CLIP-170 facilitates the formation of kinetochore–microtubule attachments

Tanenbaum, Marvin E.; Galjart, Niels; Vugt, Marcel A.T.M. van; Medema, René H.

doi:10.1038/sj.emboj.7600916

Cited by 76 publications

(104 citation statements)

References 35 publications

(68 reference statements)

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“…Although CLIP-170 depletion did not affect mitotic entry and exit, CLIP-170-depleted cells had significantly higher levels of phospho-H3-positive staining at 10, 12, and 14 h postrelease, indicating that CLIP-170 is involved in mitotic progression (data not shown). This is consistent with the published CLIP-170 loss-of-function phenotypic analysis (12). To assess the contribution of Cdc2 phosphorylation of CLIP-170 during mitosis, similar experiments were performed using U2OS cells stably expressing GFP-CLIP-170 (WT, T287A, or T287D) in the absence of endogenous CLIP-170 (Fig.…”

Section: Cdc2-mediated Phosphorylation Of Clip-170 Is Essential For Tsupporting

confidence: 71%

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Cdc2-mediated Phosphorylation of CLIP-170 Is Essential for Its Inhibition of Centrosome Reduplication

Yang

Liu

et al. 2009

Journal of Biological Chemistry

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CLIP-170, the founding member of microtubule "plus ends tracking" proteins, is involved in many critical microtubulerelated functions, including recruitment of dynactin to the microtubule plus ends and formation of kinetochore-microtubule attachments during metaphase. Although it has been reported that CLIP-170 is a phosphoprotein, neither have individual phosphorylation sites been identified nor have the associated kinases been extensively studied. Herein, we identify Cdc2 as a kinase that phosphorylates CLIP-170. We show that Cdc2 interacts with CLIP-170 mediating its phosphorylation on Thr 287 in vivo. Significantly, expression of CLIP-170 with a threonine 287 to alanine substitution (T287A) results in its mislocalization, accumulation of Plk1 and cyclin B, and block of the G2/M transition. Finally, we found that depletion of CLIP-170 leads to centrosome reduplication and that Cdc2 phosphorylation of CLIP-170 is required for the process. These results demonstrate that Cdc2-mediated phosphorylation of CLIP-170 is essential for the normal function of this protein during cell cycle progression.Microtubule dynamics consist of alternating phases of growth and shortening, a pattern of behavior known as dynamic instability (1). This process is tightly regulated by a group of proteins that bind specifically to the plus ends of the growing microtubules (2). Cytoplasmic linker protein (CLIP) 3 -170, the founding member of the microtubule plus end family (3), is composed of three separate regions: N terminus, central coiled-coil region, and C terminus. In addition to two conserved cytoskeleton-associated protein glycine-rich (CAP-Gly) domains, the N terminus has three serine-rich regions. The N-terminal domain plays an essential role in microtubule targeting (4), the long central coiled-coil domain is responsible for dimerization of the protein, and the C-terminal region, which contains two zinc-finger domains interferes with microtubule binding by interacting with the N terminus (5). Experiments in a variety of organisms have demonstrated that CLIP-170 plays an important role in microtubule dynamics (6, 7). In addition to its positive role in regulating microtubule growth in both yeast and humans (8, 9), CLIP-170 is involved in recruitment of dynactin to the microtubule plus ends and in linking microtubules to the cortex through Cdc42 and IQGAP (10, 11). The role of CLIP-170 during mitosis was recently examined by loss-offunction approaches. It was shown that CLIP-170 localizes to unattached kinetochores in prometaphase and that such localization is essential for the formation of kinetochore-microtubule attachments (12, 13).It was previously reported that CLIP-170 is a phosphoprotein and that overall phosphorylation of CLIP-170 affects its microtubule binding ability (14). More recently, metabolic labeling experiments indicated that CLIP-170 is phosphorylated at multiple sites (15). However, individual phosphorylation sites have not been identified. Moreover, the FKBP12-rapamycin-associated protein (FRAP) is the only...

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Section: Cdc2-mediated Phosphorylation Of Clip-170 Is Essential For Tsupporting

confidence: 71%

“…The role of CLIP-170 during mitosis was recently examined by loss-offunction approaches. It was shown that CLIP-170 localizes to unattached kinetochores in prometaphase and that such localization is essential for the formation of kinetochore-microtubule attachments (12,13).…”

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confidence: 99%

Cdc2-mediated Phosphorylation of CLIP-170 Is Essential for Its Inhibition of Centrosome Reduplication

Yang

Liu

et al. 2009

Journal of Biological Chemistry

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“…These results suggest that redundant pathways exist for spindle assembly and chromosome congression in mammalian cells. The prometaphase delay in myosin VI knockdown cells is very similar to the delay described recently for Clip 170 KD cells (Dujardin et al, 1998;Tanenbaum et al, 2006). Clip 170 was identified as a myosin VI binding partner in Drosophila (Lantz and Miller, 1998); however, in mammalian cells we have not been able to confirm by yeast two-hybrid or mammalian two-hybrid assays or by coimmunoprecipitation any interaction between myosin VI and Clip 170.…”

