Gab1 transduces PI3K-mediated erythropoietin signals to the Erk pathway and regulates erythropoietin-dependent proliferation and survival of erythroid cells

Cellular Signalling 21 (2009) 1775–1783

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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g

Gab1 transduces PI3K-mediated erythropoietin signals to the Erk pathway and regulates erythropoietin-dependent proliferation and survival of erythroid cells Tetsuya Fukumoto a, Yoshitsugu Kubota b,⁎, Akira Kitanaka c, Genji Yamaoka d, Fusako Ohara-Waki a, Osamu Imataki a, Hiroaki Ohnishi a, Toshihiko Ishida a, Terukazu Tanaka e a

Division of Hematology, Internal Medicine, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki, Kita-gun, Kagawa 761-0793, Japan Department of Transfusion Medicine, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki, Kita-gun, Kagawa 761-0793, Japan Department of Laboratory Medicine, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki, Kita-gun, Kagawa 761-0793, Japan d Department of Laboratory Medicine, Kagawa University Hospital, 1750-1 Ikenobe, Miki, Kita-gun, Kagawa 761-0793, Japan e Department of Environmental Health Sciences, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki, Kita-gun, Kagawa 761-0793, Japan b c

a r t i c l e

i n f o

Article history: Received 12 March 2009 Received in revised form 28 July 2009 Accepted 28 July 2009 Available online 6 August 2009 Keywords: Akt Erk Erythropoietin Gab1 Jak2 PI3K

a b s t r a c t In this study, we examined the biological functions of Gab1 in erythropoietin receptor (EPOR)-mediated signaling in vivo. Knockdown of Gab1 by the introduction of the Gab1 siRNA expression vector into F-36P human erythroleukemia (F-36P-Gab1-siRNA) cells resulted in a reduction of cell proliferation and survival in response to EPO. EPO-induced activation of Erk1/2 but not of Akt was significantly suppressed in F-36PGab1-siRNA cells compared with mock-transfected F-36P cells. The co-immunoprecipitation experiments revealed an EPO-enhanced association of Gab1 with the Grb2–SOS1 complex and SHP-2 in F-36P cells. A selective inhibitor of phosphatidylinositol 3-kinase (PI3K) LY294002 and short interfering RNA (siRNA) duplexes targeting the p85 regulatory subunit of PI3K (p85-siRNA) independently suppressed tyrosine phosphorylation of Gab1; its association with Grb2, SHP-2 and p85; and the activation of Erk in EPO-treated F-36P cells. LY294002 inhibited EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2 in human primary EPO-sensitive erythroid cells. The co-immunoprecipitation experiments using the Jak inhibitor AG490 or siRNA duplexes targeting Jak2 and in vitro binding experiments demonstrated that Jak2 regulated Gab1-mediated Erk activation through tyrosine phosphorylation of Gab1. Taken together, these results suggest that Gab1 couples PI3K-mediated EPO signals with the Ras/Erk pathway and that Gab1 plays an important role in EPOR-mediated signal transduction involved in the proliferation and survival of erythroid cells. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Erythropoietin (EPO) is a cytokine that regulates the proliferation, survival and differentiation of erythroid progenitor cells. After the binding of EPO to its cognate receptor (EPOR), EPOR is tyrosinephosphorylated by Janus kinase 2 (Jak2) and other tyrosine kinases, and thereby recruits various src homology (SH) domain-2-containing signaling molecules to stimulate downstream signaling pathways, including the Jak/signal transducers and activators of transcription 5 (Stat5), phosphatidylinositol 3-kinase (PI3K)/Akt and Ras/extracellular signal-regulated kinase (Erk) pathways [1–4]. Grb2-associated binder-1 (Gab1) is a member of the Gab/ Daughter of Sevenless (DOS) family of scaffolding adaptor molecules that includes Gab1, Gab2 and Gab3 [5–8]. Gab1 and Gab2 are

⁎ Corresponding author. Tel.: +81 87 891 2146; fax: +81 87 891 2147. E-mail addresses: [email protected] (T. Fukumoto), [email protected] (Y. Kubota), [email protected] (A. Kitanaka), [email protected] (G. Yamaoka), [email protected] (F. Ohara-Waki), [email protected] (O. Imataki), [email protected] (H. Ohnishi), [email protected] (T. Ishida), [email protected] (T. Tanaka). 0898-6568/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2009.07.013

expressed ubiquitously. Gab1 transcripts are continuously expressed throughout differentiation at least in erythroid lineage cells [9–11]. In contrast, Gab2 is expressed in mouse but not human erythroid progenitor cells [9–12]. Gab1 contains a pleckstrin homology (PH) domain in the amino-terminal region, tyrosine-based motifs which are potential binding sites for the SH2 domains of SHP-2 and the p85 regulatory subunit of PI3K, and proline-rich sequences (PXXP) interacting with the SH3 domain-containing proteins such as Grb2 [5–8]. Gab1 is located in the cytosol of unstimulated cells, and upon cell stimulation is recruited to the plasma membrane, where it becomes phosphorylated. The recruitment of Gab1 to the plasma membrane appears to be required for its function [5–7]. Gab1 is indirectly recruited to the receptor complexes via adaptor proteins such as Grb2 and Shc. Gab1 can directly bind to the hepatocyte growth factor receptor (Met) through its Met-binding domain. In addition, the binding of the Gab1 PH domain to phosphatidylinositol-3,4,5triphosphates (PIP3) in the plasma membrane is either required for initial recruitment to the receptor complexes (e.g., B-cell antigen receptor) or for sustained signaling [e.g., epidermal growth factor receptor (EGFR)] [13–15].

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Gab1 is tyrosine-phosphorylated upon the stimulation of various receptors, including receptor protein tyrosine kinases (PTKs) such as Met and EGFR, cytokine receptors such as interleukin-6 (IL-6), and Tand B-cell antigen receptors [9–16]. Recently, it was found that EPO induces tyrosine phosphorylation of Gab1 in cell lines and erythroid progenitor cells, resulting in transduction of EPO signals to the Ras/Erk and PI3K/Akt pathways through the binding of signaling molecules such as SHP-2 and p85 to tyrosine residues phosphorylated in Gab1 [9,11,17]. From a functional point of view, Gab1 is involved in multiple cell responses, including proliferation, migration, morphogenesis, transformation, and apoptosis [9,11,17,18]. However, the physiological functions of Gab1 in EPOR-mediated signal transduction remain to be elucidated. In the present study, to explore the physiological functions of Gab1 in EPO-induced signaling, we generated Gab1-deficient F-36P human erythroleukemia cells by means of the transfection of the expression vector for Gab1 siRNA (F-36P-Gab1-siRNA). We found that EPOinduced proliferation and survival of Gab1-deficient F-36P cells were significantly reduced compared to those of mock-transfected F-36P (F-36P-mock) cells. EPO-induced activation of Erk but not of PI3K was impaired in Gab1-deficient F-36P cells. Furthermore, PI3K regulated the activation of the Gab1-Erk pathway in EPO signaling.

