The Integration of Proteome-Wide PTM Data with Protein Structural and Sequence Features Identifies Phosphorylations that Mediate 14-3-3 Interactions

14-3-3s are abundant proteins that regulate essentially all aspects of cell biology, including cell cycle, motility, metabolism, and cell death. 14-3-3s work by docking to phosphorylated Ser/Thr residues on a large network of client proteins and modulating client protein function in a variety of ways. In recent years, aided by improvements in proteomics, the discovery of 14-3-3 client proteins has far outpaced our ability to understand the biological impact of individual 14-3-3 interactions. The rate-limiting step in this process is often the identification of the individual phospho-serines/threonines that mediate 14-3-3 binding, which are difficult to distinguish from other phospho-sites by sequence alone. Furthermore, trial-and-error molecular approaches to identify these phosphorylations are costly and can take months or years to identify even a single 14-3-3 docking site phosphorylation. To help overcome this challenge, we used machine learning to analyze predictive features of 14-3-3 binding sites. We found that accounting for intrinsic protein disorder and the unbiased mass spectrometry identification rate of a given phosphorylation significantly improves the identification of 14-3-3 docking site phosphorylations across the proteome. We incorporated these features, coupled with consensus sequence prediction, into a publicly available web app, called “14-3-3 site-finder”. We demonstrate the strength of this approach through its ability to identify 14-3-3 binding sites that do not conform to the loose consensus sequence of 14-3-3 docking phosphorylations, which we validate with 14-3-3 client proteins, including TNK1, CHEK1, MAPK7, and others. In addition, by using this approach, we identify a phosphorylation on A-kinase anchor protein-13 (AKAP13) at Ser2467 that dominantly controls its interaction with 14-3-3.     

Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins

Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)-labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14-3-3 proteins and specific elution with a 14-3-3-binding phosphopeptide. Purified 14-3-3-binding proteins included sucrose–phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast to the phosphorylated NR-14-3-3 complex which is activated by dissociation with 14-3-3-binding phosphopeptides, the total sugar–phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosphopeptide and the phosphopeptide-inhibited activity was reactivated by adding excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-binding status of defined target proteins change in response to extracellular stimuli.     

A 14‐3‐3 Mode‐1 Binding Motif Initiates Gap Junction Internalization During Acute Cardiac Ischemia

Altered phosphorylation and trafficking of connexin 43 (Cx43) during acute ischemia contributes to arrhythmogenic gap junction remodeling, yet the critical sequence and accessory proteins necessary for Cx43 internalization remain unresolved. 14‐3‐3 proteins can regulate protein trafficking, and a 14‐3‐3 mode‐1 binding motif is activated upon phosphorylation of Ser373 of the Cx43 C‐terminus. We hypothesized that Cx43^Ser373 phosphorylation is important to pathological gap junction remodeling. Immunofluorescence in human heart reveals the enrichment of 14‐3‐3 proteins at intercalated discs, suggesting interaction with gap junctions. Knockdown of 14‐3‐3τ in cell lines increases gap junction plaque size at cell–cell borders. Cx43^S373A mutation prevents Cx43/14‐3‐3 complexing and stabilizes Cx43 at the cell surface, indicating avoidance of degradation. Using Langendorff‐perfused mouse hearts, we detect phosphorylation of newly internalized Cx43 at Ser373 and Ser368 within 30 min of no‐flow ischemia. Phosphorylation of Cx43 at Ser368 by protein kinase C and Ser255 by mitogen‐activated protein kinase has previously been implicated in Cx43 internalization. The Cx43^S373A mutant is resistant to phosphorylation at both these residues and does not undergo ubiquitination, revealing Ser373 phosphorylation as an upstream gatekeeper of a posttranslational modification cascade necessary for Cx43 internalization. Cx43^Ser373 phosphorylation is a potent target for therapeutic interventions to preserve gap junction coupling in the stressed myocardium.      

