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Clan Analogue
Try out PMC Labs and tell us what you think. Learn More. In cytokinesis with chromatin bridges, cells delay abscission and retain actin patches at the intercellular canal to prevent chromosome breakage. In this study, we show that inhibition of Src, a protein-tyrosine kinase that regulates actin dynamics, or Chk1 kinase correlates with chromatin breakage and impaired formation of actin patches but not with abscission in the presence of chromatin bridges. Chk1 is required for optimal localization and complete activation of Src. Furthermore, Chk1 phosphorylates human Src at serine 51, and phosphorylated Src localizes to actin patches, the cell membrane, or the nucleus. Nonphosphorylatable mutation of S51 to alanine reduces Src catalytic activity and impairs formation of actin patches, whereas expression of a phosphomimicking Src-S51D protein rescues actin patches and prevents chromatin breakage in Chk1-deficient cells. We propose that Chk1 phosphorylates Src-S51 to fully induce Src kinase activity and that phosphorylated Src promotes formation of actin patches and stabilizes chromatin bridges. These results identify proteins that regulate formation of actin patches in cytokinesis. Chromatin bridges are strands of incompletely segregated chromatin that connect anaphase poles or daughter nuclei and have been linked to tumorigenesis Hoffelder et al. In the presence of chromatin bridges, eukaryotic cells delay abscission, the final cut of the narrow cytoplasmic canal that connects the daughter cells, to prevent tetraploidization by regression of the cleavage furrow or chromatin breakage Steigemann et al. In mammals, this abscission delay is called the abscission checkpoint and relies on the Aurora B protein kinase Steigemann et al. Furthermore, cells with chromatin bridges form and retain actin-rich structures called actin patches at the base of the chromatin bridge Chen and Doxsey, ; Steigemann et al. It is suggested that actin patches stabilize the intercellular canal until the DNA bridge is resolved; however, how actin patches are formed has not been previously reported. Src is a nonreceptor tyrosine kinase that is involved in a diverse spectrum of biological activities including cell proliferation, adhesion, spreading, and migration Playford and Schaller, Src is located at the plasma membrane and is also found at late endosomes, the Golgi apparatus, and the nucleus Takahashi et al. In addition, the Unique domain of Src contains phosphorylation residues that activate Src by promoting dephosphorylation of the autoinhibitory site Shenoy et al. Activating mutations in cellular Src or infection with the Src encoding Rous sarcoma virus can cause oncogenic transformation that is accompanied by dramatic changes in the actin cytoskeleton Frame, In turn, the FAK—Src signaling complex promotes changes in actin cytoskeleton and regulates focal adhesion turnover Goldberg et al. Src phosphorylates cortactin to enhance actin nucleation and binds to formins to induce formation of stress fibers Tominaga et al. In addition, Src signaling is involved in the completion of cytokinesis Kasahara et al. Chk1 kinase was first identified to regulate the DNA damage response Smith et al. Chk1 phosphorylates the mitotic kinase Aurora B in prometaphase and metaphase to induce Aurora B catalytic activity and promote correction of misattached kinetochore—microtubules Petsalaki et al. Also, Chk1 is required for successful chromosome segregation and cytokinesis and for an abscission delay in response to replication stress Peddibhotla et al. In this study, we show that Chk1 phosphorylates human Src at the newly identified site serine 51 to fully induce Src kinase activity. We also show that phosphorylated Src promotes formation of actin patches and prevents chromosome breakage in cytokinesis with chromatin bridges. Because Src and Chk1 are involved in completion of cytokinesis Kasahara et al. S1, A and B. Treatment with PP2 did not exacerbate chromatin breakage in Chk1-depleted cells, suggesting that Chk1 and Src prevent chromatin breakage by acting through the same mechanism Fig. Also, Chk1 or Src inhibition increased the frequency of chromatin bridges that were positive for phospho-Ser histone H2A. Src or Chk1-inhibition correlates with chromatin breakage. B Percentage of DNA bridges that appear broken. Intact intercellular canals are indicated by solid arrows, and broken canals are indicated by dotted arrows. Related to Videos 1, 2, and 3. Error bars show the SD from the mean from three independent experiments. A minimum of 50 fixed