The covalent modification of proteins by phosphorylation of specific amino acids has evolved as a powerful and rapid way for cells to respond to a multitude of internal and external stimuli. Traditionally, the study of phosphorylation has been restricted to a "one experiment-one protein" approach. A paper in this week’s early online PNAS shows how the advent of the mass spectrometer (MS)-which can simultaneously separate and characterize thousands of peptides in a complex sample-has made it feasible to measure the phosphorylation and dephosphorylation of proteins en masse.
Researchers at the Novartis Research Foundation and Scripps Research Institute have put this idea to the test, specifically investigating the modification of tyrosine residues. Led by Eric Peters at Novartis, joint first authors Arthur Salomon and Scott Ficarro first used antibodies to enrich cell extracts for phosphotyrosine proteins. This key treatment was essential to reduce background from phosphoserine/threonine peptides that can carry through the experimental procedure and interfere with the analysis. The authors then examined protein phosphorylation/dephosphorylation in response to two types of stimuli-the activation of human T cells by CD3 antibodies, and the response of leukemia cells to the anticancer agent Gleevec.
T cell activation has been well studied and served as a yardstick by which to measure the MS technique. The authors easily detected modification of the16 tyrosine residues that are known to be phosphorylated upon activation of these cells. Moreover, they found five additional sites on four different proteins, CAS-L, CD3, HS1, and LIM.
Next Salomon et al. turned their attention to the effect of Gleevec on chronic myeloid leukemia (CML) cells. Gleevec is an inhibitor of the fusion protein BCR-ABL, itself a tyrosine kinase; the constitutive activation of BCR-ABL leads to this type of leukemia. Activation of BCR-ABL is caused by phosphorylation of tyrosine 177 on BCR, and tyrosine 393 on the fusion protein. When Salomon et al. treated K562 human CML cells with the anticancer agent, these two sites were dephosphorylated within three hours. In total, 19 sites on nine different proteins lost their phosphate moiety while eleven new phosphotyrosines, each on a different protein, were detected.
The power of this type of global analysis is evident by the fact that the data ties together proteins and pathways previously linked by weak correlative evidence. For example, in K562 cells Gleevec is known to upregulate hemoglobin and the protein CD11b, as does activation of the granulocyte-colony stimulating factor (G-CSF) pathway. Salomon et al. were able to show that three members of the G-CSF pathway, Syk, cortactin, and SHC, are phosphorylated in response to Gleevec, thus providing hard evidence that the inhibitor and pathway are inextricably linked.—Tom Fagan
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- Salomon AR, Ficarro SB, Brill LM, Brinker A, Phung QT, Ericson C, Sauer K, Brock A, Horn DM, Schultz PG, Peters EC. Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry. Proc Natl Acad Sci U S A. 2003 Jan 21;100(2):443-8. PubMed.