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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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ADAM13-dependent regulation of calpain8 expression depends on ADAM13 phosphorylation. (A) ADAM13 was immunoprecipitated (IP) from 20 embryos using a goat polyclonal antibody g821. Noninjected embryos (NI) at stage 18 (neurula) are compared with sibling embryos injected with MO13 or MO13 plus MO-resistant mRNA encoding ADAM13-Plk/A or wild type. ADAM13 protein was detected by Western blot using 6615F. The polyvinylidene fluoride membrane was cut below 25 kDa and the two halves probed separately. The cleaved cytoplasmic domain (Cyto) is observed at 17 kDa for both wild-type and Plk/A ADAM13. Samples of the input for the IP were analyzed by Western blot with the 8C8 antibody for β1 integrin as a loading control. (B) Fluorescence images showing the localization of GFP-fusion proteins (green) expressed in Cos-7 cells along with membrane-bound mCherry (red). GFP is observed uniformly throughout the cytoplasm and nucleus, whereas GFP-C13 and the phosphodeficient mutants all accumulate strongly in the nucleus. (C, D) Analyses of targeted injection assays displaying the loss of CNC migration as a percentage of embryos. (C) MO13 + MO19 (2MO) was coinjected with mRNA encoding ADAM13 lacking its cytoplasmic domain (ΔCyto) plus either GFP-C13 wild type or phosphomutants. Values are normalized to the rescue with GFP-C13 wild type, and Student's t tests were performed to compare values to 2MO + ΔCyto. (D) 2MO was injected with ADAM13-Plk/A or ADAM13-Gsk/A mRNA alone or together with Capn8 mRNA. Inhibitions are normalized to RFP, and Student's t tests were performed against 2MO. Error bars are SD from four or more independent experiments. n, number of embryos scored. **p < 0.01, ***p < 0.005.
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Figure 6: ADAM13-dependent regulation of calpain8 expression depends on ADAM13 phosphorylation. (A) ADAM13 was immunoprecipitated (IP) from 20 embryos using a goat polyclonal antibody g821. Noninjected embryos (NI) at stage 18 (neurula) are compared with sibling embryos injected with MO13 or MO13 plus MO-resistant mRNA encoding ADAM13-Plk/A or wild type. ADAM13 protein was detected by Western blot using 6615F. The polyvinylidene fluoride membrane was cut below 25 kDa and the two halves probed separately. The cleaved cytoplasmic domain (Cyto) is observed at 17 kDa for both wild-type and Plk/A ADAM13. Samples of the input for the IP were analyzed by Western blot with the 8C8 antibody for β1 integrin as a loading control. (B) Fluorescence images showing the localization of GFP-fusion proteins (green) expressed in Cos-7 cells along with membrane-bound mCherry (red). GFP is observed uniformly throughout the cytoplasm and nucleus, whereas GFP-C13 and the phosphodeficient mutants all accumulate strongly in the nucleus. (C, D) Analyses of targeted injection assays displaying the loss of CNC migration as a percentage of embryos. (C) MO13 + MO19 (2MO) was coinjected with mRNA encoding ADAM13 lacking its cytoplasmic domain (ΔCyto) plus either GFP-C13 wild type or phosphomutants. Values are normalized to the rescue with GFP-C13 wild type, and Student's t tests were performed to compare values to 2MO + ΔCyto. (D) 2MO was injected with ADAM13-Plk/A or ADAM13-Gsk/A mRNA alone or together with Capn8 mRNA. Inhibitions are normalized to RFP, and Student's t tests were performed against 2MO. Error bars are SD from four or more independent experiments. n, number of embryos scored. **p < 0.01, ***p < 0.005.

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.


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)

ADAM13-dependent regulation of calpain8 expression depends on ADAM13 phosphorylation. (A) ADAM13 was immunoprecipitated (IP) from 20 embryos using a goat polyclonal antibody g821. Noninjected embryos (NI) at stage 18 (neurula) are compared with sibling embryos injected with MO13 or MO13 plus MO-resistant mRNA encoding ADAM13-Plk/A or wild type. ADAM13 protein was detected by Western blot using 6615F. The polyvinylidene fluoride membrane was cut below 25 kDa and the two halves probed separately. The cleaved cytoplasmic domain (Cyto) is observed at 17 kDa for both wild-type and Plk/A ADAM13. Samples of the input for the IP were analyzed by Western blot with the 8C8 antibody for β1 integrin as a loading control. (B) Fluorescence images showing the localization of GFP-fusion proteins (green) expressed in Cos-7 cells along with membrane-bound mCherry (red). GFP is observed uniformly throughout the cytoplasm and nucleus, whereas GFP-C13 and the phosphodeficient mutants all accumulate strongly in the nucleus. (C, D) Analyses of targeted injection assays displaying the loss of CNC migration as a percentage of embryos. (C) MO13 + MO19 (2MO) was coinjected with mRNA encoding ADAM13 lacking its cytoplasmic domain (ΔCyto) plus either GFP-C13 wild type or phosphomutants. Values are normalized to the rescue with GFP-C13 wild type, and Student's t tests were performed to compare values to 2MO + ΔCyto. (D) 2MO was injected with ADAM13-Plk/A or ADAM13-Gsk/A mRNA alone or together with Capn8 mRNA. Inhibitions are normalized to RFP, and Student's t tests were performed against 2MO. Error bars are SD from four or more independent experiments. n, number of embryos scored. **p < 0.01, ***p < 0.005.
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Figure 6: ADAM13-dependent regulation of calpain8 expression depends on ADAM13 phosphorylation. (A) ADAM13 was immunoprecipitated (IP) from 20 embryos using a goat polyclonal antibody g821. Noninjected embryos (NI) at stage 18 (neurula) are compared with sibling embryos injected with MO13 or MO13 plus MO-resistant mRNA encoding ADAM13-Plk/A or wild type. ADAM13 protein was detected by Western blot using 6615F. The polyvinylidene fluoride membrane was cut below 25 kDa and the two halves probed separately. The cleaved cytoplasmic domain (Cyto) is observed at 17 kDa for both wild-type and Plk/A ADAM13. Samples of the input for the IP were analyzed by Western blot with the 8C8 antibody for β1 integrin as a loading control. (B) Fluorescence images showing the localization of GFP-fusion proteins (green) expressed in Cos-7 cells along with membrane-bound mCherry (red). GFP is observed uniformly throughout the cytoplasm and nucleus, whereas GFP-C13 and the phosphodeficient mutants all accumulate strongly in the nucleus. (C, D) Analyses of targeted injection assays displaying the loss of CNC migration as a percentage of embryos. (C) MO13 + MO19 (2MO) was coinjected with mRNA encoding ADAM13 lacking its cytoplasmic domain (ΔCyto) plus either GFP-C13 wild type or phosphomutants. Values are normalized to the rescue with GFP-C13 wild type, and Student's t tests were performed to compare values to 2MO + ΔCyto. (D) 2MO was injected with ADAM13-Plk/A or ADAM13-Gsk/A mRNA alone or together with Capn8 mRNA. Inhibitions are normalized to RFP, and Student's t tests were performed against 2MO. Error bars are SD from four or more independent experiments. n, number of embryos scored. **p < 0.01, ***p < 0.005.
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.

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