Chapter 20
Electrospray Ionization Mass Spectrometric Investigation of Signal Transduction Pathways Determination of Sites of Inducible Protein Phosphorylation in Activated Τ Cells 1
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Downloaded by STANFORD UNIV GREEN LIBR on October 11, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch020
Julian D. Watts , Michael Affolter , Danielle L. Krebs , Ronald L. Wange , Lawrence E. Samelson , and Ruedi Aebersold 4
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Department of Molecular Biotechnology, University of Washington, Box 357730, Seattle, WA 98195-7730 NESTEC Ltd. Research Center, Vers-chez-les-Blanc, P.O. Box 44, CH-1000, Lausanne, Switzerland Department of Microbiology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada Cell Biology and Metabolism Branch, National Institute for Child Health and Development, National Institutes of Health, Bethesda, MD 20892 2
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We have developed methodologies for the determination of sites of protein phosphorylation by tandem microbore column chromatography, with on-line detection by electrospray ionization mass spectrometry (ESI-MS), of phosphopeptides recovered from two-dimensional (2D) phosphopeptide maps. We have applied these methods to the identification of sites of tyrosine phosphorylation induced on the lymphocyte-specific protein tyrosine kinase ZAP-70 and the Τ cell receptor (TCR) ζ subunit in vivo, following stimulation of Τ cells via their TCR. Our approach is generally applicable to the investigation of signal transduction pathways since it involves direct ESI-MS analysis of peptides recovered from 2D phosphopeptide maps, currently the analytical technique of choice for the detailed analysis of protein phosphorylation sites, and is fully adaptable for the analysis of serine- and threonine-phosphorylated peptides. It has been known for some time that a cell's ability to respond to external stimuli, be they chemical, physical or hormonal, is regulated to a large part by a wide range of cell surface receptor complexes, which in turn are linked to a variety of intracellular protein complexes and enzymes. The biochemical mechanism by which the triggering of a cell surface receptor is interpreted by the cell, resulting in a defined biological response, is often referred to as signal transduction. Of particular 5
Corresponding author 0097-6156/95/0619-0381$13.75/0 © 1996 American Chemical Society
In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry; Snyder, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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BIOLOGICAL AND BIOTECHNOLOGICAL APPLICATIONS OF ESI-MS
importance in such processes is the role of protein phosphorylation/dephosphorylation of key signal transduction pathway components, events which are carried out by protein kinases and phosphatases, respectively, with many of the critical early phosphorylation events being performed by protein tyrosine kinases (PTK). The main obstacle to unraveling signal transduction pathways is that each cell type expresses many different receptors, with each capable of eliciting a different set of biochemical responses. For example, as is shown in Figure 1, engagement of either the Τ cell receptor (TCR) or interleukin-2 (IL-2) receptor of cultured Τ cells leads to the rapid tyrosine phosphorylation of different subsets of cellular proteins. Figure 2 gives an overview of an activated TCR complex, its subunit structure, and the selection of associated PTKs and co-receptor molecules, known to be involved in TCR-mediated signaling, which are discussed below. Within Τ cells, a number of PTKs have been shown to play a role in the regulation of a range of
Figure 1: Induction of tyrosine phosphorylation in Τ cells. A) Jurkat (human) Τ cells (2 χ 106/lane) stimulated for the indicated times with the anti-(human) CD3e mAb OKT3 at 10 μg/ml essentially as described elsewhere (41, 42). B) IL-2-dependent splenic (murine) Τ cells (3.3 χ 106/lane) were prepared, starved of, and then re-stimulated with recombinant IL-2 at 100 units/ml for the indicated times as described (22). Following SDS-PAGE and transfer to nitrocellulose, tyrosine phosphorylated proteins were visualized with the anti-phosphotyrosine mAb 4G10 detected by an enhanced chemiluminescence system. The relative mobility of molecular mass standards (kDa) are also indicated.
In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry; Snyder, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
20. WATTS ET A L
ESI-MS of Signal Transduction Pathways
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biochemical and cellular responses, generated by the engagement of the TCR (reviewed in 1-3). These include members of the src family, in particular p56^* and p59/yw, which interact with the CD4/CD8 co-receptors and the TCR respectively, along with the syk family member, ZAP-70. p5&ck and p59Ù have been extensively studied in a number of model and transgenic cellular systems, and at least one or both have been shown to be vital components in multiple TCR-mediated signal transduction pathways, including positive and negative selection during thymocyte development (4-7), cell proliferation (6, 8), IL-2 production (9, 10) and TCRmediated killing (11, 12). The activation of both p56*
