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