GSK3 and Polo-like kinase regulate ADAM13 function during cranial neural crest cell migration.
Bottom Line: We further show that inhibition of either kinase blocks CNC migration and that the respective phosphomimetic forms of ADAM13 can rescue these inhibitions.However, these phosphorylations are not required for ADAM13 proteolysis of its substrates, γ-secretase cleavage, or nuclear translocation of its cytoplasmic domain.Of significance, migration of the CNC can be restored in the absence of Plk phosphorylation by expression of calpain-8a, pointing to impaired nuclear activity of ADAM13.
Affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003.Show MeSH
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Mentions: We next tested the requirement of Plk in CNC induction and migration using low doses of a dominant-negative construct, Plk-DN (Descombes and Nigg, 1998). Decreasing Plk activity by injecting 80 pg of Plk-DN mRNA at the eight-cell stage led to 52% inhibition of CNC cell migration (Figure 4A and Supplemental Movie S2). We then asked whether this migration defect could be rescued by coexpressing the ADAM13 phosphomimetic variants. In contrast to GSK3-DN, which was rescued by both phosphomimetics (Figure 3B), only the Plk-mimetic, A13-Plk/D, could restore migration of the CNC when Plk activity was reduced (Figure 4A). This is consistent with a model of successive phosphorylation of ADAM13 cytoplasmic domain by which Plk functions downstream of GSK3. To confirm the effect of Plk-DN on migration, we also performed in situ hybridization to detect the CNC markers Twist and Sox10 and observed similar results to those obtained by fluorescence in the targeted injection assays. We found that whereas CNC induction appeared unaffected by Plk-DN, there was a defect in migration caused by decreasing Plk activity, and this could be rescued by coexpressing ADAM13-Plk/D (Figure 4, B and C). Taken together, these results suggest that during CNC migration, ADAM13 is regulated by Plk and GSK3 phosphorylation and that the Plk phosphorylation is absolutely required, whereas GSK3 phosphorylation can be omitted if the Plk site is phosphorylated.
Affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003.