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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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PAR3 and CLASP2 form a complex through direct interaction. (A) RPE-1 cell lysates were precipitated with the indicated antibodies. Endogenous PAR3 was specifically coprecipitated with CLASP2 and vice versa. (B, C) Lysates of COS-7 cells expressing PAR3-GFP and Myc-CLASP isoforms were precipitated with anti-GFP antibody. PAR3 formed a complex with each CLASP. (D) The domain structures of PAR3 and its fragments. Numbers refer to amino acids. aPKC BR, aPKC binding region; CR, conserved region; PDZ, PSD95/Discs-Large/ZO-1 domain. (E) Indicated GST-fusion proteins were incubated with lysates from COS-7 cells and precipitated with glutathione beads. CLASP2 was detected in the eluates of GST-PAR3-2N and -4N. (F) Schematic of CLASP2. The domain structures of CLASP2 and its various fragments. CR, conserved region; S/R-rich, serine/arginine-rich. (G) Lysates of COS-7 cells expressing Myc-PAR3 and the indicated GFP-CLASP2 fragments were immunoprecipitated with anti-GFP antibody. Myc-PAR3 was coprecipitated with GFP–CLASP2-N2. (H) Saturable interaction of purified GST–PAR3-2N with MBP–CLASP2-N2. The indicated concentrations of MBP–CLASP2-N2 were incubated with beads coated with GST–PAR3-2N (20 pmol), and the Kd value was calculated by Scatchard analysis. The Kd value of the interaction between PAR3-2N and CLASP2-N2 was ∼6 nM. The data represent the means ± SD of more than three independent experiments.
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Figure 1: PAR3 and CLASP2 form a complex through direct interaction. (A) RPE-1 cell lysates were precipitated with the indicated antibodies. Endogenous PAR3 was specifically coprecipitated with CLASP2 and vice versa. (B, C) Lysates of COS-7 cells expressing PAR3-GFP and Myc-CLASP isoforms were precipitated with anti-GFP antibody. PAR3 formed a complex with each CLASP. (D) The domain structures of PAR3 and its fragments. Numbers refer to amino acids. aPKC BR, aPKC binding region; CR, conserved region; PDZ, PSD95/Discs-Large/ZO-1 domain. (E) Indicated GST-fusion proteins were incubated with lysates from COS-7 cells and precipitated with glutathione beads. CLASP2 was detected in the eluates of GST-PAR3-2N and -4N. (F) Schematic of CLASP2. The domain structures of CLASP2 and its various fragments. CR, conserved region; S/R-rich, serine/arginine-rich. (G) Lysates of COS-7 cells expressing Myc-PAR3 and the indicated GFP-CLASP2 fragments were immunoprecipitated with anti-GFP antibody. Myc-PAR3 was coprecipitated with GFP–CLASP2-N2. (H) Saturable interaction of purified GST–PAR3-2N with MBP–CLASP2-N2. The indicated concentrations of MBP–CLASP2-N2 were incubated with beads coated with GST–PAR3-2N (20 pmol), and the Kd value was calculated by Scatchard analysis. The Kd value of the interaction between PAR3-2N and CLASP2-N2 was ∼6 nM. The data represent the means ± SD of more than three independent experiments.

Mentions: By comprehensive screening for interactors of PAR3, we previously identified CLASP2 as a candidate interactor (Itoh et al., 2010). We first verified our finding by immunoprecipitating the complex of PAR3 and CLASP2. Endogenous CLASP2 was specifically precipitated with endogenous PAR3 and vice versa (Figure 1A). Furthermore, Myc-fused CLASP2α and CLASP2γ were precipitated with green fluorescent protein (GFP)–fused PAR3 (Figure 1B). CLASP1α was also precipitated with PAR3 (Figure 1C). These results indicate that PAR3 physiologically associates with CLASPs irrespective of its isoforms.


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)

PAR3 and CLASP2 form a complex through direct interaction. (A) RPE-1 cell lysates were precipitated with the indicated antibodies. Endogenous PAR3 was specifically coprecipitated with CLASP2 and vice versa. (B, C) Lysates of COS-7 cells expressing PAR3-GFP and Myc-CLASP isoforms were precipitated with anti-GFP antibody. PAR3 formed a complex with each CLASP. (D) The domain structures of PAR3 and its fragments. Numbers refer to amino acids. aPKC BR, aPKC binding region; CR, conserved region; PDZ, PSD95/Discs-Large/ZO-1 domain. (E) Indicated GST-fusion proteins were incubated with lysates from COS-7 cells and precipitated with glutathione beads. CLASP2 was detected in the eluates of GST-PAR3-2N and -4N. (F) Schematic of CLASP2. The domain structures of CLASP2 and its various fragments. CR, conserved region; S/R-rich, serine/arginine-rich. (G) Lysates of COS-7 cells expressing Myc-PAR3 and the indicated GFP-CLASP2 fragments were immunoprecipitated with anti-GFP antibody. Myc-PAR3 was coprecipitated with GFP–CLASP2-N2. (H) Saturable interaction of purified GST–PAR3-2N with MBP–CLASP2-N2. The indicated concentrations of MBP–CLASP2-N2 were incubated with beads coated with GST–PAR3-2N (20 pmol), and the Kd value was calculated by Scatchard analysis. The Kd value of the interaction between PAR3-2N and CLASP2-N2 was ∼6 nM. The data represent the means ± SD of more than three independent experiments.
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Related In: Results  -  Collection

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Figure 1: PAR3 and CLASP2 form a complex through direct interaction. (A) RPE-1 cell lysates were precipitated with the indicated antibodies. Endogenous PAR3 was specifically coprecipitated with CLASP2 and vice versa. (B, C) Lysates of COS-7 cells expressing PAR3-GFP and Myc-CLASP isoforms were precipitated with anti-GFP antibody. PAR3 formed a complex with each CLASP. (D) The domain structures of PAR3 and its fragments. Numbers refer to amino acids. aPKC BR, aPKC binding region; CR, conserved region; PDZ, PSD95/Discs-Large/ZO-1 domain. (E) Indicated GST-fusion proteins were incubated with lysates from COS-7 cells and precipitated with glutathione beads. CLASP2 was detected in the eluates of GST-PAR3-2N and -4N. (F) Schematic of CLASP2. The domain structures of CLASP2 and its various fragments. CR, conserved region; S/R-rich, serine/arginine-rich. (G) Lysates of COS-7 cells expressing Myc-PAR3 and the indicated GFP-CLASP2 fragments were immunoprecipitated with anti-GFP antibody. Myc-PAR3 was coprecipitated with GFP–CLASP2-N2. (H) Saturable interaction of purified GST–PAR3-2N with MBP–CLASP2-N2. The indicated concentrations of MBP–CLASP2-N2 were incubated with beads coated with GST–PAR3-2N (20 pmol), and the Kd value was calculated by Scatchard analysis. The Kd value of the interaction between PAR3-2N and CLASP2-N2 was ∼6 nM. The data represent the means ± SD of more than three independent experiments.
Mentions: By comprehensive screening for interactors of PAR3, we previously identified CLASP2 as a candidate interactor (Itoh et al., 2010). We first verified our finding by immunoprecipitating the complex of PAR3 and CLASP2. Endogenous CLASP2 was specifically precipitated with endogenous PAR3 and vice versa (Figure 1A). Furthermore, Myc-fused CLASP2α and CLASP2γ were precipitated with green fluorescent protein (GFP)–fused PAR3 (Figure 1B). CLASP1α was also precipitated with PAR3 (Figure 1C). These results indicate that PAR3 physiologically associates with CLASPs irrespective of its isoforms.

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