cells with chromatin bridges was analyzed per experiment. S1 C, and Video 1. S1 C, and Video 2. S1 C, and Video 3. These results suggest that Chk1 and Src prevent chromatin breakage in cytokinesis with chromatin bridges. Fragmented chromatin bridges can lead to the formation of micronuclei and accumulation of DNA damage Hoffelder et al. Depletion of Src or Chk1 increased the frequency of BE cells exhibiting micronuclei that were devoid of nuclear lamin B2 compared with controls Fig. These results suggest that Src and Chk1 are required to prevent formation of micronuclei and generation of DNA damage in cytokinesis with chromatin bridges. Inhibition of Src or Chk1 diminishes formation of actin patches. A minimum of cells was analyzed per experiment. D Cells were transfected as in A or treated with PP2 for 5 h. Broken DNA bridges are indicated by dotted arrows, and the bases of the intercellular canals are indicated by solid arrows. Relative actin patch intensity values are shown. E Actin patches intensity. Relative green fluorescence intensity from D is shown, and values in control were set to 1. Error bars show the SD from the mean. Control cells with chromatin bridges exhibited actin patches, i. However, Src- or Chk1-deficient cells exhibited reduced actin patches, and this was not a result of diminished total levels of actin compared with controls Fig. Control cells with relatively strong or weak DNA bridge labeling i. Also, Chk1- or Src-depleted cells exhibited reduced actin patches compared with controls regardless of the intensity of the DNA bridge signal relatively strong versus weak signal; Fig. S1 E , suggesting that the amount of DNA inside the intercellular canal does not associate with the intensity of actin patches. Actin patches were also diminished in Src- or Chk1-deficient cells with intact DNA bridges, suggesting that impaired actin patches were not a consequence of chromatin breakage Fig. S1, F—H. S1, I and J. These results show that Src and Chk1 are required for stable chromatin bridges and formation of actin patches in cytokinesis. Actin patches intensity is independent of the relative amount of DNA inside the intercellular canal. The mean fluorescence intensity of control DNA bridges was set to 0. B Actin patches intensity. Relative green fluorescence intensity from A is shown, and values in strong DNA control were set to 1. Broken DNA bridges are indicated by dotted arrows and the bases of the intercellular canals are indicated by solid arrows. Insets show 1. E Broken bridges analysis. A minimum of 30 cells with chromatin bridges was analyzed per experiment. F Actin patches intensity. S2 A , confirming that Vps4-KQ inhibits abscission under the experimental conditions used in our study Steigemann et al. These results show that chromatin breakage in Src- or Chk1-deficient cells is not caused by abscission. Expression of dominant-negative Vps4-KQ does not prevent chromatin breakage in Src or Chk1-deficient cells. A and C Examples of cells with DNA bridges exhibiting broken or intact intercellular canals after labeling with a lipophilic dye. B Frequency of cells with broken DNA bridges exhibiting intact intercellular canals. Broken DNA bridges or intercellular canals are indicated by dotted arrows. Aurora B—deficient cells exhibit premature abscission Steigemann et al. Simultaneous depletion of Chk1 or Src and Aurora B exacerbated chromatin breakage, and this correlated with reduced formation of actin patches and with increased frequency of cells with broken DNA bridges with an intact midbody i. The remaining double-depleted cells with broken DNA bridges exhibited midbody remnants, i. Control cells with intact DNA bridges also exhibited disassembled midbody microtubules midbody remnants despite the abscission delay Fig. The regulatory proteins Plk1, Mklp1, and Cep55 localize to the midbody in telophase or late cytokinesis in control cells Mishima et al. S2, B—G. It was previously reported that pharmacological inhibition of Src resulted in reduced Plk1 and increased Cep55 at the midbody in nontransformed cells Kamranvar et al. Perhaps the difference between our results and those from Kamranvar et al. We propose that Chk1 and Src stabilize chromatin bridges by promoting formation of actin patches. These results also suggest that Chk1 and Src cooperate with the Aurora B—mediated abscission checkpoint to prevent chromatin breakage in cytokinesis with DNA bridges. Chk1 or Src depletion exacerbates chromatin breakage and reduces actin patches in Aurora B—deficient cells. Broken DNA bridges and intercellular canals are indicated by dotted arrows, midbodies