX100, resuspended in 50 mL SDS sample buffer and heated at 95 C for 2 minutes. The supernatant was then subjected to Western blot analysis.
394
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
Western blot analysis SDS polyacrylamide gel electrophoresis was performed using a Protean II electrophoresis system. Cells were loaded as cell equivalents from total cell lysates. After separation by SDS polyacrylamide gel electrophoresis, proteins were transferred to polyvinylidine difluoride membranes using a Trans Blot Cell transfer system (Biorad) as per manufacturer’s instructions and probed as described previously [6].
Results BCR-ABL induces downregulation of SHIP but not SHIP2 or PTEN To confirm earlier reports that BCR-ABL reduces SHIP protein levels [4,5], we employed the Ba/F3p210tetOFF system depicted in Supplementary Figure E1A (online only, available at www.exphem.org). Using this cell system, Ba/F3p210-tetOFF cells were transferred from cytokine-free medium (since BCR-ABL expression enables these cells to grow in the absence of IL-3) into medium containing either IL-3 or IL-3 þ dox (to turn off BCR-ABL expression) and the expression of SHIP and the other two major lipid phosphatases, SHIP2 and PTEN, were monitored for up to 7 days by Western blot analysis. As shown in the top panel of Figure 1A, the addition of IL-3 þ dox dramatically reduced BCR-ABL protein levels within 24 hours and they remained low for the duration of the study. SHIP levels, in response, increased dramatically within 24 hours and plateaued at approximately fivefold the level observed in the presence of BCR-ABL for the 7 days of the study. On the other hand, the addition of IL-3 alone to these cells did not significantly increase SHIP levels. Cells from day 0 to day 3 (marked with an asterisk in Fig. 1A) were also subjected to intracellular staining with anti-SHIP Ab and confirmed that IL-3 alone did not change SHIP levels in the presence of BCR-ABL and that dox-induced reduction of BCR-ABL increased SHIP levels to the same extent in all cells (Supplementary Fig. E1B, online only, available at www.exphem.org). Interestingly, SHIP2 levels did not change upon the addition of either IL-3 þ dox or IL-3 alone (second panel of Fig. 1A). PTEN levels, on the other hand, were substantially increased (i.e., two- to threefold) when the cells were transferred into IL-3 þ dox. However, this increase was due to the addition of IL-3 rather than the loss of BCR-ABL because IL-3 alone also caused a similar increase (Fig. 1A). These results are consistent with BCR-ABL affecting SHIP but not SHIP2 or PTEN levels. Because IL-3 increased expression of PTEN, further experiments with Ba/F3p210-tetOFF cells were carried out in the presence of IL-3 to ensure that SHIP2 and PTEN levels remained constant throughout. To gain some insight into which pathways BCR-ABL used to reduce SHIP levels, Ba/F3p210-tetOFF cells were
grown in the absence of dox with different pathway inhibitors. As shown in Supplementary Figure E1C (online only, available at www.exphem.org), only PP2, a Src kinase family inhibitor, prevented the loss of SHIP protein in the presence of BCR-ABL. Because Src family members have been implicated as the kinases responsible for the tyrosine phosphorylation of SHIP [713], at least in some hematopoietic cells, this suggested that the tyrosine phosphorylation of SHIP might promote its own downregulation. Related to this, BCR-ABL’s tyrosine kinase activity has been reported to be required for reducing SHIP levels [5]. We therefore asked if BCR-ABL, directly or indirectly via a Src kinase, led to the tyrosine phosphorylation of SHIP and if SHIP’s tyrosine phosphorylation correlated with its reduced protein levels. As shown in the left lane of Figure 1B, SHIP was highly phosphorylated on its NPXY motifs (i.e., phosphorylated SHIP (pSHIP)/SHIP 5 3.1) in Ba/F3p210-tetOFF cells growing in IL-3 alone (so BCR-ABL is expressed). However, 24 hours after the addition of dox, when BCR-ABL levels were very low, we detected very low levels of pSHIP even though total SHIP levels were much higher (i.e., pSHIP/SHIP 5 0.2, a 15.5-fold drop) (right lane, Fig. 1B). Interestingly, a 24hour treatment with the Src kinase inhibitor PP2, which did not significantly affect BCR-ABL levels, resulted in total SHIP levels being higher than under normal culture conditions but lower than after dox treatment. Tyrosinephosphorylated SHIP levels, on the other hand, were lower than under normal culture conditions, but higher than