The binding selectivity of the C-terminal SH3 domain of Grb2, but not its folding pathway, is dictated by its contiguous SH2 domain.

The adaptor protein Grb2, or growth factor receptor-bound protein 2, possesses a pivotal role in the transmission of fundamental molecular signals in the cell. Despite lacking enzymatic activity, Grb2 functions as a dynamic assembly platform, orchestrating intracellular signals through its modular structure. This study delves into the energetic communication of Grb2 domains, focusing on the folding and binding properties of the C-SH3 domain linked to its neighboring SH2 domain. Surprisingly, while the folding and stability of C-SH3 remain robust and unaffected by SH2 presence, significant differences emerge in the binding properties when considered within the tandem context compared with isolated C-SH3. Through a double mutant cycle analysis, we highlighted a subset of residues, located at the interface with the SH2 domain and far from the binding site, finely regulating the binding of a peptide mimicking a physiological ligand of the C-SH3 domain. Our results have mechanistic implications about the mechanisms of specificity of the C-SH3 domain, indicating that the presence of the SH2 domain optimizes binding to its physiological target, and emphasizing the general importance of considering supramodular multidomain protein structures to understand the functional intricacies of protein-protein interaction domains.

Structural basis of binding the unique N-terminal domain of microtubule-associated protein 2c to proteins regulating kinases of signaling pathways.

Isoforms of microtubule-associated protein 2 (MAP2) differ from their homolog Tau in the sequence and interactions of the N-terminal region. Binding of the N-terminal region of MAP2c (N-MAP2c) to the dimerization/docking domains of the regulatory subunit RIIα of cAMP-dependent protein kinase (RIIDD2) and to the Src-homology domain 2 (SH2) of growth factor receptor-bound protein 2 (Grb2) have been described long time ago. However, the structural features of the complexes remained unknown due to the disordered nature of MAP2. Here, we provide structural description of the complexes. We have solved solution structure of N-MAP2c in complex with RIIDD2, confirming formation of an amphiphilic α-helix of MAP2c upon binding, defining orientation of the α-helix in the complex and showing that its binding register differs from previous predictions. Using chemical shift mapping, we characterized the binding interface of SH2-Grb2 and rat MAP2c phosphorylated by the tyrosine kinase Fyn in their complex and proposed a model explaining differences between SH2-Grb2 complexes with rat MAP2c and phosphopeptides with a Grb2-specific sequence. The results provide the structural basis of a potential role of MAP2 in regulating cAMP-dependent phosphorylation cascade via interactions with RIIDD2 and Ras signaling pathway via interactions with SH2-Grb2.

Structure and Dynamics of Drk-SH2 Domain and Its Site-Specific Interaction with Sev Receptor Tyrosine Kinase.

The Drosophila downstream receptor kinase (Drk), a homologue of human GRB2, participates in the signal transduction from the extracellular to the intracellular environment. Drk receives signals through the interaction of its Src homology 2 (SH2) domain with the phosphorylated tyrosine residue in the receptor tyrosine kinases (RTKs). Here, we present the solution NMR structure of the SH2 domain of Drk (Drk-SH2), which was determined in the presence of a phosphotyrosine (pY)-containing peptide derived from a receptor tyrosine kinase, Sevenless (Sev). The solution structure of Drk-SH2 possess a common SH2 domain architecture, consisting of three ß strands imposed between two α helices. Additionally, we interpret the site-specific interactions of the Drk-SH2 domain with the pY-containing peptide through NMR titration experiments. The dynamics of Drk-SH2 were also analysed through NMR-relaxation experiments as well as the molecular dynamic simulation. The docking simulations of the pY-containing peptide onto the protein surface of Drk-SH2 provided the orientation of the peptide, which showed a good agreement with the analysis of the SH2 domain of GRB2.

Targeting the Grb2 cSH3 domain: Design, synthesis and biological evaluation of the first series of modulators.