2.3. Analyses of cell proliferation and apoptosis A WST-1 assay (Roche Diagnostics, Mannheim, Germany) was performed to determine the level of cell proliferation, as described previously [20]. To detect apoptosis, dual staining of cells with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI) (both Roche Diagnostics) was performed according to the manufacturer's instructions. 2.4. Immunoprecipitation and immunoblotting Immunoprecipitation and immunoblotting analyses were performed as described previously [21]. Briefly, after growth factor starvation by overnight incubation, the cells were treated with EPO with or without inhibitors and lysed with the lysis buffer. Cell lysates were then subjected to immunoprecipitation followed by SDS-PAGE. Separated proteins were electrotransferred to PVDF membranes. For immunoblot analyses, PVDF membranes blocked with 5% bovine serum albumin solution were probed with specific antibodies followed by detection using an enhanced chemiluminescence system (GE Healthcare UK Ltd., Buckinghamshire, England). 2.5. Akt activation assay

2. Materials and methods 2.1. Reagents Recombinant human EPO was kindly provided by the Kirin Brewery Co. (Tokyo, Japan). AG490, LY294002 and U0126 were purchased from Calbiochem (San Diego, CA, USA). Polyclonal antibodies against EPOR, Erk1, Gab1, Grb2, Jak2, SHP-2 and SOS1, and monoclonal antibodies against Bcl-2, Grb2 and phosphotyrosine (PY) were obtained from Santa Cruz (Santa Cruz, CA, USA). Anti-p85 polyclonal antibody was obtained from Millipore (Billerica, MA, USA). Polyclonal antibodies against phospho-Erk1/2 (Thr202/Tyr204), phospho-Akt1 (Ser473), and Bcl-xL were purchased from Cell Signaling (Danvers, MA, USA). Anti-β-actin monoclonal antibody was obtained from Sigma (St. Louis, MO, USA). All other agents were purchased from commercial sources. Expression vector for the human Gab1 (pCMV-SPORT6.1-Gab1) was purchased from Open Biosystems (Huntsville, AL, USA). Expression vector for the murine wild-type Jak2 (pSRα-bsr-Jak2) was a generous gift from Dr. JN Ihle. Agarose-conjugated glutathione-S transferase (GST)-Grb2 fusion proteins were obtained from Santa Cruz.

2.2. Cell culture and transfection IL-3- and granulocyte macrophage colony-stimulating factor (GMCSF)-dependent F-36P human erythroleukemia cells were provided by RIKEN Cell Bank (Tsukuba, Japan). F-36P cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and 10 U/ml human recombinant GM-CSF (the growth medium). The target sequence for human Gab1 is 851-GAAACAGACTGCAATGATG (the number indicates the location of the first nucleotide of the sequence in the human Gab1 coding region). Oligonucleotides containing the siRNA coding sequence were cloned into the BspMI site of plasmid pcPURU6 (Takara, Otsu, Japan) to generate pGab1siRNA-851. pGab1siRNA-851 and the expression vector without any insert were electroporated into F-36P cells using a gene pulser apparatus (Bio-Rad, Richmond, CA, USA) set at 960 μF and 290 V, as described previously [19]. After the cells were cultured for 24 h in the growth medium, transformant cells were selected in the growth medium containing 1 μg/ml puromycin (Sigma).

The activation of Akt1 was determined using a phospho-Akt1 (Ser473) sandwich enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (Cell Signaling). Briefly, cell lysates were added to each well of a 96-well microplate on which anti-phospho-Akt rabbit monoclonal antibody was coated. Following extensive washing, anti-Akt1 mouse monoclonal antibody was added to detect the captured phospho-Akt1 protein. Horseradish peroxidase (HRP)-linked anti-mouse IgG antibody was then used to recognize the bound detection antibody. After HRP substrate was added to develop color, the absorbance at 450 nm was measured using a Model 680 microplate reader (Bio-Rad). The amount of Akt1 in cell lysates was measured by using a total Akt1 sandwich ELISA kit according to the manufacturer's instructions (Cell Signaling). 2.6. Short interfering RNA-mediated protein knockdown Short interfering RNA (siRNA) duplexes targeting human p85 and Jak2 (p85-siRNA and Jak2-siRNA, respectively), and siRNA duplexes with irrelevant sequences as a control (i.e., siGENOME SMARTpool targeting human p85, siGENOME SMARTpool targeting human Jak2 and siGENOME non-targeting siRNA pool) were purchased from Thermo Fisher Scientific (Yokohama, Japan). F-36P cells were transfected with siRNA duplexes using the Nucleofector technology (Lonza Cologne AG, Köln, Germany). The cells were then incubated with the growth medium for 24 h. After growth factor starvation by overnight incubation, the cells were treated with EPO and lysed with the lysis buffer. Cell lysates were then subjected to immunoprecipitation followed by immunoblotting analyses. 2.7. In vitro binding studies using GST–Grb2 fusion proteins In vitro binding studies using GST–Grb2 fusion proteins were performed as described previously [21]. Briefly, after expression vectors for Gab1 and wild-type Jak2 were co-transfected in 293T cells by using HEKFectin (Bio-Rad), the cells were incubated for 24 h. The cells were then incubated with or without AG490 for a further 16 h. Cell lysates were incubated with agarose-conjugated GST–Grb2 fusion proteins (Santa Cruz) at 4 °C for 4 h. After being washed with lysis buffer containing 1 mM Na3VO4, proteins bound to the GST–Grb2 whole molecule and GST–Grb2 SH2 domain were eluted by boiling and subjected to Western blotting analysis.

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2.8. Statistical analysis Data were analyzed by Student's t test; p b 0.05 was considered to indicate a statistically significant difference. 3. Results 3.1. Knockdown of Gab1 reduces proliferation, and promotes the apoptosis of F-36P cells To examine the physiological functions of Gab1 in EPOR-mediated signal transduction, we established several clones of F-36P-Gab1siRNA cells by means of the transfection of the expression vector carrying Gab1 siRNA as shown in Fig. 1A. The expression of Gab1 was constitutively suppressed in F-36P-Gab1-siRNA but not in F-36Pmock cells. The expression of Gab2 was not detected in F-36P cells by immunoblot analysis using a specific antibody (data not shown). We first examined the effects of knockdown of Gab1 on the proliferation of F-36P cells in response to EPO by WST-1 assay. Although both F-36P-Gab1-siRNA and F-36P-mock cells proliferated dose-dependently in response to EPO, the proliferation of F-36PGab1-siRNA cells was significantly reduced compared to that of F36P-mock cells at each concentration of EPO (0.01 U/ml–10 U/ml) (Fig. 1B). We obtained the same results from WST-1 assay using other clones of F-36P-Gab1-siRNA cells (data not shown).