in dox-treated cells (i.e., pSHIP/SHIP 51.1) (middle lane, Fig. 1B). Because PP2 has been reported to inhibit BCRABL as well as Src kinases [14], we cannot conclude from these studies that SHIP is tyrosine-phosphorylated by BCR-ABL directly or through the activation of a Src kinase. To determine how fast SHIP reaches its maximal levels after the addition of dox to Ba/F3p210-tetOFF cells, we monitored pSHIP and total and SHIP levels, as well as BCRABL protein levels, by Western analysis during a 24-hour period. As shown in Figure 1C, BCR-ABL levels started to drop steeply after 3 hours of dox treatment and were barely detectable by 24 hours. Tyrosine phosphorylated SHIP levels also decreased rapidly and showed a nice correlation with BCR-ABL levels. On the other hand, total SHIP levels only started to increase after 16 hours, reaching their maximum at 19 hours. Thus the loss of BCRABLinduced pSHIP precedes the increase in total SHIP protein levels. To gain some insight into how quickly SHIP levels drop in the presence of BCR-ABL, we preincubated Ba/F3p210-tetOFF cells growing in the absence of dox (i.e., high BCR-ABL levels) with PP2 (to maintain SHIP levels in the presence of BCR-ABL) and, after 24 hours, we washed out the PP2 and monitored total and pSHIP levels over time by Western analysis (Fig. 1D). As expected, BCR-ABL was expressed
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
395
Figure 1. BCR-ABL induces the downregulation of SH2-containing-phosphatidylinositol-50 -phosphatase (SHIP) but not SHIP2 or PTEN. (A) Ba/F3p210-tetOFF cells were incubated with interleukin (IL)-3 or IL-3 þ doxycycline (dox) for up to 7 days and Western analysis of total cell lysates (TCLs) was carried out using antibodies (Abs) to c-ABL, SHIP2, SHIP, PTEN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a loading control. (B) Ba/F3p210-tetOFF cells were treated for 24 hours 6 10 mM PP2 or 1 mg/mL dox and TCLs subjected to Western analysis using Abs to c-Abl, phosphorylated SHIP (pSHIP), SHIP, and GAPDH. (C) Ba/F3p210-tetOFF cells were incubated with 1 mg/mL dox for the indicated times and TCLs subjected to Western analysis as in (B). (D) Ba/F3p210-tetOFF cells were incubated with 10 mM PP2 for 24 hours. Cells were then washed twice to remove PP2 and TCLs taken at the indicated times for Western analysis as in (B). All blots shown are representative of at least two independent experiments.
396
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
Figure 2. BCR-ABL induces the polyubiquitination and proteasomal degradation of SH2-containing-phosphatidylinositol-50 -phosphatase (SHIP). (A) In the left panel, Ba/F3p210-tetOFF cells were treated 6 100 nM MG-132 for 24 hours and total cell lysates (TCLs) subjected to Western analysis for phosphorylated SHIP (pSHIP), SHIP, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the right panel, Ba/F3p210-tetOFF cells were treated or not with vehicle control (dimethyl sulfoxide), 100 nM MG-132, 1 mg/mL dox, 10 mM PP2, or 25 mM E64d for 24 hours and TCLs subjected to Western analysis for BCR-ABL, pSHIP, SHIP, SHIP2, PTEN, and GAPDH. (B) Ba/ F3p210-tetOFF cells were treated with 10 mM PP2, 100 nM MG-132, or 1 mg/mL doxycycline (dox) for 24 hours and SHIP immunoprecipitated with bead-bound anti-SHIP antibody (Ab). The immunoprecipitates were subjected to Western analysis with Abs to ubiquitin, BCR-ABL (left panel) and the ubiquitin blot reprobed for SHIP, using StemCell anti-SHIP #01506 (right panel). (C) Ba/F3p210-tetOFF cells were treated 6 10 mM PP2 or 1 mg/mL dox for 24 hours and TCLs subjected to Western analysis as indicated (left panel) or immunoprecipitated with bead-bound anti-SHIP Abs (right panel) and the immunoprecipitates subjected to Western analysis with antic-CBL, Cbl-b, and SHIP. All blots shown are representative of at least two independent experiments.
over the entire course of the experiment and any variation mirrored the variation in the glyceraldehyde-3-phosphate dehydrogenase loading control. After 24 hours with PP2 we found low levels of pSHIP and high total SHIP levels compared to control samples without PP2 treatment. After washing out the PP2, we observed a large increase in SHIP
phosphorylation within 30 minutes, but there was no significant drop in total SHIP levels until 3 hours, at which time the total and phosphorylated SHIP levels were back to control levels. This difference in timing suggested, once again, that the tyrosine phosphorylation of SHIP may act as a trigger for its downregulation.
