Volume 6, Issue 1, Pages 99-108 (July 2000) Site-Specific Serine Phosphorylation of the IL-3 Receptor Is Required for Hemopoietic Cell Survival  Mark A. Guthridge, Frank C. Stomski, Emma F. Barry, Wendy Winnall, Joanna M. Woodcock, Barbara J. McClure, Mara Dottore, Michael C. Berndt, Angel F. Lopez  Molecular Cell  Volume 6, Issue 1, Pages 99-108 (July 2000) DOI: 10.1016/S1097-2765(05)00002-X

Figure 1 Phosphorylation of Ser-585 of βc in Response to IL-3 and the Recruitment of 14-3-3 (A) CTL-EN cells expressing IL-3Rα and either wtβc, βcHSRSLP→EFAAAA, or βcS585G were factor deprived and stimulated with IL-3. Cells were then lysed and the βc immunoprecipitated (7 × 107 cells/IP) with the 1C1 anti-βc mAb. Immunoprecipitates were subjected to immunoblot analysis with the anti-phospho-Ser-585βc pAb, anti-14-3-3 pAb, and the 1C1 anti-βc mAb. Results were typical of three experiments. (B) MO7e cells were factor deprived for 12 hr and stimulated with IL-3. Cells were lysed, βc immunoprecipitated with the 1C1 mAb (5 × 107 cells/IP), and subjected to immunoblot analysis as for (A). (C) CTL-EN cells expressing IL-3Rα and either wtβc, βcS585G, βcRSL→AAA, or βcHSRSLP→EFAAAA were factor deprived, and IL-3 stimulated. βc immunoprecipitates (2 × 107 cells/IP) were subjected to immunoblot analysis with the 4G10 anti-phoshotyrosine mAb and the 1C1 anti-βc mAb. Results were typical of three experiments. (D) CTL-EN cells expressing IL-3Rα and either wtβc or βcS585G were 125I surface labeled using the lactoperoxidase method (Stomski et al. 1996). Surface-labeled cells were incubated in the presence or absence of IL-3 for 10 min. After cell lysis, proteins were immunoprecipitated with either anti-phospho-Ser-585βc, anti-phosphotyrosine (4G10), or anti-βc (1C1) antibodies. The immunoprecipitated proteins were subjected to SDS-PAGE and signals were visualized using a phosphorImager. Molecular Cell 2000 6, 99-108DOI: (10.1016/S1097-2765(05)00002-X)

Figure 2 PKA Phosphorylates Ser-585 of βc (A) The ability of either PKA or CKII to phosphorylate purified Hisβc445–881 was examined in an in vitro kinase assay using [γ-32P]ATP. Competitions were performed using 200 μM of the following peptides; scrambled (CLPLSGPDSHIRGPL), non-phospho-Ser-585 (CLGPPHSRSLPDILG), or phospho-Ser-585 (CLGPPHSRSPLPDILG, where SP is phosphorylated). (B) The ability of PKA and CKII to phosphorylate Ser-585 of Hisβc445–881 was examined by immunoblot analysis using the anti-phospho-Ser-585βc antibody. Filters were probed first with the anti-phospho-Ser-585βc antibody (top panel), stripped, and reprobed with an antibody that recognizes the HSRSLP motif of βc regardless of whether Ser-585 is phosphorylated (anti-HSRSLPβc) (bottom panel). (C) Determination of the Km and the Vmax for PKA phosphorylation of the βc peptide. The assay for PKA activity was performed with the indicated concentrations of non-phospho-Ser-585 peptide. Km and Vmax values for phosphorylation of the non-phospho-Ser-585 peptide have been calculated from hyperbolic regression analysis and double reciprocal Lineweaver-Burke plots (inset) using hyperbolic regression analysis software. Results were typical of two experiments. (D) Pharmacologic regulation of PKA activity and its effect on βcSer-585 phosphorylation. CTL-EN cells expressing wtβc were factor deprived and left unstimulated (nil) or stimulated with IL-3, forskolin (50 μM), or db-cAMP (100 μM) for 10 min. In addition, CTL-EN cells and MO7e cells were also factor deprived, pretreated with 10 μM H89 for 15 min, and then stimulated with IL-3 for a further 10 min (H89 + IL-3). Following treatments, cells were lysed, βc immunoprecipitated with the 1C1 mAb, and immunoblot analysis was performed using the anti-phospho-Ser-585 antibody (top panel) and the anti-βc 1C1 antibody (bottom panel). Results were typical of two experiments. Molecular Cell 2000 6, 99-108DOI: (10.1016/S1097-2765(05)00002-X)

