Cytokine receptor signaling activates an IKK-dependent phosphorylation of PUMA to prevent cell death.

P53-upregulated modifier of apoptosis (PUMA), a pro-apoptotic member of the Bcl-2 family, is transcriptionally activated by p53 and is a key effector of p53-dependent apoptosis. We show that PUMA protein is subject to rapid post-translational regulation by phosphorylation at a conserved residue, serine 10, following serum or interleukin-3 (IL-3) stimulation. Serine 10 is not within the Bcl-2 homology (BH3) domain, and PUMA phosphorylated at serine 10 retained the ability to co-immunoprecipitate with antiapoptotic Bcl-2 family members. However, phosphorylated PUMA was targeted for proteasomal degradation indicating that it is less stable than unphosphorylated PUMA. Importantly, we identified IKK1/IKK2/Nemo as the kinase complex that interacts with and phosphorylates PUMA, thereby also demonstrating that IL-3 activates NFκB signaling. The identification and characterization of this novel survival pathway has important implications for IL-3 signaling and hematopoietic cell development.

Structural and functional hot spots in cytokine receptors.

The activation of cytokine receptors is a stepwise process that depends on their specific interaction with cognate cytokines, the formation of oligomeric receptor complexes, and the initiation of cytoplasmic phosphorylation events. The recent determination of the structure of extracellular domains of several cytokine receptors allows comparison of their cytokine-binding surfaces. This comparison reveals a common structural framework that supports considerable diversity and adaptability of the binding surfaces that determine both the specificity and the orientation of subunits in the active receptor complex. These regions of the cytokine receptors have been targeted for the development of specific agonists and antagonists. The physical coupling of signaling intermediates to the intracellular domains of their receptors plays a major role in determining biological responses to cytokines. In this review, we focus principally on the receptors for cytokines of the granulocyte-macrophage colony-stimulating factor (GM-CSF) family and, where appropriate, compare them with related cytokine receptors. Several paradigms are beginning to emerge that focus on the ability of the extracellular portion of the cytokine receptor to recognize the appropriate cytokine and on a phosphorylated motif in the intracellular region of the GM-CSF receptor that couples to a specific signaling pathway.

Site-specific serine phosphorylation of the IL-3 receptor is required for hemopoietic cell survival.

In the hemopoietic compartment, IL-3, GM-CSF, and IL-5 receptors are major transducers of survival signals; however, the receptor-proximal events that determine this vital function have not been defined. We have found that IL-3 stimulation induces phosphorylation of Ser-585 of beta(c). This promotes the association of phospho-Ser-585 of beta(c) with 14-3-3 and the p85 subunit of PI 3-K. Mutation of Ser-585 specifically impairs the PI 3-K signaling pathway and reduces cell survival in response to IL-3. These results define a distinct IL-3 receptor-mediated survival pathway regulated by site-specific receptor serine phosphorylation and 14-3-3 binding and suggest that this novel mode of signaling may be utilized by disparate transmembrane receptors that have as a common theme the transduction of survival signals.

Identification of a 14-3-3 binding sequence in the common beta chain of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors that is serine-phosphorylated by GM-CSF.

The common beta chain (beta(c)) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the major signaling subunit of these receptors coupling ligand binding to multiple biological activities. It is thought that these multiple functions arise as a consequence of the recruitment of specific signaling molecules to tyrosine-phosphorylated residues in the cytoplasmic domain of beta(c). However, the contribution of serine phosphorylation in beta(c) to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of beta(c) that interacts with the adaptor protein 14-3-3zeta. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3zeta fusion protein showed that 14-3-3 directly associates with beta(c) but not the GM-CSF receptor alpha chain. C-terminal truncation mutants of beta(c) further showed that a region between amino acids 544 and 626 in beta(c) was required for its association with 14-3-3zeta. This region contains the sequence (582)HSRSLP(587), which closely resembles the RSXSXP (where S is phosphorylated) consensus 14-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of (582)HSRSLP(587) for EFAAAA completely abolished interaction of beta(c) with GST-14-3-3zeta. Furthermore, the interaction of beta(c) with GST-14-3-3 was greatly reduced in the presence of a peptide containing the 14-3-3 binding site, but only when (585)Ser was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated (585)Ser bound 14-3-3zeta with an affinity of 150 nmol/L. To study the regulation of (585)S phosphorylation in vivo, we raised antibodies that specifically recognized (585)Ser-phosphorylated beta(c). Using these antibodies, we showed that GM-CSF stimulation strongly upregulated (585)Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC-binding site ((577)Tyr) to the 14-3-3-binding site ((582)HSRSLP(587)) and their conservation between mouse, rat, and human beta(c) but not in other cytokine receptors suggest that they form a distinct motif that may subserve specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.

