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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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GSK3 and Plk can phosphorylate ADAM13. (A) Western blots for Plk1 (68 kDa) and GSK3β (47 kDa) on protein extract from 27 dissected CNC (CNC) or one total embryo equivalent at the same stage as dissection (Tot St17). (B) Western blot showing phosphorylation of ADAM13. The main forms of ADAM13 protein are represented at the top. The pro form (P) contains the prodomain and is inactive, the mature form (M) is proteolytically active, and cleavage of the metalloprotease domain results in a shorter form (D) that appears to be the principal phosphorylated form of ADAM13. Nuclear Cherry (nCherry, negative control), ADAM13 (A13), or the nonphosphorylatable mutant (A13-Plk/A) was expressed in HEK293T cells. Cells expressing ADAM13 were treated overnight with dimethyl sulfoxide (DMSO), Plk inhibitor (Plk Inh), or GSK3 inhibitor (Gsk Inh). All of the samples were first immunoprecipitated with a monoclonal antibody to ADAM13 (mAb 4A7), followed by Western blot using the phospho-ADAM13 antibody to the Plk phosphorylation site (P-A13) or the rabbit polyclonal to the ADAM13 cytoplasmic domain 15F for the total ADAM13 protein (Tot). (C) Autoradiograph of immuno­precipitated ADAM13 produced in HEK293T cells phosphorylated by purified active GSK3.
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Figure 2: GSK3 and Plk can phosphorylate ADAM13. (A) Western blots for Plk1 (68 kDa) and GSK3β (47 kDa) on protein extract from 27 dissected CNC (CNC) or one total embryo equivalent at the same stage as dissection (Tot St17). (B) Western blot showing phosphorylation of ADAM13. The main forms of ADAM13 protein are represented at the top. The pro form (P) contains the prodomain and is inactive, the mature form (M) is proteolytically active, and cleavage of the metalloprotease domain results in a shorter form (D) that appears to be the principal phosphorylated form of ADAM13. Nuclear Cherry (nCherry, negative control), ADAM13 (A13), or the nonphosphorylatable mutant (A13-Plk/A) was expressed in HEK293T cells. Cells expressing ADAM13 were treated overnight with dimethyl sulfoxide (DMSO), Plk inhibitor (Plk Inh), or GSK3 inhibitor (Gsk Inh). All of the samples were first immunoprecipitated with a monoclonal antibody to ADAM13 (mAb 4A7), followed by Western blot using the phospho-ADAM13 antibody to the Plk phosphorylation site (P-A13) or the rabbit polyclonal to the ADAM13 cytoplasmic domain 15F for the total ADAM13 protein (Tot). (C) Autoradiograph of immuno­precipitated ADAM13 produced in HEK293T cells phosphorylated by purified active GSK3.

Mentions: We then tested, first, whether the two kinases were present in the CNC and, second, whether they could phosphorylate ADAM13. To answer the first question, we performed a Western blot on CNC dissected at stage 17 using antibodies specific to either kinase. Indeed, each antibody detected the predicted size band (Figure 2A), demonstrating that both GSK3 and Plk proteins are present in the CNC during migration. We then produced an antibody against a phosphorylated peptide corresponding to the Plk site in ADAM13. The antibody was affinity purified against the phosphorylated peptide and affinity depleted with the nonphosphorylated peptide. Human HEK293T cells were transfected with either the wild-type ADAM13 or the nonphosphorylatable mutant A13-Plk/A. We found that wild-type ADAM13 was clearly phosphorylated in these cells, whereas the mutant was not (Figure 2B). In addition, treatment of the cells with either a Plk inhibitor or a GSK3 inhibitor prevented this phosphorylation, showing that in HEK293T cells, endogenous Plk did phosphorylate ADAM13. This further suggests that in the absence of GSK3, Plk did not phosphorylate ADAM13. Of interest, in these cells, the main form of ADAM13 phosphorylated is shorter (∼65 kDa) than the mature ADAM13 (M; 100 kDa), suggesting that this form may not contain the metalloprotease domain (form D). This shorter form of ADAM13 is also the one phosphorylated in vitro by purified GSK3 (Figure 2C). Phosphorylation was increased in the ADAM13 mutant A13-Gsk/D, suggesting that the third GSK3 phosphorylation site (885T) of ADAM13 can also be phosphorylated in vitro.


