ANTICANCER RESEARCH 26: 905-916 (2006)

Rin1 is a Negative Regulator of the IL3 Receptor Signal Transduction Pathways

C.M. HUNKER, A. GALVIS, M.L. VEISAGA and M.A. BARBIERI

Department of Biological Sciences, Florida International University, University Park, Miami, Florida 33199, U.S.A.

Abstract. Cytokines interact with cell-surface receptors, initiating (9:22 translocation) is often found in patients with leukemia, signaling cascades that promote cell growth while inhibiting the commonly with chronic myelogenous (>95%) and less pathways of apoptotic cells. Rin1 is a multifunctional that commonly with acute lymphocytic leukemia (<40%). Much has been shown to regulate EGF receptor signaling and research has been focused on understanding the consequence endocytosis. To examine the role of Rin1 in IL3 receptor signaling and cause of this alteration and it has been shown that the pathways, Rin1 and deletion mutants were expressed in cells using Philadelphia results in the fusion of sequences a retrovirus system. In this study, the overexpression of Rin1 from the BCR and ABL (11-18). molecules was shown to selectively block IL-3 activation of the The BCR/ABL fusion product is an activated tyrosine Ras-Erk1/2 and PI3K/Akt pathways and the IL-3-stimulated kinase that confers survival and proliferation advantages to incorporation of [3H] thymidine into DNA without a significant hematopoietic cells, thus contributing to leukemogenesis. effect on the activity of the JNK and p38K pathways. Moreover, Several common points of convergence between cytokine- the depletion of Rin1 by RNA interference induced cell growth. In growth factor signaling and BCR/ABL oncogenesis have been addition, Rin1 was also required as a downstream effector of described and cells expressing BCR/ABL no longer require BCR/ABL-induced cell proliferation. Interestingly, the expression exogenous cytokines (i.e. IL3) for their continual growth or of Rin1 selectively blocked the activation of Erk1/2 induced by the survival (4-10, 19). Thus, in cancer cells BCR/ABL BCR/ABL oncogene. These results demonstrate that Rin1 plays constitutively activates various intracellular signaling pathways, an essential and selective role in both IL3- and BCR/ABL- such as those involving Ras, Erk1/2, phosphatidylinositol 3- induced cell proliferation and highlight a new function for Rin1 kinase (PI3K), STAT5 and NFκB to regulate cell growth and in leukemic cells. prolong survival (4-10, 19). Under normal circumstances, these pathways are involved in the regulation of hematopoiesis by Cytokines interact with cell-surface receptors initiating hematopoietic cytokines and other extracellular stimuli. signaling cascades that promote cell growth and survival (1, 2). Furthermore, it has been suggested that PI3K and Ras are The JAK/STAT, Raf/Mek/Erk and PI3K/Akt signaling independently regulated by the BCR/ABL oncogene (20). pathways are activated by a variety of cytokines, including Interestingly, Ras interference 1 (Rin1), a multi-domain interleukin 3 (IL3) (1-3). Signaling through IL3/IL3-receptor protein that contains a proline rich region, has been shown to interaction has been widely studied as a prototype for how interact with c-ABL (21). However, the possible involvement cytokines regulate signal transduction pathways, which, in turn of Rin1 in a downstream signaling from BCR/ABL remains to modulate normal and abnormal hematopoiesis (4-10). The be examined. abnormality first described as the Philadelphia chromosome In this study, it is demonstrated that both overexpression and depletion of Rin1 affects both the IL3-receptor and BCR- ABL signal transduction pathways. The overexpression of Rin1 Abbreviations: Rin1, Ras interference 1; EGF, epidermal growth was found to selectively block the IL3 activation of the Ras- factor; Erk1/2, extracellular signal-regulated kinase; GEF, guanine Erk1/2 kinase and PI3K/Akt pathways and IL3-stimulated nucleotide exchange factor; GFP, green fluorescence protein; JNK, incorporation of [3H] thymidine into DNA without a Jun N-terminal kinase; p38K, p38 protein kinase; IL3, interleukin 3. significant effect on the activity of the JNK and p38K pathways. Moreover, the depletion of Rin1 by RNA Correspondence to: M.A. Barbieri, Florida International University, interference induced cell growth. In addition, Rin1 is also Department of Biological Sciences, 11200 S.W. 8th Street-OE167, required as a downstream effector of BCR/ABL-induced cell Miami, FL, 33199, U.S.A. Tel: 305-348-7535, Fax: 305-348-1986, e- mail: [email protected] proliferation. Interestingly, the expression of Rin1 selectively blocked the activation of Erk1/2 induced by the BCR/ABL Key Words: Signal transduction, Rin1, Ras, BCR/ABL. oncogene. These results demonstrate that the Rin1, PI3K/Akt