Mechanism of activation of the GM-CSF, IL-3, and IL-5 family of receptors.

The process of ligand binding leading to receptor activation is an ordered and sequential one. High-affinity binding of GM-CSF, interleukin 3 (IL-3), and IL-5 to their receptors induces a number of key events at the cell surface and within the cytoplasm that are necessary for receptor activation. These include receptor oligomerization, activation of tyrosine kinase activity, phosphorylation of the receptor, and the recruitment of SH2 (src-homology) and PTB (phosphotyrosine binding) domain proteins to the receptor. Such a sequence of events represents a recurrent theme among cytokine, growth factor, and hormone receptors; however, a number of very recent and interesting findings have identified unique features in this receptor system in terms of: A) how GM-CSF/IL-3/IL-5 bind, oligomerize, and activate their cognate receptors; B) how multiple biological responses such as proliferation, survival, and differentiation can be transduced from activated GM-CSF, IL-3, or IL-5 receptors, and C) how the presence of novel phosphotyrosine-independent signaling motifs within a specific cytoplasmic domain of betaC may be important for mediating survival and differentiation by these cytokines. This review does not attempt to be all-encompassing but rather to focus on the most recent and significant discoveries that distinguish the GM-CSF/IL-3/IL-5 receptor subfamily from other cytokine receptors.

Identification of a Cys motif in the common beta chain of the interleukin 3, granulocyte-macrophage colony-stimulating factor, and interleukin 5 receptors essential for disulfide-linked receptor heterodimerization and activation of all three receptors.

The human interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors undergo covalent dimerization of the respective specific alpha chains with the common beta subunit (betac) in the presence of the cognate ligand. We have now performed alanine substitutions of individual Cys residues in betac to identify the Cys residues involved and their contribution to activation of the IL-3, GM-CSF, and IL-5 receptors. We found that substitution of Cys-86, Cys-91, and Cys-96 in betac but not of Cys-100 or Cys-234 abrogated disulfide-linked IL-3 receptor dimerization. However, although Cys-86 and Cys-91 betac mutants retained their ability to form non-disulfide-linked dimers with IL-3Ralpha, substitution of Cys-96 eliminated this interaction. Binding studies demonstrated that all betac mutants with the exception of C96A supported high affinity binding of IL-3 and GM-CSF. In receptor activation experiments, we found that betac mutants C86A, C91A, and C96A but not C100A or C234A abolished phosphorylation of betac in response to IL-3, GM-CSF, or IL-5. These data show that although Cys-96 is important for the structural integrity of betac, Cys-86 and Cys-91 participate in disulfide-linked receptor heterodimerization and that this linkage is essential for tyrosine phosphorylation of betac. Sequence alignment of betac with other cytokine receptor signaling subunits in light of these data shows that Cys-86 and Cys-91 represent a motif restricted to human and mouse beta chains, suggesting a unique mechanism of activation utilized by the IL-3, GM-CSF, and IL-5 receptors.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor exists as a preformed receptor complex that can be activated by GM-CSF, interleukin-3, or interleukin-5.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor is expressed on normal and malignant hematopoietic cells as well as on cells from other organs in which it transduces a variety of functions. Despite the widespread expression and pleiotropic nature of the GM-CSF receptor, little is known about its assembly and activation mechanism. Using a combination of biochemical and functional approaches, we have found that the human GM-CSF receptor exists as an inducible complex, analogous to the interleukin-3 (IL-3) receptor, and also as a preformed complex, unlike the IL-3 receptor or indeed other members of the cytokine receptor superfamily. We found that monoclonal antibodies to the GM-CSF receptor alpha chain (GMR alpha) and to the common beta chain of the GM-CSF, IL-3, and IL-5 receptors (beta(c)) immunoprecipitated both GMR alpha and beta(c) from the surface of primary myeloid cells, myeloid cell lines, and transfected cells in the absence of GM-CSF. Further association of the two chains could be induced by the addition of GM-CSF. The preformed complex required only the extracellular regions of GMR alpha and beta(c), as shown by the ability of soluble beta(c) to associate with membrane-anchored GMR alpha or soluble GMR alpha. Kinetic experiments on eosinophils and monocytes with radiolabeled GM-CSF, IL-3, and IL-5 showed association characteristics unique to GM-CSF. Significantly, receptor phosphorylation experiments showed that not only GM-CSF but also IL-3 and IL-5 stimulated the phosphorylation of GMR alpha-associated beta(c). These results indicate a pattern of assembly of the heterodimeric GM-CSF receptor that is unique among receptors of the cytokine receptor superfamily. These results also suggest that the preformed GM-CSF receptor complex mediates the instantaneous binding of GM-CSF and is a target of phosphorylation by IL-3 and IL-5, raising the possibility that some of the biologic activities of IL-3 and IL-5 are mediated through the GM-CSF receptor complex.

