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: Our mutation analysis suggests that GSK3 and Polo kinases should be required for CNC migration at least to phosphorylate ADAM13. Because these kinases are critical in multiple biological processes, we next tested whether lowering their activity in the CNC would cause a defect in migration without other major developmental defects. In particular, GSK3 was shown to phosphorylate the transcription factor Twist to modify its activity (Lander et al., 2013). A nonphosphorylatable form of Twist did not bind to and inhibit Slug/Snail2, suggesting that phosphorylation by GSK3 is critical for normal Twist function during CNC development. To lower each kinase activity, we used previously characterized dominant-negative constructs and tested their ability to interfere with CNC migration without inhibiting CNC induction when injected at relatively low doses. Our results show that 300 pg of the GSK3 dominant negative (GSK3-DN; Dominguez et al., 1995) injected at the eight-cell stage in a CNC precursor perturbed CNC migration in 30% of the injected embryos, whereas it did not perturb the expression of Slug in premigratory CNC (Figure 3). We also performed grafts on embryos injected with the dominant-negative GSK3 that confirmed that migration was inhibited in these embryos (Supplemental Movie S1). We then coinjected either wild-type or the phosphomimetic forms of ADAM13 together with the GSK3-DN to test their ability to rescue migration. We found that expressing both ADAM13-Gsk/D and -Plk/D, but not the wild-type ADAM13, could rescue the loss of migration caused by GSK3-DN (Figure 3, B and C). This confirms that one of the key substrates of GSK3 during CNC migration is ADAM13 and that phosphorylations at the GSK3 and Plk sites are required for proper ADAM13 function in vivo.
Affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003.