0250-7005/2006 $2.00+.40 905 ANTICANCER RESEARCH 26: 905-916 (2006) and Raf/Erk1/2 signal transduction pathways play essential and determined using a detergent-compatible protein assay (Bio-Rad). To selective roles in both IL3- and BCR/ABL-induced cell immunoprecipitate the IL3 receptor, the cell lysates were incubated for proliferation and suggest a new function for Rin1 in both 12 h at 4ÆC with 2.5 Ìl of anti-IL3 receptor ‚-chain antibody. Immunoprecipitates were washed 3 times with the lysis buffer and twice normal and cancer cells. with the wash buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 ÌM DTT (pH 7.5) and assayed using anti-Rin1 antibodies by Western blot analysis. Materials and Methods To immunoprecipitate BCRABL, the cell lysates were incubated for 12 h at 4ÆC with anti-ABL antibody. The immunoprecipitates were Materials and cell lines. The HL-60 and K562 cells were obtained from washed 3 times with the lysis buffer and once with buffer (50 mM Tris- the American Type Culture Collection (Rockville, MD, USA). A HCl, 10 mM MgCl2, 1 ÌM DTT (pH 7.5) and were assayed using wash polyclonal antibody against GFP was obtained from Upstate. An anti- anti- Rin1 antibodies by western blot analysis. hisG monoclonal and anti-GST polyclonal antibodies came from Invitrogen (Carlsbad, CA, USA), the anti-Flag polyclonal antibody Kinase assay. The control and Rin1 cell lines were incubated in the from Sigma and the HA antibody from Usptate. The mouse presence or in the absence of IL3, as indicated in each figure legend, monoclonal and polyclonal anti-Ras and anti-Rin1 antibodies were at 37ÆC. The cells were then washed and lysed in ice-cold lysis buffer. obtained from BD Biosciences Pharmingen. The anti-IL3 receptor ‚- The lysate was clarified as described above. The protein concentration subunit antibody, anti-tyrosine (PY20), anti-ABL and anti-BCR in the cell lysates was determined using a detergent-compatible antibodies for Western blot were purchased from Transduction protein assay (Bio-Rad). Laboratories. The phospho-p42/44 (Erk1/2), phospho- Raf, phospo- For Erk1/2 activation analysis, 10 Ìg of total protein from lysates Elk1 and phospho-AKT antibodies were purchased from Cell were analyzed by 10% SDS-PAGE and transferred to a nitrocellulose Signaling Technology. The phospho-JNK and phospho-p38k membrane using a wet transfer apparatus (Bio-Rad). The membranes antibodies, as well as total anti-Erk1/2, AKT, p38k, Raf and JNK, were were probed with antibodies against total Erk1/2 and phospho-Erk1/2. purchased from Sigma-Aldrich. FuGENE6 and LipofectAMINE 2000 The immunoblots were developed using Super Signal reagents were purchased from Roche and Invitrogen, respectively. (Pierce). Recombinant human IL3 was purchased from Calbiochem and For p38K and JNK activation analysis, 10 Ìg of total protein from Upstate Biotechnology, respectively. The commercial sources for the lysates were analyzed by 10% SDS-PAGE and transferred to a electrophoresis reagents, culture media, sera, films and HRP-linked nitrocellulose membrane using a wet transfer apparatus (Bio-Rad). secondary antibodies, and the ECL detection system for immunoblot The membranes were probed with antibodies against total p38K, detection have been described previously (26). All other reagents were phospho-p38K, total JNK and phospho-JNK. The immunoblots were from Sigma unless otherwise noted. developed using Super Signal reagents (Pierce). For Akt1 activation analysis, 20 Ìg of total protein from the lysates Construction of recombinant pMX-retroviruses. The cDNAs of Rin1 and were analyzed by 10% SDS-PAGE and transferred to a nitrocellulose mutants were subcloned into the pMX-puro vector as described (22). membrane using a wet transfer apparatus (Bio-Rad). The membranes These were used for the transfection of a 60% confluent PhoA cell were probed with antibodies against total Akt1 and phospho-Akt1 and monolayer using a Fugene6-mediated procedure (Life Technologies, the immunoblots were developed using Super Signal reagents (Pierce). Inc.). The cells were maintained at 37ÆC and the media containing For PI3K activation analysis, the anti-p85 immunoprecipitates were released viruses were harvested 48 h after transfection. The virus incubated with [Á-32P] ATP and phosphatidylinositol (PI) as a stocks were aliquoted and kept frozen at –80ÆC before use. The Rin1 substrate (26). 32P-labelled phosphatidylinositol-phosphate (PIP) was mutants were generated as described earlier (23). resolved by thin layer chromatography (TLC) and visualized by Cell lines. The cell lines were generated by infecting HL-60 autoradiography. Each stop containing the PIP was then quantified by (BCR/ABL-negative cell line) and K562 (BCR/ABL-positive cell line) scintillation counting. cells with retrovirus encoding GFP, Rin1wild-type (WT) and deletion 5 mutants essentially as described earlier (22-25). Analysis of DNA synthesis. The cell lines (0.5x10 cells/ml, ~60% confluence) were seeded into 12-well dishes. The cells were then Immunoblot analysis of protein expression. The cell lysates (20 Ìg) were starved in DMEM without serum for 36 h. For the last 12 h they were analyzed by 10% SDS-PAGE, and the were transferred to a incubated in either the absence or the presence of IL3. For the last 4 nitrocellulose membrane (Millipore) using a Bio-Rad semi-dry h they were labelled with 2 µCi/ml of methyl-[3H] thymidine. After transfer apparatus. The membrane was probed with specific antibodies the incubation, the cells were washed 3 times with phosphate-buffered as described in each figure, and the immunoblot was developed using saline. Cold 10% (w/v) trichloroacetic acid was then added and the the ECL reagents (Amersham Corp). cells were solubilized with 1 M NaOH. Tritium was measured by scintillation counting. Immunoprecipitation. The cells (HL-60 and K562) were incubated in the presence or absence of IL3, as indicated in each figure legend, at RNA interference. Three pairs of 21 nucleotide sense and antisense 37ÆC. The cells were then washed and lysed in ice-cold lysis buffer (50 RNA oligonucletides protected by 2 3’-overhang (2’deoxy) thymidines mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM (dT) were synthesized by Ambion. The siRNAs synthesized EDTA, 1 mM DTT, 1 mM benzamidine, 1 mM phenylmethylsulfonyl correspond to the human Rin1 coding nucleotides AAGCGGGAGA fluoride, 1 mM Na3VO4, 30 mM NaPPi, 10 mM NaF, 100 nM okadaic AATTCAAGAGA (R1a) and AACATGT CCTGGAGAAGTCAT acid, pH 7.5 and a protease inhibitor mixture (Roche Molecular (R1b); to the human Rin2 coding nucleotides AAGAGAAGAGG Biochemicals). The lysate was clarified by centrifugation at 16,000 xg AAGATGGCA (R2a) and AACAACCGCAAGCTGTACAAG for 10 min at 4ÆC. The protein concentration in the cell lysates was (R2b); and to the human Rin3 coding nucleotides AAGCTCATT

