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Submitted on November 14, 2003
Pharmacology Dept., UT Southwestern Medical Center, Dallas, TX 75390-9196
Corresponding Author: deirdre.brekken{at}utsouthwestern.edu
A major goal of the Alliance for Cellular Signaling (AfCS) is to elaborate the components of signal transduction networks in model cell systems, including murine B lymphocytes. Due to the importance of protein phosphorylation in many aspects of cell signaling, the initial efforts have focused on the identification of phosphorylated proteins. In order to identify serine and threonine phosphorylated proteins on a proteome-wide basis, WEHI-231 cells were treated with calyculin A, a serine/threonine phosphatase inhibitor to induce high levels of protein phosphorylation. Proteins were extracted from whole cell lysates and digested with trypsin. Phosphorylated peptides were then enriched using immobilized metal affinity chromatography (IMAC) and identified by liquid chromatography-tandem mass spectrometry. A total of 107 proteins and 193 phosphorylation sites were identified using these methods. Forty-two of these proteins have been reported to be phosphorylated, but only some of them have been detected in B cells. Fifty four of the identified proteins were not previously known to be phosphorylated. The remaining 11 phosphoproteins have previously only been characterized as novel cDNA or genomic sequences. Many of the identified proteins were phosphorylated at multiple sites. The proteins identified in this study significantly expand the repertoire of proteins known to be phosphorylated in B cells. The number of newly identified phosphoproteins indicates that B cell signaling pathways utilizing protein phosphorylation are likely to be more complex than previously appreciated.
Revised on January 15, 2004
Accepted on January 17, 2004
Identification of phosphoproteins and their phosphorylation sites in the WEHI-231 B lymphoma cell line
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