and midbody remnants by arrowheads, and the bases of the intercellular canals by solid arrows. A minimum of 50 cells with chromatin bridges was analyzed per experiment. C Actin patches intensity. Relative green fluorescence from A is shown, and values in control were set to 1. D Frequency of cells with broken DNA bridges exhibiting intact midbodies. A minimum of 20 cells with chromatin bridges was analyzed per experiment. E Localization of Plk1. F Plk1 midbody intensity. Relative green fluorescence from E is shown, and values in telophase control were set to 1. Proteins involved in actin remodeling can localize to actin structures. These results indicate that actin patch formation is regulated by Src signaling. However, Chk1 was not enriched at actin patches in control cells with chromatin bridges Fig. Src localizes to actin patches in cells with chromatin bridges. Images are representative of 20 cells from two independent experiments. Depletion of Chk1 or, for comparison, treatment with the Src family kinase inhibitor PP2 impaired localization of total Src and cortactin at the base of the intercellular canal and reduced phosphorylation of Src-Y and FAK-Y which are markers of Src kinase activity compared with control cells Fig. Furthermore, Chk1-deficient interphase cells without chromatin bridges exhibited reduced levels of phosphorylated Src-Y and phosphorylated FAK-Y and diminished localization of total Src to membrane ruffles compared with controls Fig. S3, A—E. Chk1-inhibition reduces Src kinase activity. A and B Cells transfected with negative siRNA control or siChk1 were trypsinized and seeded on fibronectin-coated dishes in the absence or presence of PP2 for 1 h. NS, nonspecific. Relative band intensity values are shown, and values in control were set to 1. C Chk1 depletion reduces cell spreading. Cells were seeded on fibronectin-coated dishes and phase-contrast images taken at various times after plating. D Percentage of spread cells i. Approximately —1, cells were analyzed per experiment. E Cells were seeded on fibronectin-coated dishes and analyzed by wound healing assay. Phase-contrast images at 0 h and 12 h after wounding are shown. F Wound area covered calculated from cells as in E. Error bars show the SD from the mean from four independent experiments. Inhibition of Src kinase activity reduces cell spreading and produces a more round cell morphology Elias et al. Control or Chk1-depleted cells were trypsinized, allowed to attach on fibronectin-coated dishes in the absence or presence of PP2, and observed at various times by phase-contrast time-lapse microscopy. A relatively high percentage of Chk1-deficient or PP2-treated cells remained round and highly reflective, indicating that these cells were not able to spread properly, compared with flattened controls Fig. Also, Src kinase activity promotes migration of cells during wound healing Di Florio et al. Depletion of Chk1 decreased wound closure of a monolayer of cells compared with controls Fig. Collectively, these results show that Chk1 is required for optimal Src kinase activity in interphase cells. S3 F and S4, A and C. Only monophosphorylated peptides were observed, and of these three sites, only the avSrc-S48 Fig. S4 B is conserved in the human protein and corresponds with human Src-S Multiple sequence alignment demonstrated S51 is conserved in Src proteins from different species Fig. Mutation of human Src-S51 to alanine reduces Src catalytic activity. A Chk1 kinase assay. Top and bottom panels are from the same gel. B Alignment of Src protein sequences. Human serine 51 is marked by an asterisk. C Chk1 in vitro kinase assay. Relative band intensity values are shown, and values in the second lane from left His-Src were set to 1. Bottom: Western blot analysis of total GFP and actin. Ab, antibody. F Immunoprecipitation-kinase assay in the absence or presence of siChk1. To investigate Src-S51 phosphorylation, an antiphospho—Src-S51 antiserum was raised against the human protein sequence. We propose that Chk1 phosphorylates Src-S51 and enhances Src catalytic activity in vitro. Together, these results show that S51 phosphorylation is required for complete Src catalytic activity. One possibility is that S51 phosphorylation enhances Src catalytic activity by promoting dephosphorylation of the inhibitory site Src-Y Shenoy et al. Chk1 depletion reduces Src-S51 phosphorylation. C Cells transfected as in B were seeded on fibronectin-coated slides for 1 h. D Phosphorylated Src-S51 at membrane ruffles. Relative green fluorescence intensity from C is shown, and values in control were set to 1. E