906 Hunker et al: Rin1 and IL3 Receptor Signaling

GACACAATTGCC (R3a) and AACTGAAACAGGAGATGGTGC (R3b). The siRNA synthesized correspond to the GFP coding nucleotides AACTCT CCACTGACAGAGAATCCTGTCTC (G). Equal amounts of sense and antisense RNA oligonucleotides were mixed and annealed, according to the manufacturer’s protocol, to form RNA duplexes before transfection. The cells in 6-well plates were transfected twice (~60% confluence; 1 ml of DMEM/fetal bovine serum per well) with 4 Ìl of 20 ÌM siRNA duplex and 3 Ìl of liofectAMINE2000 reagent in 100 Ìl of Opti-MEM medium according to the manufacturer’s recommendations at 12-h intervals. For transfections in which 3 siRNA duplexes were included, the amount of each duplex was decreased so that the final siRNA concentration remained constant between experiments. The cells were placed into normal growth culture medium 6 h prior to the experiments, which were performed 3 days after the initial transfection.

Pull down assay. Small GTPase activation in cells was examined by using activation-specific probes for Ras (GST-Raf-RBD) as described previously (27). In brief, the cells were lysed and incubated with the fusion proteins pre-coupled to glutathione-agarose beads. GTP-bound small GTPase was eluted from the beads and analyzed by immunoblotting. The detected bands were analyzed by densitometry. The experiments were repeated 3 times with similar results. Figure 1. Expression of Rin1 proteins in HL-60 cells. The HL-60 cells were either infected with retrovirus encoding green fluoresence protein Results (GFP), His-Rin1: wild-type (WT), His- Rin1:N, or His-Rin1:C as described in the Materials and Methods. After selection of the infected cells Expression of Rin1 in HL-60 cells. Rin1, a multifunctional with puromycin, cell extracts (12 Ìg) were prepared and the proteins were protein, has been shown to regulate EGF receptor membrane resolved by SDS-PAGE and then transferred to nitrocellulose for immunoblotting analysis with anti-Rin1 antibodies, anti-GFP antibodies trafficking and signaling (22-24). Rin1 contains several and anti-His antibodies. domains, including an SH2 (Src homology 2) domain, a proline-rich domain, a Vps9p domain and a region involved in the binding of activated Ras (22-24). Rin1 may selectively affect IL3 receptor signaling pathways, the To examine the potential role of Rin1 in IL3 receptor activity of Erk1/2, Akt, p38K and JNK proteins in HL-60 cells signaling, a pMXpuro retrovirus system, an approach that expressing either GFP (control) or Rin1 constructs (Figure 1) allows for the efficient expression of proteins into a variety of was analyzed. The HL-60 cells were stimulated with IL3 and cells, was used. Initially, the ability of the retrovirus to express the cell lysates were analyzed by SDS-PAGE. Erk1/2, Akt, JNK Rin1:WT and mutants was assessed in HL-60 cells, which do and p38K activities were then measured by Western blotting not express the BCR/ABL oncogene. The cells infected with with phospho-specific antibodies for each kinase. The data retrovirus encoding either green fluorescence protein (GFP) shown in Figure 2A suggest that the overexpression of Rin1 alone or His-tagged Rin1:WT and deletion mutants were constructs selectively inhibits Erk1/2 activity (control cells + immunoblottled for the presence of Rin1 (Figure 1). The IL3= 100%; cells expressing Rin1:WT+IL3=36±4%; expression of endogenous Rin1 in HL-60 cells was not affected cells expressing RIN1:N+IL3=82±12%; cells expressing by the overexpression of GFP. To ensure that the antibody- RIN1:C+IL3=30±9%) without affecting JNK and p38K detected Rin1 proteins were, in fact, due to retrovirus activities (Figure 2C and D). In addition, a lesser, although still infection, these cell extracts were also immunoblottled with significant, inhibition of the Akt activity (control cells + His-specific antibody. IL3=100%; cells expressing Rin1:WT+IL3=71±6%; cells expressing RIN1:N+IL3=62±7%; cells expressing Rin1 selectively blocks IL3 activation of Erk1/2 pathways. It has RIN1:C+IL3=90±9%) was observed upon addition of IL3 been shown that the addition of IL3 leads to the activation of (Figure 2B). Our data suggest that Rin1 plays a major role Ras and, hence, to the activation of the downstream mitogenic- in the Erk1/2 and Akt pathways driven by the IL3 receptor in activated protein kinases, Erk1/2 (28, 29). It is well established HL-60 cells. that Ras plays a major role in the activation of the Erk1/2-Elk1 signaling pathway by BCR-ABL or by cytokine receptors, Rin1 does not block IL3 activation of Ras. Given the selective including the IL3 receptor (29, 30). Activation of the IL3 linkage shown above between the Rin1 and Erk1/2 pathways receptor also stimulated other signaling pathways, including the in HL-60 cells, we further examined the time-dependent IL3- PI3K/Akt pathways (20). Thus, to explore the possibility that stimulation of Erk1/2 in cells expressing GFP, Rin1:WT, or

