Supplemental Data High-Throughput Phosphotyrosine Profiling Using ...

Molecular Cell, Volume 26

Supplemental Data High-Throughput Phosphotyrosine Profiling Using SH2 Domains Kazuya Machida, Christopher M. Thompson, Kevin Dierck, Karl Jablonowski, Satu Kärkkäinen, Bernard Liu, Haimin Zhang, Piers D. Nash, Debra K. Newman, Peter Nollau, Tony Pawson, G. Herma Renkema, Kalle Saksela, Martin R. Schiller, Dong-Guk Shin, and Bruce J. Mayer

Supplemental Text Comparison of Various SH2 Profiling Platforms In the past few years, a number of technologies have been described with the overall goal of profiling SH2 domain binding sites (Table S2). The strengths and weaknesses of the various approaches are outlined here and compared to the SH2 Rosette strategy. MacBeath’s group developed a forward-phase assay using Microarrays In Microtiter plates (MIMs) technology, in which a virtually complete set of human SH2 and PTB domains was immobilized in microarrays aligned with the wells of a 96-well plate, thereby allowing simultaneous probing of multiple microarrays (Jones et al., 2006). Using synthetic phosphopeptides corresponding to potential tyrosine phosphorylation sites of EGF receptor family members, they quantitatively analyzed more than 5,000 interactions with SH2 and PTB domains. Panomics has introduced a conceptually similar membrane-based SH2 domain macro-array with 34 SH2 domains, and described its use for phosphorylated peptides, recombinant proteins and cell lysates (Panomics). Taking advantage of the Luminex fluorescent bead technology, this group also reported a forward-phase bead-based assay in which different SH2 domains are conjugated to microspheres with distinct fluorophore labels. Unlike traditional pull-down experiments, where one immobilized SH2 domain can be

analyzed per assay, this format allows competitive binding conditions (SH2s on different beads can compete with each other for same target phosphorylation site) and simultaneous (multiplexed) detection of binding for each SH2 domain probe. This assay was applied to EGF-stimulated cell lysates and distinct patterns of SH2 binding were detected (Yaoi et al., 2006). A reverse-phase multiplexed binding assay was developed by the Nollau group, based on tagging of different SH2 domains with distinct oligonucleotide tags, allowing competitive conditions in a single binding reaction and sensitive detection using PCR-based amplification (Dierck et al., 2006). This technology was used to profile leukemia patient samples and disease-specific clustering based on quantitative SH2 binding profiles was shown. Austin’s group exploited phage display methods to screen phosphopeptides against a human random cDNA phage display library, and selected high-affinity binders encoding 9 distinct SH2 domains (Videlock et al., 2004). A comprehensive SH2 protein-displayed phage library to could be used to screen peptide or purified protein targets, similar to the approach used by Saksela and colleagues for SH3 domains (Karkkainen et al., 2006). Given the availability of multiple SH2 domain profiling platforms, what factors should potential users of SH2 profiling consider for their experiments? Distinctions among these different SH2 profiling methods fall into the following categories: (i) forward-phase vs. reverse-phase; (ii) throughput; (iii) competitive vs. non-competitive binding; (iv) amount of sample needed; (v) and convenience. The most important overall criterion is that the assay must have sufficient sensitivity and signal-to-noise ratio to provide reliable quantitative binding data. This problem is particularly acute when analyzing crude whole-cell lysates, where the concentrations of tyrosine-phosphorylated proteins are very low. In forward-phase assays, SH2 domains are immobilized on a surface and incubated with a soluble analyte. The advantage of this approach is that a large number of SH2 domains (and other

