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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 aPKC regulate the interaction of CLASP2 with GCC185. (A) Control and PAR3-depleted RPE-1 cells were cross-linked with 1 mM DSP, followed by precipitation with anti-GCC185 antibody. Depletion of PAR3 increased the association between CLASP2 and GCC185. (B) The domain structures of GCC185 and its fragments. CC, Coiled-coil domain; GRIP, GRIP domain. (C, D) Lysates of COS-7 cells expressing indicated Myc-fusion proteins and GFP-fusion proteins were immunoprecipitated with anti-GFP antibody. Myc-CLASP2γ was coprecipitated with GFP–GCC185-1N (C), and Myc–GCC185-1N was coprecipitated with GFP–CLASP2-C1 (D). (E) COS-7 cells were transfected with the indicated constructs, and their lysates were pulled down with GST–GCC185-1N. The coexpression of aPKCζ catalytic kinase domain (aa 226–592, aPKCζ-cat) impaired the interaction of GCC185-1N with CLASP2γ-WT but not with -3A. All results are representative of three independent experiments.
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Figure 5: PAR3 and aPKC regulate the interaction of CLASP2 with GCC185. (A) Control and PAR3-depleted RPE-1 cells were cross-linked with 1 mM DSP, followed by precipitation with anti-GCC185 antibody. Depletion of PAR3 increased the association between CLASP2 and GCC185. (B) The domain structures of GCC185 and its fragments. CC, Coiled-coil domain; GRIP, GRIP domain. (C, D) Lysates of COS-7 cells expressing indicated Myc-fusion proteins and GFP-fusion proteins were immunoprecipitated with anti-GFP antibody. Myc-CLASP2γ was coprecipitated with GFP–GCC185-1N (C), and Myc–GCC185-1N was coprecipitated with GFP–CLASP2-C1 (D). (E) COS-7 cells were transfected with the indicated constructs, and their lysates were pulled down with GST–GCC185-1N. The coexpression of aPKCζ catalytic kinase domain (aa 226–592, aPKCζ-cat) impaired the interaction of GCC185-1N with CLASP2γ-WT but not with -3A. All results are representative of three independent experiments.

Mentions: To explore the mechanism regulating the localization of CLASP2 to the TGN dependent on PAR3 and aPKC, we examined the involvement of PAR3 and aPKC in the interaction between CLASP2 and GCC185. On the basis of a previous report (Lin et al., 2011), we used dithiobis succinimidyl propionate (DSP) as a cross-linker for the immunoprecipitation of endogenous CLASP2-GCC185 complex. Under the condition in which CLASP2 was coprecipitated with GCC185 from control cells, PAR3 depletion increased the amount of CLASP2 coprecipitated with GCC185 (Figure 5A), suggesting that PAR3 negatively regulates the interaction between CLASP2 and GCC185. The effects of aPKC depletion were inconclusive because the treatment of the cross-linker with aPKC-depleted cells drastically decreased the GCC185 solubility for the immunoprecipitation.


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 aPKC regulate the interaction of CLASP2 with GCC185. (A) Control and PAR3-depleted RPE-1 cells were cross-linked with 1 mM DSP, followed by precipitation with anti-GCC185 antibody. Depletion of PAR3 increased the association between CLASP2 and GCC185. (B) The domain structures of GCC185 and its fragments. CC, Coiled-coil domain; GRIP, GRIP domain. (C, D) Lysates of COS-7 cells expressing indicated Myc-fusion proteins and GFP-fusion proteins were immunoprecipitated with anti-GFP antibody. Myc-CLASP2γ was coprecipitated with GFP–GCC185-1N (C), and Myc–GCC185-1N was coprecipitated with GFP–CLASP2-C1 (D). (E) COS-7 cells were transfected with the indicated constructs, and their lysates were pulled down with GST–GCC185-1N. The coexpression of aPKCζ catalytic kinase domain (aa 226–592, aPKCζ-cat) impaired the interaction of GCC185-1N with CLASP2γ-WT but not with -3A. All results are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 5: PAR3 and aPKC regulate the interaction of CLASP2 with GCC185. (A) Control and PAR3-depleted RPE-1 cells were cross-linked with 1 mM DSP, followed by precipitation with anti-GCC185 antibody. Depletion of PAR3 increased the association between CLASP2 and GCC185. (B) The domain structures of GCC185 and its fragments. CC, Coiled-coil domain; GRIP, GRIP domain. (C, D) Lysates of COS-7 cells expressing indicated Myc-fusion proteins and GFP-fusion proteins were immunoprecipitated with anti-GFP antibody. Myc-CLASP2γ was coprecipitated with GFP–GCC185-1N (C), and Myc–GCC185-1N was coprecipitated with GFP–CLASP2-C1 (D). (E) COS-7 cells were transfected with the indicated constructs, and their lysates were pulled down with GST–GCC185-1N. The coexpression of aPKCζ catalytic kinase domain (aa 226–592, aPKCζ-cat) impaired the interaction of GCC185-1N with CLASP2γ-WT but not with -3A. All results are representative of three independent experiments.
Mentions: To explore the mechanism regulating the localization of CLASP2 to the TGN dependent on PAR3 and aPKC, we examined the involvement of PAR3 and aPKC in the interaction between CLASP2 and GCC185. On the basis of a previous report (Lin et al., 2011), we used dithiobis succinimidyl propionate (DSP) as a cross-linker for the immunoprecipitation of endogenous CLASP2-GCC185 complex. Under the condition in which CLASP2 was coprecipitated with GCC185 from control cells, PAR3 depletion increased the amount of CLASP2 coprecipitated with GCC185 (Figure 5A), suggesting that PAR3 negatively regulates the interaction between CLASP2 and GCC185. The effects of aPKC depletion were inconclusive because the treatment of the cross-linker with aPKC-depleted cells drastically decreased the GCC185 solubility for the immunoprecipitation.

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