Comprehensive Binary Interaction Mapping of SH2 Domains via Fluorescence Polarization Reveals Novel Functional Diversification of ErbB Receptors

<div><p>First-generation interaction maps of Src homology 2 (SH2) domains with receptor tyrosine kinase (RTK) phosphosites have previously been generated using protein microarray (PM) technologies. Here, we developed a large-scale fluorescence polarization (FP) methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB) domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry procedures. This dataset represents a rich source of testable hypotheses regarding the biological mechanisms of ErbB receptors.</p> </div

Competitive inhibition binding curves of protein-peptide interactions detected by FP.

<p>Nine ErbB phosphotyrosine sites were queried against five proteins: (<b>A</b>) RASA1-N, (<b>B</b>) SRC, (<b>C</b>) GRB7, (<b>D</b>) GRB2, and (<b>E</b>) PTK6. The predicted binding affinities of competitor peptide curves are color-coded as follows: red (<i>K_D<</i>1), purple (1≤ <i>K_D<</i>5), blue (5≤ <i>K_D<</i>20), and black (<i>K_D</i> ≥20). “OP” refers to the original rhodamine-labeled peptides and “CP” to the unlabeled competitor peptides, which have been numbered in the figure with sequences.</p

Comprehensive SH2 domain recruitment potential of the ErbB family as determined by high-throughput fluorescence polarization (HT-FP).

<p>Color-coded heat maps represent apparent dissociation constants (K_Ds) for FP interactions between SH2/PTB domains and phosphopeptides representing all potential ErbB1, ErbB2, ErbB3, and ErbB4 phosphotyrosine sites; black boxes indicate interactions that are too weak to be detected by the assay. Homologous ErbB peptides with identical amino acid residues from +1 to the +4 position relative to the phosphotyrosine (X) are indicated with an asterisk followed by the number (in order of occurrence) of the homologous receptor. Sequences of peptides used are indicated for each homologous receptor site, in which a small “d” denotes the pre-charged aspartic acid (Asp) residue on the peptide synthesis resin and not a naturally occurring Asp. NS refers to peptides that were unable to be synthesized, while NI refers to synthesized peptides that produced no positive hits in the study; therefore we cannot confirm nor deny interactions at these sites with our assay. Rows of the heatmaps for these peptides are grayed out to indicate that our FP assay could neither confirm nor deny positive or negative interactions from these peptides.</p

Characterization of unique and overlapping SH2 domain recruitment patterns by individual ErbB receptors.

<p>(<b>A</b>) SH2 recruitment potential of ErbB1 family members at different affinity thresholds. The total number of unique SH2 and PTB domains recruited over a range of affinity thresholds are depicted for each receptor. (<b>B</b>) Four-way Venn diagram (not to scale) depicts SH2 domain interactions shared by or exclusive to ErbB1, ErbB2, ErbB3, and ErbB4. (<b>C</b>) Relative binding free energies of interactions described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044471#pone-0044471-g002" target="_blank">Figure 2</a> are summed for each ErbB receptor. (<b>D</b>) Relative enrichment and depletion of binding sites for recruitment of each SH2 domain by each ErbB receptor, depicted by Z-score transforming the observed number of binding sites each receptor had for a particular domain relative to the average number of sites that bound that domain across all ErbB receptors. Domains recruited by fewer than four independent pY sites were excluded from this analysis.</p

Methodological cross-comparison of SH2 interactions determined by fluorescence polarization or in published protein microarray data for the ErbB family.

<p>(<b>A</b>) Venn diagram comparison of overall SH2 recruitment profiles revealed by FP and PMs for the ErbB family of RTKs for only peptides and proteins tested by both platforms. The red circle represents protein-peptide interactions observed by FP; the green circle represents protein-peptide interactions previously observed by PMs; and the yellow overlap represents interactions observed by both methods. (<b>B</b>) SH2 and ErbB interactions quantified over a range of binding affinity thresholds as determined previously by PMs and in this study by FP data. The red line represents interactions characterized exclusively by FP; the green line represents interactions characterized exclusively by PMs; the blue line represents interactions observed by both methods.</p

Automated high-throughput fluorescence polarization (FP) procedural schematic.

<p>(<b>A</b>) (<b>1</b>) Following synthesis and mass-directed purification, 10 µL of phospho-peptide (10 nM) labeled on the N-terminus with rhodamine is distributed equally to each well of a 96-well plate. (<b>2</b>) After expression and purification, 8 SH2 domains at an original concentration of 20 µM are serially diluted 11 times in two-fold increments into 96-well plates. (<b>3</b>) Proteins from 96-well plates are added in four pipetting steps to a single 384-well plate. To the same plate, 12 different concentrations of eight different SH2 domains are added to each quadrant of the 384-well plate. In total, 32 unique SH2 domains at 12 different concentrations are mixed with a single peptide. (<b>4</b>) Following an incubation period of 20 min, 384-well plates of SH2 domains and peptides are delivered to the Analyst GT (<b>5</b>) for measurement of FP. Data from these measurements are used for determining apparent dissociation constants (<b>6</b>). (<b>B</b>) Comparison of binding data using 13-mer- and 18-mer peptides. Heat maps depict summations of relative binding free energies (ΔΔG, kcal/mol) as a function of apparent K_Ds of SH2 and PTB domains interacting with indicated peptides (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044471#pone.0044471.e001" target="_blank">equation 1</a> in text). ΔΔG summations are color coded by binding strength.</p

Comparison of the affinity with which each ErbB family member recruits proteins representing several molecular function categories.

<p>Relative binding free energies of interactions described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044471#pone-0044471-g005" target="_blank">Figure 5C</a> were summed across all domains contained in a particular functional category <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044471#pone.0044471-Liu2" target="_blank">[55]</a> in (<b>A</b>) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044471#pone.0044471.s032" target="_blank">Table S16</a>) and divided by the number of SH2 domains represented in each class in (<b>B</b>) to determine an average recruitment potential for SH2s from each functional class. (<b>C</b>) ErbB receptor enrichment and depletion for binding sites for functional groups depicted by Z-score transformation of the raw data as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044471#pone-0044471-g005" target="_blank">Figure 5D</a>.</p

Identification of Novel Protein Expression Changes Following Cisplatin Treatment and Application to Combination Therapy

Determining the effect of chemotherapeutic treatment on changes in protein expression can provide important targets for overcoming resistance. Due to challenges in simultaneously measuring large numbers of proteins, a paucity of data exists on global changes. To overcome these challenges, we utilized microwestern arrays that allowed us to measure the abundance and modification state of hundreds of cell signaling and transcription factor proteins in cells following drug exposure. HapMap lymphoblastoid cell lines (LCLs) were exposed to cisplatin, a chemotherapeutic agent commonly used to treat testicular, head and neck, non-small cell lung, and gynecological cancers. We evaluated the expression of 259 proteins following 2, 6, and 12 h of cisplatin treatment in two LCLs with discordant sensitivity to cisplatin. Of these 259 proteins, 66 displayed significantly different protein expression changes (<i>p</i> < 0.05). Fifteen of these proteins were evaluated in a second pair of LCLs with discordant sensitivities to cisplatin; six demonstrated significant differences in expression. We then evaluated a subset of 63 proteins in a second set of LCLs with discordant sensitivity, and 40% of those that were significant in the first pair were also significant in the second part with concordant directionality (<i>p</i> < 0.05). We functionally validated one of the top proteins identified, PDK1, and demonstrated a synergistic relationship between cisplatin and a PDK1 inhibitor in multiple lung cancer lines. This study highlights the potential for identifying novel targets through an understanding of cellular changes in protein expression and modification following drug treatments