Coimmunoprecipitation from asynchronous cells. Chk1 or Src were detected by Western blotting. Confocal microscopy analysis showed that phosphorylated Src-S51 localized to actin patches in control cells with chromatin bridges Figs. Furthermore, phospho-S51 staining was impaired after incubation of the anti—pSrc-S51 antiserum with the phosphorylated peptide phospho-S51 compared with the unphosphorylated peptide S51 synthetic peptides Fig. S4 F , verifying that this reagent is specific for the phosphorylated Src-S51 in immunofluorescence. Phosphorylated Src-S51 also localized to membrane ruffles, filopodia, and the nucleus and partially colocalized with vinculin at focal adhesions in interphase cells on fibronectin, in the absence of chromatin bridges Figs. Depletion of Chk1 diminished Src-S51 phosphorylation compared with control cells in the absence or presence of DNA bridges, showing that S51 phosphorylation is Chk1-dependent Fig. These results suggest that Chk1 associates with Src and phosphorylates Src-S51 in interphase cells. In cells depleted of the endogenous Src by siSrc-2, expression of the phosphomimetic mutant S51D but not WT GFP-Src resistant to degradation by siSrc-2 prevented chromatin breakage and rescued formation of actin patches after Chk1 depletion compared with Chk1-proficient controls Fig. In contrast, expression of the nonphosphorylatable mutant Src—GFP-S51A induced chromatin breakage and diminished formation of actin patches in the absence or presence of siChk1 Fig. We propose that Chk1-mediated Src-S51 phosphorylation is required for actin patch formation and inhibition of chromatin breakage in late cytokinesis. S5, D and E , in agreement with previous studies showing that Src catalytic activity promotes filopodia formation Robles et al. Expression of the phosphomimetic Src-S51D mutant rescues formation of actin patches and prevents chromatin breakage in Chk1-deficient cells. D Actin patches intensity. E Model for the role of Chk1 and Src in cytokinesis with chromatin bridges. In this study, we show that Src, a nonreceptor tyrosine kinase that is involved in actin remodeling Frame, ; Playford and Schaller, , and Chk1, a kinase that functions in different stages of the cell cycle Zhang and Hunter, , prevent chromosome breakage and are required for formation of actin patches in cytokinesis. Chromatin breakage in Chk1- or Src-deficient cells is not caused by abscission as evidenced by the majority of cells exhibiting broken chromatin with an intact intercellular canal and also by the inability of a dominant-negative Vps4-KQ protein that inhibits abscission to prevent chromatin bridges from breaking Morita et al. Although a potential function for Chk1 and Src in DNA breakage that is independent of their role in regulating actin patches cannot be formally excluded, our results are consistent with Chk1 and Src preventing DNA bridges from breaking by promoting formation of actin patches. A minority of Chk1- or Src-deficient cells exhibit both fragmented chromatin bridges and broken intercellular canals; this could be caused by reduced stability of the intercellular canal in the absence of actin patches or by weakening of the abscission checkpoint after chromatin breakage, perhaps because of released membrane tension or the broken chromatin being reeled in from the canal Lafaurie-Janvore et al. In addition, Chk1 or Src inhibition exacerbates chromatin breakage in Aurora B—deficient cells with an impaired abscission checkpoint Steigemann et al. We propose that actin patch formation by Chk1 and Src cooperates with the Aurora B—imposed abscission delay to prevent chromatin breakage. These results also suggest that Chk1 and Src are not essential for the abscission delay in response to chromatin bridges. We also show that Chk1 is required for optimal localization and complete activation of Src, efficient cell spreading, and wound healing on fibronectin. Chk1 associates with Src and phosphorylates human Src at the conserved serine 51 inside the Src-Unique region. Phosphorylated Src-S51 localizes to membrane ruffles, filopodia, and the nucleus. Also, phosphorylated Src-S51 and Src-signaling proteins such as phosphorylated FAK-Y and cortactin localize to actin patches in control cells with chromatin bridges. In addition, expression of a phosphomimetic Src-S51D protein rescues formation of actin patches and prevents chromatin breakage in Chk1-deficient cells. On the basis of these findings, we propose the following model Fig. Furthermore, in the presence of chromatin bridges, phosphorylated Src-S51 and Src-signaling proteins promote formation of actin patches and prevent