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

Abstract

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.

The ShcA SH2 domain engages a 14-3-3/PI3′K signaling complex and promotes breast cancer cell survival

“…25 Thus, 14 --3 --3s is not likely to participate in the activation of pro-survival signaling downstream of the ShcA SH2 domain. We further demonstrate that 14 --3 --3z specifically co-immunoprecipitates with wild-type ShcA, and not the ShcR397K mutant, in mammary tumor lysates, which is consistent with the observation that 14 --3 --3z activates the PI3K/AKT pathway by recruiting the p85 regulatory subunit 19,26 (Figure 4d). However, we cannot exclude the possibility that one or more of the remaining 14 --3 --3 family members are recruited to the SH2 domain of ShcA to enhance breast cancer cell survival.…”

“…39 --40 Moreover, the ShcA SH2 domain binds Y179 of 14 --3 --3z and mutation of the ShcAbinding site (Y179F) significantly reduced AKT activation and survival of a hematopoietic cell line. 19 Thus we propose that the ShcA SH2 domain binds tyrosine phosphorylation sites within 14 --3--3z, and potentially also 14 --3 --3b, to indirectly recruit p85a-PI3K to the plasma membrane, leading to activation of PI3 0 K/AKTmediated survival signaling responses. Future studies with mice carrying mutant members of 14 --3 --3 family should allow this issue to be addressed.…”

“…A previous study suggested that the ShcA SH2 domain binds Y179 of 14 --3 --3z and that retention of this tyrosine phosphorylation site is required to activate AKT downstream of the Granulocyte-macrophage colony-stimulating factor receptor. 19 To test this possibility, we employed a quantitative proteomics approach to identify intracellular proteins that specifically couple to the SH2 domain of ShcA. MT and MT/ShcR397K tumor lysates were first immunoprecipitated with anti-FLAG antibodies, which specifically bind the ShcR397K mutant.…”

14-3-3 proteins are essential signalling hubs for beta cell survival

“…It remains to be determined whether 14-3-3ζ is critical in the regulation of cell survival. The above evidence suggests that 14-3-3 proteins serve as unique integration points to coordinate kinase activity and phospho-dependent survival cues [18]. Therefore, we tested the hypothesis that 14-3-3 proteins are essential for coordinating beta cell survival as the core of a signalling node that integrates multiple survival signals.…”

Phosphorylation-Regulated Cell Surface Expression of Membrane Proteins

“…Crystal structure of 14-3-3ζ : AANAT revealed that in addition to the phosphorylation-dependent interaction through its canonical ligand-binding groove, 14-3-3 also makes extensive contacts with AANAT via other regions of the 14-3-3 channel, although these contacts must be insufficient to form a stable complex (Obsil et al, 2001). Moreover, a recent finding by can directly interact with other proteins outside of the canonical binding groove, providing a possible molecular basis for the scaffolding function of 14-3-3 (Barry et al, 2009). 14-3-3 undergoes phosphorylation at tyrosine 179 upon cytokine stimulation and this leads to the binding of Shc protein through its SH2 domain that recognizes phosphorylated tyrosine.…”