Section: Discussionmentioning

confidence: 53%

Myosin VI Is Required for Targeted Membrane Transport during Cytokinesis

Arden

Puri

et al. 2007

MBoC

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Myosin VI plays important roles in endocytic and exocytic membrane-trafficking pathways in cells. Because recent work has highlighted the importance of targeted membrane transport during cytokinesis, we investigated whether myosin VI plays a role in this process during cell division. In dividing cells, myosin VI undergoes dramatic changes in localization: in prophase, myosin VI is recruited to the spindle poles; and in cytokinesis, myosin VI is targeted to the walls of the ingressing cleavage furrow, with a dramatic concentration in the midbody region. Furthermore, myosin VI is present on vesicles moving into and out of the cytoplasmic bridge connecting the two daughter cells. Inhibition of myosin VI activity by small interfering RNA (siRNA)-mediated knockdown or by overexpression of dominant-negative myosin VI tail leads to a delay in metaphase progression and a defect in cytokinesis. GAIP-interacting protein COOH terminus (GIPC), a myosin VI binding partner, is associated with the function(s) of myosin VI in dividing cells. Loss of GIPC in siRNA knockdown cells results in a more than fourfold increase in the number of multinucleated cells. Our results suggest that myosin VI has novel functions in mitosis and that it plays an essential role in targeted membrane transport during cytokinesis.

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“…It has been described that overexpression of p50/dynamitin, a second dynactin subunit, eliminated NE invaginations of prophase cells (1). This result suggests that p150 Glued might be also involved in NEBD, as overexpression of p50/dynamitin is a standard approach to inhibit the function of p150 Glued in a dominant-negative fashion (11). Unfortunately, we could not directly analyze the effect of depletion of p150 Glued on NEBD, as p150 Glued -depleted cells failed to enter mitosis (Fig.…”

Section: Results P150mentioning

confidence: 65%

Polo-like kinase 1 phosphorylation of p150 ^Glued facilitates nuclear envelope breakdown during prophase

Liu

Yang

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Nuclear envelope breakdown (NEBD) is an essential step during the G2/M transition in higher eukaryotic cells. Increasing evidence supports the notion that both microtubules and microtubuleassociated motor proteins are critical regulators of NEBD. Although it has been described that p150 Glued , the major component of the dynein/dynactin complex, localizes in the nuclear envelope during prophase, the exact role of p150 Glued and its regulation during NEBD are largely elusive. Polo-like kinase 1 (Plk1), the best characterized Ser/Thr kinase, is involved in mitotic entry in several systems; however, the targets of Plk1 during NEBD are unknown. Herein, we show that in mammalian cells both Plk1 and p150 Glued regulate NEBD and that Plk1 interacts with and phosphoryates p150 Glued during NEBD at prophase. Using various approaches, we showed that Plk1 phosphorylates p150 Glued at Ser-179 and that the pS179 epitope is generated at the nuclear envelope of prophase cells. Significantly, Plk1-mediated phosphorylation of p150 Glued at Ser-179 positively regulates its accumulation at the nuclear envelope during prophase. Finally, we found that cells expressing the Plk1-unphosphorylatable mutant (p150 Glued -S179A) arrest at G2, as indicated by reduced NEBD, increased levels of cyclin B and phospho-H3, but a decreased level of Cdc2 kinase activity. Taking these data together, we conclude that Plk1 phosphorylation of p150 Glued might be one major pathway of NEBD regulation. mitosis | microtubule dynamics | dynactin

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CLIP-170 facilitates the formation of kinetochore–microtubule attachments

Cited by 76 publications

References 35 publications

Cdc2-mediated Phosphorylation of CLIP-170 Is Essential for Its Inhibition of Centrosome Reduplication

Cdc2-mediated Phosphorylation of CLIP-170 Is Essential for Its Inhibition of Centrosome Reduplication

Myosin VI Is Required for Targeted Membrane Transport during Cytokinesis

Polo-like kinase 1 phosphorylation of p150 ^Glued facilitates nuclear envelope breakdown during prophase

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CLIP-170 facilitates the formation of kinetochore–microtubule attachments

Cited by 76 publications

References 35 publications

Cdc2-mediated Phosphorylation of CLIP-170 Is Essential for Its Inhibition of Centrosome Reduplication

Cdc2-mediated Phosphorylation of CLIP-170 Is Essential for Its Inhibition of Centrosome Reduplication

Myosin VI Is Required for Targeted Membrane Transport during Cytokinesis

Polo-like kinase 1 phosphorylation of p150 Glued facilitates nuclear envelope breakdown during prophase

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Polo-like kinase 1 phosphorylation of p150 ^Glued facilitates nuclear envelope breakdown during prophase