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 then tested, first, whether the two kinases were present in the CNC and, second, whether they could phosphorylate ADAM13. To answer the first question, we performed a Western blot on CNC dissected at stage 17 using antibodies specific to either kinase. Indeed, each antibody detected the predicted size band (Figure 2A), demonstrating that both GSK3 and Plk proteins are present in the CNC during migration. We then produced an antibody against a phosphorylated peptide corresponding to the Plk site in ADAM13. The antibody was affinity purified against the phosphorylated peptide and affinity depleted with the nonphosphorylated peptide. Human HEK293T cells were transfected with either the wild-type ADAM13 or the nonphosphorylatable mutant A13-Plk/A. We found that wild-type ADAM13 was clearly phosphorylated in these cells, whereas the mutant was not (Figure 2B). In addition, treatment of the cells with either a Plk inhibitor or a GSK3 inhibitor prevented this phosphorylation, showing that in HEK293T cells, endogenous Plk did phosphorylate ADAM13. This further suggests that in the absence of GSK3, Plk did not phosphorylate ADAM13. Of interest, in these cells, the main form of ADAM13 phosphorylated is shorter (∼65 kDa) than the mature ADAM13 (M; 100 kDa), suggesting that this form may not contain the metalloprotease domain (form D). This shorter form of ADAM13 is also the one phosphorylated in vitro by purified GSK3 (Figure 2C). Phosphorylation was increased in the ADAM13 mutant A13-Gsk/D, suggesting that the third GSK3 phosphorylation site (885T) of ADAM13 can also be phosphorylated in vitro.
Affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003.