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Figure 2. The effects of Rin1 on the IL3-dependent signaling pathways. The HL-60 cells expressing either GFP, Rin1: WT, Rin1: N or Rin1: C were incubated in the absence or in the presence of 50 ng/ml IL3 at 37ÆC for 6 min, washed and the cell lysates were prepared as described in the Materials and Methods. The cell proteins were separated by SDS-PAGE, blotted to nitrocellulose and antibody to (A) phospho-Erk1/2 and total Erk1/2, (B) phospho-p38 kinase and total p38 kinase, (C) phospho-JN kinase and total JN kinase and (D) phospho-Akt1 and total Akt1 were used to visualize these proteins. The experiment was repeated 3 times with similar results.

908 Hunker et al: Rin1 and IL3 Receptor Signaling

Figure 3. Activation of the Erk pathway in HL-60 cells expressing Rin1 and its mutants. The HL-60 cells expressing either GFP, Rin1: WT, Rin1: N or Rin1: C were incubated in the absence or the presence of 50 ng/ml IL3 at 37ÆC for the indicated times, washed and the cell lysates were prepared as described in the Materials and Methods. The cell proteins were separated by SDS-PAGE, blotted to nitrocellulose and (A) the Erk1/2 proteins were visualized with antibodies to phospho Erk1/2 and total Erk1/2. (B) Mek and (C) Raf were visualized with antibodies to phospho-Mek, phospho-Raf, total Mek and total Raf, respectively. (D) The activated Ras small GTPase was affinity purified from cell lysates as described in the Materials and Methods. Eluates from precipitates were subjected to Western blot analysis with anti-HRas antibodies. The relative Ras activity was quantified by densitometric analysis. The results represent the mean±SD from 3 independent experiments performed in duplicate.

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Rin1 deletion mutants by using anti-phospho-Erk1/2 antibodies growth in HL-60. The growth-promoting or mitogenic effects in HL-60 cells. In Figure 3A, the time course of inhibition and of IL3 appear to involve many of the pathways utilized by activation of protein in the MAP-kinase cascade in cells other growth factors and cytokines, including GM-CSF and expressing Rin1 constructs in response to IL3 are shown. The EPO, whose receptors do not have intrinsic tyrosine kinase time course of this cascade is shown in the control cells, where activity (28). Erk1/2 is fully activated at 3-min incubation in the presence of To determine whether the expression of Rin1 alters IL3- IL3. Furthermore, the phosphorylation of Mek (Figure 3B) induced cell-growth, the effects of Rin1 overexpression on and Raf (Figure 3C) were found to be blocked in those cells cell proliferation were investigated. HL-60 cell lines expressing Rin1:WT and Rin1:C. The expression of Rin1:N expressing vector alone, Rin1:WT and Rin1 deletion mutants shows very little effect on the activation of Erk1/2, Mek and were transfected as described in Materials and Methods. Raf (Figure 3A-C), suggesting a potential role for Rin1 just Cells expressing the Rin1 constructs were stimulated with IL3 downstream of the Ras GTP-bound form. We next examined and the incorporation of [3H] thymidine was measured. In the effect of the Rin1 constructs on the GTP-loading of Ras Figure 5A, the expression of Rin1:WT is shown to block IL3- upon stimulation with IL3. As shown in Figure 3D, treatment stimulated cell proliferation as determined by thymidine of HL-60 cells with IL3 induced the activation of Ras. incorporation. In addition, basal thymidine incorporation was However, the expression of Rin1:WT, Rin1:N and Rin1:C did not significantly inhibited by the expression of Rin1 (Figure not alter the activation of Ras (GTP-bound form) upon the 5A). To identify the domains of Rin1 that might mediate the addition of IL3. These data indicate that Rin1 plays a key role inhibition of [3H] thymidine incorporation into DNA, HL-60 during the activation of the Raf/Mek/Erk1/2 pathway, by cell lines expressing the N-and C-terminal regions of Rin1 affecting the phosphorylation and /or activation of Mek and were prepared. The N-terminal region of Rin1 contains the Raf without affecting the GTP-loading of Ras. SH2 and proline-rich domains and the C-terminal region contains the Vps9p and Ras-binding domains. Expression of Effects of Rin1 on the PI3K/Akt pathway. It is also well both Rin1:N and Rin1:C inhibited the incorporation of [3H] established that cytokines activate both PI3K and Akt proteins thymidine into DNA in cells treated with IL3 (control cells (18, 30, 31). The activation of PI3K has been associated both + IL3= 100%; cells expressing Rin1:WT+IL3=50±10%; with the interaction of the SH2 domain of the p85 subunit with cells expressing RIN1:N+IL3=80±5; cells expressing the cytokine receptor tail and with the association of the p110 RIN1:C+IL3=60±7%). subunit with GTP-bound Ras. We first examined the activity of To provide additional evidence for the role of Rin1 in IL3- PI3K in HL-60 cells expressing the Rin1 constructs. In Figure driven cell proliferation, Rin1 was depleted from 4A, the time course of the activation of PI3K upon the addition HL-60 cells by using Rin1 RNA interference, as described in of IL3 either in cells expressing GFP (control) or in cells Materials and Methods. The depletion of Rin1 by RNA expressing the Rin1 constructs is shown. IL3 stimulation of the interference clearly showed a significant enhancement in the control cells resulted in a time-dependent stimulation of PI3K incorporation of [3H] thymidine into DNA (Figure 5B). activity, where PI3K was partially activated after 1 min of Furthermore, it seems that Rin1 and Rin3, but not Rin2, might incubation. The PI3K activity was inhibited by the expression be required for IL3-induced cell growth since the depletion of of Rin1:WT and Rin1:N, but not by the expression of RIN1:C. Rin2 did not have a significant effect on the incorporation of The role of Rin1 in the time-dependent activation of Akt [3H] thymidine into DNA. by IL3 in HL-60 cells expressing GFP, Rin1:WT and mutants The effective depletion of Rin1 and Rin1-like molecules was examined by using anti-phospho Akt antibodies. The IL3 (Rin2 and Rin3) are illustrated in the Figure 5 insert. For stimulation of the GFP-infected cells resulted in a time- Rin1, more than 95% of the protein (and tagged protein dependent activation of Akt activity, where Akt was partially version) was eliminated by the 2 RNAi duplexes. Because activated after 3 min of incubation. Moreover, the addition antibodies that recognize endogenous Rin2 and Rin3 were not of IL3 partially activated Akt in cells expressing Rin1:WT and available, HA- and Flag-tagged Rin2 and Rin3 were generated Rin1:N, but not the Rin1:C construct (Figure 4B). These and expressed in the HL-60 cells. The experiment with Rin1 results suggest that Rin1 is involved in the IL3-induced validates the use of heterologously expressed tagged-proteins PI3K/Akt pathway. Taken together, these data suggest that for testing the efficiency of RNA interference. As was the case Rin1 may have a dual action as an effector for both Ras and with Rin1, the levels of Rin2 and Rin3 were reduced by more the IL3 receptor and /or activated kinases. In vivo than 99% using the 2 RNAi duplexes, as determined using associations have shown that Rin1: WT and Rin1:N, but not antibody directed against each epitope tag as described in Rin1:C, interact with the IL3 receptor tail through the N- Materials and Methods. Taken together, these observations terminal region of Rin1, which contains the SH2 domain (see suggest that the requirement of Rin1 in IL3-induced cell Figure 7). Similarly, Rin1:WT and Rin1:C, but not RIN1:N, growth might be related to the ability to alter one or more are associated with Ras. Effect of Rin1 on IL3-induced cell intracellular signaling transduction pathway.

