An alternative method of panning for rat B lymphocytes

Severson, Charles D.; Burg, Debra L.; Lafrenz, D; Feldbush, Thomas L.

doi:10.1016/0165-2478(87)90130-1

Cited by 43 publications

(16 citation statements)

References 11 publications

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“…PKD-deficient DT40 cells were a kind gift of Dr. Sharon Matthews (University of Dundee). B cells were enriched from murine spleens as described (11). Plasmids for the expression of murine Syk tagged at the C terminus with either a Mycepitope or enhanced green fluorescent protein (EGFP) were as described previously (12,13).…”

Section: Methodsmentioning

confidence: 99%

Regulation of Syk by Phosphorylation on Serine in the Linker Insert

Paris

Galán

et al. 2010

Journal of Biological Chemistry

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The Syk protein-tyrosine kinase is phosphorylated on multiple tyrosines after the aggregation of the B cell antigen receptor. However, metabolic labeling experiments indicate that Syk is inducibly phosphorylated to an even greater extent on serine after receptor ligation. A combination of phosphopeptide mapping and mass spectrometric analyses indicates that serine 291 is a major site of phosphorylation. Serine 291 lies within a 23-amino acid insert located within the linker B region that distinguishes Syk from SykB and Zap-70. The phosphorylation of serine-291 by protein kinase C enhances the ability of Syk to couple the antigen receptor to the activation of the transcription factors NFAT and Elk-1. Protein interaction studies indicate a role for the phosphorylated linker insert in promoting an interaction between Syk and the chaperone protein, prohibitin.The Syk protein-tyrosine kinase is an essential component of the protein machinery that couples receptors bearing immunoreceptor tyrosine-based activation motifs to intracellular signal transduction pathways in hematopoietic cells (for a recent review, see Ref. 1). Syk is recruited via its N-terminal pair of Src homology 2 domains to a pair of tyrosines located in the cytoplasmic immunoreceptor tyrosine-based activation motifs of immune recognition receptors that become phosphorylated after ligand-induced aggregation. For example, clustering of the B cell receptor for antigen (BCR) 4 leads to the phosphorylation of the accessory molecules CD79a and CD79b, resulting in the recruitment of Syk to the clustered BCR complex. Receptor binding leads to the activation of Syk and to its phosphorylation on tyrosines that regulate its activity and interactions with downstream effectors that bear Src homology 2 or other phosphotyrosine binding domains. This in turn couples the BCR to the activation of, among others, the phospholipase ␥/PKC, PI3K/Akt, and Ras/Raf/Erk signaling pathways. In the absence of Syk, few if any signals are sent after BCR clustering (2-4).Syk contains a stretch of 23 amino acids known as the "linker insert" that lies within the linker B region that separates the tandem pair of Src homology 2 domains from the C-terminal catalytic domain. The presence of this insert distinguishes Syk from SykB, a shorter alternatively spliced isoform, and from ZAP-70, the Syk paralog found in T cells and natural killer cells (5, 6). The role of the linker insert in modulating the properties of Syk is poorly understood, but two functions have been proposed. In one, the linker insert harbors a signal sequence that allows the kinase to transit into the nucleus (7). In another, the insert enhances the ability of the kinase to interact with immunoreceptor tyrosine-based activation motif-bearing receptors (8). Within the linker insert (TWSPGGIISRIKSYSFPKPGHKK) are several potential sites of phosphorylation. The tyrosine (Tyr-290 in the murine Syk sequence) is a site of autophosphorylation in vitro (9) but to our knowledge is not known to be phosphorylated in response to r...

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Section: Methodsmentioning

confidence: 99%

Regulation of Syk by Phosphorylation on Serine in the Linker Insert

Paris

Galán

et al. 2010

Journal of Biological Chemistry

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“…Where indicated, some experiments used pools of stably transfected, antibiotic-selected cells. Primary B cells were enriched from mouse splenocytes by a panning procedure described previously (30).…”

Section: Methodsmentioning

confidence: 99%

Nucleocytoplasmic Trafficking of the Syk Protein Tyrosine Kinase

Zhou

et al. 2006

Molecular and Cellular Biology

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The protein tyrosine kinase Syk couples the B-cell receptor (BCR) for antigen to multiple intracellular signaling pathways and also modulates cellular responses to inducers of oxidative stress in a receptorindependent fashion. In B cells, Syk is found in both the nuclear and cytoplasmic compartments but contains no recognizable nuclear localization or export signals. Through the analysis of a series of deletion mutants, we identified the presence of an unconventional shuttling sequence near the junction of the catalytic domain and the linker B region that accounts for Syk's subcellular localization. This localization is altered following prolonged engagement of the BCR, which causes Syk to be excluded from the nucleus. Nuclear exclusion requires the receptor-mediated activation of protein kinase C and new protein synthesis. Both of these processes also potentiate the activation of caspase 3 in cells in response to oxidative stress in a manner that is dependent on the localization of Syk outside of the nucleus. In contrast, restriction of Syk to the nucleus greatly diminishes the stress-induced activation of caspase 3.Syk is a 72-kDa protein tyrosine kinase with an N-terminal, tandem pair of SH2 domains separated by a long linker (linker B) from a C-terminal catalytic domain (40). Syk plays a critical role in B cells in coupling the B-cell receptor (BCR) for antigen to multiple downstream signaling pathways, including the activation of phospholipase C␥, mobilization of calcium from intracellular stores, and activation of the Ras/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase, and NF-B pathways (1,4,7,18). Upon receptor cross-linking, the tandem SH2 domains of Syk bind two phosphorylated tyrosines in the conserved immunoreceptor tyrosine-based activation motifs (ITAMs) located within the cytoplasmic domains of the immunoglobulin-␣ (CD79a) and immunoglobulin-␤ (CD79b) components of the BCR complex (3). Subsequent phosphorylation of Syk induces its activation, leading to the binding and/or phosphorylation of adaptor proteins and downstream effectors (6,7,9,13,16,20,32,33). Consequently, Syk-deficient B cells are largely nonresponsive to BCR aggregation (36) and B-cell development in Syk-deficient mice is largely blocked at the transitions from pro-B cells to pre-B cells and from immature to mature B cells (5, 39).Increasing evidence indicates that Syk also has fundamental cellular functions that are receptor and ITAM independent. Paradoxically, in B lymphocytes, the expression of Syk is required for BCR-induced apoptosis, but its presence also protects cells from apoptosis induced by ceramide, osmotic stress, or oxidative stress (9,25,27,28,35,37). While the activation of Syk by BCR engagement requires both intact SH2 domains, its participation in oxidative stress signaling does not, suggesting an ITAM-independent mechanism (8, 26).In lymphoid and epithelial cells, Syk has been reported to reside in both the nucleus and cytoplasm (21,41,43), as has its close family member . In breast canc...

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“…RPMI 2650 cells (ATCC CCL30) were cultured in minimum essential medium-10% FBS-P/S, and HeLa cells (ATCC CCL2) were grown in basal modified Eagle medium-10% FBS-P/S. Primary human B lymphocytes were isolated from 200 ml of whole blood by Ficoll-Hypaque gradient, followed by positive selection with bovine serum albumin (63), and were cultured in RPMI 1640 medium-10% FBS-P/S. All medium components were obtained from Gibco-BRL Life Technologies.…”

Section: Methodsmentioning

confidence: 99%