proteins domains) can be assayed simultaneously in a single binding reaction. There are two potential disadvantages, however. First, SH2 domains may lose binding activity upon immobilization, as they require an intact folded structure for binding. The SH2 domain is quite robust, however, and several modular protein arrays have been reported including SH2 domains as mentioned above (Espejo et al., 2002; Jones et al., 2006), so this does not seem to be a major hurdle. The second problem is potentially more severe, however. In pilot experiments we found that it was not possible to detect significant differences in tyrosine phosphorylation patterns when immobilized SH2 arrays were probed with whole cell lysates (P.N. and B.J.M., unpublished observations). Poor binding kinetics due to the very low concentration of tyrosine phosphorylated proteins in the solution phase result in relatively low signal and high nonspecific background. Thus forward-phase assays are best suited for situations where analytes such as purified peptides or phosphoproteins can be used at relatively high concentrations to drive efficient binding to the arrayed domains. In reverse-phase assays, phosphorylated samples are immobilized in the solid phase and probed with labeled SH2 domains in solution. A large number of samples can be assayed simultaneously, especially when high density protein microarrays are used (Balboni et al., 2006). Binding is driven by high concentrations of SH2 domains in solution, so sensitivity and signal to noise ratio are high even when the concentration of tyrosine phosphorylated proteins in the analyte is low. Furthermore, since SH2 domains generally bind short peptide binding sites and do not required folded protein structure, immobilization is unlikely to destroy binding sites in the reverse-phase format. For these reasons, we have chosen the reverse-phase format for SH2 profiling of whole cell lysates. In principle, the forward-phase format lends itself to quantifying the binding of a large

number of SH2 domains to a single phosphorylated target, while binding of a single SH2 domain to a large number of complex samples can best be assayed in reverse-phase format. For comprehensive proteomic studies, however, the aim is to analyze large numbers both of samples and of SH2 domains. To attain this for forward-phase assays, technologies such as MIMs and sectored arrays were developed (Chan et al., 2004; Jones et al., 2006; Yaoi et al., 2006). Similar to the MIMs approach, the Rosette assay uses a 96-well multichamber apparatus to increase the number of SH2 domain per assay in reverse-phase format (Fig. 3A). It is important to note that conditions to optimize the signal-to-noise ratio can be determined individually for each SH2 domain probe, similar to the optimization of antibodies for immunoblotting. The ability to incorporate competition among SH2 domains in a binding assay is also an important factor. Three of six above-mentioned formats use competitive binding conditions (Table S2). Competition is more likely to mimic in vivo conditions, where multiple SH2 domains compete for binding to a given tyrosine phosphorylated site (and multiple phosphoproteins compete for binding to each SH2 domain). Previously we have shown that competition with unlabeled SH2 probes resulted in more specific binding compared to the same probe without competition (Nollau and Mayer, 2001). Nevertheless we do not routinely use competitive conditions in the SH2 Rosette assay because competition reduces the sensitivity of assay. In analytical experiments using SDS-PAGE where relatively large amounts of sample can be loaded, specificity at the expense of sensitivity is acceptable, but for a high-throughput screening method such as the SH2 Rosette assay, sensitivity is critical. We have chosen to maximize sensitivity in the screening step, and confirm the specificity of detected interactions in subsequent analyses. However, competitive conditions can be useful for evaluating whether a given SH2-mediated association is favored in the presence of other SH2 domains. Therefore, we occasionally adopt

competitive binding conditions into the Rosette SH2 assay for specific applications (Fig. S3). In terms of sample requirements, we believe the SH2 Rosette assay is comparable or superior to other available SH2 profiling formats. The amount of sample required for a typical screening is about 100 µg of total lysate, equivalent to 105 cells, well within the range of most clinical samples. For example a core needle biopsy for diagnostic purposes contains approximately 105 cells (Liotta et al., 2003). Indeed, we have successfully profiled frozen breast cancer sections 1-2 mm in diameter (data not shown). The type of samples that can be used for the SH2 Rosette assay is also fairly broad, including peptides, recombinant proteins, and crude lysate. A final consideration is accessibility of the assay to the general research community. This is crucial in determining the number of investigators that can take advantage of a method and apply it to diverse biological and clinical questions. Much of the value of proteomic databases lies in their breadth of coverage—the more cell types and conditions that are analyzed using the same standardized platform, the easier it is to discern general patterns and important distinctions. Although some SH2 profiling reagents are commercially available, methods involving costly specialized equipment may not be easily or routinely accessible for many investigators. The comprehensive profiling platform described here requires no equipment beyond that available in a standard molecular biology/biochemistry lab, and thus is accessible to a wide range of investigators.