Growth factor receptor bound protein 2 (Grb2) is an adaptor protein featured by a nSH3-SH2-cSH3 domains. Grb2 finely regulates important cellular pathways such as growth, proliferation and metabolism and a minor lapse of this tight control may totally change the entire pathway to the oncogenic. Indeed, Grb2 is found overexpressed in many tumours type. Consequently, Grb2 is an attractive therapeutic target for the development of new anticancer drug. Herein, we reported the synthesis and the biological evaluation of a series of Grb2 inhibitors, developed starting from a hit-compound already reported by this research unit. The newly synthesized compounds were evaluated by kinetic binding experiments, and the most promising derivatives were assayed in a short panel of cancer cells. Five of the newly synthesized derivatives proved to be able to bind the targeted protein with valuable inhibitory concentration in one-digit micromolar concentration. The most active compound of this series, derivative 12, showed an inhibitory concentration of about 6 µM for glioblastoma and ovarian cancer cells, and an IC50 of 1.67 for lung cancer cell. For derivative 12, the metabolic stability and the ROS production was also evaluated. The biological data together with the docking studies led to rationalize an early structure activity relationship.

The intramolecular allostery of GRB2 governing its interaction with SOS1 is modulated by phosphotyrosine ligands.

Growth factor receptor-bound protein 2 (GRB2) is a trivalent adaptor protein and a key element in signal transduction. It interacts via its flanking nSH3 and cSH3 domains with the proline-rich domain (PRD) of the RAS activator SOS1 and via its central SH2 domain with phosphorylated tyrosine residues of receptor tyrosine kinases (RTKs; e.g. HER2). The elucidation of structural organization and mechanistic insights into GRB2 interactions, however, remain challenging due to their inherent flexibility. This study represents an important advance in our mechanistic understanding of how GRB2 links RTKs to SOS1. Accordingly, it can be proposed that (1) HER2 pYP-bound SH2 potentiates GRB2 SH3 domain interactions with SOS1 (an allosteric mechanism); (2) the SH2 domain blocks cSH3, enabling nSH3 to bind SOS1 first before cSH3 follows (an avidity-based mechanism); and (3) the allosteric behavior of cSH3 to other domains appears to be unidirectional, although there is an allosteric effect between the SH2 and SH3 domains.

Effect of the glycine-rich domain in GAREM2 on its unique subcellular localization upon EGF stimulation.

BACKGROUND: In mammals, there are two subtypes of Grb2-associated regulator of Erk/MAPK (GAREM), an adaptor protein that functions downstream of the cell growth factor receptor. GAREM1 is ubiquitously expressed, whereas GAREM2 is mainly expressed in the brain. However, the precise mechanism of the translocation of each GAREM subtype in growth factor-stimulated cells is still unclear. METHODS: In this study, immunofluorescence staining with specific antibodies against each GAREM subtype and time-lapse analysis using GFP fusion proteins were used to analyze the subcellular localization of each GAREM subtype in a cell growth stimulus-dependent manner. We also biochemically analyzed the correlation between its subcellular localization and tyrosine phosphorylation of GAREM2. RESULTS: We found that endogenously and exogenously expressed GAREM2 specifically aggregated and formed granules in NGF-stimulated PC-12 cells and in EGF-stimulated COS-7 cells. Based on the observed subcellular localizations of chimeric GAREM1 and GAREM2 proteins, a glycine-rich region, which is present only in GAREM2, is required for the observed granule formation. This region also regulates the degree of EGF-stimulation-dependent tyrosine phosphorylation of GAREM2. CONCLUSIONS: Our results, showing that aggregation of GAREM2 in response to EGF stimulation is dependent on a glycine-rich region, suggest that GAREM2 aggregation may be involved in neurodegenerative diseases.

High-Affinity Interactions of the nSH3/cSH3 Domains of Grb2 with the C-Terminal Proline-Rich Domain of SOS1.

Grb2 is an adaptor protein that recruits Ras-specific guanine nucleotide exchange factor, Son of Sevenless 1 (SOS1), to the plasma membrane. SOS1 exchanges GDP by GTP, activating Ras. Grb2 consists of an SH2 domain flanked by N- and C-terminal SH3 domains (nSH3/cSH3). Grb2 nSH3/cSH3 domains have strong binding affinity for the SOS1 proline-rich (PR) domain that mediates the Grb2-SOS1 interaction. The nSH3/cSH3 domains have distinct preferred binding motifs: PxxPxR for nSH3 and PxxxRxxKP for cSH3 (x represents any natural amino acid). Several nSH3-binding motifs have been identified in the SOS1 PR domain but none specific for cSH3 binding. Even though both nSH3 and cSH3 exhibit the strongest binding to the SOS1 peptide PVPPPVPPRRRP, this mutually exclusive binding combined with other potential nSH3/cSH3 binding regions in SOS1 makes understanding the Grb2-SOS1 interaction challenging. To identify the SOS1-cSH3 binding sites, we selected seven potential binding segments in SOS1. The synthesized peptides were tested for their binding to nSH3/cSH3. Our NMR data reveal that the PKLPPKTYKREH peptide has strong binding affinity for cSH3, but very weak for nSH3. The binding specificity suggests that the most likely Grb2-SOS1 binding mode is through nSH3-PVPPPVPPRRRP and cSH3-PKLPPKTYKREH interactions, which is supported by replica-exchange simulations for the Grb2-SOS1 complex models. We propose that nSH3/cSH3 binding peptides, which effectively interrupt Grb2-SOS1 association, can serve as tumor suppressors. The Grb2-SOS1 mechanism outlined here offers new venues for future therapeutic strategies for upstream mutations in cancer, such as in EGFR.

