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GSK3 and Polo-like kinase regulate ADAM13 function during cranial neural crest cell migration.

Abbruzzese G, Cousin H, Salicioni AM, Alfandari D - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003.

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Phosphorylation sites are required for ADAM13 function during CNC migration. (A) Fluorescence images showing representative embryos for the injections of RFP alone, the knockdown with 2MO, or the rescue with wild-type ADAM13. Arrows indicate the position of the three migration segments (from left to right: hyoid, branchial, mandibular). (B) Histogram of targeted injection assays testing the ability of ADAM13 phosphomutants to rescue CNC migration in ADAM13/19-deficient embryos (2MO) from at least three independent experiments. Values are percentages of embryos with no CNC migration, normalized to 2MO + wild-type ADAM13. The error bars correspond to the SD. n, number of embryos scored for each case. Statistical significance of rescue: *p < 0.05, **p < 0.01.
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Figure 1: Phosphorylation sites are required for ADAM13 function during CNC migration. (A) Fluorescence images showing representative embryos for the injections of RFP alone, the knockdown with 2MO, or the rescue with wild-type ADAM13. Arrows indicate the position of the three migration segments (from left to right: hyoid, branchial, mandibular). (B) Histogram of targeted injection assays testing the ability of ADAM13 phosphomutants to rescue CNC migration in ADAM13/19-deficient embryos (2MO) from at least three independent experiments. Values are percentages of embryos with no CNC migration, normalized to 2MO + wild-type ADAM13. The error bars correspond to the SD. n, number of embryos scored for each case. Statistical significance of rescue: *p < 0.05, **p < 0.01.

Mentions: To better understand how ADAM13 function is regulated, we identified several putative phosphorylation sites within the cytoplasmic domain using protein prediction software (ELM; Dinkel et al., 2014). To ask whether phosphorylation at any of these sites is functionally important for ADAM13 activity during CNC migration, we replaced the specific residues with either an alanine to prevent phosphorylation or a negatively charged aspartate to mimic constitutive phosphorylation. We chose targeted injection as the most direct technique to test the ability of these variants to replace the function of endogenous ADAM13 in the CNC (Figure 1A). In this technique, a single CNC precursor cell is injected with red fluorescent protein (RFP) as a lineage tracer to follow migration in vivo (McCusker et al., 2009). In these experiments, percentage inhibition represents the percentage of embryos without CNC migration rather than a decrease in migration length, which can be subjective. Because cleavage patterns can vary in different batches of embryos, we used RFP alone in each experiment to determine the targeting efficiency and normalized the results. In each experiment, the ability of the mutant to rescue migration was compared with wild-type ADAM13. For this assay, we knocked down both ADAM13 and ADAM19 using morpholino oligonucleotides (MOs), since we have shown that the cytoplasmic domains of these ADAMs have overlapping functions and can regulate calpain8-a gene expression to stimulate migration (Cousin et al., 2011). Our results show that replacing ADAM13 with nonphosphorylatable constructs for the GSK3 (A13-Gsk/A) or the Plk sites (A13-Plk/A) did not rescue CNC migration in morphant embryos. Conversely, the phosphomimetic for either of these sites (A13-Gsk/D or A13-Plk/D, respectively) is able to significantly rescue migration (Figure 1B and Supplemental Figure S1). This suggests that phosphorylation at the GSK3 and Plk sites is essential for ADAM13 function in the CNC.


GSK3 and Polo-like kinase regulate ADAM13 function during cranial neural crest cell migration.

Abbruzzese G, Cousin H, Salicioni AM, Alfandari D - Mol. Biol. Cell (2014)

Phosphorylation sites are required for ADAM13 function during CNC migration. (A) Fluorescence images showing representative embryos for the injections of RFP alone, the knockdown with 2MO, or the rescue with wild-type ADAM13. Arrows indicate the position of the three migration segments (from left to right: hyoid, branchial, mandibular). (B) Histogram of targeted injection assays testing the ability of ADAM13 phosphomutants to rescue CNC migration in ADAM13/19-deficient embryos (2MO) from at least three independent experiments. Values are percentages of embryos with no CNC migration, normalized to 2MO + wild-type ADAM13. The error bars correspond to the SD. n, number of embryos scored for each case. Statistical significance of rescue: *p < 0.05, **p < 0.01.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Phosphorylation sites are required for ADAM13 function during CNC migration. (A) Fluorescence images showing representative embryos for the injections of RFP alone, the knockdown with 2MO, or the rescue with wild-type ADAM13. Arrows indicate the position of the three migration segments (from left to right: hyoid, branchial, mandibular). (B) Histogram of targeted injection assays testing the ability of ADAM13 phosphomutants to rescue CNC migration in ADAM13/19-deficient embryos (2MO) from at least three independent experiments. Values are percentages of embryos with no CNC migration, normalized to 2MO + wild-type ADAM13. The error bars correspond to the SD. n, number of embryos scored for each case. Statistical significance of rescue: *p < 0.05, **p < 0.01.
Mentions: To better understand how ADAM13 function is regulated, we identified several putative phosphorylation sites within the cytoplasmic domain using protein prediction software (ELM; Dinkel et al., 2014). To ask whether phosphorylation at any of these sites is functionally important for ADAM13 activity during CNC migration, we replaced the specific residues with either an alanine to prevent phosphorylation or a negatively charged aspartate to mimic constitutive phosphorylation. We chose targeted injection as the most direct technique to test the ability of these variants to replace the function of endogenous ADAM13 in the CNC (Figure 1A). In this technique, a single CNC precursor cell is injected with red fluorescent protein (RFP) as a lineage tracer to follow migration in vivo (McCusker et al., 2009). In these experiments, percentage inhibition represents the percentage of embryos without CNC migration rather than a decrease in migration length, which can be subjective. Because cleavage patterns can vary in different batches of embryos, we used RFP alone in each experiment to determine the targeting efficiency and normalized the results. In each experiment, the ability of the mutant to rescue migration was compared with wild-type ADAM13. For this assay, we knocked down both ADAM13 and ADAM19 using morpholino oligonucleotides (MOs), since we have shown that the cytoplasmic domains of these ADAMs have overlapping functions and can regulate calpain8-a gene expression to stimulate migration (Cousin et al., 2011). Our results show that replacing ADAM13 with nonphosphorylatable constructs for the GSK3 (A13-Gsk/A) or the Plk sites (A13-Plk/A) did not rescue CNC migration in morphant embryos. Conversely, the phosphomimetic for either of these sites (A13-Gsk/D or A13-Plk/D, respectively) is able to significantly rescue migration (Figure 1B and Supplemental Figure S1). This suggests that phosphorylation at the GSK3 and Plk sites is essential for ADAM13 function in the CNC.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003.

Show MeSH
Related in: MedlinePlus