Src Homology 3 Binding Research Articles

An SH3 binding motif within the nucleocapsid protein of porcine reproductive and respiratory syndrome virus interacts with the host cellular signaling proteins STAMI, TXK, Fyn, Hck, and cortactin

Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important global swine disease, and has a complicated virus–host immunomodulation that often leads to a weak Th2 immune response and viral persistence. In this study, we identified a Src homology 3 (SH3) binding motif, PxxPxxP, that is conserved within the N protein of PRRSV strains. Subsequently, we identified five host cellular proteins [signal transducing adaptor molecule (STAM)I, TXK tyrosine kinase (TXK), protein tyrosine kinase fyn (Fyn), hematopoietic cell kinase (Hck), and cortactin] that interact with this SH3 motif. We demonstrated that binding of SH3 proteins with PRRSV N protein depends on at least one intact PxxP motif as disruption of P53 within the motif significantly reduced interaction of each of the 5 proteins. The first PxxP motif appears to be more important for STAMI–N protein interactions whereas the second PxxP motif was more important for Hck interaction. Both STAMI and Hck interactions with PRRSV N protein required an unhindered C-terminal domain as the interaction was only observed with STAMI and Hck proteins with N-terminal but not C-terminal fluorescent tags. We showed that the P56 residue within the SH3 motif is critical for virus lifecycle as mutation resulted in a loss of virus infectivity, however the P50 and P53 mutations did not abolish virus infectivity suggesting that these highly conserved proline residues within the SH3 motif may provide a selective growth advantage through interactions with the host rather than a vital functional element. These results have important implications in understanding PRRSV–host interactions.

Preferred SH3 Domain Partners of ADAM Metalloproteases Include Shared and ADAM-Specific SH3 Interactions

A disintegrin and metalloproteinases (ADAMs) constitute a protein family essential for extracellular signaling and regulation of cell adhesion. Catalytic activity of ADAMs and their predicted potential for Src-homology 3 (SH3) domain binding show a strong correlation. Here we present a comprehensive characterization of SH3 binding capacity and preferences of the catalytically active ADAMs 8, 9, 10, 12, 15, 17, and 19. Our results revealed several novel interactions, and also confirmed many previously reported ones. Many of the identified SH3 interaction partners were shared by several ADAMs, whereas some were ADAM-specific. Most of the ADAM-interacting SH3 proteins were adapter proteins or kinases, typically associated with sorting and endocytosis. Novel SH3 interactions revealed in this study include TOCA1 and CIP4 as preferred partners of ADAM8, and RIMBP1 as a partner of ADAM19. Our results suggest that common as well as distinct mechanisms are involved in regulation and execution of ADAM signaling, and provide a useful framework for addressing the pathways that connect ADAMs to normal and aberrant cell behavior.

The SH2 domain regulates c-Abl kinase activation by a cyclin-like mechanism and remodulation of the hinge motion.

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors.

Probing SH2-domains using Inhibitor Affinity Purification (IAP).

BackgroundMany human diseases are correlated with the dysregulation of signal transduction processes. One of the most important protein interaction domains in the context of signal transduction is the Src homology 2 (SH2) domain that binds phosphotyrosine residues. Hence, appropriate methods for the investigation of SH2 proteins are indispensable in diagnostics and medicinal chemistry. Therefore, an affinity resin for the enrichment of all SH2 proteins in one experiment would be desirable. However, current methods are unable to address all SH2 proteins simultaneously with a single compound or a small array of compounds.ResultsIn order to overcome these limitations for the investigation of this particular protein family in future experiments, a dipeptide-derived probe has been designed, synthesized and evaluated. This probe successfully enriched 22 SH2 proteins from mixed cell lysates which contained 50 SH2 proteins. Further characterization of the SH2 binding properties of the probe using depletion and competition experiments indicated its ability to enrich complexes consisting of SH2 domain bearing regulatory PI3K subunits and catalytic phosphoinositide 3-kinase (PI3K) subunits that have no SH2 domain.ConclusionThe results make this probe a promising starting point for the development of a mixed affinity resin with complete SH2 protein coverage. Moreover, the additional findings render it a valuable tool for the evaluation of PI3K complex interrupting inhibitors.