chromosome breakage in cytokinesis. Phosphorylation of serine 51 can promote Src kinase activity independently of Src-Y phosphorylation. The Src-SH3 domain also interacts with the Unique domain through a binding region in the opposite side of the SH3 peptide binding site, and the SH3—Unique domain interaction is lost after addition of a high-affinity SH3 ligand peptide that induces the active open conformation of the kinase Maffei et al. Therefore, one possibility is that S51 phosphorylation activates Src by destabilizing the interaction between the SH3 and the Unique domain to promote the open conformation. How do actin patches stabilize chromatin bridges? The oval or heart shape of many of the nuclei in telophase cells with chromatin bridges suggests that considerable pulling forces are exerted by the chromosomes in the bridge. These forces could cause chromatin breakage by mechanical rupture or induce transient nuclear envelope rupture during interphase, followed by bridge resolution by nuclease activity Ganem and Pellman, ; Maciejowski et al. Perhaps, intriguingly, breakage of chromatin bridges in cells proficient for the abscission checkpoint preferentially occurs next to centromeres at the foot of the DNA bridge, around where actin patches are formed, indicating this chromatin region is under relatively high tension Hoffelder et al. One possibility is that the dense meshwork of highly organized actin filaments inside the actin patches provides mechanical support to the nuclear envelope and underlying chromatin by increasing the stiffness and elasticity at the base of the chromatin bridge to counteract the pulling forces applied by the chromosomes in the bridge. Because cross-linkers affect the architecture and mechanical properties of the actin network, identifying how actin filaments are organized and linked together inside the patches may help us understand how such forces are acting Fletcher and Mullins, Phosphorylated FAK-Y and cortactin were also detected at actin patches. Focal adhesion proteins are involved in mechanotransduction and can promote force-dependent actin polymerization Geiger et al. Investigating which Src-signaling proteins are required for actin patch formation in response to DNA bridges may help us better understand how cells stabilize chromatin bridges. In conclusion, our study identifies novel proteins that promote formation of actin patches and protect against chromatin breakage in cytokinesis. Rabbit polyclonal antibodies against Aurora B ab and Src ab, used in Western blotting were from Abcam, rabbit polyclonal antibody against phospho—Src-Y Src \\\\\\\\\[pY\\\\\\\\\], G was from Invitrogen, and mouse monoclonal antiphospho—histone H2A. Skourides University of Cyprus, Nicosia, Cyprus. All plasmids were completely sequenced. Only the sense sequences of the siRNA duplexes are shown. Point mutations were generated by using the Q5 site-directed mutagenesis kit New England Biolabs. Human colon carcinoma BE cells a gift from S. Wilkinson and C. For expression of GFP proteins, plasmids were transfected into cells in the absence or presence of appropriate siRNA duplexes 24 h before analysis or further drug treatment using Turbofect Thermo Fisher Scientific. All cell lines used exhibited consistent morphology and growth properties and were negative for mycoplasma contamination. The method was adapted from Brunton et al. Fibronectin-coated mm dishes containing a confluent monolayer of cells were used, and a wound was created by scraping the monolayer with a yellow gel loading tip. The method was adapted from Elias et al. For peptide competitions, 2. The low-fluorescence immersion oil ; Leica was used, and imaging was performed at room temperature. Mean projections of image stacks were obtained by using the LCS Lite software. Next, the cells were permeabilized with 0. DNA bridge-fluorescence intensity signals were quantified using the LCS Lite polygon tool by analyzing an image area encompassing the entire DNA bridge or intercellular canal, and intensity values were normalized versus values obtained by analyzing an identical area outside the canal. For weak versus strong DNA staining, DNA bridge-fluorescence intensity signals from 40 control bridges were quantified, and the mean value was set to 0. GST—avSrc bands were excised from the gel and digested with trypsin, and the extracted tryptic peptides were analyzed by liquid chromatography—mass spectrometry as described previously Ducommun et al. The data were searched against an in-house database containing the avian c-Src sequence using Mascot 2. All result files were loaded into Scaffold 