Monitor receptor tyrosine kinase (RYK) activity and isolate tyrosine phosphorylated (pY) proteins using specific SH2 domains.

The Thermo Scientific SH2 Domain Phosphotyrosine Capture Kits are highly specific systems for purifying tyrosine phosphorylated proteins from mammalian cell lysates.

The interactions of proteins containing SH2 domains represent a critical interface between extracellular stimulation of receptor tyrosine kinases (RYK) and transmission of their signals to intracellular proteins.

The SH2 Domain "Pull-Down" Kits enable researchers to monitor and profile specific tyrosine phosphorylation events and identify SH2 domain protein-protein interaction networks. Analysis and profiling of the purification products can be performed by Western blot or mass spectrometry.

Page Contents

• Introduction to SH2 Domains
  • SH2 domains and receptor tyrosine kinase signaling
• Application Experiments
  • Monitoring the phospho-tyrosine state of specific proteins
  • Measuring receptor phosphorylation time-course
  • Resolving specific tyrosine phosphorylation events
  • Identifying binding interactions with MS
• The SH2 Domain Capture Kits
  • Description
  • Procedure summary
  • Menu of Kits
• References and Related Products
  

Introduction to SH2 Domains

SH2 domains and receptor tyrosine kinase signaling

Because of their affinity to specific targets involved in receptor tyrosine kinase signaling, SH2 domains can be used to selectively monitor receptor activity and effector protein binding. Tyrosine phosphorylation levels of specific cellular proteins can be analyzed by Western blotting and mass spectrometry (MS). Furthermore, MS can be used to identify SH2 domain interaction networks and receptor:cytosolic protein interactions.

The most widely studied post-translational modification is phosphorylation of serine, threonine and tyrosine residues in response to a biological signal. Approximately 30% of the phospho-proteome is transiently phosphorylated at any given time. Although phosphorylation of tyrosine accounts for only 0.1-1% of all phosphorylation events, it is the most intensely studied because deregulation of phosphotyrosine signaling is linked to many human diseases.

Src Homology 2 (SH2) domain-containing proteins are critical for mediating cellular signaling from receptor tyrosine kinases. The modular SH2 protein interaction domains function by recognizing and binding to specific phosphotyrosine (pY or p-Tyr) residues present in many signaling proteins. Consequently, SH2-pY interactions are crucial for signal transmission from receptor tyrosine kinases to downstream targets, resulting in regulation of multiple biological processes. SH2 domains have been identified in numerous human proteins that have specific biological functions, such as adaptor proteins, kinases, phophatases, cytoskeletal regulation, transcription, ubiquitination and phospholipid second messenger signaling. Deregulation of SH2 domain-containining proteins has been implicated in human disease including multiple cancers, diabetes and human immunodeficiency.

SH2 domains are comprised of approximately 100 amino acids that recognize binding partners in a highly specific manner. Binding of an SH2 domain-containing protein is dependent upon phosphorylation of a specific tyrosine residue in a particular amino acid consensus sequence (e.g., Src recognition sequence pYEEI). SH2 domains are essential for signal transmission and downstream biological processes and are present in many different protein classes including kinases, phosphatases, adaptor proteins, phospholipid second messengers and cytoskeletal regulators.

Accurately measuring phosphorylation states and signaling events is challenging because the events are transient and there are few specific detection reagents (e.g., antibodies) available. An affinity system based on recombinant SH2 domains overcomes these challenges by using the critical mediators or regulators of cell signaling events to capture specific tyrosine phosphorylation interactions from mammalian cell lysates. The improved specificity of the pull-down method based on specific SH2 domains means less non-specific binding and no antibody contamination compared with traditional immunoprecipitation experiments using anti-pY antibodies.

Application Experiments

Monitoring the phospho-tyrosine state of specific proteins

Each SH2 domain is specific for its natural target. In vivo, the phosphatase Shp2 is recruited to tyrosine 1009 of the platelet-derived growth factor receptor (PDGFR) only when it is phosphorylated. Also, PLCg is recruited to tyrosine 1021 on PDGFR in response to growth factor signaling. In quiescent cells both tyrosine 1009 and 1021 are not phosphorylated and, therefore, both domains are unable to bind.