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
BCR-ABL induces the polyubiquitination and proteasomal degradation of SHIP Because previous studies in our laboratory and others have shown that SHIP is a long-lived protein with a half-life O18 hours [5,6], its relatively short half-life of !3 hours in the presence of BCR-ABL (Fig. 1D) suggested that BCR-ABL may be inducing SHIP degradation rather than simply turning off SHIP gene transcription and/or translation. Because the proteasome is the main site of protein degradation in the cell and has been shown to play a critical role in BCR-ABLmediated pathogenesis [1521], we investigated the role of the proteasome in the BCRABLinduced reduction of SHIP levels. Specifically, we incubated Ba/F3p210-tetOFF cells, growing in IL-3 alone (so BCR-ABL is expressed), with the proteasome inhibitor MG-132 for 24 hours. As shown in the left panel of Figure 2A, both pSHIP and total SHIP levels increased dramatically with MG-132, suggesting that BCR-ABL downregulates SHIP by inducing its proteasomal degradation. However, because MG-132 has been reported, at high concentrations, to also inhibit calpains [22], we tested the calpain-specific inhibitor, E-64d, but found that even at 25 mM it did not increase SHIP or pSHIP levels (Fig. 2A, right panel). It is worthy of note in this figure, as in the left panel of Figure 2A, that in the presence of MG-132, the level of pSHIP increased significantly. In this same study, we examined the effect of MG-132, dox, and PP2 on the levels of SHIP2, PTEN and found that MG-132, dox, or PP2 did not increase SHIP2 or PTEN levels. To explore the putative proteasomal degradation of SHIP further, we immunoprecipitated SHIP from Ba/F3p210-tetOFF cells growing in IL-3 vs IL-3 plus either PP2, MG-132, or dox and carried out Western analysis using anti-ubiquitin Abs. As shown in the left panel of Figure 2B, SHIP was polyubiquitinated when BCR-ABL (IL-3 alone) or MG132 was present (to prevent proteasomal degradation of ubiquitinated SHIP), but was far less ubiquitinated when PP2 was added (consistent with the tyrosine phosphorylation of SHIP being a trigger for ubiquitination) or when dox was present (when BCR-ABL is turned off). An antiSHIP reprobe indicated the amount of SHIP in each lane (left panel of Fig. 2B) and showed that the higher molecular weight SHIP bands co-migrated with the ubiquitinated bands (right panel of Fig. 2B). Probing the same Western blot with antiBCR-ABL Abs demonstrated that SHIP was physically associated with BCR (left panel of Fig. 2B). This is consistent with, but does not prove that, BCR-ABL directly tyrosine-phosphorylates SHIP because Src family members have been reported to physically associate with BCR-ABL as well [2326]. However, attempts to show the presence of the Src family members Src, Lyn, Hck, or Fyn in reprobes of this or other similar Western blots were unsuccessful (data not shown). Because the ubiquitin ligase Cbl has been reported to interact with BCR-ABL [27], we then asked if it might
397
be involved in SHIP’s polyubiquitination. Before doing this, we first assessed the levels of Cbl, i.e., c-Cbl and Cbl-b, in total cell lysates from Ba/F3p210-tetOFF cells, treated with or without dox or PP2 for 24 hours. As shown in the left panel of Figure 2C, these two proteins were detectable under all three conditions and their levels were not affected by the presence or absence of BCR-ABL or PP2. We then immunoprecipitated SHIP (right panel of Fig. 2C) from Ba/F3p210-tetOFF cells treated with or without dox or PP2 for 24 hours and found that c-Cbl and Cbl-b were indeed present in SHIP immunoprecipitates. IL-4 directly downregulates SHIP and SHIP2 levels in BMM4s To explore the mechanism(s) involved in the downregulation of SHIP in normal primary cells, we examined IL-4induced M2 skewing of BMM4s because other studies in our laboratory demonstrated that IL-4 treatment of BMM4s downregulates SHIP levels while upregulating M2 markers, such as arginase 1 and Ym1 (Sly et al, manuscript in preparation). First, to determine if this IL-4induced reduction of SHIP occurred via IL-4 directly or through IL-4induced secretion and autocrine action of a secondary factor, we IL-4treated macrophage colony-stimulating factorderived BMM4s and examined SHIP levels during a 3-day period. As shown in Figure 3A, by 3 days, all the full-length SHIP was broken down into smaller fragments. We also found that SHIP2 was no longer detectable following 3 days of treatment with IL-4 (Fig. 3A). This was surprising because we did not observe a change in SHIP2 levels when BCR-ABL was expressed in Ba/F3p210-tetOFF cells. Thus, the mechanisms involved in reducing SHIP2 protein levels appear to vary with the cell type and/or stimulus involved. To determine if an IL4induced, autocrine-acting factor was involved in this reduction of SHIP and SHIP2, we added conditioned medium (CM) from BMM4s that had been incubated with IL-4 for 3 days to naı¨ve BMM4s in the presence or absence of IL4blocking