14-3-3:Shc scaffolds integrate phosphoserine and phosphotyrosine signaling to regulate phosphatidylinositol 3-kinase activation and cell survival.

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 14-3-3zeta is phosphorylated on Tyr(179) in response to granulocyte macrophage colony-stimulating factor. Importantly, 14-3-3zeta 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 14-3-3zeta(-/-) mice. We show that cytokine was able to promote Akt phosphorylation and viability of primary mast cells derived from 14-3-3zeta(-/-) mice when reconstituted with wild type 14-3-3zeta, 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.

Growth factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary switch.

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.

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

We have recently identified a novel mechanism of hematopoietic cell survival that involves site-specific serine phosphorylation of the common beta subunit (beta(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 beta(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 beta(c)Tyr577Phe and beta(c)Ser585Gly, respectively. Importantly, while mutants in which either Ser585 (beta(c)Ser585Gly) or all tyrosines (beta(c)F8) were substituted showed a defect in Akt phosphorylation, nuclear factor kappaB (NF-kappaB) activation, bcl-2 induction, and cell survival, the mutant beta(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-kappaB to promote bcl-2 expression.