Measuring receptor phosphorylation time-course

With SH2 domain pull-downs, it is possible to monitor timedependent binding of SH2 domains to their target. For example, PLCg, Src and Shc bind rapidly to the PDGF receptor in response to PDGF stimulation. In nature, phosphorylation is rapid and transient, as evidenced by diminished binding at 10- and 20-minute post-stimulation.

Time-specific binding of various SH2 domains to the PDGF receptor. NIH3T3 cells were rendered quiescent by serum withdrawal for 48 hours. Post-starvation cells were stimulated with PDGF (50ng/ml, CST) for the indicated times or untreated. SH2 domain pull-downs were performed with 100µg of each SH2 domain and 250µg of each cell lysate. Protein complexes were resolved by SDS-PAGE and analyzed by Western blot using a pan antibody recognizing PDGFR (CST).

Resolving specific tyrosine phosphorylation events Identifying binding interactions with MS

SH2 domain affinity provides a level of selectivity not previously attainable with generic anti-phosphotyrosine antibodies. Use different SH2 domains to uncover the mechanism of how signals are transduced or to screen multiple SH2 domains against a particular target at one time. For example, different naturally occurring SH2 domains efficiently bind to the epidermal growth factor receptor (EGFR) in response to EGF stimulation. There were strong interactions of many of the domains in the presence of epidermal growth factor (EGF) and low-level binding in quiescent, serum-starved A431 cells. RasGap binding is independent of EGF stimulation, whereas Grb2 binding is highly upregulated.

Identifying binding interactions with mass spectrometry (MS)

Pull-down samples isolated with SH2 domains are compatible with MS analysis for identifying protein interacting partners. Improved specificity with SH2 domains and the elimination of antibody contamination typically seen in traditional immunoprecipitation experiments deliver pure protein for MS analysis. Two different SH2 domains were used to pull down interacting proteins from NIH3T3 cell lysates, which were then analyzed by MS. Many of the interacting proteins identified are specific to either Fgr or PLCg.

SH2 Domain Phosphotyrosine Capture Kits

Description

Thermo Scientific SH2 Domain Phosphotyrosine Capture Kits are fast and efficient systems for purifying active tyrosine phosphorylated proteins and interactors in mammalian cell lysates.

These kits include optimal levels of purified GST-fused SH2 domains of various signaling proteins integral to cell biology. Using SH2 domains eliminates the background associated with low-specificity antibodies and enables analysis of receptor targets to which antibodies are not available. Using quality-tested purified GST-SH2 domains also ensures uniform results without variability.

The procedure involves using the GST-tagged SH2 domain and glutathione agarose resin to capture and pull down the specific binding partners. Each kit contains 600 µg of specific GST-SH2 Domain and sufficient reagents (including negative controls) to perform six pull-down reactions with approximately 500 µg of cell lysate.

Highlights

• Monitor tyrosine phosphorylation events using naturally occurring SH2 protein interaction domain
• Profile receptor tyrosine-kinase (RYK) activation
• Replace low-affinity antibodies with highly selective SH2 domains
• Identify a SH2 domain protein-protein interaction network for a particular SH2 domain
• Identify full-length endogenous proteins in a non-peptide-based format
• Capture multiple phosphorylated proteins
• Integrate into mass spectrometry
• Map the SH2 interactome

Procedure Summary

References:

1. Hanke, S. and Mann, M. (2008). The phosphotyrosine interactome of the insulin receptor family and its substrates IRS-1 and IRS-2. Mol. Cell. Proteomics Nov 11. [Epub ahead of print].
2. Liu, B.A., et al. (2006). The human and mouse complement of SH2 domain proteins-establishing the boundaries of phosphotyrosine signaling. Mol. Cell. 22(6):851-68.
3. Machida, K., et al. (2007). High-throughput phosphotyrosine profiling using SH2 domains. Mol. Cell. 26(6):899-915.
4. Major, M., et al. (2009). Capture of specific tyrosine phosphorylated proteins using SH2 domains. Previews 13(1): 9-11.
5. Major, M., et al. Exploration of the phospho-tyrosine proteome using SH2 domains. In: Proceedings of the 100th Annual Meeting of the American Association for Cancer Research; 2009 Apr 18-22; Denver, CO. AACR; 2009. Abstract nr 2625.
6. Pawson T. (2004). Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 116(2):191-203. Review.
7. Online resource: "SH2ome"  http://sh2.uchicago.edu.

Related Products:
Phosphoprotein Enrichment Kit
Phosphopeptide Isolation Kit