Cyclic Nucleotide-dependent Protein Kinases Target ARHGAP17 and ARHGEF6 Complexes in Platelets

Endothelial cells release prostacyclin (PGI₂) and nitric oxide (NO) to inhibit platelet functions. PGI₂ and NO effects are mediated by cyclic nucleotides, cAMP- and cGMP-dependent protein kinases (PKA, PKG), and largely unknown PKA and PKG substrate proteins. The small G-protein Rac1 plays a key role in platelets and was suggested to be a target of cyclic nucleotide signaling. We confirm that PKA and PKG activation reduces Rac1-GTP levels. Screening for potential mediators of this effect resulted in the identification of the Rac1-specific GTPase-activating protein ARHGAP17 and the guanine nucleotide exchange factor ARHGEF6 as new PKA and PKG substrates in platelets. We mapped the PKA/PKG phosphorylation sites to serine 702 on ARHGAP17 using Phos-tag gels and to serine 684 on ARHGEF6. We show that ARHGAP17 binds to the actin-regulating CIP4 protein in platelets and that Ser-702 phosphorylation interferes with this interaction. Reduced CIP4 binding results in enhanced inhibition of cell migration by ARHGAP17. Furthermore, we show that ARHGEF6 is constitutively linked to GIT1, a GAP of Arf family small G proteins, and that ARHGEF6 phosphorylation enables binding of the 14-3-3 adaptor protein to the ARHGEF6/GIT1 complex. PKA and PKG induced rearrangement of ARHGAP17- and ARHGEF6-associated protein complexes might contribute to Rac1 regulation and platelet inhibition.     

Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers

Assimilatory nitrate reductase (NR) of higher plants is a most interesting enzyme, both from its central function in plant primary metabolism and from the complex regulation of its expression and control of catalytic activity and degradation. Here, present knowledge about the mechanism of post-translational regulation of NR is summarized and the properties of the regulatory enzymes involved (protein kinases, protein phosphatases and 14-3-3-binding proteins) are described. It is shown that light and oxygen availability are the major external triggers for the rapid and reversible modulation of NR activity, and that sugars and/or sugar phosphates are the internal signals which regulate the protein kinase(s) and phosphatase. It is also demonstrated that stress factors like nitrate deficiency and salinity have remarkably little direct influence on the NR activation state. Further, changes in NR activity measured in vitro are not always associated with changes in nitrate reduction rates in vivo, suggesting that NR can be under strong substrate limitation. The degradation and half-life of the NR protein also appear to be affected by NR phosphorylation and 14-3-3 binding, as NR activation always correlates positively with its stability. However, it is not known whether the molecular form of NR in vivo affects its susceptibility to proteolytic degradation, or whether factors that affect the NR activation state also independently affect the activity or induction of the NR protease(s). A second and potentially important function of NR, the production of nitric oxide (NO) from nitrite is briefly described, but it remains to be determined whether NR produces NO for pathogen/stress signalling in vivo.     

Phosphorylation of Serine 1137/1138 of Mouse Insulin Receptor Substrate (IRS) 2 Regulates cAMP-dependent Binding to 14-3-3 Proteins and IRS2 Protein Degradation

Insulin receptor substrate (IRS) 2 as intermediate docking platform transduces the insulin/IGF-1 (insulin like growth factor 1) signal to intracellular effector molecules that regulate glucose homeostasis, β-cell growth, and survival. Previously, IRS2 has been identified as a 14-3-3 interaction protein. 14-3-3 proteins can bind their target proteins via phosphorylated serine/threonine residues located within distinct motifs. In this study the binding of 14-3-3 to IRS2 upon stimulation with forskolin or the cAMP analog 8-(4-chlorophenylthio)-cAMP was demonstrated in HEK293 cells. Binding was reduced with PKA inhibitors H89 or R_p-8-Br-cAMPS. Phosphorylation of IRS2 on PKA consensus motifs was induced by forskolin and the PKA activator N⁶-Phe-cAMP and prevented by both PKA inhibitors. The amino acid region after position 952 on IRS2 was identified as the 14-3-3 binding region by GST-14-3-3 pulldown assays. Mass spectrometric analysis revealed serine 1137 and serine 1138 as cAMP-dependent, potential PKA phosphorylation sites. Mutation of serine 1137/1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding. Application of cycloheximide revealed that forskolin enhanced IRS2 protein stability in HEK293 cells stably expressing IRS2 as well as in primary hepatocytes. Stimulation with forskolin did not increase protein stability either in the presence of a 14-3-3 antagonist or in the double 1137/1138 alanine mutant. Thus the reduced IRS2 protein degradation was dependent on the interaction with 14-3-3 proteins and the presence of serine 1137/1138. We present serine 1137/1138 as novel cAMP-dependent phosphorylation sites on IRS2 and show their importance in 14-3-3 binding and IRS2 protein stability.     

