PAR3 and aPKC regulate Golgi organization through CLASP2 phosphorylation to generate cell polarity.
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.
Affiliation: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.Show MeSH
Related in: MedlinePlus
Mentions: PAR3 acts as an escort protein for aPKC substrates such as Numb and Tiam1 and is required for their phosphorylation (Nishimura and Kaibuchi, 2007; Wang et al., 2012). We therefore investigated whether aPKC phosphorylates CLASP2. In in vitro phosphorylation assays, aPKCζ and aPKCλ phosphorylated histidine (His)-CLASP2γ but not His-RhoGDI, which was used as a control (Figure 2A and unpublished data). The phosphorylation sites were mapped to the serine/arginine (S/R)-rich and carboxyl (C)-terminal regions in CLASP2 (Supplemental Figure S1A and Figure 2A), which are responsible for its localization to MT ends and the TGN (Akhmanova et al., 2001; Mimori-Kiyosue et al., 2005; Efimov et al., 2007). Because aPKC depletion did not affect its localization to MT ends but did influence its localization to the TGN (see later result), we focused on the phosphorylation of the C-terminal region. aPKCζ phosphorylated GST–CLASP2-C1 (aa 872–1294) but not GST-CLASP2-C (aa 1017–1294; Figure 2B), indicating that aPKC phosphorylation occurs in the region between aa 872 and aa 1016 in CLASP2. To identify the exact phosphorylation site, we prepared a series of CLASP2-C1 point mutants whose potential PKC phosphorylation sites ([S/T]-X-[R/K], [R/K]-X-[S/T] and [R/K]-X-X-[S/T]) in this region were mutated to Ala. Ala substitutions at Ser-940 (S940A), Ser-952 (S952A), and Ser-967 (S967A) significantly reduced the phosphorylation by aPKCζ (Figure 2C), suggesting that these three Ser residues are major sites of phosphorylation by aPKC. To verify the in vivo phosphorylation of CLASP2, we attempted to generate phosphospecific antibodies against these residues and successfully produced anti–S940-P antibody. In immunoblot analysis, anti–S940-P antibody detected the phosphorylation of CLASP2-C1 by aPKCζ in vitro but not that of CLASP2-C1-S940A (Figure 2D), indicating that anti–S940-P antibody specifically detects the Ser-940 phosphorylation of CLASP2. This antibody did not detect the phosphorylation of endogenous CLASP2 in RPE-1 cells under normal growth conditions. However, treatment of the cells with calyculin-A, a phosphatase inhibitor for PP1 and PP2A, increased the phosphorylation level of CLASP2, and this increase was suppressed by pretreatment with an aPKC pseudosubstrate inhibitor (aPKC-PS; Figure 2E) and depletion of aPKC (Figure 2F; see later result). Under the same condition, depletion of PAR3 also reduced the phosphorylation of Ser-940 in CLASP2 (unpublished data). Because the phosphorylation levels of the proteins are regulated by the balance between kinases and phosphatases, these results suggest that aPKC, together with PAR3, phosphorylates CLASP2 in vivo and that the turnover of this phosphorylation is rapid.
Affiliation: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.