Ab for 2 days. BMM4s treated with CM showed a reduction in SHIP and SHIP2 that was blocked by antiIL4. We therefore conclude that IL-4 directly induces the reduction of SHIP levels in BMM4s. Given that IL-4 was directly responsible for the downregulation of SHIP and SHIP2, and that IL-4 is known to mediate many of its effects through STAT6 [28], we also asked if the presence of STAT6 was required for IL-4induced reduction of SHIP levels and, as shown in Figure 3B, this was indeed the case. Interestingly, although SHIP levels were not reduced in IL4stimulated STAT6/ BMM4s, SHIP was still tyrosinephosphorylated, suggesting that STAT6 was required for SHIP degradation downstream of SHIP’s phosphorylation. IL-4-induced degradation of SHIP and SHIP2 is blocked with PP2 and MG-132 As shown in Figure 3A, SHIP levels decreased more slowly in IL-4treated BMM4s than in BCR-ABLexpressing
398
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
Figure 3. Interleukin (IL)-4 directly downregulates SH2-containing-phosphatidylinositol-50 -phosphatase (SHIP) and SHIP2 levels in bone marrowderived macrophages (BMM4s). (A) BMM4s in BMM4 medium were incubated with 10 ng/mL IL-4 for up to 3 days. As well, conditioned medium (CM) from day 3 IL-4treated cultures was diluted such that the IL-4 5 4 ng/mL, assuming no significant loss of IL-4 during the 3-day incubation, and incubated with day 3 BMM4s 6 aIL-4 (CM þ aIL-4) for 2 days and total cell lysates (TCLs) subjected to Western analysis with antibodies to SHIP2, SHIP, and glyceraldehyde3-phosphate dehydrogenase (GAPDH). (B) BMM4s derived from wild-type (WT) or signal transducers and activators of transcription (STAT)6/ mice were incubated in BMM4 medium without IL-4 (control) or with 10 ng/mL IL-4 for 2 or 3 days and TCLs subjected to Western analysis for phosphorylated SHIP (pSHIP), SHIP, and GAPDH. Blots shown are representative of at least two independent experiments. The arrows indicate full length-SHIP. The dashed lines on the blots indicate that while the lanes to the right and left of the dashed lines were from the same blot and the same exposure, they were not adjacent and so the blot was cut to juxtapose the lanes.
Ba/F3 cells, with the biggest drop in SHIP occurring between day 2 and day 3 (Fig. 3A). To determine if this reduction in SHIP protein was due to proteasomal degradation, we added MG-132 during the last 24 hours of IL-4 treatment (i.e., from day 2 to day 3). This was necessary because MG-132, even at 10 nM, was quite toxic and we could not expose the BMM4s to it for 3 days without causing significant cell death. We found the MG-132 inhibited the degradation of full-length SHIP that occurs between day 2 and 3, i.e., MG-132 prevented further reduction of SHIP protein levels compared to control samples (left panel of Fig. 4A), without any detectable loss in cell viability (data not shown). MG-132 also prevented the loss of SHIP2 protein, as shown in the right panel of Figure 4A. Because both IL-4 in BMM4s and BCR-ABL in Ba/ F3p210-tetOFF cells induced proteasomal SHIP degradation, we tested if PP2 was also able to prevent IL-4mediated SHIP degradation in BMM4s. Specifically, we incubated BMM4s with IL-4 for 2 days with or without PP2 and, as shown in Figure 4B, the presence of PP2 resulted in more full-length SHIP protein. Because there is no BCR-ABL present in BMM4s, it therefore seems likely that IL-4 induces SHIP degradation through Src kinases in these cells, while BCR-ABL may directly induce SHIP phosphorylation and subsequent degradation in Ba/F3p210-tetOFF cells. However, treatment of BMM4s deficient in different Src kinases (i.e., Lyn, Hck, Fyn, Fyn/Lyn, or Lyn/Hck) that have been shown to play a role in the tyrosine phosphorylation of SHIP [7,10,12,2931] could not prevent IL-4induced degradation of SHIP (Fig. 4C, left and right panels), indicating that Src kinases were either able to substitute for each other or that a PP2-sensitive non-Src kinase was inducing SHIP’s degradation in BMM4s.
SHIP in BMM4s is primed for proteasomal degradation Finally, given that we had shown in earlier studies that SHIP is rapidly degraded from the C-terminus if cells are lysed with nonionic detergents (e.g., NP40 or TX100) rather than with SDS sample buffer [6], we asked if SHIP was already primed for rapid degradation via the proteasome and all that was needed was a trigger, such as Src kinase-induced tyrosine phosphoryation, to induce this process. To test this, we treated BMM4s for 2 days with or without IL-4. The cells were then washed with phosphate-buffered saline and lysed with 2% TX100 þ 0.1% NP40 in the presence of protease inhibitors to prevent nonspecific protein degradation or washed and lysed in the same way in the presence of 10 mM MG-132 to inhibit proteasomal degradation. As shown in Figure 4D, after 2 minutes of lysis at 23 C, SHIP, but not glyceraldehyde-3-phosphate dehydrogenase, levels were greatly reduced in the absence of MG-132, regardless of whether they were treated or not with IL-4.