Quality of SH2 Probes and Challenges Whichever the specific SH2 profiling format, the primary goal of the assay is the quantitative or qualitative ranking of SH2 domains for binding to a sample of interest. The results of such assays, however, must be interpreted carefully and critically. With properly chosen negative

controls, both for samples and SH2 probes (e.g. unphosphorylated sample and “empty” GST probe, respectively), positive results are likely to be meaningful. However a negative result, e.g., no detectable association between an SH2 domain and a sample, may not necessarily be significant. A substantial fraction of cloned GST-SH2 proteins form insoluble aggregates (Fig. 1A) (Jones et al., 2006), and incorporating such domains into a binding assay is a hurdle. For example it was difficult to assess the phosphorylation dependence of the Jak1 SH2 domain, the function of which is still in question (Radtke et al., 2005), because of difficulties in purification of soluble protein. This issue of probe reliability is not confined to SH2 profiling, as antibody microarrays have similar problems. For instance, it has been reported that when antibodies are arrayed on a solid support surface, only 20% of them are suitable for specific and accurate measurement of their target antigens at lower concentrations (Balboni et al., 2006), and only 5% are suitable for microarray-based analyses of cellular lysates (MacBeath, 2002). Quality assessment of SH2 domains is further complicated by their generally moderate binding specificity. Target binding sites are not well characterized for most of SH2 domains, thus specificity cannot be determined prior to binding assay; indeed, specificity determination may be a primary objective of SH2 profiling experiments. Accordingly, it is a challenge to confirm that all SH2 domains on an array or in solution are actually active. One obvious solution for this issue would be to include a positive internal standard in each assay run. Assuming all SH2 domains bind tyrosine-phosphorylated proteins, a mixture enriched in highly phosphorylated proteins, e.g. pervanadate-treated cell lysates, can serve as a positive control. Although pervanadate-treated lysate contains massive amounts of phosphotyrosine peptides, however, a lysate from a single cell type may not ensure coverage of all potentially relevant tyrosine phosphorylated sites. We are evaluating if a combined “pervanadate proteome” derived from a mixture of different cell types, as

used in Fig. 1B, can serve as a “gold standard” for all SH2s. Alternatively, a random tyrosine-phosphorylated peptide library could be used, although the relative abundance of phosphorylated sequences in such a library is likely to differ from that of naturally occurring phosphorylation sites. Because the quality of probes is critical for SH2 profiling, protein solubility is a major obstacle. In our hands about half of the recombinant SH2 domains are relatively insoluble, and highly insoluble domains tend to lack detectable binding activity. It is unlikely that this inactivity necessarily reflects inherently weak binding, because in a number of instances (such as the Stat family), domains that are insoluble/inactive in vitro have been reported to be active in their native environment in vivo (Heim et al., 1995; Nicholson et al., 1999). We have tried multiple strategies to improve the solubility of bacterially expressed GST-SH2 fusions, including growing bacteria at lower temperature, different induction conditions, and changing the domain borders and fusion site with the GST tag, but in our hands none of these was reproducibly effective for all GST-SH2 constructs. Other researchers have addressed solubility issues for individual SH2 domains. For example, Babon et al. successfully purified active SOCS3 protein containing an SH2 domain and solved the X-ray crystal structure (Babon et al., 2006). In this case it was necessary to purify protein from inclusion bodies and refold it in the presence of a phosphopeptide in order to increase the solubility. Others reported that mutagenesis or attachment of a C-terminal tag helped to generate stable and soluble SH2 proteins (Lamla et al., 2006; Welsh et al., 1994). Denaturation and refolding has been successful in generating soluble domains, but it is not clear if native binding activity is achieved in all cases (Jones et al., 2006). With a combination of individually customized solutions, however, it should be possible to increase the number of SH2 probes suitable for in vitro binding assay to allow truly comprehensive SH2 profiling.

