Signaling Scaffold Protein IQGAP1 Interacts with Microtubule Plus-end Tracking Protein SKAP and Links Dynamic Microtubule Plus-end to Steer Cell Migration.

Cell migration is orchestrated by dynamic interaction of microtubules with the plasma membrane cortex. However, the regulatory mechanisms underlying the cortical actin cytoskeleton and microtubule dynamics are less characterized. Our earlier study showed that small GTPase-activating proteins, IQGAPs, regulate polarized secretion in epithelial cells (1). Here, we show that IQGAP1 links dynamic microtubules to steer cell migration via interacting with the plus-end tracking protein, SKAP. Biochemical characterizations revealed that IQGAP1 and SKAP form a cognate complex and that their binding interfaces map to the WWIQ motif and the C-terminal of SKAP, respectively. The WWIQ peptide disrupts the biochemical interaction between IQGAP1 and SKAP in vitro, and perturbation of the IQGAP1-SKAP interaction in vivo using a membrane-permeable TAT-WWIQ peptide results in inhibition of directional cell migration elicited by EGF. Mechanistically, the N-terminal of SKAP binds to EB1, and its C terminus binds to IQGAP1 in migrating cells. Thus, we reason that a novel IQGAP1 complex orchestrates directional cell migration via coupling dynamic microtubule plus-ends to the cell cortex.

The spatiotemporal dynamics of chromatin protein HP1α is essential for accurate chromosome segregation during cell division.

Heterochromatin protein 1α (HP1α) is involved in regulation of chromatin plasticity, DNA damage repair, and centromere dynamics. HP1α detects histone dimethylation and trimethylation of Lys-9 via its chromodomain. HP1α localizes to heterochromatin in interphase cells but is liberated from chromosomal arms at the onset of mitosis. However, the structural determinants required for HP1α localization in interphase and the regulation of HP1α dynamics have remained elusive. Here we show that centromeric localization of HP1α depends on histone H3 Lys-9 trimethyltransferase SUV39H1 activity in interphase but not in mitotic cells. Surprisingly, HP1α liberates from chromosome arms in early mitosis. To test the role of this dissociation, we engineered an HP1α construct that persistently localizes to chromosome arms. Interestingly, persistent localization of HP1α to chromosome arms perturbs accurate kinetochore-microtubule attachment due to an aberrant distribution of chromosome passenger complex and Sgo1 from centromeres to chromosome arms that prevents resolution of sister chromatids. Further analyses showed that Mis14 and perhaps other PXVXL-containing proteins are involved in directing localization of HP1α to the centromere in mitosis. Taken together, our data suggest a model in which spatiotemporal dynamics of HP1α localization to centromere is governed by two distinct structural determinants. These findings reveal a previously unrecognized but essential link between HP1α-interacting molecular dynamics and chromosome plasticity in promoting accurate cell division.

Systematic analysis of the Plk-mediated phosphoregulation in eukaryotes.

Substantial evidence has confirmed that Polo-like kinases (Plks) play a crucial role in a variety of cellular processes via phosphorylation-mediated signaling transduction. Identification of Plk phospho-binding proteins and phosphorylation substrates is fundamental for elucidating the molecular mechanisms of Plks. Here, we present an integrative approach for the analysis of Plk-specific phospho-binding and phosphorylation sites (p-sites) in proteins. From the currently available phosphoproteomic data, we predicted tens of thousands of potential Plk phospho-binding and phosphorylation sites in eukaryotes, respectively. Furthermore, statistical analysis suggested that Plk phospho-binding proteins are more closely implicated in mitosis than their phosphorylation substrates. Additional computational analysis together with in vitro and in vivo experimental assays demonstrated that human Mis18B is a novel interacting partner of Plk1, while pT14 and pS48 of Mis18B were identified as phospho-binding sites. Taken together, this systematic analysis provides a global landscape of the complexity and diversity of potential Plk-mediated phosphoregulation, and the prediction results can be helpful for further experimental investigation.

Phosphorylation of microtubule-binding protein Hec1 by mitotic kinase Aurora B specifies spindle checkpoint kinase Mps1 signaling at the kinetochore.

The spindle assembly checkpoint (SAC) is a quality control device to ensure accurate chromosome attachment to spindle microtubule for equal segregation of sister chromatid. Aurora B is essential for SAC function by sensing chromosome bi-orientation via spatial regulation of kinetochore substrates. However, it has remained elusive as to how Aurora B couples kinetochore-microtubule attachment to SAC signaling. Here, we show that Hec1 interacts with Mps1 and specifies its kinetochore localization via its calponin homology (CH) domain and N-terminal 80 amino acids. Interestingly, phosphorylation of the Hec1 by Aurora B weakens its interaction with microtubules but promotes Hec1 binding to Mps1. Significantly, the temporal regulation of Hec1 phosphorylation orchestrates kinetochore-microtubule attachment and Mps1 loading to the kinetochore. Persistent expression of phosphomimetic Hec1 mutant induces a hyperactivation of SAC, suggesting that phosphorylation-elicited Hec1 conformational change is used as a switch to orchestrate SAC activation to concurrent destabilization of aberrant kinetochore attachment. Taken together, these results define a novel role for Aurora B-Hec1-Mps1 signaling axis in governing accurate chromosome segregation in mitosis.

