Anti-IgM induces up-regulation and tyrosine-phosphorylation of heterogeneous nuclear ribonucleoprotein K proteins (hnRNP K) in a Ramos B cell line
Introduction
HnRNP K protein was first discovered as a component of the hnRNP particle, and is believed to be involved in the processing of pre-mRNA by shuttling between the nucleus and cytoplasm [1], [2]. However, a wide intracellular distribution of hnRNP K in both cytoplasm and nucleus indicates that its biological roles are not limited to the function associated with hnRNP complex. In fact, hnRNP K has a variety of molecular partners, which have been implicated in signal transduction and gene expression (reviewed in references [3], [4]). For example, hnRNP K interacts with tyrosine kinases [5], [6], [7], [8] and protooncoprotein Vav [9], [10]. Structurally, hnRNP K contains a cluster of two proline-rich SH3-binding domains encompassing amino acid residues 289–315, where the SH3 domains of Src, Fyn, and Lyn have been shown to interact in vitro, and that of Vav in vivo [3]. In addition, hnRNP K can be phosphorylated by various external stimuli [6], [7], [11], [12], [13], indicating that it acts as a docking platform in signal transduction cascades.
The B cell antigen receptor (BCR) complex is composed of an antigen-recognizing membrane-bound immunoglobulin M (mIgM) and signal-transducing Igα/Igβ heterodimers [14]. Many studies using transformed and untransformed B lymphocytes have shown that cross-linking of the BCR complex leads to either proliferation or apoptosis, depending on the degree of receptor cross-linking and the stage of B cell maturation [15], [16]. The most proximal signalling step for both proliferation and apoptosis involves protein tyrosine-phosphorylation of Src family protein tyrosine kinases and Vav [14]. Vav phosphorylation is known to be a key step of the antigen receptor signal integration that controls cell cycle, differentiation and apoptosis [17].
In the present study, we employed a functional proteomic approach to identify proteins associated with BCR signalling pathway using human Burkitt lymphoma B cell line, Ramos. This cell line has been shown to undergo apoptosis upon cross-linking of BCR with anti-IgM antibody [16]. With this approach, we found that a certain hnRNP K isoform is up-regulated within a short time treatment of anti-IgM (10 min), and its level is increased until 2-h treatment. Subsequent analysis showed that hnRNP K is tyrosine-phosphorylated in response to BCR ligation. Analysis of phosphorylated proteins, which are co-immunoprecipitated with hnRNP K revealed that hnRNP K binds to phosphorylated Vav transiently after treatment of anti-IgM, and the SH3-binding domain mediates this interaction. Furthermore, Ramos cells expressing the mutant protein lacking the SH3-binding domain are less susceptible to anti-IgM-induced cell death, indicating a functional involvement of hnRNP K during BCR-mediated signalling.
Section snippets
Cell culture reagents
The human IgM-positive, Epstein Barr-negative Burkitt lymphoma cell line, Ramos, was obtained from the American Type Culture Collection (Rockville, MD). Cells were grown at 37 °C with 5% CO2 in RPMI 1640 medium supplemented with 2% sodium bicarbonate, 10% fetal bovine serum, 100 U/ml penicillin G and 50 μg/ml streptomycin. All reagents used for cell culture were purchased from Invitrogen (Grand Island, NY). Electrophoresis reagents, including acrylamide solution (40%), Tris base, glycine,
Results and discussion
To identify proteins that are altered upon BCR ligation with anti-IgM, two-dimensional (2D) protein expression patterns were compared between Ramos cells that were left untreated and that were treated with anti-IgM for various time durations. Fig. 1A shows the Coomassie Blue-stained 2D gel pattern obtained after separation of 1.5 mg protein samples from the untreated control cells. About 600 protein spots ranging from 15–150 kDa molecular mass with pI between 3 and 10 were detected. Fig. 1B is a
Acknowledgments
We thank Dr. Karol Bomsztyk (University of Washington) for supplying reagents (p18Flag-K and anti-hnRNP K antibody). This work was supported by a grant from the Korea Science and Engineering Foundation through Protein Network Research Center at Yonsei University.
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