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: The cleaved cytoplasmic domain of ADAM13 (C13) must translocate to the nucleus and increase calpain8-a (Capn8) expression to promote CNC migration (Cousin et al., 2011). Therefore we investigated whether phosphorylation by Plk is necessary to stimulate the cleavage of C13 from the membrane-bound protease, its nuclear translocation, or its activity in gene regulation. We first showed that the cytoplasmic domain of ADAM13-Plk/A is cleaved in embryos, indicating that intramembrane processing by γ-secretase is not dependent on phosphorylation by Plk (Figure 6A). We then generated the alanine substitutions at the GSK3 and Plk sites in a construct containing C13 fused to green fluorescent protein (GFP-C13) to observe their subcellular localization. When transfected into Cos-7 cells, we found that, similar to wild-type GFP-C13, both variants containing the nonphosphorylatable alanine substitutions, GFP-C13-Plk/A and GFP-C13-Gsk/A, accumulate in the nucleus (Figure 6B). Thus the requirements for nuclear translocation of C13 do not rely on phosphorylation at the GSK3 or Plk sites.
Affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003.