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Next, we examined the effects of knockdown of Gab1 on apoptosis of F-36P cells at various concentrations of EPO by flow cytometry using FITC-labeled annexin V. As the concentrations of EPO were reduced, the percentage of annexin V-positive F-36P-Gab1-siRNA and F-36P-mock cells was increased, especially at low EPO concentrations (0.01 U/ml and 0.1 U/ml) and in the absence of EPO (Table 1). Furthermore, the percentage of annexin V-positive F-36P-Gab1-siRNA cells was higher than that of F-36P-mock cells at each concentration of EPO. The reduction of the proliferation and the promotion of the apoptosis of Gab1-deficient cells were greater than those of mocktransfected cells when the concentrations of EPO were reduced, reflecting a decreased response of Gab1-deficient cells to EPO. To explore the mechanism by which knockdown of Gab1 promotes apoptosis of F-36P cells at low concentrations of EPO, we examined the expression of Bcl-xL and Bcl-2, because these molecules are known to function as anti-apoptotic effectors [2,22]. As shown in Fig. 2, the expression of Bcl-xL and Bcl-2 in F-36P-Gab1-siRNA cells was similar to that in F-36P-mock cells at a high concentration of EPO (10 U/ml). In contrast, although the expression of Bcl-xL and Bcl-2 in both F-36P-Gab1-siRNA and F-36P-mock cells was reduced at a low concentration of EPO (0.01 U/ml) similar to serum concentrations of EPO in healthy volunteers (0.005–0.04 U/ml) [23], the expression of those proteins in F-36P-Gab1-siRNA cells was significantly suppressed compared to that in F-36P-mock cells. 3.2. The effects of knockdown of Gab1 on EPO-induced activation of PI3K and Erk1/2 in F-36P cells The findings described above indicate that Gab1 is involved in EPOR-mediated proliferation and survival signal transduction. To explore the mechanism of the regulation of EPO-induced proliferation and survival signaling through Gab1, we examined whether knockdown of Gab1 would inhibit EPO-induced activation of Akt, because Gab1 is involved in the activation of PI3K in EPOR-mediated signal transduction [9,10]. Sandwich ELISA analysis using a phospho-Akt1 (Ser473) antibody showed that EPO-induced phosphorylation of Ser473 in Akt1 in F-36P-Gab1-siRNA cells was similar to that in F-36Pmock cells (Fig. 3A). This finding was confirmed by immunoblot analysis using anti-phospho-Akt1 (Ser473) antibody (data not shown). Therefore, knockdown of Gab1 did not affect EPO-induced activation of Akt in F-36P cells. We next examined the effects of Gab1 siRNA expression on the activation of Erk1/2 in response to EPO, because it is known that members of the Gab family, namely Gab1 and Gab2, couple cytokine receptor-mediated signals with the Ras/Erk pathway [16,18,24]. As shown in Fig. 3B, EPO-induced phosphorylation of Thr202 and Tyr204 in Erk1/2 was inhibited in F-36P-Gab1-siRNA cells compared to that in F-36P-mock cells. This finding indicates that EPO-induced activation of Erk1/2 is suppressed in F-36P-Gab1-siRNA cells, because phosphorylation of these sites in Erk1/2 is closely related to their activity [25].

Table 1 Knockdown of Gab1 promotes apoptosis of F-36P cells. Fig. 1. The effects of knockdown of Gab1 on EPO-dependent proliferation and survival of F-36P cells. (A) The transformants of Gab1-deficient F-36P cells were established by means of transfection of the expression vector of Gab1 siRNA (F-36P-Gab1-siRNA). The empty vector without any insert was also electroporated into F-36P cells (F-36P-mock). Cell lysates of F-36P-Gab1-siRNA and F-36P-mock cells were immunoprecipitated with anti-Gab1 (αGab1) antibody followed by Western blotting analysis using αGab1 antibody. (B) After F-36P-Gab1-siRNA and F-36P-mock cells were seeded onto each well of the flat-bottom 96-well plates at a density of 5 × 103 cells/well, the cells were incubated with increasing concentrations of EPO for 3 days. The proliferation of the cells was then analyzed by WST-1 assay. The data represent three independent experiments with triplicate samples. Bars (mean ± SD) represent the percentage cell count at the indicated concentrations of EPO relative to the cell count of F-36P-mock cells at 10 U/ml of EPO. ⁎p b 0.05; ⁎⁎p b 0.01; ns, not significant.

EPO (U/ml) Mock Gab1-siRNA

A(+) A(−) PI (+) A (+) A (−) PI (+)

(%) (%) (%) (%)

0

0.01

0.1

1

10

43.6 27.7 90.4 2.8

47.3 16.1 91.8 2.9

14.5 6.1 59.9 6.2

11.4 4.7 22.9 1.8

11.1 2.4 18.6 1.3

After F-36P-Gab1-siRNA (Gab1-siRNA) and F-36P-mock (mock) cells were cultured with increasing concentrations of EPO for 3 days, the cells were stained with FITClabeled annexin V and propidium iodide (PI). The cells were then analyzed with an EPICS XL flow cytometry system equipped with EXPO32ADC software (Beckman Coulter, Miami, FL). The data represent three independent experiments. The data represent the percentage of annexin V-positive [A (+)] or annexin V-negative and PIpositive [A (−) PI (+)] cells.

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mediated Erk activation plays an important role in the proliferation and survival signaling mediated by EPOR. To explore the involvement of Erk activity in EPOR-mediated proliferation and survival signal transduction, we examined the effects of a selective Erk inhibitor, U0126, on the EPO-dependent proliferation and survival of F-36P cells. As shown in Fig. 3C, U0126 dose-dependently inhibited the proliferation of F-36P cells in response to EPO. In addition, the expression of Bcl-2 and Bcl-xL was inhibited by U0126 in a dose-dependent manner (Fig. 3D). The inhibitory effects of U0126 on EPO-induced activation of Erk1/2 (Fig. 3E) correspond with its ability to reduce EPO-dependent proliferation and promote apoptosis of F-36P cells. Fig. 2. Analysis of anti-apoptotic proteins in F-36P cells. F-36P-Gab1-siRNA and F-36Pmock cells were cultured with EPO at 0.01 and 10 U/ml for 3 days. Whole cell lysates (40 μg) were subjected to SDS-PAGE. The expression of anti-apoptotic proteins (Bcl-2 and Bcl-xL) and β-actin was detected by Western blotting analyses using the indicated antibodies.