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)

GSK3 and Plk can phosphorylate ADAM13. (A) Western blots for Plk1 (68 kDa) and GSK3β (47 kDa) on protein extract from 27 dissected CNC (CNC) or one total embryo equivalent at the same stage as dissection (Tot St17). (B) Western blot showing phosphorylation of ADAM13. The main forms of ADAM13 protein are represented at the top. The pro form (P) contains the prodomain and is inactive, the mature form (M) is proteolytically active, and cleavage of the metalloprotease domain results in a shorter form (D) that appears to be the principal phosphorylated form of ADAM13. Nuclear Cherry (nCherry, negative control), ADAM13 (A13), or the nonphosphorylatable mutant (A13-Plk/A) was expressed in HEK293T cells. Cells expressing ADAM13 were treated overnight with dimethyl sulfoxide (DMSO), Plk inhibitor (Plk Inh), or GSK3 inhibitor (Gsk Inh). All of the samples were first immunoprecipitated with a monoclonal antibody to ADAM13 (mAb 4A7), followed by Western blot using the phospho-ADAM13 antibody to the Plk phosphorylation site (P-A13) or the rabbit polyclonal to the ADAM13 cytoplasmic domain 15F for the total ADAM13 protein (Tot). (C) Autoradiograph of immuno­precipitated ADAM13 produced in HEK293T cells phosphorylated by purified active GSK3.
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Figure 2: GSK3 and Plk can phosphorylate ADAM13. (A) Western blots for Plk1 (68 kDa) and GSK3β (47 kDa) on protein extract from 27 dissected CNC (CNC) or one total embryo equivalent at the same stage as dissection (Tot St17). (B) Western blot showing phosphorylation of ADAM13. The main forms of ADAM13 protein are represented at the top. The pro form (P) contains the prodomain and is inactive, the mature form (M) is proteolytically active, and cleavage of the metalloprotease domain results in a shorter form (D) that appears to be the principal phosphorylated form of ADAM13. Nuclear Cherry (nCherry, negative control), ADAM13 (A13), or the nonphosphorylatable mutant (A13-Plk/A) was expressed in HEK293T cells. Cells expressing ADAM13 were treated overnight with dimethyl sulfoxide (DMSO), Plk inhibitor (Plk Inh), or GSK3 inhibitor (Gsk Inh). All of the samples were first immunoprecipitated with a monoclonal antibody to ADAM13 (mAb 4A7), followed by Western blot using the phospho-ADAM13 antibody to the Plk phosphorylation site (P-A13) or the rabbit polyclonal to the ADAM13 cytoplasmic domain 15F for the total ADAM13 protein (Tot). (C) Autoradiograph of immuno­precipitated ADAM13 produced in HEK293T cells phosphorylated by purified active GSK3.
Mentions: We then tested, first, whether the two kinases were present in the CNC and, second, whether they could phosphorylate ADAM13. To answer the first question, we performed a Western blot on CNC dissected at stage 17 using antibodies specific to either kinase. Indeed, each antibody detected the predicted size band (Figure 2A), demonstrating that both GSK3 and Plk proteins are present in the CNC during migration. We then produced an antibody against a phosphorylated peptide corresponding to the Plk site in ADAM13. The antibody was affinity purified against the phosphorylated peptide and affinity depleted with the nonphosphorylated peptide. Human HEK293T cells were transfected with either the wild-type ADAM13 or the nonphosphorylatable mutant A13-Plk/A. We found that wild-type ADAM13 was clearly phosphorylated in these cells, whereas the mutant was not (Figure 2B). In addition, treatment of the cells with either a Plk inhibitor or a GSK3 inhibitor prevented this phosphorylation, showing that in HEK293T cells, endogenous Plk did phosphorylate ADAM13. This further suggests that in the absence of GSK3, Plk did not phosphorylate ADAM13. Of interest, in these cells, the main form of ADAM13 phosphorylated is shorter (∼65 kDa) than the mature ADAM13 (M; 100 kDa), suggesting that this form may not contain the metalloprotease domain (form D). This shorter form of ADAM13 is also the one phosphorylated in vitro by purified GSK3 (Figure 2C). Phosphorylation was increased in the ADAM13 mutant A13-Gsk/D, suggesting that the third GSK3 phosphorylation site (885T) of ADAM13 can also be phosphorylated in vitro.

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