Comparative proteomic analysis of <Emphasis Type="Italic">Phalaenopsis</Emphasis> leaves in the vegetative and flowering phase

Phalaenopsis, an epiphytic crassulacean acid metabolism (CAM) plant, requires moderate variations of day/night temperatures for flowering. In this study, changes in chlorophyll content, chlorophyll fluorescence, sugar components, titratable acidity and soluble protein content in Phalaenopsis leaves during flowering were observed. Comparative proteomic analysis of Phalaenopsis leaves in the vegetative and flowering phase was performed for the first time using iTRAQ (isobaric tags for relative and absolute quantification). A total of 126 proteins were differentially expressed in Phalaenopsis leaves. Analysis of potential functions revealed that the major categories of predicted function of the up-regulated proteins were protein destination (27 %), photosynthesis (15.9 %), primary metabolism (14.3 %) and defense (12.7 %) in the flowering phase, while the major categories of predicted function of the down-regulated proteins were protein destination (33.3 %), primary metabolism (20.6 %), transportation (14.3 %) and signal transduction (11.1 %). Proteome profile analysis indicated that the proteome changes were consistent with changes in sugar and protein metabolites. Some novel proteins were differentially expressed, most of which were identified as signaling proteins, including 14-3-3 proteins, fibrillin, rapid alkalinization factors (RALF), the Ras-related protein RABB1c, calreticulin and calmodulin. Histone, importin alpha, multidrug resistance proteins and the ABC transporters were also differentially expressed. These results provide insights into the mechanisms that regulate flowering in complex flowering plants.     

14-3-3蛋白对植物发育的调控作用

14-3-3蛋白是高度保守并在真核生物中普遍存在的一类调节蛋白。不同的14-3-3蛋白同工型具有不同的细胞特异性, 并通过识别特异的磷酸化序列与靶蛋白相互作用, 被称为蛋白质与蛋白质相互作用的桥梁蛋白。在植物生长发育过程中, 14-3-3蛋白通过与其它蛋白的相互作用参与多种植物激素信号转导、各种代谢调控、物质运输和光信号应答等调控过程。该文主要对近年来有关14-3-3蛋白在植物生长发育中的调控作用, 特别是14-3-3蛋白参与调控植物激素信号转导等方面的研究进展进行综述。     

Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose

Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown. In this paper, we show that TPS5, 6 and 7 are phosphoproteins that bind to 14-3-3 proteins, by using 14-3-3 affinity chromatography, 14-3-3 overlay assays, and by co-immunoprecipitating TPS5 and 14-3-3 isoforms from cell extracts. GST-TPS5 bound to 14-3-3s after in vitro phosphorylation at Ser22 and Thr49 by either mammalian AMP-activated protein kinase (AMPK) or partially purified plant Snf1-related protein kinase 1 (SnRK1s). Dephosphorylation of TPS5, or mutation of either Ser22 or Thr49, abolished binding to 14-3-3s. Ser22 and Thr49 are both conserved in TPS5, 7, 9 and 10. When GST-TPS5 was expressed in human HEK293 cells, Thr49 was phosphorylated in response to 2-deoxyglucose or phenformin, stimuli that activate the AMPK via the upstream kinase LKB1. 2-deoxyglucose stimulated Thr49 phosphorylation of endogenous TPS5 in Arabidopsis cells, whereas phenformin did not. Moreover, extractable SnRK1 activity was increased in Arabidopsis cells in response to 2-deoxyglucose. The plant kinase was inactivated by dephosphorylation and reactivated by phosphorylation with human LKB1, indicating that elements of the SnRK1/AMPK pathway are conserved in Arabidopsis and human cells. We hypothesize that coordinated phosphorylation and 14-3-3 binding of nitrate reductase (NR), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP) and class II TPS isoforms mediate responses to signals that activate SnRK1.     