3.3. Suppression of Erk activity leads to growth inhibition and induction of apoptosis in F-36P cells As described above, the suppression of EPO-induced activation of Erk in Gab1-deficient F-36P cells is consistent with the promotion of apoptosis and growth inhibition of the cells, indicating that Gab1-

3.4. EPO induces the association of Gab1 with Grb2, SHP-2 and p85 in F-36P cells To explore the molecular mechanism of Erk activation through Gab1 in EPOR-mediated signaling, we first examined the association of Gab1 with Grb2 and SHP-2 in response to EPO, because Grb2 and SHP-2 have been found to transmit the signals mediated by diverse cytokine receptors to the Ras/Erk pathway [22,24–29]. Time course analysis (Fig. 4A) showed that the level of tyrosine phosphorylation of Gab1 was increased 1 min after EPO treatment, and reached a maximum level at 5 min after EPO stimulation. Concomitant with the increase in Gab1 tyrosine phosphorylation, the

Fig. 3. Erk1/2 but not Akt are involved in the EPO-dependent proliferation and survival of F-36P cells. (A) Analysis of EPO-induced activation of Akt activity in Gab1-deficient F-36P cells. After growth factor starvation, the cells were treated with 100 U/ml EPO at 37 °C for 10 min. Phosphorylation of Ser 473 in Akt1 in the lysates was analyzed using a solid phase sandwich ELISA. The data (mean ± SD) represent the ratio of the amount of pAkt1 (Ser473) or Akt1 in lysates from EPO-treated cells relative to that from cells without EPO treatment. The experiments with triplicate samples were performed independently at least three times. (B) Analyses of EPO-induced activation of Erk1/2 activity in Gab1-deficient F36P cells. F-36P-Gab1-siRNA and F-36P-mock cells were treated as described above. Whole cell lysates (15 μg) were then subjected to Western blotting analyses using anti-phosphoErk (Thr202/Tyr204) (αpErk) and αErk antibodies. NSB: a non-specific band. (C) Inhibitory effects of U0126 on EPO-dependent proliferation of F-36P cells. F-36P cells were cultured with increasing concentrations of U0126 in the presence of 5 U/ml EPO. The proliferation of the cells was then analyzed by WST-1 assay. The data represent three independent experiments with triplicate samples. Bars (mean ± SD) represent the percentage of cell growth with U0126 relative to parallel control cultures without U0126. (D) U0126 dosedependently inhibits the expression of anti-apoptotic proteins in F-36P cells. F-36P cells were treated with increasing concentrations of U0126 in the presence of 5 U/ml of EPO for 3 days. Whole cell lysates (40 μg) were subjected to Western blotting analyses using the indicated antibodies. (E) Inhibitory effects of U0126 on EPO-induced Erk1/2 activation. F-36P cells were treated as described above. Whole cell lysates (15 μg) were subjected to Western blotting analyses using αpErk (Thr202/Tyr204) and αErk antibodies.

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whether PI3K regulates the Gab1-mediated activation of Erk in response to EPO, we examined the effects of a selective PI3K inhibitor LY294002 on EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2, SHP-2 and p85. LY294002 reduced EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2, SHP-2 and p85 in F-36P cells (Fig. 5A). Furthermore, EPO-induced phosphorylation of Thr202/Tyr204 in Erk1/2 was diminished in F-36P cells pretreated with LY294002 (Fig. 5B). These results indicate that activation of PI3K is required for tyrosine phosphorylation of Gab1 and its association with Grb2, SHP-2 and p85 in EPO-treated F-36P cells, and that PI3K lies upstream of Erk1/2 in EPOR-mediated signaling and regulates the Erk1/2 activation. 3.7. p85-siRNA reduces EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2 and SHP-2, and Erk activation in F-36P cells

Fig. 4. Time course analysis of EPO-induced association of Gab1 with Grb2, SHP-2 and p85, and constitutive association of Grb2 with SOS1. (A) After growth factor starvation, F-36P cells were treated with 100 U/ml EPO at 37 °C for the indicated times. Cell lysates were then subjected to immunoprecipitation with αGab1 antibody followed by Western blotting analyses using the indicated antibodies. (B) After growth factor starvation, F-36P cells were treated with EPO (100 U/ml) at 37 °C for 10 min. Cell lysates were then subjected to immunoprecipitation with αGrb2 and αSOS1 antibodies followed by Western blotting analyses using the indicated antibodies.

binding of SHP-2 and Grb2 to Gab1 reached its maximum levels at 5 and 10 min after EPO stimulation, respectively, and then gradually decreased. In addition, because Grb2 but not SHP-2 was detected in anti-Gab1 immunoprecipitates from lysates of unstimulated F-36P cells, it appears that Grb2 was constitutively associated with Gab1 at a basal level. In addition, p85 was constitutively associated with Gab1 at a basal level, and the binding of p85 to Gab1 paralleled the levels of tyrosine phosphorylation of Gab1 in response to EPO. 3.5. SOS1 is constitutively associated with Grb2 Because Grb2 is found to transmit the signals mediated by diverse cytokine receptors to the Ras/Erk pathway through its binding to guanine nucleotide exchange factor SOS1 [26–28], we next examined the EPO-induced association of Grb2 and SOS1 in F-36P cells. The amount of Grb2 in anti-SOS1 immunoprecipitates from lysates of EPO-treated F-36P cells was similar to that of F-36P cells without EPO treatment (Fig. 4B). In addition, EPO did not affect the amount of SOS1 co-immunoprecipitated with Grb2. 3.6. LY294002 inhibits EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2, SHP-2 and p85, and Erk activation in F-36P cells The findings described above indicate that the Grb2–SOS1 complex is recruited to the vicinity of Ras through Grb2 bound to Gab1 when Gab1 is translocated to the plasma membrane after EPO treatment, leading to Ras activation and resulting in Erk activation. One of the mechanisms of recruitment of Gab1 to the plasma membrane is to bind the PH domain of Gab1 to PIP3, which is produced by EPO-activated PI3K [13–15]. Therefore, to determine

