AfCS Research Reports

Addition of phosphate to LSP1 should cause an increase in negative charge and shift the protein to more acidic isoelectric points. This hypothesis was tested by determining the distribution of the LSP1 spots in response to PMA treatment, a known stimulator of LSP1 phosphorylation(8). WEHI-231 cells were either left unlabeled and untreated (control) or labeled with ³²P inorganic phosphate and then stimulated with PMA (treated). Following lysis in IEF lysis buffer, proteins from control cells were labeled with Cy3 minimal maleimide dye and proteins in the ³²P-labeled, treated lysate were labeled with Cy5 dye. Aliquots from the two lysates were mixed, and the proteins were resolved on the same 2-D gels. Following electrophoresis, images of the Cy3 and Cy5 fluorescence were obtained. The gels were then fixed and dried between pieces of cellophane. The dried gel was then placed in contact with a phosphorimager screen and held in place with tape to detect ³²P. After exposure, the dried gel and phosphor screen were placed together on the surface of a phosphorimager capable of scanning Cy5 fluorescence (Molecular Dynamics Storm 860). An image of the phosphor screen was obtained, followed by imaging of the Cy5 fluorescence to obtain overlapping images of the ³²P-labeled and fluorescently labeled proteins. This combination provided two sets of overlaid images. One set was used to compare the mobilities of the Cy3 and Cy5-labeled LSP1 protein spots in control and treated samples. The other set was used to compare the fluorescently labeled spots with the ³²P-labeled spots from the treated sample.

Fig. 3. LSP1 phosphorylation is enhanced by PMA stimulation. WEHI-231 cells were either unlabeled and untreated (control) or labeled with ³²P inorganic phosphate and stimulated for 10 min with 1mM PMA (treated). Following lysis in IEF lysis buffer, proteins from control cells were labeled with Cy3, and proteins from the treated lysate were labeled with Cy5. Aliquots containing 10mg of each fluorescently labeled fraction were mixed, and proteins were resolved by 2-D gel electrophoresis. Codetection of Cy3 and Cy5, and Cy5 and ³²P, in the same gels was performed as described under Methods and Protocols. Panels A and B show codetected Cy3 and Cy5 images. Panels C and D show a three-dimensional representation of the images shown in A and B. Panels E and F show codetected Cy5 and ³²P images from the same gel. The images show the region of the 2-D gel containing the LSP1 spots and correspond to the area of the gel boxed in Fig. 1A. The pH gradient in this area ranges from pH 4.2 (on the left) to 4.8 (on the right). The lowercase letters a and c in panels A, B, and E refer to reference spots for which mobility was the same in both samples. These are the same reference spots indicated in Fig. 1C. The LSP1 spots are numbered 1 through 6 from left (more acidic) to right (less acidic). The LSP1 numbering system corresponds to that used in Fig. 1.

Stimulation of WEHI-231 cells with PMA resulted in an acidic shift in the LSP1 spots. Four Cy3-labeled LSP1 spots were detected in control cells (Fig. 3A). Following stimulation with PMA, six Cy5-labeled LSP1 spots were detected (Fig. 3B). Fewer total spots were detected in the fluorescent labeling experiments compared to the preparative gels shown in Fig. 1 due to differences in the lysis buffer, the amounts of protein loaded, and the fluorescent labeling of proteins. The second row of faint spots migrating above the main LSP1 spots in the PMA-stimulated sample were likely due to an artifact of the fluorescent labeling. These spots were not observed when the PMA-stimulated sample was labeled with Cy3 rather than Cy5. Since these samples were run on the same 2-D gel, the relative positions of the spots could be directly compared. Analysis of the images with DeCyder software showed that PMA stimulation resulted in an increase in the intensities of spot 3 and the appearance of two new spots (labeled 1 and 2 in Fig. 3B). The increase in the intensity of the acidic spots coincided with a decrease in the relative intensities of the basic spots (labeled 5 and 6 in Fig. 3A and 3B). The PMA-induced mobility shifts could be readily seen in a three-dimensional plot of the fluorescent image data (Fig. 3C and 3D). In Fig. 3C and 3D, a colored line is drawn around spot 4 that was detected in both the Cy3 and Cy5 images. Co-imaging of the Cy5 fluorescence and ³²P showed that each of the six protein-staining spots present in PMA-stimulated WEHI-231 cells coincided with a ³²P-labeled spot (Fig. 3E and 3F). A similar comparison showed that the four spots detected in the control lysate were also phosphorylated (not shown). These patterns of protein and ³²P-labeled spots indicate that LSP1 is phosphorylated at multiple sites under basal conditions. Stimulation of PKC with phorbol ester caused phosphorylation at additional sites, resulting in increased negative charge and an acidic (leftward) shift in mobility in the IEF dimension. Increased phosphorylation of LSP1 also appeared to cause a slight reduction in mobility in the SDS dimension. Based on the number of spots detected under basal and PMA- stimulated conditions, at least six phosphorylated forms of LSP1 were present in WEHI-231 cells. With higher protein loads, a seventh spot (L7) was detected (Fig. 1). These observations are consistent with previous 2-D gel data from murine T cells, indicating that LSP1 has at least six phosphorylation sites(10). With the amounts of protein loaded in this experiment, only the long splice variant of LSP1 was detected in WEHI-231 cells by either fluorescent- or ³²P-labeling.