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Profiling Phosphorylation of Receptor Tyrosine Kinases Using Antibody Arrays
by David Finkel, James Rivard, and Richard Krzyzek
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Figure 1, top. PDGF ligands induce tyrosine phosphorylation of PDGF receptors. Arrays were incubated with 100 µg of lysate prepared from immortalized human fibroblasts (CCD 1070sk) that were untreated or treated with 100 ng/ml of PDGF-AA, PDGF-AB, or PDGF-BB for 5 minutes. Key: EGF R (1), PDGF RΑ (2), PDGF RΒ (3), and FGF R3 (4).
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Introduction
Multicellular organisms have evolved complex intercellular signaling pathways to coordinate the activities of individual cells. Cytokines are extracellular signaling molecules that transduce their signals across cellular membranes by activating transmembrane receptors, such as Receptor Tyrosine Kinases (RTKs). RTKs possess intrinsic tyrosine kinase activity that is activated after ligand binding and receptor dimerization, resulting in phosphorylation of specific tyrosine residues in its own cytoplasmic domain and associated downstream signaling proteins. The reversible phosphorylation of tyrosine residues in RTKs plays a key role in regulating a number of different biological processes including proliferation, differentiation, migration, cell survival, and angiogenesis.(1) While RTK tyrosine kinase activity is tightly controlled in normal cells, receptor overexpression or mutations causing constitutive activation can lead to uncontrolled RTK signaling and the subsequent development and progression of certain cancers. Many of the 58 known human RTKs are proto-oncogenes and some are associated with the progression of different types of cancers.
The ability to identify all RTKs activated in a particular cell type provides a framework to better understand signaling mechanisms in normal and disease states. For example, a comprehensive RTK phospho-tyrosine profile of tumor cells is essential to understanding their full oncogenic potential and responsiveness to various chemotherapeutic reagents. In addition, profiling RTK tyrosine phosphorylation could facilitate the study of RTK transactivation or "crosstalk". Although reverse transcriptase-polymerase chain reaction has been used for global profiling of tyrosine kinase mRNA expression,(2) detecting the tyrosine phosphorylation of RTKs would be a more accurate predictor of function.
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Figure 2, bottom. IP-Western and ELISA analysis are consistent with array data. The same lysates used in Figure 1 were tested by array (A), IP-Western (B), and ELISA (C) analysis. IP-Western blots were done using 50 µg of lysate, an anti-PDGF RΒ Mab, and anti-mouse IgG agarose. Immunoblots were incubated with biotinylated anti-phospho-tyrosine monoclonal antibody followed by Streptavidin-HRP and visualized by chemiluminescent detection. ELISA for human phospho-PDGF RΒ was done using 20 µg of lysate. ELISA results are expressed as optical density ± S.D. for duplicate wells.
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Several methods have been used for profiling protein tyrosine phosphorylation. Standard proteomics methods such as two-dimensional gel electrophoresis followed by mass spectrometry (MS)(3) or newer MS methods(4) require the use of radioisotopes or enrichment steps in order to achieve the sensitivity required to detect low abundance phosphorylated regulatory proteins. These methods are well suited for determining sites of tyrosine phosphorylation and do not require a phospho-specific detection tool, such as an antibody. However, these methods may not be readily available to most laboratories and they are not currently suitable for high throughput analyses.(5) The most commonly used technique for detection of protein phosphorylation is immunoprecipitation (IP) followed by Western blot analysis. Although this traditionally used method may not be amenable to profiling phosphorylation of a large number of proteins, it is a powerful and readily available tool for detecting the relative level of phosphorylation for an individual protein. In a typical IP-Western, immunoprecipitation is performed with a protein-specific antibody followed by immunoblot analysis using an antibody that recognizes either phosphorylated tyrosine, serine or threonine to detect phosphorylation of the target protein. However, profiling the phosphorylation of many proteins using IP-Western blot analysis is tedious and requires a significant quantity of sample. Finally, enzyme-linked immunosorbent assays (ELISAs) have also been used to quantitatively measure protein phosphorylation(6,7) and thus have become a viable alternative to IP-Western blots.