Supplemental Experimental Procedures Cell Culture and Sample Preparation A431 cells and human hepatoma cell line HepG2 expressing PDGFRβ (obtained from A. Kazlauskas, Schepens Eye Research Institute, Boston MA), and NIH-3T3 cells stably expressing v-Abl, v-Src, v-Fps, CrkI or CrkII were maintained in DMEM supplemented with 10% FBS. Human multiple myeloma cell line MR20 (obtained from B. Lin and K. C. Anderson, Dana-Farber Cancer Institute, Boston MA) was maintained in RPMI supplemented with 10% FBS. Cells, with or without treatment with 50mM pervanadate for 30 min, were lysed by homogenization in KLB buffer (150 mM NaCl, 25 mM Tris-HCl pH 7.4, 5 mM ethylene diamine tetraacetic acid (EDTA), 1 mM phenyl methyl sulfonyl fluoride (PMSF), 1% Triton X-100, 10% glycerol, 0.1% sodium pyrophosphate, 10 mM β-glycerophosphate, 10 mM NaF, 5 µg/ml of Aprotinin (Sigma A6279), 50µM pervanadate). Insoluble material was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad).

Cell Adhesion and Suspension Experiments SYF cells and SYF-Src cells were grown in DMEM with 10% FCS until confluence. Cells were trypsinized and approximately 106 cells were cultured in 100mm cell culture dishes (Corning) or untreated 100mm petri dishes (Falcon) for indicated times. Cells were washed once with PBS, then lysed on ice with 200µl of KLB buffer. For IP-Western experiments (Fig. 7), 200 µg of pre-cleared lysate were incubated with antibodies against p130Cas (rabbit polyclonal, gift of A. Bouton, University of Virginia Medical School), FAK (SC-558, Santa Cruz) and paxillin (monoclonal, gift

of C. Turner, SUNY Upstate Medical Center) for 2h. Protein A- or protein G-conjugated agarose beads were added and incubated 4 h at 4oC. Beads were washed three times with lysis buffer and boiled in Laemmli sample buffer for 3 min, and loaded on the gel (50µg of total lysate or the immunoprecipitate from 100µg lysate per lane).

Supplemental References Babon, J.J., McManus, E.J., Yao, S., DeSouza, D.P., Mielke, L.A., Sprigg, N.S., Willson, T.A., Hilton, D.J., Nicola, N.A., Baca, M., et al. (2006). The structure of SOCS3 reveals the basis of the extended SH2 domain function and identifies an unstructured insertion that regulates stability. Mol Cell 22, 205-216. Balboni, I., Chan, S.M., Kattah, M., Tenenbaum, J.D., Butte, A.J., and Utz, P.J. (2006). Multiplexed protein array platforms for analysis of autoimmune diseases. Annu Rev Immunol 24, 391-418. Chan, S.M., Ermann, J., Su, L., Fathman, C.G., and Utz, P.J. (2004). Protein microarrays for multiplex analysis of signal transduction pathways. Nat Med 10, 1390-1396. Dierck, K., Machida, K., Voigt, A., Thimm, J., Horstmann, M., Fiedler, W., Mayer, B.J., and Nollau, P. (2006). Quantitative multiplexed profiling of cellular signaling networks using phosphotyrosine-specific DNA-tagged SH2 domains. Nat Methods 3, 737-744. Espejo, A., Cote, J., Bednarek, A., Richard, S., and Bedford, M.T. (2002). A protein-domain microarray identifies novel protein-protein interactions. Biochem J 367, 697-702. Heim, M.H., Kerr, I.M., Stark, G.R., and Darnell, J.E., Jr. (1995). Contribution of STAT SH2 groups to specific interferon signaling by the Jak-STAT pathway. Science 267, 1347-1349. Jones, R.B., Gordus, A., Krall, J.A., and MacBeath, G. (2006). A quantitative protein interaction network for the ErbB receptors using protein microarrays. Nature 439, 168-174. Karkkainen, S., Hiipakka, M., Wang, J.H., Kleino, I., Vaha-Jaakkola, M., Renkema, G.H., Liss, M., Wagner, R., and Saksela, K. (2006). Identification of preferred protein interactions by phage-display of the human Src homology-3 proteome. EMBO Rep 7, 186-191. Lamla, T., Hoerer, S., and Bauer, M.M. (2006). Screening for soluble expression constructs using