910 Hunker et al: Rin1 and IL3 Receptor Signaling

Figure 4. Activation of PI3K and Akt1 in HL-60 cells expressing Rin1 and its mutants. (A) The HL-60 cells expressing either GFP, Rin1: WT, Rin1: N or Rin1: C were incubated in the absence or the presence of 50 ng/ml IL3 at 37ÆC for the indicated time, washed and the cell lysates were prepared as described in Materials and Methods. The cell proteins were separated by SDS-PAGE, blotted to nitrocellulose and the Akt1 proteins were visualized with antibodies to phospho-Akt1 and total Atk1. (B) The activity of PI3-kinase was analyzed by TLC as described in Materials and Methods. The relative PI3K activity was analyzed by measuring the amount of radioactivity (32P) incorporated into phosphatidylinositol (PI). The results represent the mean±SD from 3 independent experiments performed in duplicate.

911 ANTICANCER RESEARCH 26: 905-916 (2006)

Figure 5. Rin1 is required for cell proliferation in HL-60 cells. (A) The HL-60 cells expressing either GFP, Rin1: WT, Rin1: N or Rin1: C were incubated in the absence or the presence of 10 ng/ml IL3 at 37ÆC for 24 h and the incorporation of [3H] thymidine into the DNA was determined as described in Materials and Methods. The results represent the mean±SD from 3 independent experiments performed in duplicate. (B) The HL-60 cells expressing GFP, His-Rin1:WT, HA-Rin2:WT or Flag-Rin3:WT were transfected with siRNAs ( R1a, R2a, R3b and G) as indicated in Materials and Methods. The transfected cells were then incubated in the absence or in the presence of 10 ng/ml IL3 at 37ÆC for 24 h and the [3H] thymidine incorporation into DNA was determined as described in Materials and Methods. The results represent the mean SD from 2 independent determinations performed in duplicates. Insert: The cell proteins were separated by SDS-PAGE, blotted to nitrocellulose and anti-GFP, anti-His, anti-HA and anti-Flag antibodies were used to visualize these proteins. The experiment was repeated 3 times with similar results.

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Figure 6. Rin1 interacts with BCR/ABL in K562 cells. (A) The K562 cell line expressing Rin1:WT, Rin1:N or Rin1:C was incubated in the absence or in the presence of 50 ng/ml IL3 for 8 min at 37ÆC. The cells were lysed and immunoprecipitated (IP) with anti-ABL antibody. The proteins were then separated by SDS-PAGE and blotted to nitrocellulose. The immunoprecipitated cells were subjected to Western blotting (WB) with Rin1 antibodies. (B) The cell lysates obtained from the K562 and HL-60 cells, incubated in the absence or the presence of IL3, were immuno-precipitated (IP) with anti-IL3 receptor ‚-chain antibody. The proteins were then separated by SDS-PAGE and blotted to nitrocellulose. The immunoprecipitated cells were subject to Western blotting (WB) with Rin1 and anti-IL3 receptor ‚-chain. The positions where molecular mass standards ran on the gel are indicated.