To confirm the findings obtained by the experiments using LY294002, we examined the effects of p85-siRNA on EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2 and SHP-2, and Erk activation in F-36P cells. As shown in Fig. 5C, the expression of p85 was suppressed in F-36P cells transfected with p85-siRNA. Suppression of p85 expression by transfection of p85-siRNA in F-36P cells reduced EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2 and SHP-2 (Fig. 5C). EPO-induced association of Gab1 with p85 could be barely detected in F-36P cells transfected with p85-siRNA because the expression of p85 was significantly suppressed in those cells. Furthermore, EPO-induced phosphorylation of Thr202/Tyr204 in Erk1/2 was diminished in F-36P cells transfected with p85-siRNA but not in those with non-targeting siRNA (Fig. 5D). These results confirm that activation of PI3K is required for tyrosine phosphorylation of Gab1 and its association with Grb2 and SHP-2, and Erk activation in EPO-treated F-36P cells. 3.8. LY294002 inhibits EPO-induced tyrosine phosphorylation of Gab1 in human primary EPO-sensitive erythroid cells To confirm that activation of PI3K is required for tyrosine phosphorylation of Gab1 in EPOR-mediated signaling, we examined the effect of LY294002 on EPO-induced tyrosine phosphorylation of Gab1 in human primary EPO-sensitive erythroid cells. In this experiment, after informed consent was obtained from healthy volunteers, hematopoietic progenitor cells were mobilized to the peripheral blood by treatment with granulocyte colony-stimulating factor (G-CSF). The mononuclear cells containing hematopoietic progenitor cells mobilized to the peripheral blood were then collected using a continuous blood flow separation technique. For the analysis of PI3K-dependent tyrosine phosphorylation of Gab1, we used the peripheral mononuclear cells collected from G-CSF-treated healthy volunteers, which contained 518 ± 87 colonies of erythroid colonyforming units (CFU-E)/105 of mononuclear cells. As shown in Fig. 5E, Gab1 was tyrosine-phosphorylated after treatment with EPO and LY294002 inhibited EPO-induced tyrosine phosphorylation of Gab1 in human primary erythroid cells. Furthermore, Grb2 was constitutively associated with Gab1 at a basal level. EPO enhanced the association of Grb2 with Gab1, whereas the EPOinduced association between Grb2 and Gab1 was diminished to a basal level by pretreatment with LY294002. 3.9. Jak2 inhibitor AG490 diminishes tyrosine phosphorylation of Gab1 and its association with Grb2, SHP-2 and p85 in response to EPO Tyrosine phosphorylation of Gab1 is critical to mediate EPO signals to downstream pathways through Gab1 [9,11]. Because Jak2 plays a

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Fig. 5. PI3K regulates the association of Gab1 with Grb2, SHP-2 and p85, and Erk1/2 activity in EPOR-mediated signal transduction. (A) LY294002 reduces tyrosine phosphorylation of Gab1 and its association with Grb2, SHP-2 and p85 in F-36P cells after EPO treatment. Growth factor-starved F-36P cells were treated with 50 μM LY294002 or the same volume of dimethyl sulfoxide (DMSO; vehicle) as a control for 1 h followed by stimulation with 100 U/ml of EPO at 37 °C for 10 min. Cell lysates were then subjected to immunoprecipitation with αGab1 antibody followed by Western blotting analyses using the indicated antibodies. IgL, immunoglobulin light chain. (B) Inhibitory effects of LY294002 on EPO-induced Erk1/2 activation in F-36P cells. Whole cell lysates were prepared as described above. The lysates (15 μg) were subjected to SDS-PAGE followed by Western blotting analyses using αpErk (Thr202/Tyr204) and αErk antibodies. (C) Suppression of p85 expression by transfection of p85-siRNA inhibits EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2 and SHP-2 in F-36P cells. p85-siRNA (p85, 2 μg) and non-targeting siRNA (C, 2 μg) as a control were introduced in F-36P cells by Nucleofector technology as described in the Materials and methods. The cells were then incubated with the growth medium for 24 h. After growth factor starvation by overnight incubation, the cells were treated with 100 U/ml of EPO at 37 °C for 10 min. The cell lysates were then subjected to immunoprecipitation with αGab1 antibody followed by Western blotting analyses using the indicated antibodies. The expression of p85 in the lysates prepared as described above was detected by Western blotting analysis using αp85 antibody. WCL, whole cell lysates. (D) Inhibitory effects of p85-siRNA on EPO-induced Erk1/2 activation in F-36P cells. Cell lysates were prepared as described above. The lysates (15 μg) were subjected to SDS-PAGE followed by Western blotting analyses using αpErk (Thr202/Tyr204) and αErk antibodies. C, non-targeting siRNA (2 μg) as a control; p85, p85-siRNA (2 μg). (E) Inhibitory effects of LY294002 on EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2 in human erythroid cells. After informed consent was obtained from healthy volunteers, hematopoietic progenitor cells were mobilized to the peripheral blood by treatment with G-CSF. The mononuclear cells containing hematopoietic progenitor cells mobilized were then collected using a continuous blood flow separation technique. In this experiment, we used peripheral mononuclear cells collected from G-CSF-treated healthy volunteers after confirming that 1 × 105 mononuclear cells contained 518 ± 87 colonies of CFU-E. After growth factor starvation, the peripheral mononuclear cells (5 × 108) were treated with 50 μM LY294002 or the same volume of dimethyl sulfoxide (DMSO; vehicle) as a control for 1 h followed by stimulation with 100 U/ml of EPO at 37 °C for 10 min. Cell lysates were then subjected to immunoprecipitation with αGab1 antibody followed by Western blotting analyses using the indicated antibodies.

central role in the tyrosine phosphorylation of EPOR and signaling molecules, we examined the effects of a selective Jak tyrosine kinase inhibitor, AG490, on EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2, SHP-2 and p85 in F-36P cells (Fig. 6A). EPO-induced tyrosine phosphorylation of Gab1 was inhibited by AG490. SHP-2 was only slightly detected in anti-Gab1 immunopre-

cipitates from the lysates of AG490-treated F-36P cells. EPOenhanced association of Gab1 with Grb2 and p85 in AG490-treated F-36P cells was reduced to a basal level similar to that in F-36P cells without EPO treatment. These results indicate that Jak2 plays a crucial role in the tyrosine phosphorylation of Gab1 and the binding of the SH2 domains of Grb2, SHP-2 and p85 to Gab1 in EPORmediated signaling.

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3.10. Jak2-siRNA reduces EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2, SHP-2 and p85 in F-36P cells

3.11. Gab1 tyrosine-phosphorylated by Jak2 is associated with the SH2 domain of Grb2 in vitro

To confirm that Jak2 regulates Gab1-mediated EPO signaling, we examined the effects of Jak2-siRNA on tyrosine phosphorylation of Gab1 and its association with Grb2, SHP-2 and p85 in EPO-treated F36P cells. As shown in Fig. 6B, the expression of Jak2 was suppressed in F36P cells transfected with Jak2-siRNA. Knockdown of Jak2 by transfection of Jak2-siRNA in F-36P cells diminished EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2, SHP-2 and p85 (Fig. 6B). Thus, taken together with the findings obtained by the experiments using AG490, these results confirm that Jak2 regulates the interaction of Gab1 with signaling molecules such as Grb2, SHP-2 and p85 through tyrosine phosphorylation of Gab1 in EPO signaling.