Profiling tyrosine phosphorylation using antibody arrays
Proteome Profiler Human Phospho-RTK Array (R&D Systems, Inc., Minneapolis, MN) can simultaneously profile 42 of the 58 known RTKs in the human genome. Each array contains a panel of 42 carefully selected anti-RTK capture antibodies as well as relevant positive and negative controls spotted in duplicate on a nitrocellulose membrane. Using this array, both phosphorylated and unphosphorylated RTKs bind specifically to their cognate capture antibodies on the array and phosphorylated tyrosine residues are then detected with a pan anti-phosphotyrosine antibody covalently conjugated to horseradish peroxidase (HRP). Array signals are visualized by chemiluminescent detection using X-ray film. The assay conserves time and samples because a large number of RTKs can be evaluated simultaneously using the same quantity of sample typically used for a single sample in an IP-Western blot experiment.
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Figure 3. Global RTK tyrosine phosphorylation profiling using the Human Phospho-RTK Array. Arrays were incubated with 100 µg of lysates prepared from immortalized human fibroblasts (A) and human breast cancer cells (MDA-MB-453) (B) that were treated with 1 mM pervanadate for 5 minutes. Key: EGF R (1), ErbB2 (2), ErbB3 (3), ErbB4 (4), FGF R3 (5), FGF R4 (6), Axl (7), HGF R (8), PDGF RΑ (9), PDGF Rß (10), c-Ret (11), and EphA4 (12).
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The Proteome Profiler Human Phospho-RTK Array was validated by profiling the tyrosine phosphorylation of platelet-derived growth factor receptors (PDGF Rα and PDGF Rβ) in response to three specific PDGF ligands. Array analysis of lysates prepared from immortalized human fibroblasts treated with PDGF-AA, -AB, or -BB showed that PDGF Rβ is strongly activated by PDGF-BB, moderately activated by PDGF-AB, and not activated by PDGF-AA (Figure 1). In contrast, PDGF RΑ was activated to a lower, but similar extent by all three ligands. These observations are consistent with the known receptor-ligand binding specificity and relative level of phospho-tyrosine activation.(8) A high level of constitutive tyrosine phosphorylation of the epidermal growth factor receptor (EGF R) and low constitutive levels of phosphorylation for both PDGF receptors was also observed (Figure 1, untreated). The tyrosine phosphorylation of PDGF Rß that was observed using arrays was confirmed by using two independent methods, IP-Western blot and ELISA (Figure 2).
One possible application of the Proteome Profiler Human Phospho-RTK Array is to compare the global RTK phospho-tyrosine profiles of different cells. As an example, we tested lysates prepared from a human fibroblast cell line (Figure 3A) and a human breast cancer cell line (Figure 3B) that were both treated with the phospho-tyrosine phosphatase inhibitor pervanadate. Inhibition of tyrosine phosphatases causes an increase in the level of RTK tyrosine phosphorylation that is ligand independent, thereby allowing the detection of multiple RTKs present in a cell line using a single sample. Graphical representation of array data shows that these two cell lines possess distinct phospho-tyrosine RTK profiles (Figure 3).
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Figure 4. Profiling RTK transactivation using the Human Phospho-RTK Array.
Arrays were incubated with 200 µg of lysates prepared from the human liver cell line, HepG2, that were untreated (A) or treated (B) with 1 µg/ml of Insulin for 5 minutes. Array signals were quantitated by densitometry and graphed (C). Key: EGF R (1), INS R (2), and IGF-I R (3).
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Another useful application of the Proteome Profiler Human Phospho-RTK Array is to detect cross-communication between different cellular signaling systems. EGF R is a well-characterized target of receptor transactivation, receiving signals from G-protein coupled receptors, cytokine receptors, and other RTKs.(9) Using the human liver cell line, HepG2, we demonstrated that tyrosine phosphorylation of the EGF R was increased 3-fold upon insulin-treatment (Figure 4), most likely due to transactivation by the activated insulin receptor (INS R) and insulin-like growth factor-I receptor (IGF-I R) heterodimer complex.(10)
Conclusion
The data presented here are some practical examples on the use of R&D Systems Proteome Profiler Human Phospho-RTK Array to profile RTK tyrosine phosphorylation as well as to detect changes in RTK tyrosine phosphorylation that may occur during cell signaling. This antibody array is another example of how this technology is emerging as an important tool to complement traditional proteomics methods for profiling tyrosine phosphorylation. Adoption of such a tool in the lab will facilitate investigations into RTK tyrosine phosphorylation and at the same time provide a more comprehensive picture of how these changes impact cellular behavior.
About the authors
David Finkel is the Team Leader and James Rivard is the Group Leader for Antibody Arrays, and Richard Krzyzek is the Chief Scientific Officer at R&D Systems, Inc. For more information regarding this product or other R&D Systems products, please visit www.RnDSystems.com. The authors wish to acknowledge the contributions of many R&D Systems scientists.
References
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