Limits...
TTBK2 with EB1/3 regulates microtubule dynamics in migrating cells through KIF2A phosphorylation.

Watanabe T, Kakeno M, Matsui T, Sugiyama I, Arimura N, Matsuzawa K, Shirahige A, Ishidate F, Nishioka T, Taya S, Hoshino M, Kaibuchi K - J. Cell Biol. (2015)

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.

View Article: PubMed Central - HTML - PubMed

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

Show MeSH

Related in: MedlinePlus

TTBK2 tracks MT plus ends in an EB-dependent manner. (A) Schematic diagram of TTBK1 and TTBK2. Kinase, protein kinase domain; SxIP, EB-binding motif. The red X indicates the mutation site in the SxIP motif. (B) Immunoprecipitation (IP) between TTBK2 and EB3 using COS-7 cells. R IgG indicates rabbit IgG. (C) COS-7 cells expressing mGFP-TTBK2 were lysed and incubated with GST-fused proteins. mGFP-TTBK2 coprecipitated with EB3-FL and EB3 lacking 3 aa from the C terminus but not with the further deletion mutant EB3 1–251 aa. Note that GST–EB3 1–251 aa and GST–EB3 Δ3 aa contain a 13-aa linker after the GST tag (see Protein purification and biochemistry in Materials and methods). IB, immunoblotting. CBB, Coomassie Brilliant blue staining. (D) Mutation of both SxIP motifs diminished the association between TTBK2 and EB3. (E) COS-7 cells expressing TTBK2 were imaged using epifluorescence microscopy. mGFP–TTBK2-WT accumulated at EB3-positive MT plus ends, but the mutant TTBK2-m1/2 did not. Bar, 10 µm. (F) mGFP–TTBK2-C colocalized with EB3 at the MT ends. Bar, 5 µm. (G) In vitro reconstitution of TTBK2 tracking. Purified mGFP–TTBK2-FL tracked the MT plus ends only in the presence of EB3. Protein concentrations are as follows: 15-µM tubulin (containing 3.2% rhodamine-labeled tubulin), 25-nM FLAG–mGFP-TTBK2, and 400-nM EB3. Kymograph images are presented. Horizontal and vertical bars, 5 µm and 50 s, respectively. See Fig. S1 for identification of the TTBKs as EB-binding proteins.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4555816&req=5

fig1: TTBK2 tracks MT plus ends in an EB-dependent manner. (A) Schematic diagram of TTBK1 and TTBK2. Kinase, protein kinase domain; SxIP, EB-binding motif. The red X indicates the mutation site in the SxIP motif. (B) Immunoprecipitation (IP) between TTBK2 and EB3 using COS-7 cells. R IgG indicates rabbit IgG. (C) COS-7 cells expressing mGFP-TTBK2 were lysed and incubated with GST-fused proteins. mGFP-TTBK2 coprecipitated with EB3-FL and EB3 lacking 3 aa from the C terminus but not with the further deletion mutant EB3 1–251 aa. Note that GST–EB3 1–251 aa and GST–EB3 Δ3 aa contain a 13-aa linker after the GST tag (see Protein purification and biochemistry in Materials and methods). IB, immunoblotting. CBB, Coomassie Brilliant blue staining. (D) Mutation of both SxIP motifs diminished the association between TTBK2 and EB3. (E) COS-7 cells expressing TTBK2 were imaged using epifluorescence microscopy. mGFP–TTBK2-WT accumulated at EB3-positive MT plus ends, but the mutant TTBK2-m1/2 did not. Bar, 10 µm. (F) mGFP–TTBK2-C colocalized with EB3 at the MT ends. Bar, 5 µm. (G) In vitro reconstitution of TTBK2 tracking. Purified mGFP–TTBK2-FL tracked the MT plus ends only in the presence of EB3. Protein concentrations are as follows: 15-µM tubulin (containing 3.2% rhodamine-labeled tubulin), 25-nM FLAG–mGFP-TTBK2, and 400-nM EB3. Kymograph images are presented. Horizontal and vertical bars, 5 µm and 50 s, respectively. See Fig. S1 for identification of the TTBKs as EB-binding proteins.