To explore the binding of an SH2 domain of Grb2 to Gab1, the in vitro binding of GST–Grb2 fusion proteins to Gab1 was examined. As shown in Fig. 6C, the GST–Grb2 SH2 domain bound to Gab1 tyrosinephosphorylated by Jak2 co-expressed with Gab1 in 293T cells but was not associated with unphosphorylated Gab1 expressed alone in the cells. AG490 inhibited the Jak2-induced association of Gab1 with the GST–Grb2 SH2 domain. Therefore, the Grb2 SH2 domain directly binds to Gab1 tyrosine-phosphorylated by Jak2. 4. Discussion In the present study, we have demonstrated that Gab1 couples PI3K-mediated signals with Erk1/2 in EPOR-mediated signal transduction and that Gab1-mediated Erk activation plays a crucial role in the proliferation and survival signaling mediated by EPOR. Furthermore, down-regulation of Bcl-xL and Bcl-2 was detected in Gab1deficient F-36P cells at the low concentration of EPO. Bcl-xL expression was found to be regulated by both the Stat5-dependent and Erk-dependent pathways in EPO signaling [2,30–33]. In addition, Erk was found to regulate Bcl-2 expression through CREB phosphorylation [28,34]. However, several studies using knockout mice and erythroid cells have indicated that expression of Bcl-xL but not Bcl-2 is essential for the survival of erythroid cells [30–33,35,36]. Taken together with these findings, the present results suggest that Gab1mediated Erk activation is involved in the EPOR-mediated survival signaling through up-regulation of Bcl-xL, although Bcl-2 expression may also be involved in the EPO-induced anti-apoptotic effects on erythroid cells. The EPO-induced association of Gab1 with Grb2 and SHP-2 was consistent with Erk activation in EPO-treated F-36P cells. Furthermore, the dissociation of Gab1 from Grb2 and SHP-2 by LY294002 was correlated with the inhibitory effects of LY294002 on EPO-induced activation of Erk1/2. In addition, suppression of p85 expression by transfection of p85-siRNA into F-36P cells diminished the EPOinduced association of Gab1 with Grb2 and SHP-2 as well as Erk activation. These results suggest that Grb2 and SHP-2 intervene in transducing Gab1-mediated signals to Erk1/2. Since Grb2 is constitutively associated with SOS1, SOS1 translocates to the vicinity of Ras through Grb2 bound to Gab1, which is recruited to the plasma membrane after EPO treatment, resulting in activation of the Ras/Erk pathway. Furthermore, although the precise mechanisms by which

Fig. 6. Jak2 regulates the association of Gab1 with Grb2, SHP-2 and p85 in EPORmediated signal transduction. (A) AG490 inhibits EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2, SHP-2 and p85 in F-36P cells. Growth factorstarved F-36P cells were treated with 100 μM AG490 or the same volume of vehicle for 16 h prior to EPO (100 U/ml) stimulation at 37 °C for 10 min. Cell lysates were immunoprecipitated with αGab1 antibody followed by Western blotting analyses using the indicated antibodies. (B) Knockdown of Jak2 by transfection of Jak2-siRNA inhibits EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2, SHP-2 and p85 in F-36P cells. F-36P cells transfected with Jak2-siRNA (Jak2, 2 μg) and non-targeting siRNA (C, 2 μg) were incubated with the growth medium for 24 h. After growth factor starvation by overnight incubation, the cells were treated with 100 U/ml of EPO at 37 °C for 10 min. Cell lysates were then subjected to immunoprecipitation with αGab1 antibody followed by Western blotting analyses using the indicated antibodies. The expression of Jak2 was detected by Western blotting analysis using αJak2 antibody after cell lysates prepared as described above were immunoprecipitated with αJak2 antibody. (C) Jak2 tyrosine-phosphorylates Gab1 and induces the association of Gab1 with GST–Grb2 SH2 domain in vitro. Gab1 was transiently expressed with or without Jak2 in 293T cells. The cells were treated with 100 μM AG490 for 16 h prior to cell harvest. Two days after transfection, cell lysates were prepared as described above. The lysates were incubated with agarose-conjugated GST–Grb2 SH2 domain (GST-SH2) and GST–Grb2 whole molecule (GST–Grb2) at 4 °C for 4 h. GSTagarose (GST control) was used as a control. The proteins precipitated with the agarose beads were analyzed by Western blotting analysis using αPY antibody.