Specific association of Rin1 with the IL3 receptor and BCR/ABL not shown). Consistent with this observation, Rin1 was in K562 cells. K562, a cell line that expresses BCR/ABL, was constitutively associated with the BCR/ABL oncoprotein in the either left untreated or treated with IL3 and the cell lysates K562 cells. These results suggest that Rin1 is selectively bound were immunoprecipitated with an anti-ABL antibody and were to the IL3 receptor in the BCR/ABL-negative cell line. then immunoblotted with anti-ABL and anti-Rin1 antibodies. However, in the BCR/ABL-positive cell line, Rin1 was A 210-kDa protein band corresponding to BCR/ABL was associated with BCR/ABL but not with the the IL3 receptor, detected. This protein was tyrosine phosphorylated (data not even in the presence of IL3. shown). In addition to the 210 kDa band, another band of ~90 kDa was also detected and there was a very marked Effect of Rin1 on BCR/ABL-induced cell growth in K562. interaction even in the absence of IL3. By probing this Several points of convergence between cytokine signaling and membrane with anti-Rin1 antibody, we demonstrated that the BCR-ABL oncogenesis have been described and cells ~90 kDa band corresponded to Rin1. Thus, the association expressing BCR-ABL no longer require exogenous cytokines of Rin1 with BCR/ABL occurs independently of the addition (IL3 or GM-CSF) for their proliferation and/or survival by IL3 (Figure 6A). Furthermore, Rin1:N, but not Rin1:C, also activation of several signaling molecules, including kinases interacted with BCR/ABL. Again, this association occured and GTPases (28). independently of IL3. To determine whether the expression of Rin1 or its mutants The next investigation focussed on whether Rin1 interacts affects cell growth induced by BCR-ABL, the effects of Rin1 with the IL3 receptor in the K562 and HL-60 cell lines. In HL- overexpression on cell proliferation were investigated. The 60 cells, we observed that, upon addition of IL3, Rin1 was co- K652 cell line expressing the vector alone, Rin1:WT or Rin1 immunoprecipitated with the IL3 receptor. However, to our mutants was transfected as described in Materials and surprise, Rin1 was not associated with the IL3 receptor even Methods. The cells expressing Rin1:WT and Rin1:C, but not in the presence of IL3 (Figure 6B). In addition, the IL3 Rin1:N, were incubated either in the presence or in the receptor was tyrosine phosphorylated in both cell lines (data absence of IL3, and the incorporation of [3H] thymidine was

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Figure 7. Rin1 blocks the BCR/ABL activation of cell growth. (A) The K652 cells expressing either GFP, Rin1: WT, Rin1: N or Rin1: C were incubated in the absence or the presence of 10 ng/ml IL3 at 37ÆC for 24 h and the incorporation of [3H] thymidine into DNA was determined, as described in Materials and Methods. The results represent the mean±SD from 3 independent experiments performed in duplicate. (B) K652 cells expressing GFP, His- Rin1:WT, HA-Rin2:WT or Flag-Rin3:WT were transfected with siRNA as indicated in Materials and Methods. The transfected cells were then incubated in the absence or in the presence of 10 ng/ml IL3 at 37ÆC for 24 h and [3H] thymidine incorporation into DNA was determined as described in Materials and Methods. The results represent the mean±SD from 2 independent experiments performed in duplicate. (C) The activation of Erk1/2 and Akt in K652 cells expressing either GFP or Rin1:WT was analyzed by antibody to phospho-Erk1/2 and total Erk1/2 and (D) phospho-Akt1 and total Akt1, respectively. The experiment was repeated 3 times with similar results. measured. As expected, the addition of IL3 did not affect the shown), blocked only the activation of Erk1/2 but not that of proliferation of the K652 control cells (Figure 7A). We also Akt (Figure 7D). These results suggest that Rin1, and probably found that the expression of Rin1:WT and Rin1:C blocked Rin3, are required for BCR/ABL-induced cell growth. BCR/ABL-induced cell proliferation, as determined by thymidine incorporation. The expression of Rin1:N also Discussion significantly inhibited thymidine incorporation (control cells + IL3= 100%; control cells -IL3=95±6%; cells expressing The present study provides evidence for the role of Rin1 in the Rin1:WT -IL3=42±10%; cells expressing Rin1:N - IL3 receptor- and BCR/ABL-dependent signaling pathways. IL3=78±6%; cells expressing Rin1:C -IL3=39±6%). We identified Rin1 as a key element in both signaling pathways. To provide additional evidence for the role of Rin1 in This observation is supported by the fact that Rin1 was shown BCR/ABL-driven cell proliferation, Rin1 was depleted from to alter the Raf/Mek/Erk1/2 pathway without affecting the K562 cells by using Rin1 RNA interference, as described in activity of the JN- and p38-kinase pathways. Furthermore, Rin1 Materials and Methods. The depletion of Rin1 by RNAi clearly played a role in the activation of the PI3K/Akt pathway showed a significant enhancement in the incorporation of [3H] regulated by IL3. In contrast, Rin1 only inhibited the thymidine into DNA (Figure 7B). Similar results were also Raf/Mek/Erk1/2 signaling pathway stimulated by BCR/ABL. found by the depletion of Rin3, but not of Rin2, proteins. In The use of the pMX-puro retrovirus system to generate cell addition, the effectiveness of the depletion of Rin1 and Rin1- lines was important because they express endogenous levels of like molecules (Rin2 and Rin3) was also confirmed by Western the IL3-receptor and BCR-ABL fusion protein, and blot analysis (data not shown). Furthermore, the expression of proliferation in these cells is mainly driven by these signaling Rin1:WT (Figure 7C) and Rin1:C, but not Rin1:N (data not molecules. The IL3- receptor has served as model for signal

