[Show abstract][Hide abstract] ABSTRACT: Primary cilia are formed by extending the microtubule-based axoneme from the mother centriole-derived basal body. Recruitment of Tau tubulin kinase-2 (TTBK2) to the mother centriole and subsequent removal of CP110 and its interactor Cep97 are crucial for the initiation of ciliogenesis. We analyzed the roles of two TTBK2-binding proteins, EB1 and Cep164, in centriolar localization of TTBK2. TTBK2 bound EB1 and Cep164 through its SxIP motifs and a proline-rich motif, respectively. Using TTBK2 variants that contained mutations in the SxIP or proline-rich motifs, we obtained evidence that Cep164, but not EB1, is essential for centriolar localization of TTBK2. Depletion of TTBK2 inhibited CP110 removal and ciliogenesis, whereas expression of wild-type TTBK2, but not non-Cep164-binding mutants, rescued CP110 removal and ciliogenesis in TTBK2-depleted cells. Therefore, Cep164 binding is essential for the function of TTBK2 in promoting CP110 removal and ciliogenesis. We also provide evidence that TTBK2 has the potential to effectively phosphorylate Cep164 and Cep97 and inhibits the interaction between Cep164 and its binding partner Dishevelled-3 (an important regulator of ciliogenesis) in a kinase activity-dependent manner.
[Show abstract][Hide abstract] ABSTRACT: The structure and function of microtubules (MTs) are regulated by post-translational modifications of tubulin subunits, such as acetylation of the Lys-40 residue of α-tubulin. Regulation of the organization and dynamics of MTs is essential for the precise formation of the mitotic spindle. Spindle MTs are highly acetylated, but the mechanism regulating this acetylation is largely unknown. Furry (Fry) is an evolutionarily conserved protein that binds to MTs and colocalizes with acetylated MTs in the mitotic spindle. In this study, we examined the role of Fry in the acetylation of MTs in the mitotic spindle. Depletion of Fry significantly reduced the level of MT acetylation in the mitotic spindle. Expression of the N-terminal fragment of Fry induced hyperacetylation of MTs in both mitotic and interphase cells. These results indicate that Fry promotes MT acetylation in the mitotic spindle. We also found that Fry binds to the tubulin-deacetylase SIRT2, preferentially in mitotic cells. Cell-free experiments revealed that the N-terminal region of Fry is the domain responsible for binding to and inhibiting the tubulin-deacetylase activity of SIRT2. AGK2, a specific inhibitor of SIRT2, increased the level of MT acetylation in the mitotic spindle, indicating that SIRT2 is involved in the deacetylation of spindle MTs. Furthermore, AGK2 reversed the decrease in MT acetylation induced by Fry depletion. In summary, these results suggest that Fry plays a crucial role in promoting the level of MT acetylation in the mitotic spindle by inhibiting the tubulin-deacetylase activity of SIRT2.
[Show abstract][Hide abstract] ABSTRACT: Primary cilia are antenna-like sensory organelles protruding from the plasma membrane. Defects in ciliogenesis cause diverse genetic disorders. NDR2 was identified as the causal gene for a canine ciliopathy, early retinal degeneration, but its role in ciliogenesis remains unknown. Ciliary membranes are generated by transport and fusion of Golgi-derived vesicles to the pericentrosome, a process requiring Rab11-mediated recruitment of Rabin8, a GDP-GTP exchange factor (GEF) for Rab8, and subsequent Rab8 activation and Rabin8 binding to Sec15, a component of the exocyst that mediates vesicle tethering. This study shows that NDR2 phosphorylates Rabin8 at Ser-272 and defects in this phosphorylation impair preciliary membrane assembly and ciliogenesis, resulting in accumulation of Rabin8-/Rab11-containing vesicles at the pericentrosome. Rabin8 binds to and colocalizes with GTP-bound Rab11 and phosphatidylserine (PS) on pericentrosomal vesicles. The phospho-mimetic S272E mutation of Rabin8 decreases affinity for PS but increases affinity for Sec15. These results suggest that NDR2-mediated Rabin8 phosphorylation is crucial for ciliogenesis by triggering the switch in binding specificity of Rabin8 from PS to Sec15, thereby promoting local activation of Rab8 and ciliary membrane formation.
No preview · Article · Feb 2013 · The EMBO Journal
[Show abstract][Hide abstract] ABSTRACT: Mps one binder 1a (MOB1A) and MOB1B are key components of the Hippo signaling pathway and are mutated or inactivated in many human cancers. Here we show that intact Mob1a or Mob1b is essential for murine embryogenesis and that loss of the remaining WT Mob1 allele in Mob1aΔ/Δ1btr/+ or Mob1aΔ/+1btr/tr mice results in tumor development. Because most of these cancers resembled trichilemmal carcinomas, we generated double-mutant mice bearing tamoxifen-inducible, keratinocyte-specific homozygous-null mutations of Mob1a and Mob1b (kDKO mice). kDKO mice showed hyperplastic keratinocyte progenitors and defective keratinocyte terminal differentiation and soon died of malnutrition. kDKO keratinocytes exhibited hyperproliferation, apoptotic resistance, impaired contact inhibition, enhanced progenitor self renewal, and increased centrosomes. Examination of Hippo pathway signaling in kDKO keratinocytes revealed that loss of Mob1a/b altered the activities of the downstream Hippo mediators LATS and YAP1. Similarly, YAP1 was activated in some human trichilemmal carcinomas, and some of these also exhibited MOB1A/1B inactivation. Our results clearly demonstrate that MOB1A and MOB1B have overlapping functions in skin homeostasis, and exert their roles as tumor suppressors by regulating downstream elements of the Hippo pathway.