The apoptosis-inducing granulocyte-macrophage colony-stimulating factor (GM-CSF) analog E21R functions through specific regions of the heterodimeric GM-CSF receptor and requires interleukin-1beta-converting enzyme-like proteases.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) analog E21R induces apoptosis of hemopoietic cells. We examined the GM-CSF receptor subunit requirements and the signaling molecules involved. Using Jurkat T cells transfected with the GM-CSF receptor we found that both receptor subunits were necessary for E21R-induced apoptosis. Specifically, the 16 membrane-proximal residues of the alpha subunit were sufficient for apoptosis. This sequence could be replaced by the corresponding sequence from the interleukin-2 receptor common gamma subunit, identifying this as a conserved cytokine motif necessary for E21R-induced apoptosis. Cells expressing the alpha subunit and truncated betac mutants showed that the 96 membrane-proximal residues of betac were sufficient for apoptosis. E21R, in contrast to GM-CSF, did not alter tyrosine phosphorylation of betac, suggesting that receptor-associated tyrosine kinases were not activated. Consistent with this, E21R decreased the mitogen-activated protein kinase ERK (extracellular signal-regulated kinase). E21R-induced apoptosis was independent of Fas/APO-1 (CD95) and required interleukin-1beta-converting enzyme (ICE)-like proteases. In contrast, Bcl-2, which protects cells from growth factor deprivation-induced cell death, did not prevent this apoptosis. These findings demonstrate the GM-CSF receptor and ICE-like protease requirements for E21R-induced apoptosis.

Roles of the N and C terminal domains of the interleukin-3 receptor alpha chain in receptor function.

The interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and IL-5 receptor alpha chains are each composed of three extracellular domains, a transmembrane domain and a short intracellular region. Domains 2 and 3 constitute the cytokine receptor module (CRM), typical of the cytokine receptor superfamily; however, the function of the N-terminal domain is not known. We have investigated the functions of the N-terminal and C-terminal domains of the IL-3 receptor (IL-3R) alpha chain. We find that cells transfected with the receptor beta chain (h beta c) and a truncated IL-3R alpha that is devoid of the intracellular region fail to proliferate or to activate STAT5 in response to human IL-3, despite binding the IL-3 with affinity indistinguishable from that of full-length receptor. In addition, IL-3-induced phosphorylation of h beta c was not detected. Thus, the IL-3R alpha intracellular region does not contribute detectably to stabilization of the receptor/ligand complex, but is essential for signal propagation. In contrast, a truncated IL-3R alpha with the N-terminal domain deleted interacts functionally with the beta chain; mouse cells transfected with these receptor chains proliferate in response to human IL-3 and STAT5 transcription factor is activated. High- and low-affinity binding sites are retained, although the affinity for IL-3 is decreased 15-fold, indicating a significant role for the N-terminal domain in IL-3 binding.

Human interleukin-3 (IL-3) induces disulfide-linked IL-3 receptor alpha- and beta-chain heterodimerization, which is required for receptor activation but not high-affinity binding.

