Ligand-Induced Phosphorylation of LSP1

While the phosphorylation of LSP1 is modulated in response to several stimuli in T cells and neutrophils, pathways leading to LSP1 phosphorylation have not been as well studied in B cells. To further characterize the phosphorylation of LSP1, we examined the effects of physiological stimuli in addition to PMA. In order to obtain more quantitative measures of LSP1 phosphorylation, the ³²P contents of individual LSP1 spots were determined. WEHI-231 cells were labeled with ³²P and stimulated for 10 minutes with either PMA, anti-IgM to activate the B-cell receptor, or with interleukin-4 (IL-4). The ³²P-labeled proteins from stimulated and control cells were then resolved by 2-D gel electrophoresis. The ³²P-labeled spots corresponding to LSP1 were detected and quantitated using Progenesis software.

Fig. 4. Quantitative analysis of ligand-induced changes in LSP1 phosphorylation in WEHI-231 cells. WEHI-231 cells were labeled with 32P inorganic phosphate for 3 hr and stimulated with IL-4, anti-IgM, or PMA for 10 min. Proteins were resolved by 2-D gel electrophoresis and the dried gels were exposed to phosphor screens for 72 hr. Following imaging of the phosphor screens, the 32P-labeled spots corresponding to LSP1 were detected and quantitated with Progenesis software. The region of the 2-D gels containing the LSP1 spots from control cells (A), cells treated with IL-4 (B), cells treated with anti-IgM (C), and cells treated with PMA (D) are shown in the upper section of the figure. Spots detected by the software are labeled with blue crosses. Spots labeled L1 to L7 correspond to the different charge forms of WEHI-231 LSP1. The 32P spots labeled a through d are reference spots for which intensities and mobilities did not change under the different conditions. Panel E shows a graph of the normalized volumes of spots L1 to L7 under the different conditions. The data shown are the mean ± standard deviation of four separate gels.

Seven ³²P-labeled spots with mobilities corresponding to LSP1 were detected in WEHI-231 cells. The most basic, least phosphorylated, spot (L7) was faint and only observed in control cells or cells treated with IL-4 (Fig. 4, panels A and B). Stimulation of WEHI-231 cells with IL-4 caused little change in LSP1 phosphorylation. The volumes of spots L5, L6, and L7 were slightly decreased, while the volume of spot L2 was increased (Fig. 4B and 4E). Stimulation of the B-cell receptor with anti-IgM caused a significant increase in LSP1 phosphorylation. The most basic spot (L7) disappeared, and the volumes of spots L5 and L6 were reduced relative to the controls. The volume of acidic spot L3 increased and two new acidic spots (L1 and L2) appeared (Fig. 4C and 4E). PMA-stimulation caused the greatest increase in LSP1 phosphorylation (Fig. 4D and 4E). Spots L6 and L7 were barely detectable, while the volumes of spots L1 and L2 were greatly enhanced. The similarities in LSP1 phosphorylation in response to anti-IgM and PMA are consistent with a role for PKC in phosphorylation of LSP1.

Fig. 5. Detection of ligand-induced changes in LSP1 phosphorylation in primary B cells. Primary B cells were labeled with 32P inorganic phosphate and stimulated for 10 min with IL-4, anti-IgM, or PMA. Proteins were resolved by 2-D gel electrophoresis and analyzed as described for Fig. 4. Regions of the 2-D gel images containing LSP1 from control (A), IL-4-stimulated (B), anti-IgM-stimulated (C), and PMA-stimulated (D) cells are shown. The 32P spots detected by Progenesis software are marked by a blue cross. Spots labeled L1 to L7 and S1 to S6 correspond to the different charge forms of the long and short splice variants of LSP1, respectively. Spots labeled a, b, and c are reference spots that had similar mobilities under all conditions.

The effects of IL-4, anti-IgM, and PMA on LSP1 phosphorylation were also tested in primary B cells. Isolated B cells were cultured for 3 hours in serum-free medium containing ³²P inorganic phosphate and then stimulated with ligands for 10 minutes. The effects of stimulation on phosphorylation of the long and short forms of LSP1 were quantitated as described for WEHI-231 cells. In contrast to WEHI-231 cells, detectable levels of phosphorylation were observed in both the long and short variants of LSP1. The predominant ³²P-labeled spots in non-stimulated B cells were L3 and L4, as opposed to L4 and L5 in WEHI-231 cells, indicating that the level of LSP1 phosphorylation in resting B cells was quite high. Stimulation with IL-4 had minimal if any effect on LSP1 (Fig. 5B). Stimulation with anti IgM caused an increase in phosphorylation, as observed by the appearance of new acidic spots (L1 and S1); an increase in the intensities of spots L2, L3, S2, and S3; and a decrease in the intensities of spots L6, L7, S5, and S6 (Fig. 5C). PMA treatment caused the greatest increase in phosphorylation, as judged by the increased intensities of acidic spots and decreased intensities of basic spots. Overall, the effects of ligand and PMA stimulation of primary B cells were very similar to those seen in WEHI-231 cells.