The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch

The Raf-1 protein kinase is a major activator of the ERK MAPK pathway, which links signaling by a variety of cell surface receptors to the regulation of cell proliferation, survival, differentiation and migration. Signaling by Raf-1 is regulated by a complex and poorly understood interplay between phosphorylation events and protein–protein interactions. One important mode of Raf-1 regulation involves the phosphorylation-dependent binding of 14-3-3 proteins. Here, we have examined the mechanism whereby the C-terminal 14-3-3 binding site of Raf-1, S621, controls the activation of MEK-ERK signaling. We show that phosphorylation of S621 turns over rapidly and is enriched in the activated pool of endogenous Raf-1. The phosphorylation on this site can be mediated by Raf-1 itself but also by other kinase(s). Mutations that prevent the binding of 14-3-3 proteins to S621 render Raf-1 inactive by specifically disrupting its capacity to bind to ATP, and not by gross conformational alteration as indicated by intact MEK binding. Phosphorylation of S621 correlates with the inhibition of Raf-1 catalytic activity in vitro, but 14-3-3 proteins can completely reverse this inhibition. Our findings suggest that 14-3-3 proteins function as critical cofactors in Raf-1 activation, which induce and maintain the protein in a state that is competent for both ATP binding and MEK phosphorylation.     

14-3-3 proteins: regulation of signal-induced events

The field of signal transduction has experienced a significant paradigm shift as a result of an increased understanding of the roles of 14-3-3 proteins. There are many cases where signal-induced phosphorylation itself may cause a change in protein function. This simple modification is, in fact, the primary basis of signal transduction events in many systems. There are a large and growing number of cases, however, where simple phosphorylation is not enough to effect a change in protein function. In these cases, the 14-3-3 proteins can be required to complete the change in function. Therefore signal transduction can be either the relatively simple process where phosphorylation alters target activity, or it can be a more complex, multistep process with the 14-3-3 proteins playing the major role of bringing the signal transduction event to completion. This makes 14-3-3-modulated signal transduction a more complicated process with additional avenues for regulation and variety. Adding further complexity to the process is the fact that 14-3-3 proteins are present as multigene families in most organisms (Aitken et al. Trends Biochem Sci 17: 498–501, 1992; Ferl Annu Rev Plant Physiol Plant Molecular Biology 47: 49–73, 1996), with each member of the family being differentially expressed in various tissues and with potentially differential affinity for various target proteins. This review focuses on the 14-3-3 family of Arabidopsis as a model for further developing understanding of the roles of the 14-3-3 proteins as modulators of signal transduction events in plants. The primary approaches to these questions are not unlike the approaches that would be used in the functional dissection of any multigene family, but the interpretation of these data will have wide implications since the 14-3-3 s physically interact with other protein families.     