cell-free protein synthesis. Int J Biol Macromol. Liotta, L.A., Espina, V., Mehta, A.I., Calvert, V., Rosenblatt, K., Geho, D., Munson, P.J., Young, L., Wulfkuhle, J., and Petricoin, E.F., 3rd (2003). Protein microarrays: meeting analytical challenges for clinical applications. Cancer Cell 3, 317-325. Liu, B.A., Jablonowski, K., Raina, M., Arce, M., Pawson, T., and Nash, P.D. (2006). The Human and Mouse Complement of SH2 Domain Proteins-Establishing the Boundaries of Phosphotyrosine Signaling. Mol Cell 22, 851-868. MacBeath, G. (2002). Protein microarrays and proteomics. Nat Genet 32 Suppl, 526-532. Nicholson, S.E., Willson, T.A., Farley, A., Starr, R., Zhang, J.G., Baca, M., Alexander, W.S., Metcalf, D., Hilton, D.J., and Nicola, N.A. (1999). Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction. Embo J 18, 375-385. Nollau, P., and Mayer, B.J. (2001). Profiling the global tyrosine phosphorylation state by Src homology 2 domain binding. Proc Natl Acad Sci U S A 98, 13531-13536. Panomics http://www.panomics.com/MA3040.cfm. Radtke, S., Haan, S., Jorissen, A., Hermanns, H.M., Diefenbach, S., Smyczek, T., Schmitz-Vandeleur, H., Heinrich, P.C., Behrmann, I., and Haan, C. (2005). The Jak1 SH2 domain does not fulfill a classical SH2 function in Jak/STAT signaling but plays a structural role for receptor interaction and up-regulation of receptor surface expression. J Biol Chem 280, 25760-25768. Videlock, E.J., Chung, V.K., Mohan, M.A., Strok, T.M., and Austin, D.J. (2004). Two-dimensional diversity: screening human cDNA phage display libraries with a random diversity probe for the display cloning of phosphotyrosine binding domains. J Am Chem Soc 126, 3730-3731.

Welsh, M., Mares, J., Karlsson, T., Lavergne, C., Breant, B., and Claesson-Welsh, L. (1994). Shb is a ubiquitously expressed Src homology 2 protein. Oncogene 9, 19-27. Yaoi, T., Chamnongpol, S., Jiang, X., and Li, X. (2006). Src homology 2 domain-based high throughput assays for profiling downstream molecules in receptor tyrosine kinase pathways. Mol Cell Proteomics 5, 959-968.

Figure S1. Phosphotyrosine-Dependent SH2 Domain Binding (A) Binding specificity of SH2 domains determined by pull-down assay. Lysates of untreated (-) and pervanadate-treated (+) HepG2 cells expressing PDGFR were precipitated with GST-SH2

probes as indicated, and tyrosine-phosphorylated binding proteins were detected by immunoblotting with anti-phosphotyrosine antibody. (B) FLVRES motif-dependent binding of SH2 domains. Pervanadate (POV)-treated or untreated cell lysate (mixture of NIH-3T3, A431, HepG2-PDGFR, and MR20) was separated on gradient gels and identical blots probed with GST-SH2 domain probes, corresponding R to K mutants of the conserved FLVRES motif (RK), GST, anti-phosphotyrosine antibody (anti-pTyr), and anti-tubulin as a loading control. Asterisks indicate loss of specific binding of SH2 mutants.

Figure S2. Agreement of Sequence Similarity and Binding Patterns Tree positions are directly compared without distance metric evaluation. Linear position of SH2 domains on each axis reflects phylogenetic tree (Fig. 1A, (Liu et al., 2006)) horizontally and SH2 binding pattern-based tree (Fig. 2A) vertically. For each SH2 domain, the intersection point of the two axes is indicated in green; closely clustered spots indicate families that are highly similar in both sequence and binding specificity (red circles).

Figure S3. Phosphotyrosine Profiling of PECAM-1 ITIM Motifs in Competitive Binding Conditions Competitive binding assay was performed on samples described in Fig. 4. Indicated amounts of labeled GST-SH2 probes were incubated in absence or presence of a competitor cocktail, consisting of a mixture of 15 different unlabeled probes in equimolar amounts, including the homologous competitor. Competition-tolerant binding of SHP-2 (N) probe to pY663 is indicated by arrow. Binding of SH2 domains to pY686 is dramatically reduced in competitive conditions (indicated by arrowhead).