The human interleukin-3 receptor (IL-3R) is a heterodimer that comprises an IL-3 specific alpha chain (IL-3R alpha) and a common beta chain (beta C) that is shared with the receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5. These receptors belong to the cytokine receptor superfamily, but they are structurally and functionally more related to each other and thus make up a distinct subfamily. Although activation of the normal receptor occurs only in the presence of ligand, the underlying mechanisms are not known. We show here that human IL-3 induces heterodimerization of IL-3R alpha and beta c and that disulfide linkage of these chains is involved in receptor activation but not high-affinity binding. Monoclonal antibodies (MAb) to IL-3R alpha and beta c were developed which immunoprecipitated, in the absence of IL-3, the respective chains from cells labelled with 125I on the cell surface. However, in the presence of IL-3, each MAb immunoprecipitated both IL-3R alpha and beta c. IL-3-induced receptor dimers were disulfide and nondisulfide linked and were dependent on IL-3 interacting with both IL-3R alpha and beta c. In the presence of IL-3 and under nonreducing conditions, MAb to either IL-3R alpha or beta c immunoprecipitated complexes with apparent molecular weights of 215,000 and 245,000 and IL-3R alpha and beta c monomers. Preincubation with iodoacetamide prevented the formation of the two high-molecular-weight complexes without affecting noncovalent dimer formation or high-affinity IL-3 binding. Two-dimensional gel electrophoresis and Western blotting (immunoblotting) demonstrated the presence of both IL-3R alpha and beta c in the disulfide-linked complexes. IL-3 could also be coimmunoprecipitated with anti-IL-3R alpha or anti-beta c MAB, but it was not covalently attached to the receptor. Following IL-3 stimulation, only the disulfide-linked heterodimers exhibited reactivity with antiphosphotyrosine antibodies, with beta c but not IL-3R alpha being the phosphorylated species. A model of IL-3R activation is proposed which may be also applicable to the related GM-CSF and IL-5 receptors.

Monoclonal antibody 7G3 recognizes the N-terminal domain of the human interleukin-3 (IL-3) receptor alpha-chain and functions as a specific IL-3 receptor antagonist.

The human interleukin-3 receptor (IL-3R) is expressed on myeloid, lymphoid, and vascular endothelial cells, where it transduces IL-3-dependent signals leading to cell activation. Although IL-3R activation may play a role in hematopoiesis and immunity, its aberrant expression or excessive stimulation may contribute to pathologic conditions such as leukemia, lymphoma, and allergic reactions. We describe here the generation and characterization of a monoclonal antibody (MoAb), 7G3, which specifically binds to the IL-3R alpha-chain and completely abolishes its function. MoAb 7G3 immunoprecipitated and recognized in Western blots the IL-3R alpha-chain expressed by transfected cells and bound to primary cells expressing IL-3R alpha. MoAb 7G3 bound the IL-3R alpha-chain with a kd of 900 pmol/L and inhibited 125I-IL-3 binding to high- and low-affinity receptors in a dose-dependent manner. Conversely, IL-3 but not granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited 125I-7G3 binding to high- and low-affinity IL-3Rs, indicating that MoAb 7G3 and IL-3 bind to common or adjacent sites. In keeping with the inhibition of IL-3 binding, MoAb 7G3 antagonized IL-3 biologic activities, namely stimulation of TF-1 cell proliferation, basophil histamine release, and IL-6 and IL-8 secretion from human endothelial cells. Two other anti-IL-3R alpha-chain MoAbs failed to inhibit IL-3 binding or function. Epitope mapping experiments using truncated IL-3R alpha-chain mutants and IL-3R alpha/GM-CSFR alpha chimeras revealed that 31 amino acids in the N-terminus of IL-3R alpha were required for MoAb 7G3 binding. MoAb 7G3 may be of clinical significance for antagonizing IL-3 in pathologic conditions such as some myeloid leukemias, follicular B-cell lymphoma, and allergy. Furthermore, these results implicate the N-terminal domain of IL-3R alpha in IL-3 binding. Since this domain is unique to the IL-3/GM-CSF/IL-5 receptor subfamily, it may represent a novel and common binding feature in these receptors.

Adhesion to thrombospondin by human embryonic fibroblasts is mediated by multiple receptors and includes a role for glycoprotein 88 (CD36).

Fetal embryonic fibroblasts attach and spread on thrombospondin (TSP). Adhesion is tight and focal adhesion plaques and "spots" are formed. We have investigated the receptors responsible for this adhesion. Unstimulated cells express the vitronectin receptor on their surface and this beta 3 integrin molecule contributes to adhesion. Another putative receptor for TSP, termed glycoprotein (GP) 88, which exists as a cytoplasmic pool in unstimulated cells becomes surface expressed when these cells are plated on TSP and localizes to areas of cell adhesion. Western blot analysis of cell lysate confirms GP88 as a TSP binding protein. Studies with fucoidan indicate that the heparan sulfate proteoglycan, known to function as a receptor for TSP, appears to contribute substantially to the TSP attachment of these cells and may be the receptor most important in the initial phases of TSP interaction.

Blood leukocytes bind platelet glycoprotein (IIb-IIIa)' but do not express the vitronectin receptor.