Discussion Considering the potential importance of BCR-ABLinduced and IL-4induced downregulation of SHIP, very little is known as yet about how they are regulated. Taken together, our data with Ba/F3p210tetOFF cells suggest a model of BCR-ABLinduced SHIP degradation, shown in Figure 5, in which BCR-ABL forms a complex with SHIP, c-Cbl, and Cbl-b and either tyrosine-phosphorylates SHIP directly or via a Src family member. The lack of highly specific Src family inhibitors and the fact that BCR-ABL has been shown to physically associate with Src family members [24,32] make this a difficult issue to resolve. Nonetheless, we propose that the tyrosine phosphorylation of SHIP triggers its
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
399
Figure 4. Interleukin (IL)-4induced degradation of SH2-containing-phosphatidylinositol-50 -phosphatase (SHIP) is blocked with PP2 and MG-132. (A) In the left panel, bone marrowderived macrophages (BMM4s) in BMM4 medium were incubated without IL-4 (control) or with 10 ng/mL IL-4 for the days indicated. For one sample, 10 nM MG-132 was added for 24 hours from day 2 to 3 and total cell lysates (TCLs) subjected to Western analysis for SHIP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Similar results were obtained when MG-132 was used at 0.1 mM and 1 mM (not shown). In the right panel, BMM4s in BMM4 medium were incubated without IL-4 (control) or with 10 ng/mL IL-4 for the days indicated. For one sample, 10 nM MG-132 was added for 24 hours from day 2 to 3 and TCLs subjected to Western analysis for SHIP2, SHIP, and GAPDH. (B) BMM4s in BMM4 medium were incubated without IL-4 (control) or with 10 ng/mL IL-4 for up to 2 days or with 10 ng/mL IL-4 þ 10 mM PP2 for 2 days. (C) BMM4s from either wild-type (WT), Lyn/, Hck/, or Lyn/Hck/ mice (left panel) or from WT, Fyn/, and Fyn/Lyn/ mice (right panel) were incubated in BMM4 medium without IL-4 (control) or with 10 ng/mL IL-4 6 10 mM PP2 for 2 days and phosphorylated SHIP (pSHIP) and SHIP levels determined by Western analysis. (D) BMM4s in BMM4 medium were incubated without IL-4 (control) or with 10 ng/mL IL-4 for 2 days. The cells were then either washed 1 with phosphate-buffered saline (PBS) and lysed with phosphorylation solubilization buffer (PSB) þ 2% TX100 þ 0.1% NP40 for 2 minutes at 23 C in the presence of protease inhibitors (-MG-132 lysis) or washed and lysed the same way but in the presence of 10 mM MG-132 (þMG-132 lysis). All blots shown are representative of at least two independent experiments. The arrows indicate full length SHIP. The dashed lines on the blots indicate that while the lanes to the left and right of the dashed lines were from the same blot and the same exposure, they were not adjacent and so the blot was cut to juxtapose the lanes.
400
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
Figure 5. Model of BCR-ABL- and interleukin (IL)-4induced proteasomal degradation of SH2-containing-phosphatidylinositol-50 -phosphatase (SHIP). BCR-ABL and other oncogenic tyrosine kinases (e.g., constitutively active c-kit), directly or indirectly via Src family kinases, tyrosine phosphorylate SHIP and this acts as a trigger for SHIP to be polyubiquitinated, possibly by c-Cbl and/or Cbl-b, and degraded in the proteasome. IL-4 treatment of bone marrowderived macrophages (BMM4s) also leads to the tyrosine phosphorylation of SHIP, via src family members, and its signal transducers and activators of transcription (STAT)6-dependent degradation in the proteasome. Dashed arrows indicate uncertain pathways.