914 Hunker et al: Rin1 and IL3 Receptor Signaling

Rin1:C inhibited IL3-stimulated [3H] thymidine incorporation into DNA. In addition, the activation of Elk-1 was also affected by the expression of Rin1 (data not shown). The activation of Elk-1 by growth factors/cytokines has been demonstrated in leukemic cells (29). However, the effect on Rin1 in BCR/ABL- regulated signal transduction pathways has been restricted to the activation of Raf/Mek/Erk1/2 signaling molecules. Our data showed that Rin1 is tightly linked to both the IL3 receptor through the PI3K/Akt and Raf/Erk1/2 pathways, and to the BCR-ABL oncogene through the Raf/Mek/Erk1/2 pathway. These two pathways are known to be involved in cellular growth and proliferation. Both the N- and C-terminal regions of Rin1 appeared to be involved in optimal growth inhibition. It also seems reasonable to assume that the effects of Rin1:N and Rin1:C are mediated by separate molecular mechanisms. Rin1:C may interact with Ras via its Ras-binding domain, thereby blocking Ras action on downstream pathways. Our observations also suggest that Rin1 might alter the Ras-Raf interaction. This hypothesis is further supported by the observation that the Ras binding domain of Rin1 was able to block interaction of GTP-bound Ras and Raf (12) and also by the fact that the expression of Rin1 affected both IL3- dependent and BCR/ABL-dependent Raf phosphorylation. Even though both Rin1:WT and Rin1:N, but not Rin1:C, were found to be associated with BCR/ABL in BCR/ABL-positive cell lines, it seems reasonable that Rin1 may link BCR/ABL directly to signals downstream of Ras. On the other hand, it is likely that the expression of Rin1:N, which contains both SH2 and proline-rich domains, interferes with the recruitment of adaptor proteins, including full-length Rin1 to activated IL3 receptor, thereby interfering with IL3 receptor signal transduction. Consistent with our hypothesis, Rin1:WT and Rin1:N, but not Rin1:C, were found to interact with the IL3 receptor in BCR/ABL-negative cell lines. Several observations also support the selective effect of Rin1 Figure 8. Model for the role of Rin1 in BCR/ABL-positive and -negative on the PI3K/Akt pathway; first, Rin1 was associated with leukemic cells. (A) Autophosporylation of BCR-ABL generates BCR/CBL in K526 but not in HL-60 cells (21); second, phosphorylated tyrosines (i.e. tyr 177), which in turn bind Grb2 molecules. BCR/ABL independently regulated both Ras and PI3K activity This leads to the binding and phosprylation of Gab2. This activates Gab2 (20); and finally, mutations in the Ras effector loop reveal a signals to recruit molecules, including PI3K, which is required for Akt activation. On the other hand, Grb2 will interact with SOS, which surprising observation. The Ras mutation (C40-V12) retained activates Ras. The activation of Ras leads to the activation of Erk1/2. (B) affinity with PI3K, but not with Rin1 or Raf (29). In the Activation of the IL3 receptor by IL3 induces the activity of the JAK/STAT BCR/ABL-negative cells, Rin1:WT and Rin1:N, but not (not shown), Raf/Erk1/2 and PI3K/Akt pathways. Both Akt and Erk1/2 Rin1:C, were associated with the IL3 receptor (Figure 7), then send signals to regulate survival/proliferation/transformation. The site which may interfere with the recruitment of adaptor proteins, of Rin1 action is indicated in this figure. including PI3K. Consistent with our hypothesis, we found that the expression of Rin1 altered the PI3K activity associated with transduction studies and many IL3-receptor-associated the IL3 receptor (data not shown). Clearly, more work has to molecules have been characterized (28). be done to fully understand the molecular mechanisms of Rin1 As shown in this study, Rin1 altered both Erk1/2 and Akt in IL3 receptor signaling pathways. It is likely that this selective pathways, suggesting a specific and selective role of Rin1 in and specific association of Rin1 with different molecules in IL3-dependent Erk1/2 and Akt activation. Consistent with this both BCR/ABL-negative and -positive cell lines may help to observation, we found that the expression of Rin1:WT and explain, in part, our observations.