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.


TTBK2 with EB1/3 regulates microtubule dynamics in migrating cells through KIF2A phosphorylation.

Watanabe T, Kakeno M, Matsui T, Sugiyama I, Arimura N, Matsuzawa K, Shirahige A, Ishidate F, Nishioka T, Taya S, Hoshino M, Kaibuchi K - J. Cell Biol. (2015)

TTBK2 tracks MT plus ends in an EB-dependent manner. (A) Schematic diagram of TTBK1 and TTBK2. Kinase, protein kinase domain; SxIP, EB-binding motif. The red X indicates the mutation site in the SxIP motif. (B) Immunoprecipitation (IP) between TTBK2 and EB3 using COS-7 cells. R IgG indicates rabbit IgG. (C) COS-7 cells expressing mGFP-TTBK2 were lysed and incubated with GST-fused proteins. mGFP-TTBK2 coprecipitated with EB3-FL and EB3 lacking 3 aa from the C terminus but not with the further deletion mutant EB3 1–251 aa. Note that GST–EB3 1–251 aa and GST–EB3 Δ3 aa contain a 13-aa linker after the GST tag (see Protein purification and biochemistry in Materials and methods). IB, immunoblotting. CBB, Coomassie Brilliant blue staining. (D) Mutation of both SxIP motifs diminished the association between TTBK2 and EB3. (E) COS-7 cells expressing TTBK2 were imaged using epifluorescence microscopy. mGFP–TTBK2-WT accumulated at EB3-positive MT plus ends, but the mutant TTBK2-m1/2 did not. Bar, 10 µm. (F) mGFP–TTBK2-C colocalized with EB3 at the MT ends. Bar, 5 µm. (G) In vitro reconstitution of TTBK2 tracking. Purified mGFP–TTBK2-FL tracked the MT plus ends only in the presence of EB3. Protein concentrations are as follows: 15-µM tubulin (containing 3.2% rhodamine-labeled tubulin), 25-nM FLAG–mGFP-TTBK2, and 400-nM EB3. Kymograph images are presented. Horizontal and vertical bars, 5 µm and 50 s, respectively. See Fig. S1 for identification of the TTBKs as EB-binding proteins.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4555816&req=5

fig1: TTBK2 tracks MT plus ends in an EB-dependent manner. (A) Schematic diagram of TTBK1 and TTBK2. Kinase, protein kinase domain; SxIP, EB-binding motif. The red X indicates the mutation site in the SxIP motif. (B) Immunoprecipitation (IP) between TTBK2 and EB3 using COS-7 cells. R IgG indicates rabbit IgG. (C) COS-7 cells expressing mGFP-TTBK2 were lysed and incubated with GST-fused proteins. mGFP-TTBK2 coprecipitated with EB3-FL and EB3 lacking 3 aa from the C terminus but not with the further deletion mutant EB3 1–251 aa. Note that GST–EB3 1–251 aa and GST–EB3 Δ3 aa contain a 13-aa linker after the GST tag (see Protein purification and biochemistry in Materials and methods). IB, immunoblotting. CBB, Coomassie Brilliant blue staining. (D) Mutation of both SxIP motifs diminished the association between TTBK2 and EB3. (E) COS-7 cells expressing TTBK2 were imaged using epifluorescence microscopy. mGFP–TTBK2-WT accumulated at EB3-positive MT plus ends, but the mutant TTBK2-m1/2 did not. Bar, 10 µm. (F) mGFP–TTBK2-C colocalized with EB3 at the MT ends. Bar, 5 µm. (G) In vitro reconstitution of TTBK2 tracking. Purified mGFP–TTBK2-FL tracked the MT plus ends only in the presence of EB3. Protein concentrations are as follows: 15-µM tubulin (containing 3.2% rhodamine-labeled tubulin), 25-nM FLAG–mGFP-TTBK2, and 400-nM EB3. Kymograph images are presented. Horizontal and vertical bars, 5 µm and 50 s, respectively. See Fig. S1 for identification of the TTBKs as EB-binding proteins.
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.

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.

View Article: PubMed Central - HTML - PubMed

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

Show MeSH
Related in: MedlinePlus