The development of cell therapeutics from embryonic stem (ES) cells will require technologies that direct cell differentiation to specific somatic cell lineages in response to defined factors. The initial step in formation of the somatic lineages from ES cells, differentiation to an intermediate, pluripotent primitive ectoderm-like cell, can be achieved in vitro by formation of early primitive ectoderm-like (EPL) cells in response to a biological activity contained within the conditioned medium MEDII. Fractionation of MEDII has identified two activities required for EPL cell formation, an activity with a molecular mass of <3 kDa and a second, much larger species. Here, we have identified the low-molecular-weight activity as l-proline. An inhibitor of l-proline uptake, glycine, prevented the differentiation of ES cells in response to MEDII. Supplementation of the culture medium of ES cells with >100 M l-proline and some l-proline-containing peptides resulted in changes in colony morphology, cell proliferation, gene expression, and differentiation kinetics consistent with differentiation toward a primitive ectoderm-like cell. This activity appeared to be associated with l-proline since other amino acids and analogs of proline did not exhibit an equivalent activity. Activation of the mammalian target of rapamycin (mTOR) signaling pathway was found to be necessary but not sufficient for l-proline activity; addition of other activators of the mTOR signaling pathway failed to alter the ES cell phenotype. This is the first report describing a role for amino acids in the regulation of pluripotency and cell differentiation and identifies a novel role for the imino acid l-proline.
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...
Integrated cascades of protein tyrosine and serine/threonine phosphorylation play essential roles in transducing signals in response to growth factors and cytokines. How adaptor or scaffold proteins assemble signaling complexes through both phosphotyrosine and phosphoserine/threonine residues to regulate specific signaling pathways and biological responses is unclear. We show in multiple cell types that endogenous 14-3-3 is phosphorylated on Tyr 179 in response to granulocyte macrophage colony-stimulating factor. Importantly, 14-3-3 can function as an intermolecular bridge that couples to phosphoserine residues and also directly binds the SH2 domain of Shc via Tyr 179 . The assembly of these 14-3-3:Shc scaffolds is specifically required for the recruitment of a phosphatidylinositol 3-kinase signaling complex and the regulation of CTL-EN cell survival in response to cytokine. The biological significance of these findings was further demonstrated using primary bone marrow-derived mast cells from 14-3-3 ؊/؊ mice. We show that cytokine was able to promote Akt phosphorylation and viability of primary mast cells derived from 14-3-3 ؊/؊ mice when reconstituted with wild type 14-3-3, but the Akt phosphorylation and survival response was reduced in cells reconstituted with the Y179F mutant. Together, these results show that 14-3-3:Shc scaffolds can act as multivalent signaling nodes for the integration of both phosphoserine/threonine and phosphotyrosine pathways to regulate specific cellular responses.
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