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In addition, the depletion of Rin1 by RNA interference 12 Sawyers CL: Chronic myeloid leukemia. N Engl J Med 340(17): revealed an important aspect of Rin1 function in IL3 receptor 1330-1340, 1999. signaling and proliferation. Moreover, the depletion of Rin3, 13 Faderl S et al: The biology of chronic myeloid leukemia. N Engl J Med 341(3): 164-172, 1999. but not Rin2, suggests that the elimination of a combination 14 Frank DA and Varticovski L: BCR/Abl leads to the constitutive of at least 2 molecules (Rin1 and Rin3) is necessary to reveal activation of Stat proteins, and shares an epitope with tyrosine the role of these molecules in both the IL3 receptor and phosphorylated Stats. Leukemia 10(11): 1724-1730, 1996. BCR/ABL signaling pathways. Finally, a model of the possible 15 Sawyers CL, Denny CT and Witte ON: Leukemia and the sites of inhibition of Rin1 in the BCR/ABL and IL3 cytokine disruption of normal hematopoiesis. Cell 64(2): 337-350, 1991. 16 Calabretta B and Skorski T: regulatory mechanisms operative signal transduction pathways is presented in Figure 8. on hematopoietic cells: proliferation, differentiation, and neoplasia. In summary, this study lends support to the importance of Crit Rev Eukaryot Gene Expr 7(1-2): 117-124, 1997. the Ras/Rin1 pathway in cell proliferation driven by the IL3 17 Tenen DG et al: Transcription factors, normal myeloid receptor and the BCR/ABL oncogene and raises the possibility development, and leukemia. Blood 90(2): 489-519, 1997. that interference with this pathway might be a rational 18 Donato NJ et al: Down-regulation of interleukin-3/granulocyte- macrophage colony-stimulating factor receptor beta-chain in BCR- therapeutic strategy for leukemia characterized by oncogenic ABL(+) human leukemic cells: association with loss of cytokine- activation of tyrosine kinases. mediated Stat-5 activation and protection from apoptosis after BCR-ABL inhibition. Blood 97(9): 2846-2853, 2001. Acknowledgements 19 Luo JM et al: Possible dominant-negative mutation of the SHIP gene in acute myeloid leukemia. Leukemia 17(1): 1-8, 2003. We thank Dr. P. D. Stahl for the generous gifts of experimental 20 Sattler M et al: Critical role for Gab2 in transformation by materials and suggestions. This work was support by the Jose Carreras BCR/ABL. Cancer Cell 1(5): 479-492, 2002. International Leukemia Foundation (E.D. Thomas Fellowship 21 Afar DE et al: Regulation of the oncogenic activity of BCR-ABL Program) at Florida International University, U.S.A. by a tightly bound substrate protein RIN1. Immunity 6(6): 773- 782, 1997. 22 Barbieri MA et al: Role of rab5 in EGF receptor-mediated signal References transduction. Eur J Cell Biol 83(6): 305-314, 2004. 23 Tall GG et al: Ras-activated endocytosis is mediated by the Rab5 1 Lee JT Jr and McCubrey JA: The Raf/MEK/ERK signal guanine nucleotide exchange activity of RIN1. Dev Cell 1(1): 73- transduction cascade as a target for chemotherapeutic intervention 82, 2001. in leukemia. Leukemia 16(4): 486-507, 2002. 24 Barbieri MA et al: The SRC homology 2 domain of Rin1 mediates 2 McCubrey JA et al: Interactions between the PI3K and Raf its binding to the epidermal growth factor receptor and regulates signaling pathways can result in the transformation of receptor endocytosis. J Biol Chem 278(34): 32027-3236, 2003. hematopoietic cells. Cancer Detect Prev 25(4): 375-393, 2001. 25 Barbieri MA et al: Epidermal growth factor and membrane 3 Hantschel O and Superti-Furga G: Regulation of the c-Abl and trafficking. EGF receptor activation of endocytosis requires Rab5a. Bcr-Abl tyrosine kinases. Nat Rev Mol Cell Biol 5(1): 33-44, 2004. J Cell Biol 151(3): 539-550, 2000. 4 Nieborowska-Skorska M et al: Signal transducer and activator of 26 Traina F et al: BCR-ABL binds to IRS-1 and IRS-1 transcription (STAT)5 activation by BCR/ABL is dependent on phosphorylation is inhibited by imatinib in K562 cells. FEBS Lett intact Src homology (SH)3 and SH2 domains of BCR/ABL and is 535(1-3): 17-22, 2003. required for leukemogenesis. J Exp Med 189(8): 1229-1242, 1999. 27 Bone H and Welham MJ: Shc associates with the IL-3 receptor beta 5 Neshat MS et al: The survival function of the Bcr-Abl oncogene is subunit, SHIP and Gab2 following IL-3 stimulation. Contribution mediated by Bad-dependent and -independent pathways: roles for of Shc PTB and SH2 domains. Cell Signal 12(3): 183-194, 2000. phosphatidylinositol 3- kinase and Raf. Mol Cell Biol 20(4): 1179- 28 Steelman LS et al: JAK/STAT, Raf/MEK/ERK, PI3K/Akt and 1186, 2000. BCR-ABL in cell cycle progression and leukemogenesis. Leukemia 6 Pendergast AM et al: SH1 domain autophosphorylation of P210 18(2): 189-218, 2004. BCR/ABL is required for transformation but not growth factor 29 Mizuchi D et al: BCR/ABL activates Rap1 and B-Raf to stimulate independence. Mol Cell Biol 13(3): 1728-36, 1993. the MEK/Erk signaling pathway in hematopoietic cells. Biochem 7 Pendergast AM et al: BCR sequences essential for transformation Biophys Res Commun 326(3): 645-651, 2005. by the BCRABL oncogene bind to the ABL SH2 regulatory 30 Skorski T et al: Transformation of hematopoietic cells by BCR/ABL domain in a non-phosphotyrosine-dependent manner. Cell 66(1): requires activation of a PI-3k/Akt-dependent pathway. EMBO J 161-71, 1991. 16(20): 6151-6161, 1997. 8 Sirard C, Laneuville P and Dick JE: Expression of bcr-abl abrogates 31 McFarlin DR, Lindstrom MJ and Gould MN: Affinity with Raf is factor-dependent growth of human hematopoietic M07E cells by an sufficient for Ras to efficiently induce rat mammary carcinomas. autocrine mechanism. Blood 83(6): 1575-1585, 1994. Carcinogenesis 24(1): 99-105, 2003. 9 Sillaber C et al: STAT5 activation contributes to growth and viability in Bcr/Abl-transformed cells. Blood 95(6): 2118-2125, 2000. 10 de Groot RP et al: STAT5 activation by BCR-Abl contributes to transformation of K562 leukemia cells. Blood 94(3): 1108-1112, 1999. 11 Ben-Neriah Y et al: The chronic myelogenous leukemia-specific P210 protein is the product of the bcr/abl hybrid gene. Science Received October 10, 2005 233(4760): 212-214, 1986. Accepted December 13, 2005

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