polyubiquitination, via c-Cbl and/or Cbl-b, and subsequent proteasomal degradation. Our finding that, in the presence of BCR-ABL, SHIP’s tyrosine phosphorylation precedes its degradation and that PP2 reduces SHIP’s polyubiquitination and degradation lends support to this model. Interestingly, protein levels of SHIP2 and PTEN, two other lipid phosphatases that act as negative regulators of the PI3K pathway in hematopoietic cells, appear to be unaffected by the presence of BCR-ABL but PTEN levels are increased with IL-3. This increase in PTEN might serve as a negative feedback mechanism to limit IL-3induced PI3K activation and subsequent cell proliferation. The specific susceptibility of SHIP to BCR-ABLinduced degradation demonstrates that SHIP, SHIP2, and PTEN protein levels are regulated, at least in part, by different mechanisms in Ba/F3p210tetOFF cells. To understand how SHIP is downregulated by normal physiological regulators, we also investigated IL-4treated BMM4s and found that IL-4 induces the proteasomal degradation of SHIP directly and not through the secretion and autocrine action of a secondary factor (as observed in lipopolysaccharide (LPS)-induced upregulation of SHIP by secreted transforming growth factorb [2] and, as found
with BCR-ABLinduced degradation, it could be prevented with PP2 or MG-132. Moreover, we found in IL-4treated STAT6/ BMM4s that SHIP still becomes tyrosinephosphorylated but not degraded, suggesting that STAT6 is required either for polyubiquitination or a subsequent step prior to proteasomal degradation (Fig. 5). However, unlike in Ba/F3p210tetOFF cells, we found that treatment of BMM4s with IL-4 leads to reduced levels of SHIP2 as well, indicating that the regulation of SHIP2 varies with cell type and/or stimulus. Interestingly, in this regard, both SHIP and SHIP2 contain PEST domains, i.e., domains rich in prolines (P), glutamic acids (E), serines (S), and threonines (T) [33], and these domains have been shown to direct eukaryotic proteins for proteasomal degradation [34]. Our results further suggest that SHIP may be present in a complex in BMM4s that allows for rapid degradation through the proteasome, because the lysis of BMM4s with TX-100 and NP-40, instead of SDS sample buffer, results in breakdown of SHIP that is prevented when MG-132 is present in the lysis buffer. Such an effect was not seen for glyceraldehyde-3-phosphate dehydrogenase, suggesting that this was not due to nonspecific degradation and might
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
suggest that disruption of intracellular compartments by nonionic detergents might allow for Src family or other tyrosine kinases to phosphorylate SHIP, and perhaps other susceptible proteins, leading to proteasomal degradation. Because BCR-ABL was not present in our BMM4s, we hypothesize that the block in IL-4induced SHIP breakdown with PP2 is due to inhibition of a Src family kinase. However, as shown in Figure 4C, IL-4 treatment of BMM4s derived from mice lacking Lyn, Fyn, Hck, Fyn/ Lyn, or Lyn/Hck results in SHIP degradation to the same extent as in WT cells. Importantly, though, treatment with PP2 reduced the phosphorylation and prevented degradation. Thus we conclude that either other Src family members are able to substitute for the one(s) knocked out or PP2 inhibits a non-Src kinase that phosphorylates the critical tyrosine(s) involved in degradation. Relevant to the first possibility, c-Src has been shown to be upregulated in LPS-stimulated Lyn/Fgr/Hck triple knockout macrophages and compensates for the loss of these other Src family members in triggering nitric oxide and tumor necrosis factora secretion [35]. Moreover, a recent report showing that Lyn//Hck/ mice, which have a very similar phenotype to SHIP/ mice, can be rescued by expression of a membrane-bound form of SHIP suggests that the tyrosine phosphorylation of SHIP by Lyn plus Hck acts as a trigger for SHIP’s relocation [29]. This is supported by an earlier report showing that SHIP translocates to the actin cytoskeleton upon Src-mediated tyrosine phosphorylation in activated human platelets [36]. However, our current results show that tyrosine phosphorylation is also involved in SHIP degradation because this can be blocked by PP2. Thus, a dual role for SHIP’s tyrosine phosphorylation seems most likely. Related to this, the anti-pSHIP Abs we employed in this study were against one of the two NPXpY motifs and recent studies have shown that SHIP becomes tyrosine-phosphorylated following stimulation on other tyrosines as well [37], and it is conceivable that the two phosphorylated NPXY motifs, which are attracted to proteins with phosphotyrosine binding domains [38], are strictly involved in the translocation of SHIP to the plasma membrane to hydrolyze PI-3,4,5-P3, while some of these other tyrosines might be involved in triggering polyubiquitination and subsequent proteasomal degradation. If this is the case, it will be interesting to elucidate which tyrosinephosphorylation patterns induce SHIP’s translocation vs degradation. Acknowledgments We would like to thank Dr. R. Hodes (National Institutes of Health, Bethesda, MD, USA) for c-Cbl deficient bones and Christine Kelly for preparing the manuscript. This work was supported by the Terry Fox Foundation (Vancouver, BC, Canada) and the Canadian Cancer Society (Toronto, Ontario, Canada), with core support from the BC Cancer Foundation (Vancouver, BC, Canada) and the BC Cancer Agency (Vancouver, BC, Canada).
401
Conflict of Interest Disclosure G.K. is a founding member and Chief Scientific Advisor of Aquinox Pharmaceuticals Inc (Vancouver, BC, Canada), which is dedicated to identifying small molecule activators and inhibitors of SHIP.