Characterization of GAB3 and its association with NNV resistance in the Asian seabass.

Understanding the functions of genes related to disease resistance and identifying polymorphisms in these genes are essential in molecular breeding for disease resistance. Viral nervous necrosis (VNN) is one of the major diseases in the Asian seabass, Lates calcarifer. Our previous works on QTL mapping, GWAS and cell-line transcriptome analysis of the Asian seabass after NNV challenge revealed that the gene GAB3 might be a candidate gene for VNN resistance. In this study, we cloned and characterized GAB3, and identified SNPs in the gene of the Asian seabass. The cDNA of the gene was 2165 bp, containing an ORF of 1674 bp encoding 557 amino acids. The gene consisted of 10 exons and nine introns. It was ubiquitously expressed in normal fish. An analysis of the association between two SNPs in the second intron and NNV resistance in 1035 fish descended from 43 families revealed that the two SNPs were significantly associated with VNN resistance. After NNV infection, the expression of GAB3 was significantly increased in the brain, spleen, muscle and gut, and was suppressed in the liver. The GAB3 protein was localized in the nucleus. Overexpression of GAB3 with specific GAB3-pcDNA was positively correlated to increased viral RNA and titer in NNV-infected Asian seabass cells. Our study provides new evidence to support that GAB3 may be an important gene related to NNV resistance. In addition, the SNPs provide DNA markers for the selection of candidate genes resistance to NNV at the juvenile stage of Asian seabass.

SOS1 interacts with Grb2 through regions that induce closed nSH3 conformations.

Grb2 is an adaptor protein connecting the epidermal growth factor receptor and the downstream Son of sevenless 1 (SOS1), a Ras-specific guanine nucleotide exchange factor (RasGEF), which exchanges GDP by GTP. Grb2 contains three SH domains: N-terminal SH3 (nSH3), SH2, and C-terminal SH3 (cSH3). The C-terminal proline-rich (PR) domain of SOS1 regulates nSH3 open/closed conformations. Earlier, several nSH3 binding motifs were identified in the PR domain. More recently, we characterized by nuclear magnetic resonance and replica exchange simulations possible cSH3 binding regions. Among them, we discovered a cSH3-specific binding region. However, how PR binding at these sites regulates the nSH3/cSH3 conformation has been unclear. Here, we explore the nSH3/cSH3 interaction with linked and truncated PR segments using molecular dynamics simulations. Our 248 µs simulations include 620 distinct trajectories, each 400 ns. We construct the effective free energy landscape to validate the nSH3/cSH3 binding sites. The nSH3/cSH3-SOS1 peptide complex models indicate that strong peptide binders attract the flexible nSH3 n-Src loop, inducing a closed conformation of nSH3; by contrast, the cSH3 conformation remains unchanged. Inhibitors that disrupt the Ras-SOS1 interaction have been designed; the conformational details uncovered here may assist in the design of polypeptides inhibiting Grb2-SOS1 interaction, thus SOS1 recruitment to the membrane where Ras resides.

High-Throughput Phosphotyrosine Protein Complexes Screening by Photoaffinity-Engineered Protein Scaffold-Based Forward-Phase Protein Array.