Figure S4. pY194 Dependency of Sap Binding Confirmed by Surface Plasmon Resonance Analysis Equal amounts of wild type and mutant C-terminally His-tagged PAK proteins were immobilized on each of the two channels of the Biacore X Ni-NTA sensor chip. GST-SH2 domains of Sap and Eat2 were used as analyte. Sensorgrams of the association and dissociation phases for Sap and Eat2 SH2 binding to PAK2N proteins are shown. Because of the slow dissociation phase, the chip was regenerated using EDTA after each Sap or Eat2 binding and PAK2N proteins were reloaded. For each of the analytes, the runs for WT1 and Y130F, WT2 and Y139F, WT3 and Y194F, and WT4 and 3F were performed as pairs on the same surface. Y194 dependence of Sap SH2 binding is indicated by arrow. RU, resonance units.

Figure S5. SH2 Profiling of SYF Cells in Adherent and Suspended States SYF or c-Src reconstituted SYF (SYF-Src) mouse embryo fibroblast cells were cultured in adhesion or suspension conditions for 45 min or 24 h (photomicrographs, upper left). Whole cell lysates were spotted in duplicate as shown diagrammatically (top right). Identical spots were probed with different SH2 domain probes, GST control, anti-pTyr, and stained with Ponceau S as indicated to right (lower panels).

Table S1. Summary of Phosphotyrosine-Binding Properties of GST-SH2 Domain Probes Phosphotyrosine dependence of SH2 probe binding was determined by pull-down assays coupled to immunoblotting with anti-phosphotyrosine antibody (Experiments 1and 2), and far-Western blotting (Experiment 3, see text). Note that an SH2 protein or SH2 probe may contain two SH2 domains, so total number of SH2 proteins (110) is less than total number of SH2 domains (120).

Table S2. Properties of SH2 Domain Probes Used in This Study (See the online Excel document entitled Table S2) GST-SH2 probes were generated by PCR and subcloning into pGEX fusion vectors. Name of SH2 domain containing proteins (probe), alternate names, Gene ID, source organism, and amino acid residues included (AA) are shown. In addition to SH2 domains, the CblA, CblB, and CblC probes contain a TKB domain, the Grb7, Grb10, and Grb14 probes contain a PIR domain, and the GAP probe contains an SH3 domain, respectively. Solubility of proteins was estimated in a small-scale assay. Briefly, 0.4 ml overnight bacterial culture was inoculated into 1.6 ml fresh pre-warmed LB-amp and grown at 37° for 1 h, IPTG was added to a final concentration of 0.1 mM and incubated for 1h. Bacterial pellet was resupended and sonicated in 0.4 ml of BXB buffer (PBS with 100 mM EDTA, 1% triton X-100 1mM PMSF, and 1% aprotinin). Whole cell fraction and soluble fraction were separated by centrifugation for 5 min and solubility (soluble fraction divided by whole cell fraction) was determined by immunoblotting with anti-GST antibody or gel staining by Coomassie Brilliant Blue (indicated by asterisks). “+” indicates clear difference in binding between pervanadate treated (POV) and untreated (no POV) samples, or in presence or absence of phenylphosphate. Optimized probe concentrations used for far-Western blotting are indicated in the right column. Conditions were optimized for each probe to provide roughly equivalent overall

binding to positive controls, and to minimize nonspecific binding to negative controls.

Table S3

Table S3. The SH2 Domain-Based Phosphoproteomic Profiling Approaches SH2 microarrays, fabricated forward-phase microarrays in 96 well microtiter plates (MIMs) containing nearly entire set of SH2 domains and PTB domains (Jones et al., 2006); SH2 domain array, membrane based forward-phase SH2 domain array of 34 SH2 domains (Panomics); SH2 Rosette assay, far-Western-based dot blotting in a multi-chamber plate with nearly entire complement of SH2 domains (described in this report); Multiplexed fluorescent microsphere assay, multiplexed pull-down assay with 25 SH2-conjugated fluorescent microspheres (Yaoi et al., 2006); Oligonucleotide-tagged multiplex assay, multiplexed SH2 (~10) binding assay in combination with a sensitive PCR-ELISA for detection (Dierck et al., 2006); Phage display, a screening of human (entire or SH2) cDNA phage display libraries against random or fixed phosphopeptides (Videlock et al., 2004).