4. Protein thresholds were set to For the in vitro Chk1 kinase assays in Figs. Radioactive labeling of Chk1 substrates was determined by autoradiography. The in vitro Chk1 kinase assay in Fig. For the in vitro kinase assays in Figs. For GFP immunoprecipitations Fig. Cells were lysed as described in the previous section. For actin patches fluorescence in Figs. For intact intercellular canals indices in Fig. For intact midbodies indices in Fig. For filopodia formation Fig. No statistical method was used to predetermine the sample size. S1 shows the fluorescence intensity of actin patches in control, Chk1-deficient, or Src-deficient cells with intact DNA bridges. S1 also shows an example of control cells with a broken chromatin bridge, and Src-depleted cells with relatively strong or weak DNA bridge staining. S2 shows localization of Mklp1 and Cep55 to the midbody in control, Chk1-deficient, or Src-deficient cells in telophase or late cytokinesis. S4 also shows phosphorylations of Src-S51 and Src-Y in vitro in the presence of purified Chk1 and Src proteins, and specificity of the antiphospho-S51 antiserum in immunofluorescence. S5 shows localization of phosphorylated Src-S51 at focal adhesions and filopodia in interphase cells without chromatin bridges. We thank D. Gerlich, M. Resh, P. Skourides, W. Sundquist, and N. Yamagushi for sharing reagents. Petsalaki was supported by a postdoctoral fellowship from the Bodossaki Foundation, and M. Dandoulaki was supported by Worldwide Cancer Research. Author contributions: E. Petsalaki, M. Dandoulaki, and G. Zachos performed experiments and analyzed the results. Sumpton and S. Zanivan performed the mass spectrometry analysis. Zachos designed the study and wrote the paper. National Center for Biotechnology Information , U. Journal List J Cell Biol v. J Cell Biol. Author information Article notes Copyright and License information Disclaimer. Correspondence to George Zachos: rg. This article has been cited by other articles in PMC. Abstract In cytokinesis with chromatin bridges, cells delay abscission and retain actin patches at the intercellular canal to prevent chromosome breakage. Graphical Abstract. Open in a separate window. Introduction Chromatin bridges are strands of incompletely segregated chromatin that connect anaphase poles or daughter nuclei and have been linked to tumorigenesis Hoffelder et al. Results Src and Chk1 prevent chromatin breakage in cytokinesis Because Src and Chk1 are involved in completion of cytokinesis Kasahara et al. Figure 1. Figure 2. Src and Chk1 are required for actin patch formation in cytokinesis with chromatin bridges Control cells with chromatin bridges exhibited actin patches, i. Figure 3. Figure 4. Figure 5. Src and Src-signaling proteins localize to actin patches Proteins involved in actin remodeling can localize to actin structures. Figure 6. Chk1 is required for optimal Src localization and kinase activity Depletion of Chk1 or, for comparison, treatment with the Src family kinase inhibitor PP2 impaired localization of total Src and cortactin at the base of the intercellular canal and reduced phosphorylation of Src-Y and FAK-Y which are markers of Src kinase activity compared with control cells Fig. Figure 7. Figure 8. Phosphorylation at S51 promotes Src kinase activity To investigate Src-S51 phosphorylation, an antiphospho—Src-S51 antiserum was raised against the human protein sequence. Phosphorylation at S51 enhances Src catalytic activity in the absence of Y phosphorylation One possibility is that S51 phosphorylation enhances Src catalytic activity by promoting dephosphorylation of the inhibitory site Src-Y Shenoy et al. Figure 9. Chk1 is required for Src-S51 phosphorylation in cultured cells Confocal microscopy analysis showed that phosphorylated Src-S51 localized to actin patches in control cells with chromatin bridges Figs. Phosphorylated Src-S51 prevents chromatin breakage In cells depleted of the endogenous Src by siSrc-2, expression of the phosphomimetic mutant S51D but not WT GFP-Src resistant to degradation by siSrc-2 prevented chromatin breakage and rescued formation of actin patches after Chk1 depletion compared with Chk1-proficient controls Fig. Figure Mutagenesis Point mutations were generated by using the Q5 site-directed mutagenesis kit New England Biolabs. Cell culture and treatments Human colon carcinoma BE cells a gift from S. Wound healing assay Fibronectin-coated mm dishes containing a confluent monolayer of cells were used, and a wound was created by scraping the monolayer with a yellow gel loading tip. Indirect immunofluorescence microscopy For intercellular canal