Low-abundance phosphotyrosine (pTyr)-mediated signaling protein complexes play critical roles in cancer signaling. The precise and comprehensive profiling of these pTyr-mediated protein complexes remains challenging because of their dynamic nature and weak binding affinity. Taking advantage of the SH2 domains modified with trifunctional chemical probes and genetic mutations (termed Photo-pTyr-scaffold), we developed a Photo-pTyr-scaffold-based forward-phase protein array that can be used to specifically capture complexes by developing an engineered SH2 domain, photoaffinity cross-linking, and antibody-based measuring weak pTyr-mediated protein complexes from complex biological samples in a 96-well microplate format. This platform demonstrated good precision for quantitation (R2 = 0.99) and high sensitivity by which only 5 µg of whole cell lysates is needed. We successfully applied the technology for profiling the dynamic EGF-stimulation-dependent EGFR signaling protein complexes across four different time courses (i.e., 0, 2, 5, 10, and 30 min) in a high-throughput manner. We further evaluated the modulation of EGFR-GRB2-SHC1 protein complexes by FDA-approved EGFR kinase inhibitor erlotinib, demonstrating the feasibility of this approach for high-throughput drug screening. The Photo-pTyr-scaffold-based forward-phase protein array could be generically applicable for exploring the dynamic pTyr signaling complexes in various biological systems and screening for related drugs in a high-throughput manner.

Cell penetrating peptides to dissect host-pathogen protein-protein interactions in Theileria-transformed leukocytes.

One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way to explore their contribution to a particular cellular phenotype in a given disease context. Cell-penetrating peptides can be synthetically constrained through various chemical modifications that stabilize a given structural fold with the potential to improve competitive binding to specific targets. Theileria-transformed leukocytes display high PKA activity, but PKA is an enzyme that plays key roles in multiple cellular processes; consequently genetic ablation of kinase activity gives rise to a myriad of confounding phenotypes. By contrast, ablation of a specific kinase-substrate interaction has the potential to give more refined information and we illustrate this here by describing how surgically ablating PKA interactions with BAD gives precise information on the type of glycolysis performed by Theileria-transformed leukocytes. In addition, we provide two other examples of how ablating specific protein-protein interactions in Theileria-infected leukocytes leads to precise phenotypes and argue that constrained penetrating peptides have great therapeutic potential to combat infectious diseases in general.

Protein-ligand binding enthalpies from near-millisecond simulations: Analysis of a preorganization paradox.

Calorimetric studies of protein-ligand binding sometimes yield thermodynamic data that are difficult to understand. Today, molecular simulations can be used to seek insight into such calorimetric puzzles, and, when simulations and experiments diverge, the results can usefully motivate further improvements in computational methods. Here, we apply near-millisecond duration simulations to estimate the relative binding enthalpies of four peptidic ligands with the Grb2 SH2 domain. The ligands fall into matched pairs, where one member of each pair has an added bond that preorganizes the ligand for binding and thus may be expected to favor binding entropically, due to a smaller loss in configurational entropy. Calorimetric studies have shown that the constrained ligands do in fact bind the SH2 domain more tightly than the flexible ones, but, paradoxically, the improvement in affinity for the constrained ligands is enthalpic, rather than entropic. The present enthalpy calculations yield the opposite trend, as they suggest that the flexible ligands bind more exothermically. Additionally, the small relative binding enthalpies are found to be balances of large differences in the energies of structural components such as ligand and the binding site residues. As a consequence, the deviations from experiment in the relative binding enthalpies represent small differences between these large numbers and hence may be particularly susceptible to error, due, for example, to approximations in the force field. We also computed first-order estimates of changes in configurational entropy on binding. These too are, arguably, paradoxical, as they tend to favor binding of the flexible ligands. The paradox is explained in part by the fact that the more rigid constrained ligands reduce the entropy of binding site residues more than their flexible analogs do, at least in the simulations. This result offers a rather general counterargument to the expectation that preorganized ligands should be associated with more favorable binding entropies, other things being equal.

Dynamic Scaling Analysis of Molecular Motion within the LAT:Grb2:SOS Protein Network on Membranes.

Biochemical signaling pathways often involve proteins with multiple, modular interaction domains. Signaling activates binding sites, such as by tyrosine phosphorylation, which enables protein recruitment and growth of networked protein assemblies. Although widely observed, the physical properties of the assemblies, as well as the mechanisms by which they function, remain largely unknown. Here we examine molecular mobility within LAT:Grb2:SOS assemblies on supported membranes by single-molecule tracking. Trajectory analysis reveals a discrete temporal transition to subdiffusive motion below a characteristic timescale, indicating that the LAT:Grb2:SOS assembly has the dynamical structure of a loosely entangled polymer. Such dynamical analysis is also applicable in living cells, where it offers another dimension on the characteristics of cellular signaling assemblies.