Novel nucleotide mutation leading to a recurrent amino acid alteration in SH3BP2 in a patient with cherubism

Cherubism is a rare genetic disorder characterized by progressive facial deformity caused by non-neoplastic bone lesions in the mandible and/or the maxilla. Src homology-3 binding protein 2 gene (SH3BP2) has been found to be the responsible gene, with alterations in six amino acids noted in patients with this condition. Recently, mutations in this domain have been found to cause stabilization of SH3BP2 by uncoupling with tankyrase. In this study, we identified a new 2-bp mutation that led to a recurrent amino acid change in a sporadic case of cherubism. Our findings indicate that it is important to understand the pattern of progress in typical cherubism.

In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity

Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors.

Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR) Domain-containing Proteins Leads to GTP-dependent Membrane Scission*

Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.

SH3 interactome conserves general function over specific form.

Src homology 3 (SH3) domains bind peptides to mediate protein-protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.

Transient Structure and SH3 Interaction Sites in an Intrinsically Disordered Fragment of the Hepatitis C Virus Protein NS5A

Understanding the molecular mechanisms involved in virus replication and particle assembly is of primary fundamental and biomedical importance. Intrinsic conformational disorder plays a prominent role in viral proteins and their interaction with other viral and host cell proteins via transiently populated structural elements. Here, we report on the results of an investigation of an intrinsically disordered 188-residue fragment of the hepatitis C virus non-structural protein 5A (NS5A), which contains a classical poly-proline Src homology 3 (SH3) binding motif, using sensitivity- and resolution-optimized multidimensional NMR methods, complemented by small-angle X-ray scattering data. Our study provides detailed atomic-resolution information on transient local and long-range structure, as well as fast time scale dynamics in this NS5A fragment. In addition, we could characterize two distinct interaction modes with the SH3 domain of Bin1 (bridging integrator protein 1), a pro-apoptotic tumor suppressor. Despite being largely disordered, the protein contains three regions that transiently adopt α-helical structures, partly stabilized by long-range tertiary interactions. Two of these transient α-helices form a noncanonical SH3-binding motif, which allows low-affinity SH3 binding. Our results contribute to a better understanding of the role of the NS5A protein during hepatitis C virus infection. The present work also highlights the power of NMR spectroscopy to characterize multiple binding events including short-lived transient interactions between globular and highly disordered proteins.

Analysis of Conserved Structural Features of Selenoprotein K

Selenium plays important roles in human health and these roles may be exerted through its presence in selenoproteins. Among the 25 selenoproteins in human is selenoprotein K (SelK) whose exact function is still unclear. Here, we investigated the conserved structural features of SelK using bioinformatics as an approach to understand the function and regulation of SelK in mammals. Our data showed that mammalian SelK sequences have more than 90% identity relative to the human SelK. Using RT-PCR assays, we found that SelK is expressed in various rat organs including liver, testes, brain, skeletal muscles, kidneys, spleen, lungs and heart. The selenocysteine (Sec) residue in animal SelK is located three amino acid residues from the C-terminal end of the protein. This Sec residue (U) occurs in a conserved GGUGR sequence in animal SelK. We also found SelK Cys homologues occurring in plants that contain cysteine residues in a conserved CGSCCG sequence at the C-terminal end of the protein. Sequences of SelK in various mammals were found to have disordered region that contains conserved sequences of Src homology 3 (SH3) binding motifs, preceded by a potential serine phosphorylation site at position 58. These SH3 binding motifs may be involved in binding of SelK to protein partners containing SH3 binding domains. In conclusion, the conserved GGUGR sequence in animal SelK may be involved in the role of SelK in redox homeostasis. SelK may be involved in protein-protein interactions through its SH3 binding motifs in mammals and this role may be regulated by phosphorylation of the serine residue at position 58. Keywords : Bioinformatics, protein - protein interaction ; SECIS element; SelK; SelK Cys homologues; SH3 binding, bioinformatics

SH3 Domain-Mediated Recruitment of Host Cell Amphiphysins by Alphavirus nsP3 Promotes Viral RNA Replication

Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication.

