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PAR3 and aPKC regulate Golgi organization through CLASP2 phosphorylation to generate cell polarity.

Matsui T, Watanabe T, Matsuzawa K, Kakeno M, Okumura N, Sugiyama I, Itoh N, Kaibuchi K - Mol. Biol. Cell (2014)

Bottom Line: CLASP2 is known to localize to the TGN through its interaction with the TGN protein GCC185.This interaction was inhibited by the aPKC-mediated phosphorylation of CLASP2.Furthermore, the nonphosphorylatable mutant enhanced the colocalization of CLASP2 with GCC185, thereby perturbing the Golgi organization.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.

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The interaction of CLASP2 with PAR3 is essential for the organization of the Golgi ribbon. (A) Schematic presentation of CLASP2 mutants and their binding abilities to PAR3-2N, -4N, EB1, and IQGAP1. CLASP2-DN bound to PAR3-2N and -4N but neither EB1 nor IQGAP1 (see Supplemental Figure S3). Asterisks indicate the locations of EB-binding motif. The red X indicates the mutation site in EB-binding motif. (B) GST- or GST-PAR3-2N– or -4N–immobilized beads were incubated with COS-7 cell lysates expressing GFP-CLASP2γ in the presence or absence of MBP-fusion proteins. The bound proteins were analyzed by immunoblotting. Increasing amounts of MBP–CLASP2-DN inhibited the interaction of CLASP2 with PAR3-2N and -4N. (C) To examine the effect of the expression of CLASP2-DN on CLASP2 localization, RPE-1 cells expressing HA–CLASP2-N1 or -DN were fixed with methanol at −30°C for 20 min, followed by immunostaining with anti-HA, anti-CLASP2, and anti-GM130 antibodies. The ectopic expression of CLASP2-DN increased the accumulation of CLASP2 at the Golgi apparatus. Bars, 10 µm. (D) To monitor the expression level of CLASP2-DN and examine its effect on the Golgi organization, the indicated RPE-1 cells were fixed with 2% paraformaldehyde and permeabilized with 0.2% Triton X-100, followed by immunostaining with anti-HA (gray), anti-GCC185 (magenta), and anti-GM130 (cyan). Bars, 10 µm. Right, graph of circularity index of the Golgi morphology in indicated cells. The ectopic expression of CLASP2-DN induced the circular appearance of the Golgi. n > 30. ***p < 0.001. All results are representative of three independent experiments.
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Figure 4: The interaction of CLASP2 with PAR3 is essential for the organization of the Golgi ribbon. (A) Schematic presentation of CLASP2 mutants and their binding abilities to PAR3-2N, -4N, EB1, and IQGAP1. CLASP2-DN bound to PAR3-2N and -4N but neither EB1 nor IQGAP1 (see Supplemental Figure S3). Asterisks indicate the locations of EB-binding motif. The red X indicates the mutation site in EB-binding motif. (B) GST- or GST-PAR3-2N– or -4N–immobilized beads were incubated with COS-7 cell lysates expressing GFP-CLASP2γ in the presence or absence of MBP-fusion proteins. The bound proteins were analyzed by immunoblotting. Increasing amounts of MBP–CLASP2-DN inhibited the interaction of CLASP2 with PAR3-2N and -4N. (C) To examine the effect of the expression of CLASP2-DN on CLASP2 localization, RPE-1 cells expressing HA–CLASP2-N1 or -DN were fixed with methanol at −30°C for 20 min, followed by immunostaining with anti-HA, anti-CLASP2, and anti-GM130 antibodies. The ectopic expression of CLASP2-DN increased the accumulation of CLASP2 at the Golgi apparatus. Bars, 10 µm. (D) To monitor the expression level of CLASP2-DN and examine its effect on the Golgi organization, the indicated RPE-1 cells were fixed with 2% paraformaldehyde and permeabilized with 0.2% Triton X-100, followed by immunostaining with anti-HA (gray), anti-GCC185 (magenta), and anti-GM130 (cyan). Bars, 10 µm. Right, graph of circularity index of the Golgi morphology in indicated cells. The ectopic expression of CLASP2-DN induced the circular appearance of the Golgi. n > 30. ***p < 0.001. All results are representative of three independent experiments.

