Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific alpha subunit and a betac subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

The process of ligand binding leading to receptor activation is an ordered and sequential one. Highaffinity 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 β C 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.

The 14-3-3 proteins play a central role in the regulation of cell growth, cycling, and apoptosis by modulating the functional activities of key signaling proteins. Through binding to a phosphoserine motif, 14-3-3 alters target proteins activities by sequestering them, relocalizing them, conformationally altering their functional activity, or by promoting interaction with other proteins. These functions of 14-3-3 are facilitated by, if not dependent on, its dimeric structure. We now show that the dimeric status of 14-3-3 is regulated by site-specific serine phosphorylation. We found that a sphingosine-dependent kinase phosphorylates 14-3-3 in vitro and in vivo on a serine residue (Ser 58 ) located within the dimer interface. Furthermore, by developing an antibody that specifically recognizes 14-3-3 phosphorylated on Ser 58 and employing native-PAGE and cross-linking techniques, we found that 14-3-3 phosphorylated on Ser 58 is monomeric both in vitro and in vivo. Phosphorylated 14-3-3 was detected solely as a monomer, indicating that phosphorylation of a single monomer within a dimer is sufficient to disrupt the dimeric structure. Significantly, phosphorylation-induced monomerization did not prevent 14-3-3 binding to a phosphopeptide target. We propose that this regulated monomerization of 14-3-3 controls its ability to modulate the activity of target proteins and thus may have significant implications for 14-3-3 function and the regulation of many cellular processes.

Complex neuropsychiatric disorders are believed to arise from multiple synergistic deficiencies within connected biological networks controlling neuronal migration, axonal pathfinding and synapse formation. Here, we show that deletion of 14-3-3ζ causes neurodevelopmental anomalies similar to those seen in neuropsychiatric disorders such as schizophrenia, autism spectrum disorder and bipolar disorder. 14-3-3ζ-deficient mice displayed striking behavioural and cognitive deficiencies including a reduced capacity to learn and remember, hyperactivity and disrupted sensorimotor gating. These deficits are accompanied by subtle developmental abnormalities of the hippocampus that are underpinned by aberrant neuronal migration. Significantly, 14-3-3ζ-deficient mice exhibited abnormal mossy fibre navigation and glutamatergic synapse formation. The molecular basis of these defects involves the schizophrenia risk factor, DISC1, which interacts isoform specifically with 14-3-3ζ. Our data provide the first evidence of a direct role for 14-3-3ζ deficiency in the aetiology of neurodevelopmental disorders and identifies 14-3-3ζ as a central risk factor in the schizophrenia protein interaction network.

Pleiotropism is a hallmark of cytokines and growth factors; yet, the underlying mechanisms are not clearly understood. We have identified a motif in the granulocyte macrophage-colony-stimulating factor receptor composed of a tyrosine and a serine residue that functions as a binary switch for the independent regulation of multiple biological activities. Signalling occurs either through Ser585 at lower cytokine concentrations, leading to cell survival only, or through Tyr577 at higher cytokine concentrations, leading to cell survival as well as proliferation, differentiation or functional activation. The phosphorylation of Ser585 and Tyr577 is mutually exclusive and occurs via a unidirectional mechanism that involves protein kinase A and tyrosine kinases, respectively, and is deregulated in at least some leukemias. We have identified similar Tyr/Ser motifs in other cell surface receptors, suggesting that such signalling switches may play important roles in generating specificity and pleiotropy in other biological systems.

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...

Pathological drug withdrawal syndrome is linked to accumulation of JAK2 phosphorylation in V617F myelofibrosis.

Engagement of the adhesion receptor glycoprotein (GP) Ib-IX-V by von Willebrand factor (VWF) mediates platelet adhesion to damaged vessels and triggers platelet activation and thrombus formation in heart attack and stroke. GPIb-IX-V contains distinct 14-3-3ζ–binding sites at the GPIbα C-terminus involving phosphorylation of Ser609, an upstream site involving phosphorylated Ser587/Ser590, and a protein kinase A (PKA)–dependent site on GPIbβ involving Ser166. 14-3-3ζ regulates the VWF-binding affinity of GPIb-IX-V and inhibiting 14-3-3ζ association blocks receptor signaling, suggesting a key functional role for 14-3-3ζ. We used deletion mutants of GPIbα expressed in Chinese hamster ovary (CHO) cells to define the relationship of 14-3-3ζ binding to another GPIb-IX-V–associated signaling protein, phosphoinositide 3-kinase (PI3-kinase). Pull-down experiments involving glutathione S-transferase (GST)–PI3-kinase/p85-subunit and GST–14-3-3ζ indicated that both proteins interacted with contiguous GPIbα sequences 580 to 590/591 to 610. Deleting these, but not upstream sequences of GPIbα expressed in CHO cells, inhibited VWF/ristocetin-dependent Akt phosphorylation, relative to wild-type receptor, confirming this region encompassed a functional PI3-kinase–binding site. Pull-down experiments with GST-p85 truncates indicated the GPIbα-binding region involved the p85 breakpoint cluster region (BCR) domain, containing RSXSXP. However, pull-down of GPIb-IX was unaltered by mutation/deletion/phosphorylation of this potential 14-3-3ζ–binding sequence in mutant constructs of GST-p85, suggesting PI3-kinase bound GPIbα independently of 14-3-3ζ; 14-3-3ζ inhibitor peptide R18 also blocked pull-down of receptor by GST-14-3-3ζ but not GST-p85, and GST-p85 pull-downs were unaffected by excess 14-3-3ζ. Together, these data suggest the GPIbα C-terminus regulates signaling through independent association of 14-3-3ζ and PI3-kinase.