Interactions of the HIV/SIV Pathogenicity Factor Nef with SH3 Domain-Containing Host Cell Proteins

A bewildering number of host cell proteins associated with Nef can be found in the literature and in the public protein interaction databases. However, only in a few of these cases, including binding of Nef to certain Src homology-3 (SH3) domain proteins, is the interaction understood in any molecular detail or even known to be direct. Indeed, SH3 binding capacity by Nef is required for many of the other protein interactions to take place, suggesting that a large proportion of the latter is indirectly coupled to Nef via an SH3 protein. Accordingly, the proline-rich SH3 binding site, the "PxxP motif", is one of the key functional determinants of Nef. It is highly conserved among the lentiviral Nef proteins, and mutations disrupting it abrogate the majority of the known effects of Nef on host cell physiology. This review summarizes the current understanding as well as the outstanding gaps in our knowledge regarding the relevant SH3 protein partners and SH3-dependent cellular functions of Nef. The roles of these interactions in the pathogenesis of AIDS and their potential as targets for antiviral drug development are also discussed.

Sprouty-related Ena/Vasodilator-stimulated Phosphoprotein Homology 1-Domain-containing Protein (SPRED1), a Tyrosine-Protein Phosphatase Non-receptor Type 11 (SHP2) Substrate in the Ras/Extracellular Signal-regulated Kinase (ERK) Pathway

SHP2 is a tyrosine phosphatase involved in the activation of the Ras/ERK signaling pathway downstream of a number of receptor tyrosine kinases. One of the proposed mechanisms involving SHP2 in this context is to dephosphorylate and inactivate inhibitors of the Ras/ERK pathway. Two protein families bearing a unique, common domain, Sprouty and SPRED proteins, are possible candidates because they have been reported to inhibit the Ras/ERK pathway upon FGF activation. We tested whether any of these proteins are likely substrates of SHP2. Our findings indicate that Sprouty2 binds to the C-terminal tail of SHP2, which is an unlikely substrate binding site, whereas SPRED proteins bind to the tyrosine phosphatase domain that is known to be the binding site for its substrates. Overexpressed SHP2 was able to dephosphorylate SPREDs but not Sprouty2. Finally, we found two tyrosine residues on SPRED1 that are required, when phosphorylated, to inhibit Ras/ERK activation and identified Tyr-420 as a specific dephosphorylation target of SHP2. The evidence obtained indicates that SPRED1 is a likely substrate of SHP2, whose tyrosine dephosphorylation is required to attenuate the inhibitory action of SPRED1 in the Ras/ERK pathway.

The Structure of the Plakin Domain of Plectin Reveals a Non-canonical SH3 Domain Interacting with Its Fourth Spectrin Repeat

Plectin belongs to the plakin family of cytoskeletal crosslinkers, which is part of the spectrin superfamily. Plakins contain an N-terminal conserved region, the plakin domain, which is formed by an array of spectrin repeats (SR) and a Src-homology 3 (SH3), and harbors binding sites for junctional proteins. We have combined x-ray crystallography and small angle x-ray scattering (SAXS) to elucidate the structure of the central region of the plakin domain of plectin, which corresponds to the SR3, SR4, SR5, and SH3 domains. The crystal structures of the SR3-SR4 and SR4-SR5-SH3 fragments were determined to 2.2 and 2.95 Å resolution, respectively. The SH3 of plectin presents major alterations as compared with canonical Pro-rich binding SH3 domains, suggesting that plectin does not recognize Pro-rich motifs. In addition, the SH3 binding site is partially occluded by an intramolecular contact with the SR4. Residues of this pseudo-binding site and the SR4/SH3 interface are conserved within the plakin family, suggesting that the structure of this part of the plectin molecule is similar to that of other plakins. We have created a model for the SR3-SR4-SR5-SH3 region, which agrees well with SAXS data in solution. The three SRs form a semi-flexible rod that is not altered by the presence of the SH3 domain, and it is similar to those found in spectrins. The flexibility of the plakin domain, in analogy with spectrins, might contribute to the role of plakins in maintaining the stability of tissues subject to mechanical stress.