Identification of 14-3-3zeta as a protein kinase B/Akt substrate

Protein kinase B/Akt (PKB/Akt) is a member of the ACG kinase family, which also includes protein kinase C, that phosphorylates a number of 14-3-3-binding proteins. 14-3-3 protein regulation of protein kinase C activity is modulated by 14-3-3 phosphorylation. We examined the hypothesis that PKB/Akt interacts with and phosphorylates 14-3-3zeta, leading to modulation of dimerization. By glutathione S-transferase pull-down, Akt precipitated recombinant 14-3-3zeta and endogenous 14-3-3zeta from HEK293 cell lysates. Recombinant active PKB/Akt phosphorylated recombinant 14-3-3zeta in an in vitro kinase assay. Transfection of active PKB/Akt into HEK293 cells resulted in phosphorylation of 14-3-3zeta. Based on a motif search of 14-3-3zeta, a potential PKB/Akt phosphorylation site, Ser-58, was mutated to alanine. PKB/Akt was unable to phosphorylate this mutant protein. Incubation of 14-3-3zeta with recombinant active PKB/Akt resulted in phosphorylation of 45% of the protein, as determined by a pI shift on two-dimensional electrophoresis, but 14-3-3zeta dimerization was not altered. These data indicate that PKB/Akt phosphorylates Ser-58 on 14-3-3zeta both in vitro and in intact cells. The functional relevance of this phosphorylation remains to be determined.     

Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport

The members of the RGK small GTP-binding protein family, Kir/Gem, Rad, Rem and Rem2, are multifunctional proteins that regulate voltage-gated calcium channel activity and cell shape remodeling. Calmodulin (CaM) or CaM 14-3-3 are regulators of RGK functions and their association defines the subcellular localization of RGK proteins. Abolition of CaM association results in the accumulation of RGK proteins in the nucleus, whereas 14-3-3 binding maintains them in the cytoplasm. Kir/Gem possesses nuclear localization signals (NLS) that mediate nuclear accumulation through an importin alpha5-dependent pathway (see Mahalakshmi RN, Nagashima K, Ng MY, Inagaki N, Hunziker W, Béguin P. Nuclear transport of Kir/Gem requires specific signals and importin alpha5 and is regulated by Calmodulin and predicted service phosphorylations. Traffic 2007; doi: 10.1111/j.1600-0854.2007.00598.x). Because the extent of nuclear localization depends on the RGK protein and the cell type, the mechanism and regulation of nuclear transport may differ. Here, we extend our analysis to the other RGK members and show that Rem also binds importin alpha5, whereas Rad associates with importins alpha3, alpha5 and beta through three conserved NLS. Predicted phosphorylation of a serine residue within the bipartite NLS affects, as observed for Kir/Gem, nuclear accumulation of Rem, but not that of Rad or Rem2. We also identify an additional regulatory phosphorylation for all RGK proteins that prevents binding of 14-3-3 and thereby interferes with their cytosolic relocalization by 14-3-3. Functionally, nuclear localization of RGK proteins contributes to the suppression of RGK-mediated cell shape remodeling. Importantly, we show that endogenous RGK proteins are localized predominantly in the nucleus of individual cells of the brain cortex 'in situ' as well as in primary hippocampal cells, indicating that transport between the nucleus and their site of action in the cytoplasm (i.e., cytoskeleton, endoplasmic reticulum or plasma membrane) is of physiological relevance for the regulation of RGK protein function.     

14-3-3 binding regulates catalytic activity of human Wee1 kinase.

The mitotic inducer Cdc2 is negatively regulated, in part, by phosphorylation on tyrosine 15. Human Wee1 is a tyrosine-specific protein kinase that phosphorylates Cdc2 on tyrosine 15. Human Wee1 is subject to multiple levels of regulation including reversible phosphorylation, proteolysis, and protein-protein interactions. Here we have investigated the contributions made by 14-3-3 binding to human Wee1 regulation and function. We report that the interactions of 14-3-3 proteins with human Wee1 are reduced during mitosis and are stable in the presence of the protein kinase inhibitor UCN-01. A mutant of Wee1 that is incapable of binding to 14-3-3 proteins has lower enzymatic activity, and this likely accounts for its reduced potency relative to wild-type Wee1 in inducing a G(2) cell cycle delay when overproduced in vivo. These findings indicate that 14-3-3 proteins function as positive regulators of the human Wee1 protein kinase.     

