The phosphoserine-585–dependent pathway of the GM-CSF/IL-3/IL-5 receptors mediates hematopoietic cell survival through activation of NF-κB and induction of bcl-2

Abstract

We have recently identified a novel mechanism of hematopoietic cell survival that involves site-specific serine phosphorylation of the common beta subunit (␤ c ) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors. However, the downstream components of this pathway are not known, nor is its relationship to survival signals triggered by tyrosine phosphorylation of the receptor clear. We have now found that phosphorylation of Ser585 of ␤ c in response to GM-CSF recruited 14-3-3 and phosphatidyl inositol 3-OH kinase (PI 3-kinase) to the receptor, while phosphorylation of the neighboring Tyr577 within this "viability domain" promoted the activation of both Src homology and collagen (Shc) and Ras. These are independent processes as demonstrated by the intact reactivity of phosphospecific anti-Ser585 and anti-Tyr577 antibodies on the cytotoxic T-lymphocyte-ecotrophic retroviral receptor neomycin (CTL-EN) mutants ␤ c Tyr577Phe and ␤ c Ser585Gly, respectively. Importantly, while mutants in which either Ser585 (␤ c Ser585Gly) or all tyrosines (␤ c F8) were substituted showed a defect in Akt phosphorylation, nuclear factor B (NF-B) activation, bcl-2 induction, and cell survival, the mutant ␤ c Tyr577Phe was defective in Shc, Ras, and extracellular signal-related kinase (ERK) activation, but supported CTL-EN cell survival in response to GM-CSF. These results demonstrate that both serine and tyrosine phosphorylation pathways play a role in hematopoietic cell survival, are initially independent of each other, and converge on NF-B to promote bcl-2 expression. IntroductionHematopoiesis is a dynamic process undergoing constant flux where the enormous proliferative capacity of hematopoietic cells is precisely balanced against cell death programs. At the heart of this process lie the hematopoietic cytokines, which are central regulators of both cell proliferation and survival. Many hematopoietic cell types remain poised to activate intrinsic cell death programs and require constant survival signals provided by cytokines. There are 3 such cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5, that are potent regulators of not only myeloid cell proliferation but also cell survival through their ability to suppress apoptotic programs and as such can play a role also in certain inflammatory conditions and leukemia. 1,2 The receptors for GM-CSF, IL-3, and IL-5 share a subunit, ␤ c , which is required for most, if not all, of the signaling including cell survival. 1,2 However, the molecular basis and signaling cascades that emanate from ␤ c and underpin this prosurvival effect are not fully understood. While some experiments have implicated tyrosine phosphorylation of ␤ c (below), we noted that Ser585 was phosphorylated in response to ligand, resulting in the recruitment of the adaptor molecule 14-3-3. 3 Importantly, we demonstrated using mutations at this position that Ser585 was required for phosphatidyl inositol 3-OH kinase (PI 3-k...

Interleukin-3 receptor in acute leukemia

“…25 One additional important target for the PI3K/AKT pathway is represented by NFkB: this transcription factor is held in an inactive, latent state in the cytoplasm by the inhibitor protein IkB; AKT phosphorylates IkB kinase that in turn phosphorylates IkB with its consequent degradation by the proteasome, allowing NFkB to translocate to the nucleus and regulate the expression of a broad of genes such as bcl-2, bcl-X L and A1, all involved in cell survival. 26 This pathway is activated by IL-3 via phosposerine-585 bc phosphorylation. 26 According to all these observations it was proposed that the antiapoptotic activity of IL-3 could be related to the induction of the Bcl-2 family genes, bcl-2, bcl-X L , A1 and mcl-1.…”

“…26 This pathway is activated by IL-3 via phosposerine-585 bc phosphorylation. 26 According to all these observations it was proposed that the antiapoptotic activity of IL-3 could be related to the induction of the Bcl-2 family genes, bcl-2, bcl-X L , A1 and mcl-1. 27 The screening by microarray gene expression profiling of genes downmodulated by IL-3 deprivation in an IL-3-dependent cell line following IL-3 starvation provided evidence that pim-2 is the gene most modulated by IL-3.…”

14-3-3:Shc Scaffolds Integrate Phosphoserine and Phosphotyrosine Signaling to Regulate Phosphatidylinositol 3-Kinase Activation and Cell Survival

“…The cells were lysed in either RIPA buffer or Nonidet P-40 lysis buffer as previously described (9). Immunoprecipitations were performed using 1 g of antibody absorbed to protein A-Sepharose (Amersham Biosciences).…”

“…The anti-active-ERK pAb (Promega) was used at 1:5000. The anti-Tyr(P) 179 antibody was generated by coupling KKS-VFY(pY)EILNSC to keyhole limpet hemocyanin and injecting into New Zealand White rabbits (9,12) and used at 1:1000.…”

Concepts of human leukemic development

“…Thus, upregulation of IL-3Ra may be a common survival mechanism in AML. Stimulation of IL-3R initiates numerous signaling pathways with effects including the activation of the Bcl-2 family member Mcl-1, as well as the antiapoptotic transcription factor NFkB (Wang et al, 2003;Guthridge et al, 2004). NFkB is constitutively activated in the majority of primary AML samples, and notably in quiescent LSC populations, but not in normal HSC.…”