References 1. Antignano F, Ruschmann J, Hamilton M, et al. The Src homology 2 containing inositol 5’ phosphatases. In: Bradsaw RA, Dennis EA, eds. Handbook of Cell Signalling. San Diego: Elsevier Inc; 2009. p. 1065–1084. 2. Sly LM, Rauh MJ, Kalesnikoff J, Song CH, Krystal G. LPS-induced upregulation of SHIP is essential for endotoxin tolerance. Immunity. 2004;21:227–239. 3. Sly LM, Hamilton MJ, Kuroda E, et al. SHIP prevents lipopolysaccharide from triggering an anti-viral response in mice. Blood. 2009;113:2945– 2954. 4. Sattler M, Salgia R, Shrikhande G, et al. The phosphatidylinositol polyphosphate 5-phosphatase SHIP and the protein tyrosine phosphatase SHP-2 form a complex in hematopoietic cells which can be regulated by BCR/ABL and growth factors. Oncogene. 1997;15:2379–2384. 5. Sattler M, Verma S, Byrne CH, et al. BCR/ABL directly inhibits expression of SHIP, an SH2-containing polyinositol-5-phosphatase involved in the regulation of hematopoiesis. Mol Cell Biol. 1999;19: 7473–7480. 6. Damen JE, Liu L, Ware MD, Ermolaeva M, Majerus PW, Krystal G. Multiple forms of SHIP are generated by C-terminal truncation. Blood. 1998;92:1199–1205. 7. Gardai S, Whitlock BB, Helgason C, et al. Activation of SHIP by NADPH oxidase-stimulated Lyn leads to enhanced apoptosis in neutrophils. J Biol Chem. 2002;277:5236–5246. 8. Malbec O, Attal JP, Fridman WH, Daeron M. Negative regulation of mast cell proliferation by FcgRIIB. Mol Immunol. 2002;38:1295–1299. 9. Hibbs ML, Harder KW, Armes J, et al. Sustained activation of Lyn tyrosine kinase in vivo leads to autoimmunity. J Exp Med. 2002; 196:1593–1604. 10. Baran CP, Tridandapani S, Helgason CD, Humphries RK, Krystal G, Marsh CB. The inositol 5’-phosphatase SHIP-1 and the Src kinase lyn negatively regulate macrophage colony-stimulating factor-induced Akt activity. J Biol Chem. 2003;278:38628–38636. 11. Harder KW, Quilici C, Naik E, et al. Perturbed myelo/erythropoiesis in Lyn-deficient mice is similar to that in mice lacking the inhibitory phosphatases SHP-1 and SHIP-1. Blood. 2004;104:3901–3910. 12. Hernandez-Hansen V, Smith AJ, Surviladze Z, et al. Dysregulated FcgRI signaling and altered Fyn and SHIP activities in Lyn-deficient mast cells. J Immunol. 2004;173:100–112. 13. Xiao W, Nishimoto H, Hong H, et al. Positive and negative regulation of mast cell activation by Lyn via the FcgRI. J Immunol. 2005;175: 6885–6892. 14. Tatton L, Morley GM, Chopra R, Khwaja A. The Src-selective kinase inhibitor PP1 also inhibits Kit and Bcr-Abl tyrosine kinases. J Biol Chem. 2003;278:4847–4853. 15. Jagani Z, Song K, Kutok JL, et al. Proteasome inhibition causes regression of leukemia and abrogates BCR-ABL-induced evasion of apoptosis in part through regulation of forkhead tumor suppressors. Cancer Res. 2009;69:6546–6555. 16. Crawford LJ, Windrum P, Magill L, et al. Proteasome proteolytic profile is linked to Bcr-Abl expression. Exp Hematol. 2009;37:357–366. 17. Magill L, Lynas J, Morris TC, Walker B, Irvine AE. Proteasome proteolytic activity in hematopoietic cells from patients with chronic myeloid leukemia and multiple myeloma. Haematologica. 2004;89:1428–1433. 18. Gatto S, Scappini B, Pham L, et al. The proteasome inhibitor PS-341 inhibits growth and induces apoptosis in Bcr/Abl-positive cell lines sensitive and resistant to imatinib mesylate. Haematologica. 2003; 88:853–863.
402
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
19. Tauchi T, Yoshimura A, Ohyashiki K. CIS1, a cytokine-inducible SH2 protein, suppresses BCR/ABL-mediated transformation. Involvement of the ubiquitin proteasome pathway. Exp Hematol. 2001;29: 356–361. 20. Dou QP, McGuire TF, Peng Y, An B. Proteasome inhibition leads to significant reduction of Bcr-Abl expression and subsequent induction of apoptosis in K562 human chronic myelogenous leukemia cells. J Pharmacol Exp Ther. 1999;289:781–790. 21. Dai Z, Quackenbush RC, Courtney KD, et al. Oncogenic Abl and Src tyrosine kinases elicit the ubiquitin-dependent degradation of target proteins through a Ras-independent pathway. Genes Dev. 1998;12: 1415–1424. 22. Kojima M, Oguro K, Sawabe K, et al. Rapid turnover of tryptophan hydroxylase is driven by proteasomes in RBL2H3 cells, a serotonin producing mast cell line. J Biochem. 2000;127:121–127. 23. Danhauser-Riedl S, Warmuth M, Druker BJ, Emmerich B, Hallek M. Activation of Src kinases p53/56 lyn and p59 hck by p210 bcr/abl in myeloid cells. Cancer Res. 1996;56:3589–3596. 24. Warmuth M, Bergmann M, Priess A, Hauslmann K, Emmerich B, Hallek M. The Src family kinase Hck interacts with Bcr-Abl by a kinaseindependent mechanism and phosphorylates the Grb2-binding site of Bcr. J Biol Chem. 1997;272:33260–33270. 25. Lionberger JM, Wilson MB, Smithgall TE. Transformation of myeloid leukemia cells to cytokine independence by Bcr-Abl is suppressed by kinase-defective Hck. J Biol Chem. 2000;275:18581–18585. 26. Wilson MB, Schreiner SJ, Choi HJ, Kamens J, Smithgall TE. Selective pyrrolo-pyrimidine inhibitors reveal a necessary role for Src family kinases in Bcr-Abl signal transduction and oncogenesis. Oncogene. 2002;21:8075–8088. 27. Brehme M, Hantschel O, Colinge J, et al. Charting the molecular network of the drug target Bcr-Abl. Proc Natl Acad Sci U S A. 2009;106:7414–7419. 28. Takeda K, Tanaka T, Shi W, et al. Essential role of Stat6 in IL-4 signalling. Nature. 1996;380:627–630.
