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

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Rin1 Is a Negative Regulator of the IL3 Receptor Signal Transduction Pathways 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 protein 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 chromosome results in the fusion of sequences a retrovirus system. In this study, the overexpression of Rin1 from the BCR and ABL genes (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 proteins 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.
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