The F-box protein ß-TrCP promotes ubiquitination of TRF1 and regulates the ALT-associated PML bodies formation in U2OS cells.

The telomeric repeat binding protein 1 (TRF1) is a major factor of the mammalian telosome/shelterin and negatively regulates telomere length by inhibiting access of telomerase at telomere termini in telomerase-positive cells. In telomerase-negative cancer cells, TRF1 also plays a critical role in the mechanism called alternative lengthening of telomeres (ALT) and is essential for formation of the ALT-associated PML bodies (APBs). It was reported that TRF1 can be degraded by the ubiquitin-proteasome pathway, involving in two regulation factors, Fbx4 and RLIM. Here, we reported that ß-TrCP1, a member of the F-box family protein with ubiquitin ligase activity, is a novel TRF1-associating protein. ß-TrCP1 interacts with TRF1 in vivo and in vitro and promotes its ubiquitination. Overexpression of ß-TrCP1 reduces endogenous TRF1 protein levels, while inhibition of ß-TrCP1 by siRNA stabilizes TRF1. Moreover, we found that ß-TrCP1 is essential for regulation of promyelocytic leukemia body recruitment of TRF1 in U2OS cells. These results reveal that ß-TrCP1 is involved in the negative regulation of TRF1 and represents a new pathway for APB formation in telomerase-negative cells.

Phosphorylation of Tara by Plk1 is essential for faithful chromosome segregation in mitosis.

Trio-associated repeat on actin (Tara) is an F-actin binding protein and regulates actin cytoskeletal organization. In our previous study, we have found that Tara associates with telomeric repeat binding factor 1 (TRF1) and mediates the function of TRF1 in mitotic regulation. We also found that overexpression HECTD3, a member of HECT E3 ubiquitin ligases, enhances the ubiquitination of Tara in vivo and promotes the degradation of Tara, and such degradation of Tara facilitates cell cycle progression. However, less is known about the post-translational modification of Tara in mitosis. Here we show that Tara is a novel Polo-like kinase 1 (Plk1) target protein. Plk1 interacts with and phosphorylates Tara in vivo and in vitro. Actually, the Thr-457 in Tara was a bona fide in vivo phosphorylation site for Plk1. Interestingly, we found that the centrosomal localization of Tara depended on the Thr-457 phosphorylation and the kinase activity of Plk1. Furthermore, overexpression of non-phosphorylatable mutant of Tara caused aberrant mitosis delay in HeLa cells. Our study demonstrated that Plk1-mediated phospho-dependent centrosomal localization of Tara is important for faithful chromosome segregation, and provided novel insights into understanding on the role of Plk1 in cooperation with Tara in mitotic progression.

PhosSNP for systematic analysis of genetic polymorphisms that influence protein phosphorylation.

We are entering the era of personalized genomics as breakthroughs in sequencing technology have made it possible to sequence or genotype an individual person in an efficient and accurate manner. Preliminary results from HapMap and other similar projects have revealed the existence of tremendous genetic variations among world populations and among individuals. It is important to delineate the functional implication of such variations, i.e. whether they affect the stability and biochemical properties of proteins. It is also generally believed that the genetic variation is the main cause for different susceptibility to certain diseases or different response to therapeutic treatments. Understanding genetic variation in the context of human diseases thus holds the promise for "personalized medicine." In this work, we carried out a genome-wide analysis of single nucleotide polymorphisms (SNPs) that could potentially influence protein phosphorylation characteristics in human. Here, we defined a phosphorylation-related SNP (phosSNP) as a non-synonymous SNP (nsSNP) that affects the protein phosphorylation status. Using an in-house developed kinase-specific phosphorylation site predictor (GPS 2.0), we computationally detected that approximately 70% of the reported nsSNPs are potential phosSNPs. More interestingly, approximately 74.6% of these potential phosSNPs might also induce changes in protein kinase types in adjacent phosphorylation sites rather than creating or removing phosphorylation sites directly. Taken together, we proposed that a large proportion of the nsSNPs might affect protein phosphorylation characteristics and play important roles in rewiring biological pathways. Finally, all phosSNPs were integrated into the PhosSNP 1.0 database, which was implemented in JAVA 1.5 (J2SE 5.0). The PhosSNP 1.0 database is freely available for academic researchers.

