Protein-tyrosine kinase, Syk, is required for CXCL12-induced polarization of B cells

https://doi.org/10.1016/j.bbrc.2005.01.076Get rights and content

Abstract

Cell polarization and migration in response to CXCL12 is essential for hematopoiesis. To investigate the role of Syk in CXCL12/CXCR4-induced signaling, wild-type Syk or its dominant-negative form (DN-Syk) was introduced in mouse pro-B cells, BAF3. With CXCL12 stimulation, BAF3 cells became polarized with the formation of a leading edge and contractile uropod at the rear end with increased motility. Overexpression of wild-type Syk caused enhanced polarization, whereas DN-Syk inhibited cell polarity due to the loss of contractile structure at the rear end, and the altered phenotype was enhanced after CXCL12 stimulation. Motility of mutant BAF3 containing DN-Syk increased independent of CXCL12 stimulation. As β1 integrin-mediated cell adhesion was inhibited, decreased adhesion might promote motility. CXCL12 stimulation led to prompt activation of RhoA, but expression of DN-Syk suppressed RhoA activation. These results demonstrate that Syk participates in CXCL12-induced cell polarization, which occurs in concert with cell adhesion mediated by β1 integrin.

Section snippets

Materials and methods

Cells and cell culture. BAF3, a mouse IL-3-dependent pro-B cell line, and its mutant cell clones were cultured in RPMI1640 medium (Sigma, St. Louis, MO, USA) containing 10% fetal calf serum (FCS), 50 μM β-mercaptoethanol, and IL-3 in 5% CO2 humidified air at 37 °C.

Plasmids and transfection. Human syk cDNA, provided by Dr. Muller [11], was modified as previously described [12]. The PCR product of Flag-epitope-tagged (DYKDDDDK) human syk cDNA and the PCR product of Flag-tagged dominant-negative

CXCL12 induces tyrosine phosphorylation of Syk kinase

Previous studies have shown that CXCR4 signaling induced by CXCL12 participates in the retention of normal hematopoietic stem cells within the bone marrow [14], [15], [16], and Syk is potentially involved in hematopoiesis of the B cell lineage [17], [18]. To determine whether Syk is involved in hematopoiesis via CXCL12/CXCR4-induced signaling, BAF3 cells were stimulated with CXCL12, and tyrosine phosphorylation of whole cell lysates and Syk protein was analyzed. Rapid tyrosine phosphorylation

Acknowledgments

This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, the 21st Century COE Program of the Ministry of Education, and The Osaka Medical Research Foundation for Incurable Diseases.

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