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SHP-2 transduces Gab1-mediated signals to the Ras/Erk pathway remain to be elucidated, it is possible that SHP-2 negatively regulates the recruitment of p120-RasGAP, a GTPase-activating protein, to Gab1 by dephosphorylating the p120-RasGAP binding site(s) in Gab1, resulting in activation of the Ras/Erk pathway [29]. We have shown that LY294002 and suppression of p85 expression by transfection of p85-siRNA independently inhibited EPO-induced tyrosine phosphorylation of Gab1 and its interaction with Grb2 and SHP-2 as well as the activation of Erk1/2. These findings indicate that PI3K lies upstream of Gab1 and plays a role in the activation of Erk1/2 by regulating the tyrosine phosphorylation of Gab1 and its interaction with the Grb2–SOS1 complex and SHP-2 in EPO signaling. Several studies have shown the regulation of Erk activity by PI3K in EPORmediated signaling [37–40]. Klingmüller et al. [37] have shown that the binding site of PI3K in Tyr479 of mouse EPOR is important for the activation of Erk activity, because EPO-induced activation of Erk1/2 is impaired in Ba/F3 cells expressing the mutant EPOR lacking the PI3Kbinding site. In addition, several groups have also shown that the selective PI3K inhibitors LY294002 and wortmannin inhibit EPOinduced activation of Erk1/2 in Ba/F3 and 32D cells expressing the wild-type EPOR and erythroid progenitor cells [37–40]. However, the signaling molecules which couple PI3K with the Erk pathway remain to be elucidated, although experiments using Ba/F3 cells expressing EPOR and rat fetal liver cells have suggested that protein kinase C is a possible mediator connecting PI3K with the Erk cascade [37,38]. In the present study, we have clearly demonstrated that Gab1 is one of the mediators which link PI3K to the Erk pathway in EPOR-mediated signal transduction. Recently, van der Akker et al. [12] have shown that EPO-induced tyrosine phosphorylation of Gab2 but not of Gab1 was dependent on the activation of PI3K in mouse fetal liver erythroblasts and erythroblast cell line R1 and that the tyrosine kinase RON was required for EPO-induced tyrosine phosphorylation of Gab1. However, the authors have also indicated that tyrosine phosphorylation of Gab1 induced by EPO was partially affected by inhibitors of PI3K. In contrast, we have shown PI3K-dependent tyrosine phosphorylation of Gab1 in EPOR-mediated signaling in human primary erythroid cells and in erythroleukemia cell line F-36P. Gab2 was not expressed in human erythroid cells as described previously [9–11]. Therefore, Gab1 may compensate for the function of Gab2 in human erythroid cells, as EPO-induced tyrosine phosphorylation of Gab2 was enhanced in Gab1- or RON-deficient mouse erythroblasts [12]. The difference in the mechanism of Gab1 phosphorylation between human and mouse erythroblasts may be dependent on the origin of the cells, although the precise reason for this discrepancy is unknown. The recruitment of Gab1 to the plasma membrane where Gab1 becomes phosphorylated is required to mediate EPO signals downstream of Gab1. Based on the findings that the inhibition of EPOinduced activation of PI3K by LY294002 and suppression of p85 expression by transfection of p85-siRNA led to the suppression of tyrosine phosphorylation of Gab1 and its associations with Grb2 and SHP-2, which transduce EPO signals to the Erk pathway, Gab1 is likely to be recruited to the plasma membrane through the binding of its PH domain to PIP3 produced by PI3K activated after EPO treatment, as described previously [6,14,41]. Because EPO-induced association of Gab1 with EPOR could not be detected in F-36P cells, as described previously (data not shown) [11], Gab1 is unlikely to be recruited to the membrane through binding to EPOR. Tyrosine phosphorylation of Gab1 plays an important role in its interaction with signaling molecules including Grb2, SHP-2 and p85, resulting in the transmission of signals downstream to the Ras/Erk and PI3K/Akt pathways [5–7,9,11,17]. Studies using AG490 and Jak2siRNA indicate that Jak2 is involved in EPO-induced tyrosine phosphorylation of Gab1 and its interaction with Grb2, SHP-2 and p85, as Gab2 phosphorylation was dependent on Jak2 activity in GCSFR signaling [24]. The finding that EPO-induced tyrosine phosphor-

ylation of Gab1 was correlated with the enhancement of the association of Gab1 with Grb2 indicates that the SH2 domain of Grb2 binds to tyrosine residue(s) in Gab1 phosphorylated by EPO treatment. In vitro binding experiments further demonstrated the interaction of Grb2 with Gab1 tyrosine-phosphorylated by Jak2 through the Grb2 SH2 domain. Furthermore, Gab1 was found to be constitutively associated with Grb2 in vitro and in vivo, as Lewitzky et al. [42] have demonstrated that Grb2 constitutively binds to Gab1 through the C-terminal SH3 domain of Grb2. The co-expression experiments indicate that Jak2 can directly tyrosine-phosphorylate Gab1. Recently, it has been demonstrated that RON plays a crucial role in EPO-induced tyrosine phosphorylation of Gab1 in mouse erythroblasts and that Jak2 regulates RON activity [12]. Therefore, it is possible that Jak2 tyrosine-phosphorylates Gab1 through the activation of RON activity. Several reports have demonstrated that Gab1 plays a role in the activation of PI3K in EPOR-mediated signaling through the p85 SH2 domain(s) bound to tyrosine residues phosphorylated in Gab1 [9,11]. In the present study, we have also shown that EPO-induced tyrosine phosphorylation of Gab1 is correlated with the association of Gab1 with p85 in F-36P cells. However, Gab1-mediated activation of PI3K in EPO-treated F-36P cells is unlikely to play a central role in the EPOinduced activation of PI3K, because knockdown of Gab1 in F-36P cells failed to inhibit the activation of PI3K in response to EPO. Because PI3K is activated by p85 bound to EPOR and IRS-2 [1,2,11,21,43], these signaling pathways may compensate for Gab1-mediated activation of PI3K. In conclusion, Gab1 plays an important role in transducing EPORmediated proliferation and survival signals to the Erk pathway. In addition, PI3K activity controls the Gab1-mediated activation of Erk1/2 by regulating the tyrosine phosphorylation of Gab1 and its association with the Grb2–SOS1 complex and SHP-2 in EPOR-mediated signal transduction. A model of the Gab1-mediated Erk activation in EPO signaling is as follows: After EPO binds to its receptors, EPOR is phosphorylated on tyrosine residues to provide the binding site(s) for PI3K. PIP3, which is produced by EPO-activated PI3K, recruits Gab1 to the plasma membrane through the Gab1 PH domain bound to PIP3. Since Gab1 is constitutively associated with the Grb2–SOS1 complex at a basal level, the Grb2–SOS1 complex is recruited in the vicinity of Ras, leading to Ras/Erk activation. After the recruitment of Gab1 to the membrane, Gab1 is tyrosine-phosphorylated by PTKs including Jak2. Then more Grb2–SOS1 complexes and SHP-2 can bind to Gab1, thereby resulting in the enhancement of EPO-induced activation of the Ras/Erk pathway.

Acknowledgements We would like to thank Dr. JN Ihle for providing the Jak2 cDNA. We would also like to thank the Kirin Brewery Co. for providing recombinant human erythropoietin. Contributions: T.F. and Y.K. designed and performed research, analyzed data, and wrote the paper; A.K. designed research, analyzed data, and commented on the paper; G.Y. performed research; F.O.W., O.I. and H.O. analyzed data; T.I. supervised the research; T.T. supervised the research and commented on the paper.

References [1] [2] [3] [4] [5] [6] [7] [8]

T.D. Richmond, M. Chohan, D.L. Barber, Trends Cell. Biol. 15 (2005) 146. U. Testa, Leukemia 18 (2004) 1176. T. Bittorf, R. Jaster, B. Lüdtke, B. Kamper, J. Brock, Cell. Signal. 9 (1997) 85. A. Schaefer, F. Kósa, T. Bittorf, M. Magócsi, A. Rosche, Y. Ramirez-Chávez, S. Marotzki, H. Marquardt, Cell. Signal. 16 (2004) 223. Y. Liu, L.R. Rohrschneider, FEBS Lett. 515 (2002) 1. H. Gu, B.G. Neel, Trends Cell. Biol. 13 (2003) 122. G. Sármay, A. Angyal, A. Kertész, M. Maus, D. Medgyesi, Immunol. Lett. 104 (2006) 76. K. Nishida, T. Hirano, Cancer Sci. 94 (2003) 1029.