MiCroKit 3.0: an integrated database of midbody, centrosome and kinetochore.

During cell division/mitosis, a specific subset of proteins is spatially and temporally assembled into protein super complexes in three distinct regions, i.e. centrosome/spindle pole, kinetochore/centromere and midbody/cleavage furrow/phragmoplast/bud neck, and modulates cell division process faithfully. Although many experimental efforts have been carried out to investigate the characteristics of these proteins, no integrated database was available. Here, we present the MiCroKit database (http://microkit.biocuckoo.org) of proteins that localize in midbody, centrosome and/or kinetochore. We collected into the MiCroKit database experimentally verified microkit proteins from the scientific literature that have unambiguous supportive evidence for subcellular localization under fluorescent microscope. The current version of MiCroKit 3.0 provides detailed information for 1489 microkit proteins from seven model organisms, including Saccharomyces cerevisiae, Schizasaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, Mus musculus and Homo sapiens. Moreover, the orthologous information was provided for these microkit proteins, and could be a useful resource for further experimental identification. The online service of MiCroKit database was implemented in PHP + MySQL + JavaScript, while the local packages were developed in JAVA 1.5 (J2SE 5.0).

Dimerization of CPAP orchestrates centrosome cohesion plasticity.

Centrosome cohesion and segregation are accurately regulated to prevent an aberrant separation of duplicated centrosomes and to ensure the correct formation of bipolar spindles by a tight coupling with cell cycle machinery. CPAP is a centrosome protein with five coiled-coil domains and plays an important role in the control of brain size in autosomal recessive primary microcephaly. Previous studies showed that CPAP interacts with tubulin and controls centriole length. Here, we reported that CPAP forms a homodimer during interphase, and the fifth coiled-coil domain of CPAP is required for its dimerization. Moreover, this self-interaction is required for maintaining centrosome cohesion and preventing the centrosome from splitting before the G(2)/M phase. Our biochemical studies show that CPAP forms homodimers in vivo. In addition, both monomeric and dimeric CPAP are required for accurate cell division, suggesting that the temporal dynamics of CPAP homodimerization is tightly regulated during the cell cycle. Significantly, our results provide evidence that CPAP is phosphorylated during mitosis, and this phosphorylation releases its intermolecular interaction. Taken together, these results suggest that cell cycle-regulated phosphorylation orchestrates the dynamics of CPAP molecular interaction and centrosome splitting to ensure genomic stability in cell division.

PinX1 is a novel microtubule-binding protein essential for accurate chromosome segregation.

Mitosis is an orchestration of dynamic interactions between spindle microtubules and chromosomes, which is mediated by protein structures that include the kinetochores, and other protein complexes present on chromosomes. PinX1 is a potent telomerase inhibitor in interphase; however, its function in mitosis is not well documented. Here we show that PinX1 is essential for faithful chromosome segregation. Deconvolution microscopic analyses show that PinX1 localizes to nucleoli and telomeres in interphase and relocates to the periphery of chromosomes and the outer plate of the kinetochores in mitosis. Our deletion analyses mapped the kinetochore localization domain of PinX1 to the central region and its chromosome periphery localization domain to the C terminus. Interestingly, the kinetochore localization of PinX1 is dependent on Hec1 and CENP-E. Our biochemical characterization revealed that PinX1 is a novel microtubule-binding protein. Our real time imaging analyses show that suppression of PinX1 by small interference RNA abrogates faithful chromosome segregation and results in anaphase chromatid bridges in mitosis and micronuclei in interphase, suggesting an essential role of PinX1 in chromosome stability. Taken together, the results indicate that PinX1 plays an important role in faithful chromosome segregation in mitosis.

GPS 2.0, a tool to predict kinase-specific phosphorylation sites in hierarchy.

Identification of protein phosphorylation sites with their cognate protein kinases (PKs) is a key step to delineate molecular dynamics and plasticity underlying a variety of cellular processes. Although nearly 10 kinase-specific prediction programs have been developed, numerous PKs have been casually classified into subgroups without a standard rule. For large scale predictions, the false positive rate has also never been addressed. In this work, we adopted a well established rule to classify PKs into a hierarchical structure with four levels, including group, family, subfamily, and single PK. In addition, we developed a simple approach to estimate the theoretically maximal false positive rates. The on-line service and local packages of the GPS (Group-based Prediction System) 2.0 were implemented in Java with the modified version of the Group-based Phosphorylation Scoring algorithm. As the first stand alone software for predicting phosphorylation, GPS 2.0 can predict kinase-specific phosphorylation sites for 408 human PKs in hierarchy. A large scale prediction of more than 13,000 mammalian phosphorylation sites by GPS 2.0 was exhibited with great performance and remarkable accuracy. Using Aurora-B as an example, we also conducted a proteome-wide search and provided systematic prediction of Aurora-B-specific substrates including protein-protein interaction information. Thus, the GPS 2.0 is a useful tool for predicting protein phosphorylation sites and their cognate kinases and is freely available on line.