Quantifying the Interaction between EGFR Dimers and Grb2 in Live Cells.

Adaptor proteins are a class of cytoplasmic proteins that bind to phosphorylated residues in receptor tyrosine kinases and trigger signaling cascades that control critically important cellular processes, such as cell survival, growth, differentiation, and motility. Here, we seek to characterize the interaction between epidermal growth factor receptor (EGFR) and the cytoplasmic adaptor protein growth factor receptor-bound protein 2 (Grb2) in a cellular context. To do so, we explore the utility of a highly biologically relevant model system, mammalian cells under reversible osmotic stress, and a recently introduced Förster resonance energy transfer microscopy method, fully quantified spectral imaging. We present a method that allows us to quantify the stoichiometry and the association constant of the EGFR-Grb2 binding interaction in the plasma membrane, in the presence and absence of activating ligand. The method that we introduce can have broad utility in membrane protein research, as it can be applied to different membrane protein-cytoplasmic protein pairs.

Genetically Encoded 2-Aryl-5-carboxytetrazoles for Site-Selective Protein Photo-Cross-Linking.

The genetically encoded photo-cross-linkers promise to offer a temporally controlled tool to map transient and dynamic protein-protein interaction complexes in living cells. Here we report the synthesis of a panel of 2-aryl-5-carboxytetrazole-lysine analogs (ACTKs) and their site-specific incorporation into proteins via amber codon suppression in Escherichia coli and mammalian cells. Among five ACTKs investigated, N-methylpyrroletetrazole-lysine (mPyTK) was found to give robust and site-selective photo-cross-linking reactivity in E. coli when placed at an appropriate site at the protein interaction interface. A comparison study indicated that mPyTK exhibits higher photo-cross-linking efficiency than a diazirine-based photo-cross-linker, AbK, when placed at the same location of the interaction interface in vitro. When mPyTK was introduced into the adapter protein Grb2, it enabled the photocapture of EGFR in a stimulus-dependent manner. The design of mPyTK along with the identification of its cognate aminoacyl-tRNA synthetase makes it possible to map transient protein-protein interactions and their interfaces in living cells.

Synthesis of Grb2 SH2 Domain Proteins for Mirror-Image Screening Systems.

Grb2 is an adaptor protein that mediates cellular signal transduction. Grb2 contains an SH2 domain that interacts with phosphotyrosine-containing sequences in EGFR and other signaling molecules, and it is a promising molecular target for anticancer agents. To identify novel inhibitors of the Grb2 SH2 domain from natural products and their mirror-image isomers, screening systems using both enantiomers of a synthetic Grb2 SH2 domain protein were established. A pair of synthetic procedures for the proteins were investigated: one employed a single native chemical ligation (NCL) of two segment peptides, and the other used the N-to-C-directed NCL of three segment peptides for easier preparation. Labeling at the N-terminus or the Ala115 residue of the Grb2 SH2 domain provided functional probes to detect binding to a phosphotyrosine-containing peptide. The resulting synthetic-protein-based probes were applied to bioassays, including chemical array analysis and enzyme-linked immunosorbent assays.

Structural basis of focal adhesion targeting domain-mediated signaling in cardiac hypertrophy.

The focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK) exists in monomeric closed (c) or arm exchanged (ae) dimeric state. FAT interaction with Grb2 necessitates an intermediate open (o) state that interacts with Grb2 and activates signaling pathways leading to pathological cardiac hypertrophy. Targeted molecular dynamics (TMD) simulation was carried out in order to capture the structure of the intermediate formed by opening of Helix1 (H1) from monomeric cFAT leading to the formation of monomeric aeFAT. During TMD, H1 separated from the four helices bundle of cFAT, completely unfolded and performed a full turn before folding back to a helix inclined at an acute angle to the helical bundle in aeFAT. The entire transition can be described in six distinct intermediate structural stages. The most significant correlation of H1 motion was observed with Loop3 (L3) and is the likely reason for the complete disruption of the FAT interaction with paxillin during the transition. High-affinity analogs of the paxillin LD4 region can be a promising strategy to drive the equilibrium towards cFAT, thus antagonizing FAT-Grb2 association. During transition, the overall shift in orientation of all the four helices rejects paxillin binding and approves Grb2 association. Exposure and ß-turn conformation of the YENV motif (residues 925-928) in oFAT-facilitated phosphorylation and Grb2 binding. Docking, MD simulation and conservation analysis of oFAT-Grb2 complex provided insight into the structural determinants of binding and specificity. Our work provides a structural basis for pharmacological modulation of dynamic conformational changes and interactions of FAT.