Full-text · Article · Nov 2012 · The Journal of clinical investigation
[Show abstract][Hide abstract] ABSTRACT: Bipolar mitotic spindle organization is fundamental to faithful chromosome segregation. Furry (Fry) is an evolutionarily conserved
protein implicated in cell division and morphology. In human cells, Fry localizes to centrosomes and spindle microtubules
in early mitosis, and depletion of Fry causes multipolar spindle formation. However, it remains unknown how Fry controls bipolar
spindle organization. This study demonstrates that Fry binds to polo-like kinase 1 (Plk1) through the polo-box domain of Plk1
in a manner dependent on the cyclin-dependent kinase 1-mediated Fry phosphorylation at Thr-2516. Fry also binds to Aurora
A and promotes Plk1 activity by binding to the polo-box domain of Plk1 and by facilitating Aurora A-mediated Plk1 phosphorylation
at Thr-210. Depletion of Fry causes centrosome and centriole splitting in mitotic spindles and reduces the kinase activity
of Plk1 in mitotic cells and the accumulation of Thr-210-phosphorylated Plk1 at the spindle poles. Our results suggest that
Fry plays a crucial role in the structural integrity of mitotic centrosomes and in the maintenance of spindle bipolarity by
promoting Plk1 activity at the spindle poles in early mitosis.
Full-text · Article · Jun 2012 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Proper attachment of microtubules to kinetochores is essential for accurate chromosome segregation. Here, we report a novel protein involved in kinetochore-microtubule attachment, chromosome alignment-maintaining phosphoprotein (CAMP) (C13orf8, ZNF828). CAMP is a zinc-finger protein containing three characteristic repeat motifs termed the WK, SPE, and FPE motifs. CAMP localizes to chromosomes and the spindle including kinetochores, and undergoes CDK1-dependent phosphorylation at multiple sites during mitosis. CAMP-depleted cells showed severe chromosome misalignment, which was associated with the poor resistance of K-fibres to the tension exerted upon establishment of sister kinetochore bi-orientation. We found that the FPE region, which is responsible for spindle and kinetochore localization, is essential for proper chromosome alignment. The C-terminal region containing the zinc-finger domains negatively regulates chromosome alignment, and phosphorylation in the FPE region counteracts this regulation. Kinetochore localization of CENP-E and CENP-F was affected by CAMP depletion, and by expressing CAMP mutants that cannot functionally rescue CAMP depletion, placing CENP-E and CENP-F as downstream effectors of CAMP. These data suggest that CAMP is required for maintaining kinetochore-microtubule attachment during bi-orientation.
[Show abstract][Hide abstract] ABSTRACT: The precise alignment of chromosomes on the metaphase plate prior to the onset of anaphase is essential for ensuring equal segregation of sister chromatids into two daughter cells, and defects in this process potentially cause chromosome instability and tumor progression [1-3]. NDR1 is an evolutionarily conserved serine/threonine kinase whose activity is regulated by MST kinases, Furry (Fry), and MOB . Although the NDR1 signaling pathway is implicated in cell division and morphogenesis in yeast and invertebrates [4-16], the mechanisms of NDR1 activation and the functional significance of the NDR1 pathway in mammalian cells are largely unknown. Here, we show that NDR1 is required for accurate chromosome alignment at metaphase in HeLa cells; depletion of NDR1, Fry, or MST2 caused mitotic chromosome misalignment. Chromosome misalignment in MST2-depleted cells was corrected by expression of active NDR1. The kinase activity of NDR1 increased in early mitotic phase and was dependent on Fry and MST2. We also provide evidence that Fry binds to microtubules, localizes on the spindle, acts as a scaffold that binds to both NDR1 and MOB2, and synergistically activates NDR1 with MOB2. Our findings suggest that MST2-, Fry-, and MOB2-mediated activation of NDR1 is crucial for the fidelity of mitotic chromosome alignment in mammalian cells.
Full-text · Article · Apr 2009 · Current biology: CB
[Show abstract][Hide abstract] ABSTRACT: Cofilin mediates lamellipodium extension and polarized cell migration by stimulating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by phosphorylation at Ser-3 and reactivated by cofilin-phosphatase Slingshot-1L (SSH1L). Little is known of signaling mechanisms of cofilin activation and how this activation is spatially regulated. Here, we show that cofilin-phosphatase activity of SSH1L increases approximately 10-fold by association with actin filaments, which indicates that actin assembly at the leading edge per se triggers local activation of SSH1L and thereby stimulates cofilin-mediated actin turnover in lamellipodia. We also provide evidence that 14-3-3 proteins inhibit SSH1L activity, dependent on the phosphorylation of Ser-937 and Ser-978 of SSH1L. Stimulation of cells with neuregulin-1beta induced Ser-978 dephosphorylation, translocation of SSH1L onto F-actin-rich lamellipodia, and cofilin dephosphorylation. These findings suggest that SSH1L is locally activated by translocation to and association with F-actin in lamellipodia in response to neuregulin-1beta and 14-3-3 proteins negatively regulate SSH1L activity by sequestering it in the cytoplasm.
Full-text · Article · Jun 2004 · The Journal of Cell Biology