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Phosphorylation-dependent ubiquitination of paraxial protocadherin (PAPC) controls gastrulation cell movements.

Kai M, Ueno N, Kinoshita N - PLoS ONE (2015)

Bottom Line: Using Xenopus embryos, we found that PAPC is also regulated at the protein level and is degraded and excluded from the plasma membrane in the axial mesoderm by the late gastrula stage.Regulation of PAPC requires poly-ubiquitination that is dependent on phosphorylation.PAPC is phosphorylated by GKS3 in the evolutionarily conserved cytoplasmic domain, and this in turn is necessary for poly-ubiquitination by an E3 ubiquitin ligase β-TrCP.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji-cho, Okazaki, Aichi 444-8585, Japan.

ABSTRACT
Paraxial protocadherin (PAPC) has been shown to be involved in gastrulation cell movements during early embryogenesis. It is first expressed in the dorsal marginal zone at the early gastrula stage and subsequently restricted to the paraxial mesoderm in Xenopus and zebrafish. Using Xenopus embryos, we found that PAPC is also regulated at the protein level and is degraded and excluded from the plasma membrane in the axial mesoderm by the late gastrula stage. Regulation of PAPC requires poly-ubiquitination that is dependent on phosphorylation. PAPC is phosphorylated by GKS3 in the evolutionarily conserved cytoplasmic domain, and this in turn is necessary for poly-ubiquitination by an E3 ubiquitin ligase β-TrCP. We also show that precise control of PAPC by phosphorylation/ubiquitination is essential for normal Xenopus gastrulation cell movements. Taken together, our findings unveil a novel mechanism of regulation of a cell adhesion protein and show that this system plays a crucial role in vertebrate embryogenesis.

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GSK3 is involved in the regulation of PAPC localization and stability.(A) Inhibiting GSK3 activity by expression of dominant-negative GSK3 (dnGSK3), or treating explants with a GSK3 inhibitor BIO one hour prior to observation, led to extensive aggregation of PAPC in the cytoplasm in animal cap cells (arrowheads). A putative phosphorylation-mimic mutant PAPC-SD, which carries S816 and S820 to aspartic acid mutations, was resistant to dnGSK3 expression and remained on the plasma membrane. (B) PAPC was not efficiently degraded in DMZ explants expressed with dominant-negative GSK3 (dnGSK3) at stage 15 and formed cytoplasmic aggregates. (C) GST-tagged PAPC was expressed in the dorsal marginal zone. When embryos reached the gastrula stage, PAPC-GST was partially purified with Glutathione Sepharose 4B. Phosphorylated PAPC (pPAPC) was detected by anti-phosopho-PAPC antibody, raised against phosphorylated DSR peptide as an antigen. The pPAPC signal was abolished by treating the extract with lambda protein phosphatase (PPase). The recombinant C-terminal domain of PAPC was phosphorylated in vitro in the presence of recombinant GSK3, as confirmed by a Western blot using anti-pPAPC antibody. Total input of PAPC was estimated by anti-GST antibody.
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pone.0115111.g004: GSK3 is involved in the regulation of PAPC localization and stability.(A) Inhibiting GSK3 activity by expression of dominant-negative GSK3 (dnGSK3), or treating explants with a GSK3 inhibitor BIO one hour prior to observation, led to extensive aggregation of PAPC in the cytoplasm in animal cap cells (arrowheads). A putative phosphorylation-mimic mutant PAPC-SD, which carries S816 and S820 to aspartic acid mutations, was resistant to dnGSK3 expression and remained on the plasma membrane. (B) PAPC was not efficiently degraded in DMZ explants expressed with dominant-negative GSK3 (dnGSK3) at stage 15 and formed cytoplasmic aggregates. (C) GST-tagged PAPC was expressed in the dorsal marginal zone. When embryos reached the gastrula stage, PAPC-GST was partially purified with Glutathione Sepharose 4B. Phosphorylated PAPC (pPAPC) was detected by anti-phosopho-PAPC antibody, raised against phosphorylated DSR peptide as an antigen. The pPAPC signal was abolished by treating the extract with lambda protein phosphatase (PPase). The recombinant C-terminal domain of PAPC was phosphorylated in vitro in the presence of recombinant GSK3, as confirmed by a Western blot using anti-pPAPC antibody. Total input of PAPC was estimated by anti-GST antibody.