29. Xiao W, Hong H, Kawakami Y, Lowell CA, Kawakami T. Regulation of myeloproliferation and M2 macrophage programming in mice by Lyn/Hck, SHIP, and Stat5. J Clin Invest. 2008;118:924–934. 30. Chari R, Kim S, Murugappan S, Sanjay A, Daniel JL, Kunapuli SP. Lyn, PKC-d, SHIP-1 interactions regulate GPVI-mediated platelet-dense granule secretion. Blood. 2009;114:3056–3063. 31. Xu R, Abramson J, Fridkin M, Pecht I. SH2 domain-containing inositol polyphosphate 5’-phosphatase is the main mediator of the inhibitory action of the mast cell function-associated antigen. J Immunol. 2001; 167:6394–6402. 32. Ptasznik A, Urbanowska E, Chinta S, et al. Crosstalk between BCR/ABL oncoprotein and CXCR4 signaling through a Src family kinase in human leukemia cells. J Exp Med. 2002;196:667–678. 33. Backers K, Blero D, Paternotte N, Zhang J, Erneux C. The termination of PI3K signalling by SHIP1 and SHIP2 inositol 5-phosphatases. Adv Enzyme Regul. 2003;43:15–28. 34. Schnupf P, Portnoy DA, Decatur AL. Phosphorylation, ubiquitination and degradation of listeriolysin O in mammalian cells: role of the PEST-like sequence. Cell Microbiol. 2006;8:353–364. 35. Leu TH, Charoenfuprasert S, Yen CK, Fan CW, Maa MC. Lipopolysaccharide-induced c-Src expression plays a role in nitric oxide and TNFalpha secretion in macrophages. Mol Immunol. 2006;43:308–316. 36. Giuriato S, Bodin S, Erneux C, et al. pp60c-src associates with the SH2-containing inositol-5-phosphatase SHIP1 and is involved in its tyrosine phosphorylation downstream of aIIbb3 integrin in human platelets. Biochem J. 2000;348:107–112. 37. Cao L, Yu K, Banh C, et al. Quantitative time-resolved phosphoproteomic analysis of mast cell signaling. J Immunol. 2007;179:5864– 5876. 38. DiNitto JP, Lambright DG. Membrane and juxtamembrane targeting by PH and PTB domains. Biochim Biophys Acta. 2006;1761:850–867. 39. Dugray A, Geay JF, Foudi A, et al. Rapid generation of a tetracyclineinducible BCR-ABL defective retrovirus using a single autoregulatory retroviral cassette. Leukemia. 2001;15:1658–1662.
J. Ruschmann et al./ Experimental Hematology 2010;38:392–402
402.e1
Supplementary Figure E1. (A) Ba/F3p210-tetOFF cells were derived from Ba/F3 cells as described in data from [39]. In the absence of doxycycline (dox) both BCR-ABL and the tTa (Tet repressor-VP16 fusion protein) get expressed. Increasing levels of tTa lead to higher activity of the minimal cytomegalovirus (CMV) (CMVmin) promoter, which, in turn, leads to increasing BCR-ABL and tTa levels. Upon addition of dox, tTa cannot bind to the CMVmin promoter and BCR-ABL as well as tTa levels decrease. (B) Ba/F3p210-tetOFF cells were transferred into medium containing IL-3 only (þIL-3) or IL-3 and dox (þIL-3/ dox) for up to 3 days and the cells intracellularly stained for SH2-containing-phosphatidylinositol-50 -phosphatase (SHIP). (C) Left panel, Ba/F3p210-tetOFF cells were incubated with IL-3 alone (þIL-3), IL-3 þ dox or IL-3 þ the indicated inhibitors for 24 hours and total cell lysates (TCLs) subjected to Western analysis. The blot shown is representative of two independent experiments. Right panel, list of inhibitors, their targets, and the final concentration used (based on studies showing these concentrations blocked the targeted pathways in Ba/F3 cells [unpublished]).