Characterization of molecular recognition of STAT3 SH2 domain inhibitors through molecular simulation

Signal transducer and activator of transcription 3 (STAT3) is an anti-cancer target protein due to its over-activation in tumor cells. The Tyr705-phosphorylated (pTyr) STAT3 binds to the pTyr-recognition site of its Src Homology 2 (SH2) domain of another STAT3 monomer to form a homo-dimer, which then causes cellular anti-apoptosis, proliferation, and tumor invasion. Recently, many STAT3 SH2 dimerization inhibitors have been discovered via both computational and experimental methods. To systematically assess their binding affinities and specificities, for eight representative inhibitors, we utilized molecular docking, molecular dynamics simulation, and ensuing energetic analysis to compare their binding characteristics. The inhibitors' binding free energies were calculated via MMPB(GB)SA, and the STAT3 SH2 binding "hot spots" were evaluated through binding energy decomposition and hydrogen bond (H-bond) distribution analysis. Several conclusions can be drawn: (1) the overall enthalpy-entropy compensation paradigm is preserved for the STAT3 SH2/ligand binding thermodynamics; (2) at one end of the binding spectrum, two compounds bind to SH2 due to their minimum entropic penalties that result from their relative rigidities and increased dynamics of SH2 upon their binding; at the other end of the binding spectrum, one compound shows a typical weak binder behavior due to its loose binding in the SH2's strongest enthalpy-contributing binding subsite; (3) hydrogen bonding seems a strong indicator to evaluate the SH2/ligand binding potency, which echoes a finding that CH/π non-classical H-bond is responsible for some pTyr peptides binding to their corresponding SH2 domains; (4) STAT3 SH2 domain possesses three binding "hot spots": pTyr705-binding pocket with polar residues and contributing the largest binding enthalpy (two-thirds); Leu706 subsite which is the most dynamic and hardest to target; a hydrophobic side pocket which is unique to STAT3 and very targetable, which may offer unique opportunity to design STAT3-specific inhibitors, particularly with fragment-based approach.

Role of the Kaposi's Sarcoma-Associated Herpesvirus K15 SH3 Binding Site in Inflammatory Signaling and B-Cell Activation

The Kaposi's sarcoma-associated herpesvirus (KSHV) contains several open reading frames (ORFs) that encode proteins capable of initiating and modulating cellular signaling pathways. Among them is ORF K15, encoding a 12-transmembrane-spanning protein with a cytoplasmic C-terminal domain. Through conserved binding motifs, such as Src homology 2 (SH2) and SH3 binding sites, K15 interacts with cellular proteins, activates the NF-kappaB, MEK/Erk, and Jun N-terminal protein kinase (JNK) pathways, and induces the expression of several inflammatory and angiogenic genes. In this study, we investigated the role of an SH3 domain binding site centered on a PPLP motif in K15. We screened libraries of cellular SH3 domains to identify signaling molecules interacting with the KSHV PPLP motif. We found its affinities for two Src kinase family members, Lyn and Hck, to exceed those of other viral proteins. While the SH2 binding motif YEEV is essential for the inflammatory response induced by KSHV K15, recruitment of Lyn and Hck to the K15 PPLP motif seems to be dispensable for this inflammatory response. However, the PPLP motif is essential for the decrease in B-cell receptor-mediated signaling induced by K15, as measured by calcium mobilization assays.

Alternative splicing of ADAM15 regulates its interactions with cellular SH3 proteins

A Disintegrin And Metalloprotease (ADAM15) is a member of the adamalysin protein family and has been associated with cancer, possibly via its role in ectodomain shedding of cadherins. Alternative mRNA splicing generates several ADAM15 isoforms containing different combinations of putative Src homology-3 (SH3) domain binding sites in their cytosolic tails. Here we present a comprehensive characterization of SH3 binding potential of different ADAM15 isoforms. Alternative use of ADAM15 exons was found to profoundly influence selection of SH3-containing cellular partner proteins, including the avid interactions with nephrocystin and sorting nexin-33 (SNX33 a.k.a. SNX30). Specifically, strong co-precipitation of nephrocystin from cell lysates was specific to ADAM15 isoforms i4, i5, and i6. These isoforms contain one or both of the two almost identical proline-rich regions encoded by exons 20 and 21, wherein the residues RxLPxxP were found to be indispensable for nephrocystin SH3 binding. Similarly, robust cellular association with SNX33 was observed only for ADAM15 isoforms containing the most carboxyterminal proline cluster lacking in isoforms i1 and i3. Thus, alternative mRNA splicing provides a versatile mechanism for regulation of intracellular protein interactions and thereby likely the cellular functions of ADAM15, which could explain the association with cancer of some but not all ADAM15 isoforms.