Figure 3 14-3-3 Binding to βc in Response to IL-3 Stimulation Is Required for the Recruitment and Activation of PI 3-K (A) Analysis of PI 3-K activity. CTL-EN cells expressing IL-3Rα and either wtβc, βcHSRSLP→EFAAAA, or βcS585G were factor deprived and stimulated with IL-3. Cells were then lysed, phosphotyrosine-containing proteins were immunoprecipitated (2 × 107 cells/IP) using the 4G10 antibody, and PI 3-K activity of the immunoprecipitates was measured. Where indicated, cells were pretreated for 30 min with 2 μM Wortmannin (+wort) or 16 μM LY294002 (+LY) and then stimulated with IL-3 for 5 min. In addition, p85 was immunoprecipitated from nonstarved CTL-EN cells (2 × 106 cells/IP) expressing wtβc, βcHSRSLP→EFAAAA, or βcS585G. Shown are 32P-labeled phosphatidylinositols (PIP) and the origin. Results were typical of three experiments. (B) CTL-EN cells were examined for the association of βc with p85. CTL-EN cells expressing either wtβc, βcHSRSLP→EFAAAA, or βcS585G were factor deprived and then stimulated with IL-3. Cells were then lysed and the βc immunoprecipitated (2 × 107 cells/IP) with the 1C1 anti-βc-specific mAb. Immunoprecipitates were then subjected to immunoblot analysis with the anti-phospho-Ser-585βc, anti-p85, and the 1C1 anti-βc antibodies. Results were typical of three experiments. (C) The association of recombinant Hisβc445–881 with p85 and 14-3-3 was assessed in pulldown experiments. Pulldown experiments were performed using COS-7 lysates using Sepharose alone (lane 1), unphosphorylated Sepharose–Hisβc445–881 (10 μg) (lane 2), and Sepharose–Hisβc445–881 phosphorylated on Ser-585 with PKA (lanes 3–6). Peptide competitions were also performed with 200 μM competing peptides (lane 4, Ser-585-Ala; lane 5, non-phospho-Ser-585; lane 6, phospho-Ser-585). The resin was then examined for associated p85 and 14-3-3 with the anti-p85 antibody (top) and anti-14-3-3 antibody (bottom). Results were typical of two experiments. Molecular Cell 2000 6, 99-108DOI: (10.1016/S1097-2765(05)00002-X)

Figure 4 14-3-3 Binding to βc Is Required for Akt Activation but Not STAT5, ERK, JNK, or JAK2 Activation (A) Phosphorylation of Akt, STAT5, and ERK2. CTL-EN cells expressing IL-3Rα and either wtβc or βcHSRSLP→EFAAAA were factor deprived and stimulated with IL-3. Whole-cell lysates were subjected to immunoblot analysis with anti-phospho Akt pAb, anti-phosphorylated STAT5 mAb, anti-active MAPK pAb, and anti-ERK pAb. (B) Activation of JNK activity. CTL-EN cells expressing IL-3Rα and either wtβc or βcHSRSLP→EFAAAA were factor deprived and stimulated with IL-3. Cells were lysed, and JNK immunoprecipitates (2 × 107 cells/IP) were then subjected to an in vitro kinase assay using [γ-32P]ATP and 1 μg GST-jun1–79 as a substrate. Kinase assays were subjected to SDS-PAGE and transferred to a nitrocellulose filter. An autoradiogram of the filters is shown with 32P-labeled GST-jun indicated. (C) Tyrosine phosphorylation of JAK2. CTL-EN cells expressing either wtβc or βcHSRSLP→EFAAAA were starved and stimulated with IL-3. Cells were then lysed and JAK2 immunoprecipitated (2 × 107 cells/IP). Immunoprecipitates were then subjected to immunoblot analysis with the 4G10 anti-phosphotyrosine mAb and the anti-JAK2 pAb. Molecular Cell 2000 6, 99-108DOI: (10.1016/S1097-2765(05)00002-X)