T. Fukumoto et al. / Cellular Signalling 21 (2009) 1775–1783 [9] A. Wickrema, S. Uddin, A. Sharma, F. Chen, Y. Alsayed, S. Ahmad, S.T. Sawyer, G. Krystal, T. Yi, K. Nishada, M. Hibi, T. Hirano, L.C. Platanias, J. Biol. Chem. 274 (1999) 24469. [10] D. Bouscary, C. Lecoq-Lafon, S. Chrétien, S. Zompi, S. Fichelson, O. Muller, F. Porteu, I. Dusanter-Fourt, S. Gisselbrecht, P. Mayeux, C. Lacombe, Oncogene 20 (2001) 2197. [11] D. Bouscary, F. Pene, Y.E. Claessens, O. Muller, S. Chrétien, M. Fontenay-Roupie, S. Gisselbrecht, P. Mayeux, C. Lacombe, Blood 101 (2003) 3436. [12] E. van der Akker, T. van Dijk, M. Parren-van Amelsvoort, K.S. Grossmann, U. Schaeper, K. Toney-Earley, S.E. Waltz, B. Löwenberg, M. von Lindern, Blood 103 (2004) 4457. [13] K. Nishida, Y. Yoshida, M. Itoh, T. Fukada, T. Ohtani, T. Shirogane, T. Atsumi, M. Takahashi-Tezuka, K. Ishihara, M. Hibi, T. Hirano, Blood 93 (1999) 1809. [14] G.A. Rodrigues, M. Falasca, Z. Zhang, S.H. Ong, J. Schlessinger, Mol. Cell. Biol. 20 (2000) 1448. [15] R.J. Ingham, L. Santos, M. Dang-Lawson, M. Holgado-Madruga, P. Dudek, C.R. Maroun, A.J. Wong, L. Matsuuchi, M.R. Gold, J. Biol. Chem. 276 (2001) 12257. [16] M. Takahashi-Tezuka, Y. Yoshida, T. Fukada, T. Ohtani, Y. Yamanaka, K. Nishida, K. Nakajima, M. Hibi, T. Hirano, Mol. Cell. Biol. 18 (1998) 4109. [17] C. Lecoq-Lafon, F. Verdier, S. Fichelson, S. Chrétien, S. Gisselbrecht, C. Lacombe, P. Mayeux, Blood 93 (1999) 2578. [18] M. Itoh, Y. Yoshida, K. Nishida, M. Narimatsu, M. Hibi, T. Hirano, Mol. Cell. Biol. 20 (2000) 3695. [19] Y. Okutani, A. Kitanaka, T. Tanaka, H. Kamano, H. Ohnishi, Y. Kubota, T. Ishida, J. Takahara, Oncogene 20 (2001) 6643. [20] N. Ichihara, Y. Kubota, A. Kitanaka, T. Tanaka, T. Taminato, Cancer Lett. 260 (2008) 155. [21] Y. Kubota, T. Tanaka, A. Kitanaka, H. Ohnishi, Y. Okutani, M. Waki, T. Ishida, H. Kamano, EMBO J. 20 (2001) 5666. [22] P.P. Ruvolo, X. Deng, W.S. May, Leukemia 15 (2001) 515. [23] A.W. Wognum, V. Lam, R. Goudsmit, G. Krystal, Blood 76 (1990) 1323. [24] L. Wang, J. Xue, E.V. Zadorozny, L.J. Robinson, Cell. Signal. 20 (2008) 1890.

1783

[25] D.M. Payne, A.J. Rossomando, P. Martino, A.K. Erickson, J.H. Her, J. Shabanowitz, D.F. Hunt, M.J. Weber, T.W. Sturgill, EMBO J. 10 (1991) 885. [26] L. Buday, Biochim. Biophys. Acta. 1422 (1999) 187. [27] L.S. Steelman, S.C. Pohnert, J.G. Shelton, R.A. Franklin, F.E. Bertrand, J.A. McCubrey, Leukemia 18 (2004) 189. [28] L.S. Steelman, S.L. Abrams, J. Whelan, F.E. Bertrand, D.E. Ludwig, J. Bäsecke, M. Libra, F. Stivala, M. Milella, A. Tafuri, P. Lunghi, A. Bonati, A.M. Martelli, J.A. McCubrey, Leukemia 22 (2008) 686. [29] M. Dance, A. Montagner, J.P. Salles, A. Yart, P. Raynal, Cell. Signal 20 (2008) 453. [30] N. Motoyama, F. Wang, K.A. Roth, H. Sawa, K. Nakayama, N. Nakayama, I. Negishi, S. Senju, Q. Zhang, S. Fujii, D.Y. Loh, Science 267 (1995) 1506. [31] M. Silva, A. Benito, C. Sanz, F. Prosper, D. Ekhterae, G. Nuñez, J.L. Fernandez-Luna, J. Biol. Chem. 274 (1999) 22165. [32] M. Socolovsky, H. Nam, M.D. Fleming, V.H. Haase, C. Brugnara, H.F. Lodish, Blood 98 (2001) 3261. [33] M. Mori, M. Uchida, T. Watanabe, K. Kirito, K. Hatake, K. Ozawa, N. Komatsu, J. Cell. Physiol. 195 (2003) 290. [34] J.A. McCubrey, W.S. May, V. Duronio, A. Mufson, Leukemia 14 (2000) 9. [35] P.A. Gregoli, M.C. Bondurant, Blood 90 (1997) 630. [36] D.J. Veis, C.M. Sorenson, J.R. Shutter, S.J. Korsmeyer, Cell 75 (1993) 229. [37] U. Klingmüller, H. Wu, J.G. Hsiao, A. Toker, B.C. Duckworth, L.C. Cantley, H.F. Lodish, Proc. Natl. Acad. Sci. U S A 94 (1997) 3016. [38] E. Devemy, C. Billat, B. Haye, Cell. Signal. 1 (1997) 41. [39] A. Arai, E. Kanda, O. Miura, Oncogene 21 (2002) 2641. [40] E.K. Schmidt, S. Fichelson, S.M. Feller, BMC Biol. 2 (2004) 7. [41] C.R. Maroun, M. Holgado-Madruga, I. Royal, M.A. Naujokas, T.M. Fournier, A.J. Wong, M. Park, Mol. Cell. Biol. 19 (1999) 1784. [42] M. Lewitzky, C. Kardinal, N.H. Gehring, E.K. Schmidt, B. Konkol, M. Eulitz, W. Birchmeier, U. Schaeper, S.M. Feller, Oncogene 20 (2001) 1052. [43] J.E. Damen, R.L. Cutler, H. Jiao, T. Yi, G. Krystal, J. Biol. Chem. 270 (1995) 23402.