Comparing pharmacophore models derived from crystallography and NMR ensembles.

NMR and X-ray crystallography are the two most widely used methods for determining protein structures. Our previous study examining NMR versus X-Ray sources of protein conformations showed improved performance with NMR structures when used in our Multiple Protein Structures (MPS) method for receptor-based pharmacophores (Damm, Carlson, J Am Chem Soc 129:8225-8235, 2007). However, that work was based on a single test case, HIV-1 protease, because of the rich data available for that system. New data for more systems are available now, which calls for further examination of the effect of different sources of protein conformations. The MPS technique was applied to Growth factor receptor bound protein 2 (Grb2), Src SH2 homology domain (Src-SH2), FK506-binding protein 1A (FKBP12), and Peroxisome proliferator-activated receptor-γ (PPAR-γ). Pharmacophore models from both crystal and NMR ensembles were able to discriminate between high-affinity, low-affinity, and decoy molecules. As we found in our original study, NMR models showed optimal performance when all elements were used. The crystal models had more pharmacophore elements compared to their NMR counterparts. The crystal-based models exhibited optimum performance only when pharmacophore elements were dropped. This supports our assertion that the higher flexibility in NMR ensembles helps focus the models on the most essential interactions with the protein. Our studies suggest that the "extra" pharmacophore elements seen at the periphery in X-ray models arise as a result of decreased protein flexibility and make very little contribution to model performance.

Regions outside of conserved PxxPxR motifs drive the high affinity interaction of GRB2 with SH3 domain ligands.

SH3 domains are evolutionarily conserved protein interaction domains that control nearly all cellular processes in eukaryotes. The current model is that most SH3 domains bind discreet PxxPxR motifs with weak affinity and relatively low selectivity. However, the interactions of full-length SH3 domain-containing proteins with ligands are highly specific and have much stronger affinity. This suggests that regions outside of PxxPxR motifs drive these interactions. In this study, we observed that PxxPxR motifs were required for the binding of the adaptor protein GRB2 to short peptides from its ligand SOS1. Surprisingly, PxxPxR motifs from the proline rich region of SOS1 or CBL were neither necessary nor sufficient for the in vitro or in vivo interaction with full-length GRB2. Together, our findings show that regions outside of the consensus PxxPxR sites drive the high affinity association of GRB2 with SH3 domain ligands, suggesting that the binding mechanism for this and other SH3 domain interactions may be more complex than originally thought.

Conformation-Dependent Human p52Shc Phosphorylation by Human c-Src.

Phosphorylation of the human p52Shc adaptor protein is a key determinant in modulating signaling complex assembly in response to tyrosine kinase signaling cascade activation. The underlying mechanisms that govern p52Shc phosphorylation status are unknown. In this study, p52Shc phosphorylation by human c-Src was investigated using purified proteins to define mechanisms that affect the p52Shc phosphorylation state. We conducted biophysical characterizations of both human p52Shc and human c-Src in solution as well as membrane-mimetic environments using the acidic lipid phosphatidylinositol 4-phosphate or a novel amphipathic detergent (2,2-dihexylpropane-1,3-bis-ß-D-glucopyranoside). We then identified p52Shc phosphorylation sites under various solution conditions, and the amount of phosphorylation at each identified site was quantified using mass spectrometry. These data demonstrate that the p52Shc phosphorylation level is altered by the solution environment without affecting the fraction of active c-Src. Mass spectrometry analysis of phosphorylated p52Shc implies functional linkage among phosphorylation sites. This linkage may drive preferential coupling to protein binding partners during signaling complex formation, such as during initial binding interactions with the Grb2 adaptor protein leading to activation of the Ras/MAPK signaling cascade. Remarkably, tyrosine residues involved in Grb2 binding were heavily phosphorylated in a membrane-mimetic environment. The increased phosphorylation level in Grb2 binding residues was also correlated with a decrease in the thermal stability of purified human p52Shc. A schematic for the phosphorylation-dependent interaction between p52Shc and Grb2 is proposed. The results of this study suggest another possible therapeutic strategy for altering protein phosphorylation to regulate signaling cascade activation.