Figure 5 14-3-3 Binding to βc in Response to IL-3 Stimulation Promotes Cellular Survival by Suppressing Apoptosis (A) Cellular viability. CTL-EN cells expressing IL-3Rα and either wtβc (circle, square) or βcHSRSLP→EFAAAA (inverted triangle, triangle) were plated at 5 × 105 cells/ml in DMEM containing 0.1% FCS and either 10 ng/ml IL-3 (square, triangle) or 10 ng/ml IL-2 (circle, inverted triangle). Viable cells were counted using the trypan blue exclusion method over 3 days. Results shown were typical of two experiments. (B) Metabolic activity. CTL-EN cells expressing IL-3Rα and either wtβc (inverted triangle, square) or βcHSRSLP→EFAAAA (diamond, triangle) were plated in DMEM containing 0.1% FCS containing no IL-3 (inverted triangle, diamond) or 10 ng/ml IL-3 (square, triangle). Metabolic activity was measured (490 nm) each day using the MTS reduction method. Results were typical of two experiments. (C) DNA laddering. CTL-EN cells expressing wtβc or βcHSRSLP→EFAAAA were plated out in 20 ng/ml IL-2, no factor (NF), or 50 ng/ml IL-3. DNA was isolated from cells after 48 hr, electrophoresed on an 0.8% agarose gel, and stained with ethidium bromide. (D) Annexin V and propidium iodide staining. CTL-EN cells expressing IL-3Rα and either wtβc, βcS585G, βcRSL→AAA, or βcHSRSLP→EFAAAA were plated in either 20 ng/ml IL-2, no factor or 50 ng/ml IL-3 for 30 hr. Cells were then stained with annexin V and propidium iodide and subjected to flow cytometry. The percentage of viable cells (annexin V and propidium iodide negative) and apoptotic cells (annexin V and propidium iodide positive) is indicated in each histogram. Results were typical of two experiments. Molecular Cell 2000 6, 99-108DOI: (10.1016/S1097-2765(05)00002-X)

Figure 6 14-3-3 Binding to βc Is Not Required for Cell Cycle Progression (A) The distribution of cells in G0/G1, S, and G2/M phases was analyzed by the DNA content of propidium iodide-stained cells as determined by flow cytometry. CTL-EN cells expressing IL-3Rα and either wtβc or βcHSRSLP→EFAAAA were examined for cell cycle distribution under three different conditions: (1) asynchronous, cells growing in 10 ng/ml IL-2; (2) starved, cells factor-deprived for 24 hr; (3) +IL-3, factor-deprived cells were stimulated for 24 hr with 50 ng/ml IL-3. Results are typical of two experiments. (B) c-Myc induction in response to IL-3 stimulation. CTL-EN cells were starved and stimulated for 2 hr with 50 ng/ml IL-3. Total RNA was extracted and subjected to Northern blot analysis. Filters were probed sequentially with 32P-labeled cDNAs for c-Myc and 18S rRNA. Molecular Cell 2000 6, 99-108DOI: (10.1016/S1097-2765(05)00002-X)

Figure 7 Proposed Model for the Regulation of Survival by IL-3 Binding of IL-3 to the IL-3 receptor, composed of a ligand-specific α chain (α) and a common β chain (βc), results in receptor oligomerization (only one α chain and one β chain are shown for simplicity). Receptor oligomerization results in the activation of tyrosine kinases, which results in tyrosine phosphorylation of βc (P-Y) and the recruitment of SH2 and PTB binding proteins (data not shown). In addition, activation of PKA results in the phosphorylation of Ser-585 of βc (S-P). Ser-585 phosphorylation allows the recruitment of 14-3-3, which in turn recruits PI 3-K either directly through the p110 or p85 subunits or indirectly through an additional adaptor molecule(s). These receptor-proximal events identified in the current studies that result in the activation of PI 3-K are then likely to couple to a downstream pathway involving Akt and BAD. As described in the Discussion, BAD phosphorylation results in 14-3-3 binding and sequestration of BAD in the cytoplasm and suppression of apoptosis (survival). In the absence of cytokine, BAD remains unphosphorylated and translocates to the mitochondria where the events leading to apoptosis are triggered (death). Molecular Cell 2000 6, 99-108DOI: (10.1016/S1097-2765(05)00002-X)