labeling with FM FX, see Intercellular canal labeling. Mass spectrometry GST—avSrc bands were excised from the gel and digested with trypsin, and the extracted tryptic peptides were analyzed by liquid chromatography—mass spectrometry as described previously Ducommun et al. In vitro kinase assays For the in vitro Chk1 kinase assays in Figs. Protein coimmunoprecipitations and GST-pulldown assays Cells were lysed as described in the previous section. Online supplemental material Fig. Video 1: Click here to view. Video 2: Click here to view. Video 3: Click here to view. Acknowledgments We thank D. The authors declare no competing financial interests. References Amata I. Phosphorylation of unique domains of Src family kinases. Plk1 negatively regulates Cep55 recruitment to the midbody to ensure orderly abscission. Cell Biol. Structural characterization of the active and inactive states of Src kinase in solution by small-angle X-ray scattering. Src and FAK kinases cooperate to phosphorylate paxillin kinase linker, stimulate its focal adhesion localization, and regulate cell spreading and protrusiveness. III, Sawyer T. Identification of Src-specific phosphorylation site on focal adhesion kinase: dissection of the role of Src SH2 and catalytic functions and their consequences for tumor cell behavior. Cancer Res. The chromosomal passenger complex controls the function of endosomal sorting complex required for transport-III Snf7 proteins during cytokinesis. Open Biol. A last-minute rescue of trapped chromatin. Src family kinase activity regulates adhesion, spreading and migration of pancreatic endocrine tumour cells. Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets: identification of mitochondrial fission factor as a new AMPK substrate. Polyamine-dependent activation of Rac1 is stimulated by focal adhesion-mediated Tiam1 activation. Cell Adhes. Cell mechanics and the cytoskeleton. Src in cancer: deregulation and consequences for cell behaviour. Linking abnormal mitosis to the acquisition of DNA damage. Environmental sensing through focal adhesions. Src phosphorylates Cas on tyrosine to promote migration of transformed cells. Src and cortactin promote lamellipodia protrusion and filopodia formation and stability in growth cones. Resolution of anaphase bridges in cancer cells. Membrane tension maintains cell polarity by confining signals to the leading edge during neutrophil migration. Substrate specificity determinants of the checkpoint protein kinase Chk1. FEBS Lett. Chromosome segregation errors as a cause of DNA damage and structural chromosome aberrations. Cytokinesis breaks dicentric chromosomes preferentially at pericentromeric regions and telomere fusions. Genes Dev. Chromothripsis and Kataegis Induced by Telomere Crisis. ATR and a Chk1-Aurora B pathway coordinate postmitotic genome surveillance with cytokinetic abscission. Focal adhesion kinase: in command and control of cell motility. EMBO J. Trends Cell Biol. The DNA-damage effector checkpoint kinase 1 is essential for chromosome segregation and cytokinesis. Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism. Chk1 and Mps1 jointly regulate correction of merotelic kinetochore attachments. Cell Sci. Clks 1, 2 and 4 prevent chromatin breakage by regulating the Aurora B-dependent abscission checkpoint. Phosphorylation at serine is required for Aurora B activation. Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src. The interplay between Src and integrins in normal and tumor biology. Src-dependent tyrosine phosphorylation at the tips of growth cone filopodia promotes extension. Src protein-tyrosine kinase structure, mechanism, and small molecule inhibitors. Role of p34cdc2-mediated phosphorylations in two-step activation of pp60c-src during mitosis. Aurora B-mediated abscission checkpoint protects against tetraploidization. Cdc2-mediated modulation of pp60c-src activity. Nuclear localization of Src-family tyrosine kinases is required for growth factor-induced euchromatinization. Cell Res. Src phosphorylation of cortactin enhances actin assembly. Coupling of human circadian and cell cycles by the timeless protein. Emergence of micronuclei and their effects on the fate of cells under replication stress. PLoS One. Accuracy and precision in quantitative fluorescence microscopy. Three-dimensional structure of the tyrosine kinase c-Src. Crystal structures of c-Src reveal features of its autoinhibitory mechanism. Chk1 is required for spindle checkpoint function. Roles of Chk1 in cell biology and cancer therapy. Support Center Support Center. External link. 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