Mentions: Because S816 and S820 in the DSR domain of Xenopus PAPC conform to the GSK3 consensus motif (Ser-X-X-X-Ser[p]), where downstream D824 may serve as a constitutive priming residue,, we next investigated whether GSK3 is involved in the regulation of PAPC. Expression of a dominant-negative GSK3 (dnGSK3) [16] reduced the PAPC-GFP signal on the plasma membrane (Fig. 4A, upper right panel), and induced the formation of large aggregates in the cytoplasm (arrowheads in Fig. 4A, upper middle panel), in a similar manner to PAPC-SA1-GFP. As expressing dnGSK3 from the earliest stages of development by mRNA injection is expected to cause pleiotropic effects, we treated the explants with a GSK3 inhibitor BIO one hour prior to the observation, and confirmed it too led to PAPC-GFP aggregates formation (Fig. 4A, upper right panel). On the other hand, a ‘phosphorylation-mimic’ PAPC mutant, in which S816 and S820 are replaced with aspartic acid (PAPC-SD; Fig. 2A), was resistant to dnGSK3 expression and whose wild-type-like localization was not altered (Fig. 4A, lower right panel). Degradation of PAPC in DMZ explants at stage 15 was also blocked by dnGSK3 expression (Fig. 4B). By using an antibody specifically raised to recognize phosphorylation at these residues (anti-pPAPC Ab), we confirmed that PAPC is indeed phosphorylated in Xenopus embryos at stage 11 (Fig. 4C). In vitro analysis further revealed that purified GSK3 is able to phosphorylate the cytoplasmic domain of PAPC expressed in E. coli (Fig. 4C). These results strongly support the argument that phosphorylation at S816 and/or S820 by GSK3 is essential for normal PAPC regulation during Xenopus gastrulation.


Phosphorylation-dependent ubiquitination of paraxial protocadherin (PAPC) controls gastrulation cell movements.

Kai M, Ueno N, Kinoshita N - PLoS ONE (2015)

GSK3 is involved in the regulation of PAPC localization and stability.(A) Inhibiting GSK3 activity by expression of dominant-negative GSK3 (dnGSK3), or treating explants with a GSK3 inhibitor BIO one hour prior to observation, led to extensive aggregation of PAPC in the cytoplasm in animal cap cells (arrowheads). A putative phosphorylation-mimic mutant PAPC-SD, which carries S816 and S820 to aspartic acid mutations, was resistant to dnGSK3 expression and remained on the plasma membrane. (B) PAPC was not efficiently degraded in DMZ explants expressed with dominant-negative GSK3 (dnGSK3) at stage 15 and formed cytoplasmic aggregates. (C) GST-tagged PAPC was expressed in the dorsal marginal zone. When embryos reached the gastrula stage, PAPC-GST was partially purified with Glutathione Sepharose 4B. Phosphorylated PAPC (pPAPC) was detected by anti-phosopho-PAPC antibody, raised against phosphorylated DSR peptide as an antigen. The pPAPC signal was abolished by treating the extract with lambda protein phosphatase (PPase). The recombinant C-terminal domain of PAPC was phosphorylated in vitro in the presence of recombinant GSK3, as confirmed by a Western blot using anti-pPAPC antibody. Total input of PAPC was estimated by anti-GST antibody.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4291225&req=5