Mentions: To examine whether the PAR3-CLASP2 interaction regulates the localization of CLASP2 to the TGN and the organization of the Golgi ribbon, we attempted to generate a CLASP2 mutant that inhibits the PAR3-CLASP2 interaction in a dominant-negative manner. Because the CLASP2-N2 region is also responsible for interacting with EBs, IQGAP1, and MTs (Mimori-Kiyosue et al., 2005; Watanabe et al., 2009), we sought to identify the CLASP2 region that specifically interacted with PAR3. CLASP2-N2-1 (aa 481–550) bound to PAR3-2N and -4N but not to IQGAP1 (Figure 4A and Supplemental Figure S3). CLASP2-N2-1 has two EB-binding motifs (Ser-X-Ile-Pro; Honnappa et al., 2009), and replacement of the Ile-Pro with Ser-Ser impaired the interaction with EB1 but had no effect on the interactions with PAR3-2N and -4N (Figure 4A and Supplemental Figure S3). These results indicate that the CLASP2-N2-1 mutant that is defective in EB-binding, called CLASP2-DN, specifically interacts with PAR3. MBP–CLASP2-DN inhibited the interaction of CLASP2 with GST–PAR3-2N or -4N in a dose-dependent manner (Figure 4B). Thus CLASP2-DN appears to function as a dominant-negative mutant by competing with the PAR3-CLASP2 binding. The expression of hemagglutinin (HA)–CLASP2-DN in RPE-1 cells increased the accumulation of CLASP2 at the TGN concomitantly with disruption of the Golgi ribbon, whereas that of HA–CLASP2-N1, used as a control, had little effect (Figure 4, C and D). The analysis of the circularity index of the Golgi confirmed that the CLASP2-DN expression significantly disrupted the Golgi ribbon morphology (Figure 4D). These results suggest that the PAR3-CLASP2 interaction regulates the localization of CLASP2 to the TGN and the organization of the Golgi ribbon.


PAR3 and aPKC regulate Golgi organization through CLASP2 phosphorylation to generate cell polarity.

Matsui T, Watanabe T, Matsuzawa K, Kakeno M, Okumura N, Sugiyama I, Itoh N, Kaibuchi K - Mol. Biol. Cell (2014)

