The 14-3-3ζ Protein Binds to the Cell Adhesion Molecule L1, Promotes L1 Phosphorylation by CKII and Influences L1-Dependent Neurite Outgrowth
Figure 4
14-3-3ζ supports CKII-catalyzed L1ICD phosphorylation.
A. L1ICD was preincubated in the presence or absence of GST-14-3-3ζ followed by incubation with CKII. At different time points, CKII phosphorylation was stopped by adding SDS loading buffer, and samples subjected to SDS-PAGE. Western blot analysis with the 74-5H7 anti-L1 antibody revealed that the band intensities of phosphorylated L1 (phospho-L1ICD) increased over time when L1ICD was preincubated with GST-14-3-3ζ (compare C). B. Upper panel, Comparison of L1ICD phosphorylation by CKII in the presence of GST-14-3-3ζ or GST. The phospho-L1ICD and L1ICD bands at time points 0 and 180 min (representative example given in panel A) were quantified and the ratio of phospho-L1ICD/total L1ICD at t = 0 min and t = 180 min in the presence of GST-14-3-3ζ or GST was calculated. Error bars denote ±SEM based on 3 independent experiments. Lower panel, GST Western Blot analysis of phosphorylation assay samples taken at the indicated time points confirms that equal amounts of GST or GST-14-3-3ζ were present in the reactions. C. L1ICD S1181A mutant was subjected to the same CKII phosphorylation assay as described in A. No band presumably representing newly phosphorylated L1ICD could be observed. D. Samples incubated with CKII for 180 min were subjected to subsequent treatment with λ protein phosphatase 1 (λPP1). Changes in the L1ICD band pattern were analyzed by Western Blot with the 74-5H7 antibody.