Molecular Dynamics Simulations Reveal that Tyr-317 Phosphorylation Reduces Shc Binding Affinity for Phosphotyrosyl Residues of Epidermal Growth Factor Receptor

The Src homology 2 (SH2) and collagen domain protein Shc plays a pivotal role in signaling via tyrosine kinase receptors, including epidermal growth factor receptor (EGFR). Shc binding to phospho-tyrosine residues on activated receptors is mediated by the SH2 and phospho-tyrosine binding (PTB) domains. Subsequent phosphorylation on Tyr-317 within the Shc linker region induces Shc interactions with Grb2-Son of Sevenless that initiate Ras-mitogen-activated protein kinase signaling. We use molecular dynamics simulations of full-length Shc to examine how Tyr-317 phosphorylation controls Shc conformation and interactions with EGFR. Our simulations reveal that Shc tyrosine phosphorylation results in a significant rearrangement of the relative position of its domains, suggesting a key conformational change. Importantly, computational estimations of binding affinities show that EGFR-derived phosphotyrosyl peptides bind with significantly more strength to unphosphorylated than to phosphorylated Shc. Our results unveil what we believe is a novel structural phenomenon, i.e., tyrosine phosphorylation of Shc within its linker region regulates the binding affinity of SH2 and PTB domains for phosphorylated Shc partners, with important implications for signaling dynamics.

Proline Isomerization Preorganizes the Itk SH2 Domain for Binding to the Itk SH3 Domain

We report here the NMR-derived structure of the binary complex formed by the interleukin-2 tyrosine kinase (Itk) Src homology 3 (SH3) and Src homology 2 (SH2) domains. The interaction is independent of both a phosphotyrosine motif and a proline-rich sequence, the classical targets of the SH2 and SH3 domains, respectively. The Itk SH3/SH2 structure reveals the molecular details of this nonclassical interaction and provides a clear picture for how the previously described prolyl cis/ trans isomerization present in the Itk SH2 domain mediates SH3 binding. The higher-affinity cis SH2 conformer is preorganized to form a hydrophobic interface with the SH3 domain. The structure also provides insight into how autophosphorylation in the Itk SH3 domain might increase the affinity of the intermolecular SH3/SH2 interaction. Finally, we can compare this Itk complex with other examples of SH3 and SH2 domains engaging their ligands in a nonclassical manner. These small binding domains exhibit a surprising level of diversity in their binding repertoires.

Phosphoproteomic study of breast cancer: quantitative analysis of SH2 binding proteins.

Abstract Abstract #2034 Background: An important biological process involved in breast cancer cell is tyrosine phosphorylation. Particularly, two tyrosine kinases, Src and HER2, are over expressed in invasive breast cancer tissues and both are considered excellent prognostic markers and therapeutic targets. Since tyrosine kinases transduce downstream signals by phosphorylating substrate proteins, leading to cellular responses, we assume the status of tyrosine-phosphorylated proteins in tumors can also provide molecular markers for breast cancer. Recently we established a method termed SH2 profiling, in which a battery of Src Homology 2 (SH2) domains are used to profile proteomes based on interaction between SH2 domains and tyrosine phosphorylated proteins. In this study we developed a semi-automated method to quantify SH2 binding proteins on multiple blots, and explored the feasibility of SH2 binding patterns as potential molecular markers for breast cancer. Material and Methods: Protein lysates from 10 breast cancer patient tissues as well as cancer cell lines were prepared and loaded into multiple precast gels. Proteins were separated using a SDS-PAGE multigel apparatus and transferred onto nitrocellulose membranes. Blocked membranes were incubated with HRP-labeled GST-SH2 domain probes, and SH2 binding patterns were detected by chemiluminescence with a phosphorimager. To correct blot-to-blot variation, we produced a semi-automated imaging tool for multiple blots. SH2 binding proteins were individually quantified based on their molecular sizes on blots, and different breast cancer samples were compared using the SH2 binding profiles. Results and discussion: 1. We have developed a method to quantify and incorporate data from multiple protein blots, a task that is always challenging due to blot-to-blot variation. 2. Using the semi-automated quantitation approach, large numbers of SH2 binding proteins in breast cancer samples were analyzed. SH2 domains including Vav2 and Eat2 bound strongly to proteins even in HER-2 negative tumors suggesting activation of HER-2 independent tyrosine kinase signaling pathways in breast cancer. 3. Further development of the quantitation method is underway to determine if specific SH2 binding patterns are potential prognostic molecular markers for breast cancer. This study is supported by the Connecticut Breast Health Initiative, Inc. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2034.