The interaction of CLASP2 with PAR3 is essential for the organization of the Golgi ribbon. (A) Schematic presentation of CLASP2 mutants and their binding abilities to PAR3-2N, -4N, EB1, and IQGAP1. CLASP2-DN bound to PAR3-2N and -4N but neither EB1 nor IQGAP1 (see Supplemental Figure S3). Asterisks indicate the locations of EB-binding motif. The red X indicates the mutation site in EB-binding motif. (B) GST- or GST-PAR3-2N– or -4N–immobilized beads were incubated with COS-7 cell lysates expressing GFP-CLASP2γ in the presence or absence of MBP-fusion proteins. The bound proteins were analyzed by immunoblotting. Increasing amounts of MBP–CLASP2-DN inhibited the interaction of CLASP2 with PAR3-2N and -4N. (C) To examine the effect of the expression of CLASP2-DN on CLASP2 localization, RPE-1 cells expressing HA–CLASP2-N1 or -DN were fixed with methanol at −30°C for 20 min, followed by immunostaining with anti-HA, anti-CLASP2, and anti-GM130 antibodies. The ectopic expression of CLASP2-DN increased the accumulation of CLASP2 at the Golgi apparatus. Bars, 10 µm. (D) To monitor the expression level of CLASP2-DN and examine its effect on the Golgi organization, the indicated RPE-1 cells were fixed with 2% paraformaldehyde and permeabilized with 0.2% Triton X-100, followed by immunostaining with anti-HA (gray), anti-GCC185 (magenta), and anti-GM130 (cyan). Bars, 10 µm. Right, graph of circularity index of the Golgi morphology in indicated cells. The ectopic expression of CLASP2-DN induced the circular appearance of the Golgi. n > 30. ***p < 0.001. All results are representative of three independent experiments.
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Figure 4: The interaction of CLASP2 with PAR3 is essential for the organization of the Golgi ribbon. (A) Schematic presentation of CLASP2 mutants and their binding abilities to PAR3-2N, -4N, EB1, and IQGAP1. CLASP2-DN bound to PAR3-2N and -4N but neither EB1 nor IQGAP1 (see Supplemental Figure S3). Asterisks indicate the locations of EB-binding motif. The red X indicates the mutation site in EB-binding motif. (B) GST- or GST-PAR3-2N– or -4N–immobilized beads were incubated with COS-7 cell lysates expressing GFP-CLASP2γ in the presence or absence of MBP-fusion proteins. The bound proteins were analyzed by immunoblotting. Increasing amounts of MBP–CLASP2-DN inhibited the interaction of CLASP2 with PAR3-2N and -4N. (C) To examine the effect of the expression of CLASP2-DN on CLASP2 localization, RPE-1 cells expressing HA–CLASP2-N1 or -DN were fixed with methanol at −30°C for 20 min, followed by immunostaining with anti-HA, anti-CLASP2, and anti-GM130 antibodies. The ectopic expression of CLASP2-DN increased the accumulation of CLASP2 at the Golgi apparatus. Bars, 10 µm. (D) To monitor the expression level of CLASP2-DN and examine its effect on the Golgi organization, the indicated RPE-1 cells were fixed with 2% paraformaldehyde and permeabilized with 0.2% Triton X-100, followed by immunostaining with anti-HA (gray), anti-GCC185 (magenta), and anti-GM130 (cyan). Bars, 10 µm. Right, graph of circularity index of the Golgi morphology in indicated cells. The ectopic expression of CLASP2-DN induced the circular appearance of the Golgi. n > 30. ***p < 0.001. All results are representative of three independent experiments.
Mentions: To examine whether the PAR3-CLASP2 interaction regulates the localization of CLASP2 to the TGN and the organization of the Golgi ribbon, we attempted to generate a CLASP2 mutant that inhibits the PAR3-CLASP2 interaction in a dominant-negative manner. Because the CLASP2-N2 region is also responsible for interacting with EBs, IQGAP1, and MTs (Mimori-Kiyosue et al., 2005; Watanabe et al., 2009), we sought to identify the CLASP2 region that specifically interacted with PAR3. CLASP2-N2-1 (aa 481–550) bound to PAR3-2N and -4N but not to IQGAP1 (Figure 4A and Supplemental Figure S3). CLASP2-N2-1 has two EB-binding motifs (Ser-X-Ile-Pro; Honnappa et al., 2009), and replacement of the Ile-Pro with Ser-Ser impaired the interaction with EB1 but had no effect on the interactions with PAR3-2N and -4N (Figure 4A and Supplemental Figure S3). These results indicate that the CLASP2-N2-1 mutant that is defective in EB-binding, called CLASP2-DN, specifically interacts with PAR3. MBP–CLASP2-DN inhibited the interaction of CLASP2 with GST–PAR3-2N or -4N in a dose-dependent manner (Figure 4B). Thus CLASP2-DN appears to function as a dominant-negative mutant by competing with the PAR3-CLASP2 binding. The expression of hemagglutinin (HA)–CLASP2-DN in RPE-1 cells increased the accumulation of CLASP2 at the TGN concomitantly with disruption of the Golgi ribbon, whereas that of HA–CLASP2-N1, used as a control, had little effect (Figure 4, C and D). The analysis of the circularity index of the Golgi confirmed that the CLASP2-DN expression significantly disrupted the Golgi ribbon morphology (Figure 4D). These results suggest that the PAR3-CLASP2 interaction regulates the localization of CLASP2 to the TGN and the organization of the Golgi ribbon.

Bottom Line: CLASP2 is known to localize to the TGN through its interaction with the TGN protein GCC185.This interaction was inhibited by the aPKC-mediated phosphorylation of CLASP2.Furthermore, the nonphosphorylatable mutant enhanced the colocalization of CLASP2 with GCC185, thereby perturbing the Golgi organization.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.

Show MeSH
Related in: MedlinePlus