TTBK2 with EB1/3 regulates microtubule dynamics in migrating cells through KIF2A phosphorylation.
Bottom Line: TTBK2 depletion reduced MT lifetime (facilitated shrinkage and suppressed rescue) and impaired HeLa cell migration, and these phenotypes were partially restored by KIF2A co-depletion.Expression of nonphosphorylatable KIF2A, but not wild-type KIF2A, reduced MT lifetime and slowed down the cell migration.These findings indicate that TTBK2 with EB1/3 phosphorylates KIF2A and antagonizes KIF2A-induced depolymerization at MT plus ends for cell migration.
Affiliation: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Showa, Nagoya 466-8550, Japan.Show MeSH
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Mentions: EB1 and EB3 play crucial roles in recruiting other +TIPs to regulate MT dynamics (Akhmanova and Steinmetz, 2008). We sought to isolate the +TIPs that interact with EB1 and EB3 with a pull-down assay using whole rat brain lysates and mass spectrometry. We identified TTBK1 and TTBK2 as EB-binding proteins (Fig. S1), similar to results reported by others (Jiang et al., 2012). Both TTBKs display similar domain organization: a kinase domain at the N terminus and two EB-binding SxIP motifs in the C terminus (Fig. 1 A). Then, we characterized the EB3-binding and end-tracking properties of TTBK2. The formation of complexes between TTBKs and EB3 was confirmed by immunoprecipitation (Fig. 1 B). A pull-down assay using GST-EB3 demonstrated the common binding properties of the SxIP motifs in TTBK2 (Fig. 1 C): EB3 full length (FL) and EB3 Δ3 aa, which lacks the final 3 aa that recognize the cytoskeleton-associated protein glycine-rich domain (Komarova et al., 2005; Mishima et al., 2007), were associated with monomeric GFP (mGFP)–TTBK2, but this association was diminished after further deletion of the C-terminal region of EB3, which is responsible for its binding to the SxIP motif (Honnappa et al., 2009). Furthermore, mutation of both SxIP motifs in TTBK2 almost completely abolished its binding to EB3, although each SxIP motif differentially contributed to EB3 binding (Fig. 1 D). Consistently, the accumulation of mGFP-TTBK2 at MT ends was lost when both SxIP motifs were mutated (Fig. 1 E). The C-terminal region of TTBK2 (TTBK2-C; Fig. 1 A), which harbors the two SxIP motifs, was sufficient for end-tracking (Fig. 1 F). In addition, purified mGFP-TTBK2 required EB3 for its accumulation at the growing ends of MTs in vitro (Fig. 1 G). Collectively, the data indicate that TTBK2 acts as a typical SxIP motif–containing +TIP.
Affiliation: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Showa, Nagoya 466-8550, Japan.