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 14-3-3ζ is phosphorylated on Tyr¹⁷⁹ in response to granulocyte macrophage colony-stimulating factor. Importantly, 14-3-3ζ can function as an intermolecular bridge that couples to phosphoserine residues and also directly binds the SH2 domain of Shc via Tyr¹⁷⁹. 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 14-3-3ζ^-/- mice. We show that cytokine was able to promote Akt phosphorylation and viability of primary mast cells derived from 14-3-3ζ^-/- mice when reconstituted with wild type 14-3-3ζ, 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 ability of a cell to respond to extrinsic stimuli critically hinges on its ability to regulate specific intracellular protein-protein interactions in a reversible manner. Such signals are relayed within the cell through the assembly of signaling complexes that are built using protein scaffolds. One important mechanism by which this occurs is via the binding of Src homology 2 (SH2)⁵ or phosphotyrosine-binding (PTB) domains to phosphotyrosine residues (1, 2). Importantly, the ability of individual SH2 or PTB domains to recognize specific phosphotyrosine motifs in different proteins enables the assembly of purpose-built signaling complexes that promote signaling via specific pathways (3). In some cases, signaling proteins not only contain more than one SH2 and/or PTB domain but are also themselves tyrosine-phosphorylated, leading to a network of phosphotyrosine-mediated protein-protein interactions.Although less well studied, phosphoserine/threonine-binding proteins are also important for the assembly of signaling complexes. For example, the 14-3-3 family of proteins is able to bind phosphoserine/threonine residues in a sequence-specific context (RSX(S/T)XP and RXXX(S/T)XP, where (S/T) is phosphoserine/threonine) (4, 5). The 14-3-3 proteins have been proposed to function as “modifiers” or “sequestrators”; however, because of their dimeric structure, they have also been proposed to function as “adaptor” or “scaffold” proteins through their ability to bring together two serine/threonine phosphorylated proteins (4–7). Additionally, a number of phosphoserine/threonine-binding modules such as tryptophan-tryptophan (WW), Forkhead-associated (FHA), Polo box (PBD), and BRCA1 C-terminal (BRCT) domains have been shown to interact with phosphoserine/threonine residues within a sequence-specific context and have also been proposed to be important for the assembly of multi-protein signaling complexes (8).The genes/cassettes encoding each phosphotyrosine- and phosphoserine/threonine-binding protein/module arose as a separate evolutionary event, and the DNA encoding these modules has been subject to frequent duplication and shuffling. For example, the 14-3-3 family of proteins is ubiquitously expressed in mammalian tissues and is composed of seven different isoforms, each encoded by a separate gene (6). In addition, duplication and shuffling of SH2, PTB, WW, FHA, PBD, and BCRT cassettes has led to their wide distribution among signaling proteins. Yet, despite the frequent duplication and shuffling of the DNA encoding these domains throughout evolution, proteins that contain both a phosphotyrosine-binding cassette (e.g. SH2 or PTB) and a phosphoserine/threonine-binding cassette (e.g. 14-3-3, WW, FHA, PBD, and BCRT) have not been identified. This is perhaps surprising given the highly integrated nature of phosphotyrosine and phosphoserine/threonine signaling and would suggest that alternative strategies to regulate integration are at play.We show here that 14-3-3ζ is tyrosine-phosphorylated, enabling it to interact with Shc and provide a scaffold for the assembly of signaling complexes via both phosphoserine/threonine and phosphotyrosine residues. Our results show that Tyr¹⁷⁹ of 14-3-3ζ directly binds to the SH2 domain of Shc and that this interaction is critical for the assembly of a phosphatidylinositol (PI) 3-kinase signaling complex in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation. Moreover, we show that Tyr¹⁷⁹ of 14-3-3ζ is necessary and sufficient for the ability of GM-CSF to regulate PI 3-kinase and cell survival in the CTL-EN line. Furthermore, reconstitution of primary mast cells derived from 14-3-3ζ^-/- mice with wild type (wt) or mutant 14-3-3ζ demonstrated an important role for Tyr¹⁷⁹ in cytokine-mediated Akt phosphorylation and cell survival. These multivalent 14-3-3:Shc scaffolds provide a novel mechanism by which phosphoserine/threonine and phosphotyrosine pathways can be integrated for the regulation of specific cellular responses.