Within the integrin family of Arg-Gly-Asp(RGD)-binding adhesion receptors, the subfamily defined by the beta chain known as beta-3 or glycoprotein (GP)IIIa is known to contain two individual receptors. These are the GPIIb-IIIa complex of platelets, where the alpha chain of the heterodimer is GPIIb, and the vitronectin receptor (VnR) containing the alpha V subunit. The presence of either GPIIb-IIIa and/or the VnR on blood leukocytes has been controversial. We have investigated this problem by performing immunoprecipitation and immunoblotting studies with rabbit and monoclonal antibodies (mAb) to each of the subunits of GPIIb-IIIa and the VnR. On cultured cells of different origin, it was established that almost all expressed the VnR but none had GPIIb-IIIa, and the only polypeptide associated with beta 3 was alpha V. Platelets expressed predominantly GPIIb-IIIa, and weakly, the VnR. Monocytes and neutrophils freshly isolated from blood did not express the VnR but bore on their surface a modified form of GPIIb-IIIa. This molecule appeared identical to GPIIb-IIIa but an epitope on GPIIb was masked on the intact cell and was only revealed after immunoblotting. We have termed this modified form of GPIIb-IIIa, GP(IIb-IIIa)'. With differentiation in culture, monocytes rapidly lost surface GP(IIb-IIIa)' and concurrently began to express the VnR. Evidence is presented that GP(IIb-IIIa)' is derived from particles released by activated platelets and is bound firmly to the leukocyte membrane. Its primary function does not seem to be to mediate attachment to matrix proteins; thus, although U937 cells bearing platelet-derived GP(IIb-IIIa)' bound fibrinogen in an RGD-dependent manner, isolated blood monocytes did not. It is suggested that this transfer of membrane proteins from platelets to monocytes and neutrophils may regulate the expression of the leukocyte VnR and also serve as a means of facilitating leukocyte procoagulant activity.

Identification of a novel integrin beta subunit expressed on cultured monocytes (macrophages). Evidence that one alpha subunit can associate with multiple beta subunits.

The vitronectin receptor (VnR) is one member of a subset of cell adhesion receptors within the integrin supergene family which shares the beta 3 subunit (IIIa). We show here that the VnR is absent from the surface of monocytes freshly isolated from blood but is expressed on these cells after a period of in vitro culture. Such cultured monocytes (macrophages) from a patient with type I Glanzmann's thrombasthenia, however, failed to express the VnR. Instead, immunoprecipitation with a monoclonal antibody directed to the VnR alpha chain (alpha v) revealed a novel integrin comprising alpha v associated noncovalently with a 100-kDa beta subunit (beta 3b), immunologically unrelated to the VnR beta subunit (beta 3a). This same novel integrin complex was also identified on 10-day-old macrophages from healthy donors, but on these cells, the beta 3b subunit was co-expressed with the classical VnR complex of alpha v beta 3a. The novel beta 3b subunit was not identified by monoclonal or polyclonal antibodies to IIIa (beta 3a) nor by a monoclonal antibody to the classical VnR complex. The beta 3b subunit could be distinguished from beta 3a by its relatively greater migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction, by its distinct isoelectric point upon two-dimensional gel electrophoresis, and by one-dimensional peptide mapping. Neither platelets nor B lymphoblasts from this patient with Glanzmann's thrombasthenia expressed any VnR on their surface, whereas control cells from a normal donor expressed the classical VnR but not the beta 3b subunit. The two beta chains, and hence also the combined receptor complexes, appeared to be differentially regulated. These findings provide the first example of an integrin alpha chain complexed with more than a single beta chain in the same cell. Furthermore, the differential regulation of expression of the different beta subunits that associate with the VnR alpha chain on cultured monocytes suggests a role for the novel receptor complex during monocyte/macrophage differentiation.

Cancer of the breast and beta 2 microglobulin.

In carcinoma of the breast the disease stage at diagnosis determines therapy and is closely related to prognosis. To date no single biochemical marker of disseminated breast cancer has been described although beta 2 microglobulin values in the serum have been suggested as a discriminant between localized and systemic disease. Using a new method of beta 2 microglobulin estimation, we carried out a prospective study of levels in 53 patients with breast cancer who were studied repetitively over a 2 yr period. No relationship could be determined between beta 2 microglobulin values and the stage of the disease. Moreover beta 2 microglobulin levels, even when elevated, did not predict early metastasis. Repeated beta 2 microglobulin estimations during treatment of metastatic disease had limited usefulness in that patients with responsive disease usually showed a fall in beta 2 microglobulin, whereas there was generally no change in non-responsive patients. These changes were, however, often within the normal range and seemed to offer a marginal improvement in assessment.