Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry

Abstract

The reversible phosphorylation of tyrosine residues is an important mechanism for modulating biological processes such as cellular signaling, differentiation, and growth, and if deregulated, can result in various types of cancer. Therefore, an understanding of these dynamic cellular processes at the molecular level requires the ability to assess changes in the sites of tyrosine phosphorylation across numerous proteins simultaneously as well as over time. Here we describe a sensitive approach based on multidimensional liquid chromatography͞ mass spectrometry that enables the rapid identification of numerous sites of tyrosine phosphorylation on a number of different proteins from human whole cell lysates. We used this methodology to follow changes in tyrosine phosphorylation patterns that occur over time during either the activation of human T cells or the inhibition of the oncogenic BCR-ABL fusion product in chronic myelogenous leukemia cells in response to treatment with STI571 (Gleevec). Together, these experiments rapidly identified 64 unique sites of tyrosine phosphorylation on 32 different proteins. Half of these sites have been documented in the literature, validating the merits of our approach, whereas motif analysis suggests that a number of the undocumented sites are also potentially involved in biological pathways. This methodology should enable the rapid generation of new insights into signaling pathways as they occur in states of health and disease.M any cellular processes are directly controlled through the reversible phosphorylation of protein tyrosine residues. These regulatory functions are ultimately affected through the coordinated phosphorylation of numerous tyrosine residues across multiple proteins over time. Clearly, there are benefits to individually characterizing specific components of a particular pathway, such as identifying a site of phosphorylation on a given protein, the kinase responsible for the modification, or the identity of subsequently interacting proteins. Ultimately, though, a thorough understanding of these signaling pathways at the molecular level requires the wide-scale, simultaneous evaluation of these phosphorylation events as they occur over time.To date, two-dimensional gel electrophoresis (2D-GE) remains the most common methodology for assessing wide-scale changes in phosphorylation (1). However, this methodology is relatively slow, and suffers from a number of well documented operational limitations. For example, 2D-GE has been shown to be poorly suited for the direct detection and analysis of medium to low abundance proteins from whole cell lysates, a particular concern in the case of regulatory proteins such as kinases, which often exist at very low copy numbers per cell (2). Even with the improved dynamic range afforded by multiple 2D-GE runs of prefractionated samples, the individual gelisolated proteins still require further characterization by using methods such as two-dimensional tryptic phosphopeptide mapping (3), Edman degradation (4), or precursor ion scan...

SDF1α‐induced interaction of the adapter proteins Nck and HS1 facilitates actin polymerization and migration in T cells

“…In detail, the HS1 sites Y 378 and Y 397 are phosphorylated by Syk in vitro [36] and presumably by ZAP70 upon TCR ligation [27]. Besides these tyrosine motifs, in vitro phosphorylation of Y 222 (YKKT) [36][37][38] and Y 198 (YGIQ) [39] was documented. However, since the sequence of these phospho-sites pretty much differs from the Nck SH2 consensus-binding sequence, an interaction with the Nck SH2 domain is rather unlikely.…”

SHEP1 Function in Cell Migration Is Impaired by a Single Amino Acid Mutation That Disrupts Association with the Scaffolding Protein Cas but Not with Ras GTPases

“…In addition to tyrosine 635 in the CA-GEF domain, our mass spectrometry experiments identified tyrosines 121 and 126 in the SHEP1 SH2 domain as phosphorylation sites downstream of EphB2. Tyrosine 121 was also identified as a SHEP1 phosphorylation site downstream of Bcr-Abl in myeloid leukemia cells (56). Phosphorylation of tyrosine 121, which is in strand ␤E of the SH2 domain, may regulate the binding affinity and specificity of the SHEP1 SH2 domain-like phosphorylation of the similarly located tyrosine in the Src and Lck SH2 domains (57,58).…”

Large‐scale proteomic analysis of tyrosine‐phosphorylation induced by T‐cell receptor or B‐cell receptor activation reveals new signaling pathways

“…Immunoaffinity purification based on PTMs such as phosphorylation, ubiquitylation, and acetylation allows the enrichment of proteins targeted for such modification. The combination of such purification with online LC-MS/MS analysis then allows the identification of large sets of proteins with specific PTMs [3][4][5][6][7][8][9][10][11][12]. Furthermore, methods for tagging with stable isotopes, such as stable isotope labeling in cell culture (SILAC), ICAT, or iTRAQ, provide quantitative information about proteins or peptides from MS or MS/MS spectra [13][14][15][16].…”