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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 of ADAM13 does not affect its proteolytic activity. (A) Western blot from Cos-7 cells transfected with the protocadherin PAPC and various ADAM13 constructs. The glycoproteins purified from the conditioned media were probed with an antibody to the extracellular domain of PAPC or with 7C9, a monoclonal antibody to the cysteine-rich domain of ADAM13. E/A is a catalytically inactive variant of ADAM13. (B) The histogram represents the percentage of embryos displaying no migration from targeted injection of cadherin-11 mRNA alone or together with the ADAM13 variants. Values are normalized to RFP alone. Error bars are SD. n, number of embryos scored from at least three independent experiments. *p < 0.05, **p < 0.01.
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Figure 5: Phosphorylation of ADAM13 does not affect its proteolytic activity. (A) Western blot from Cos-7 cells transfected with the protocadherin PAPC and various ADAM13 constructs. The glycoproteins purified from the conditioned media were probed with an antibody to the extracellular domain of PAPC or with 7C9, a monoclonal antibody to the cysteine-rich domain of ADAM13. E/A is a catalytically inactive variant of ADAM13. (B) The histogram represents the percentage of embryos displaying no migration from targeted injection of cadherin-11 mRNA alone or together with the ADAM13 variants. Values are normalized to RFP alone. Error bars are SD. n, number of embryos scored from at least three independent experiments. *p < 0.05, **p < 0.01.

Mentions: To determine the effect of GSK3 and Plk phosphorylations on ADAM13 proteolytic activity, we tested the ability of the nonphosphorylatable mutants to cleave substrates both in vitro and in vivo. When cotransfected with the protocadherin PAPC in Cos-7 cells, both nonphosphorylatable ADAM13 variants Plk/A and Gsk/A are able to shed the extracellular domain of PAPC from the cell surface as efficiently as wild-type ADAM13, whereas the protease active-site mutant ADAM13-E/A cannot (Cousin et al., 2011; Figure 5A). Similarly, the phosphodeficient variants are also able to undergo autoproteolysis to shed their own ectodomain into the medium (Gaultier et al., 2002; Figure 5A). In addition, coexpressing Plk-DN or GSK3-DN does not prevent wild-type ADAM13 from shedding PAPC (unpublished data).


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 of ADAM13 does not affect its proteolytic activity. (A) Western blot from Cos-7 cells transfected with the protocadherin PAPC and various ADAM13 constructs. The glycoproteins purified from the conditioned media were probed with an antibody to the extracellular domain of PAPC or with 7C9, a monoclonal antibody to the cysteine-rich domain of ADAM13. E/A is a catalytically inactive variant of ADAM13. (B) The histogram represents the percentage of embryos displaying no migration from targeted injection of cadherin-11 mRNA alone or together with the ADAM13 variants. Values are normalized to RFP alone. Error bars are SD. n, number of embryos scored from at least three independent experiments. *p < 0.05, **p < 0.01.
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Related In: Results  -  Collection

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Figure 5: Phosphorylation of ADAM13 does not affect its proteolytic activity. (A) Western blot from Cos-7 cells transfected with the protocadherin PAPC and various ADAM13 constructs. The glycoproteins purified from the conditioned media were probed with an antibody to the extracellular domain of PAPC or with 7C9, a monoclonal antibody to the cysteine-rich domain of ADAM13. E/A is a catalytically inactive variant of ADAM13. (B) The histogram represents the percentage of embryos displaying no migration from targeted injection of cadherin-11 mRNA alone or together with the ADAM13 variants. Values are normalized to RFP alone. Error bars are SD. n, number of embryos scored from at least three independent experiments. *p < 0.05, **p < 0.01.
Mentions: To determine the effect of GSK3 and Plk phosphorylations on ADAM13 proteolytic activity, we tested the ability of the nonphosphorylatable mutants to cleave substrates both in vitro and in vivo. When cotransfected with the protocadherin PAPC in Cos-7 cells, both nonphosphorylatable ADAM13 variants Plk/A and Gsk/A are able to shed the extracellular domain of PAPC from the cell surface as efficiently as wild-type ADAM13, whereas the protease active-site mutant ADAM13-E/A cannot (Cousin et al., 2011; Figure 5A). Similarly, the phosphodeficient variants are also able to undergo autoproteolysis to shed their own ectodomain into the medium (Gaultier et al., 2002; Figure 5A). In addition, coexpressing Plk-DN or GSK3-DN does not prevent wild-type ADAM13 from shedding PAPC (unpublished data).

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