pone.0115111.g004: GSK3 is involved in the regulation of PAPC localization and stability.(A) Inhibiting GSK3 activity by expression of dominant-negative GSK3 (dnGSK3), or treating explants with a GSK3 inhibitor BIO one hour prior to observation, led to extensive aggregation of PAPC in the cytoplasm in animal cap cells (arrowheads). A putative phosphorylation-mimic mutant PAPC-SD, which carries S816 and S820 to aspartic acid mutations, was resistant to dnGSK3 expression and remained on the plasma membrane. (B) PAPC was not efficiently degraded in DMZ explants expressed with dominant-negative GSK3 (dnGSK3) at stage 15 and formed cytoplasmic aggregates. (C) GST-tagged PAPC was expressed in the dorsal marginal zone. When embryos reached the gastrula stage, PAPC-GST was partially purified with Glutathione Sepharose 4B. Phosphorylated PAPC (pPAPC) was detected by anti-phosopho-PAPC antibody, raised against phosphorylated DSR peptide as an antigen. The pPAPC signal was abolished by treating the extract with lambda protein phosphatase (PPase). The recombinant C-terminal domain of PAPC was phosphorylated in vitro in the presence of recombinant GSK3, as confirmed by a Western blot using anti-pPAPC antibody. Total input of PAPC was estimated by anti-GST antibody.
Mentions: Because S816 and S820 in the DSR domain of Xenopus PAPC conform to the GSK3 consensus motif (Ser-X-X-X-Ser[p]), where downstream D824 may serve as a constitutive priming residue,, we next investigated whether GSK3 is involved in the regulation of PAPC. Expression of a dominant-negative GSK3 (dnGSK3) [16] reduced the PAPC-GFP signal on the plasma membrane (Fig. 4A, upper right panel), and induced the formation of large aggregates in the cytoplasm (arrowheads in Fig. 4A, upper middle panel), in a similar manner to PAPC-SA1-GFP. As expressing dnGSK3 from the earliest stages of development by mRNA injection is expected to cause pleiotropic effects, we treated the explants with a GSK3 inhibitor BIO one hour prior to the observation, and confirmed it too led to PAPC-GFP aggregates formation (Fig. 4A, upper right panel). On the other hand, a ‘phosphorylation-mimic’ PAPC mutant, in which S816 and S820 are replaced with aspartic acid (PAPC-SD; Fig. 2A), was resistant to dnGSK3 expression and whose wild-type-like localization was not altered (Fig. 4A, lower right panel). Degradation of PAPC in DMZ explants at stage 15 was also blocked by dnGSK3 expression (Fig. 4B). By using an antibody specifically raised to recognize phosphorylation at these residues (anti-pPAPC Ab), we confirmed that PAPC is indeed phosphorylated in Xenopus embryos at stage 11 (Fig. 4C). In vitro analysis further revealed that purified GSK3 is able to phosphorylate the cytoplasmic domain of PAPC expressed in E. coli (Fig. 4C). These results strongly support the argument that phosphorylation at S816 and/or S820 by GSK3 is essential for normal PAPC regulation during Xenopus gastrulation.

Bottom Line: Using Xenopus embryos, we found that PAPC is also regulated at the protein level and is degraded and excluded from the plasma membrane in the axial mesoderm by the late gastrula stage.Regulation of PAPC requires poly-ubiquitination that is dependent on phosphorylation.PAPC is phosphorylated by GKS3 in the evolutionarily conserved cytoplasmic domain, and this in turn is necessary for poly-ubiquitination by an E3 ubiquitin ligase β-TrCP.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji-cho, Okazaki, Aichi 444-8585, Japan.

ABSTRACT
Paraxial protocadherin (PAPC) has been shown to be involved in gastrulation cell movements during early embryogenesis. It is first expressed in the dorsal marginal zone at the early gastrula stage and subsequently restricted to the paraxial mesoderm in Xenopus and zebrafish. Using Xenopus embryos, we found that PAPC is also regulated at the protein level and is degraded and excluded from the plasma membrane in the axial mesoderm by the late gastrula stage. Regulation of PAPC requires poly-ubiquitination that is dependent on phosphorylation. PAPC is phosphorylated by GKS3 in the evolutionarily conserved cytoplasmic domain, and this in turn is necessary for poly-ubiquitination by an E3 ubiquitin ligase β-TrCP. We also show that precise control of PAPC by phosphorylation/ubiquitination is essential for normal Xenopus gastrulation cell movements. Taken together, our findings unveil a novel mechanism of regulation of a cell adhesion protein and show that this system plays a crucial role in vertebrate embryogenesis.

Show MeSH
Related in: MedlinePlus