Systematic study of protein sumoylation: Development of a site-specific predictor of SUMOsp 2.0.

Protein sumoylation is an important reversible post-translational modification on proteins, and orchestrates a variety of cellular processes. Recently, computational prediction of sumoylation sites has attracted much attention for its cost-efficiency and power in genomic data mining. In this work, we developed SUMOsp 2.0, an accurate computing program with an improved group-based phosphorylation scoring algorithm. Our analysis demonstrated that SUMOsp 2.0 has greater prediction accuracy than SUMOsp 1.0 and other existing tools, with a sensitivity of 88.17% and a specificity of 92.69% under the medium threshold. Previously, several large-scale experiments have identified a list of potential sumoylated substrates in Saccharomyces cerevisiae and Homo sapiens; however, the exact sumoylation sites in most of these proteins remain elusive. We have predicted potential sumoylation sites in these proteins using SUMOsp 2.0, which provides a great resource for researchers and an outline for further mechanistic studies of sumoylation in cellular plasticity and dynamics. The online service and local packages of SUMOsp 2.0 are freely available at: http://sumosp.biocuckoo.org/.

Proteome-wide prediction of PKA phosphorylation sites in eukaryotic kingdom.

Protein phosphorylation is one of the most essential post-translational modifications (PTMs), and orchestrates a variety of cellular functions and processes. Besides experimental studies, numerous computational predictors implemented in various algorithms have been developed for phosphorylation sites prediction. However, large-scale predictions of kinase-specific phosphorylation sites have not been successfully pursued and remained to be a great challenge. In this work, we raised a "kiss farewell" model and conducted a high-throughput prediction of cAMP-dependent kinase (PKA) phosphorylation sites. Since a protein kinase (PK) should at least "kiss" its substrates and then run away, we proposed a PKA-binding protein to be a potential PKA substrate if at least one PKA site was predicted. To improve the prediction specificity, we reduced false positive rate (FPR) less than 1% when the cut-off value was set as 4. Successfully, we predicted 1387, 630, 568 and 912 potential PKA sites from 410, 217, 173 and 260 PKA-interacting proteins in Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens, respectively. Most of these potential phosphorylation sites remained to be experimentally verified. In addition, we detected two sites in one of PKA regulatory subunits to be conserved in eukaryotes as potentially ancient regulatory signals. Our prediction results provide an excellent resource for delineating PKA-mediated signaling pathways and their system integration underlying cellular dynamics and plasticity.

The E3 ubiquitin ligase HECTD3 regulates ubiquitination and degradation of Tara.

Tara was identified as an interacting partner of guanine nucleotide exchange factor Trio and TRF1. Tara is proposed to be involved in many important fundamental cellular processes, ranging from actin remodeling, directed cell movement, to cell cycle regulation. Yet, its exact roles required further elucidation. Here, we identify a novel Tara-binding protein HECTD3, a putative member of HECT E3 ubiquitin ligases. HECTD3 directly binds Tara in vitro and forms a complex with Tara in vivo. Overexpression of HECTD3 enhances the ubiquitination of Tara in vivo and promotes the turnover of Tara, whereas depletion of HECTD3 by small interfering RNA decreases Tara degradation. Furthermore, depletion of HECTD3 leads to multipolar spindle formation. All these findings suggest that HECTD3 may facilitate cell cycle progression via regulating ubiquitination and degradation of Tara.

Computational analyses of TBC protein family in eukaryotes.

Tre-2/Bub2/Cdc16 domain-containing proteins (TBC proteins) participate in wide range cellular processes. With computational approaches, 137 non-redundant TBC proteins from five model organisms were identified and classified into 13 subfamilies base on molecular evolutionary tree. This phylogenetic analysis provides useful functional annotation of newly-identified TBC proteins and guides for further experimentation.

NBA-Palm: prediction of palmitoylation site implemented in Naïve Bayes algorithm.

BACKGROUND: Protein palmitoylation, an essential and reversible post-translational modification (PTM), has been implicated in cellular dynamics and plasticity. Although numerous experimental studies have been performed to explore the molecular mechanisms underlying palmitoylation processes, the intrinsic feature of substrate specificity has remained elusive. Thus, computational approaches for palmitoylation prediction are much desirable for further experimental design. RESULTS: In this work, we present NBA-Palm, a novel computational method based on Naïve Bayes algorithm for prediction of palmitoylation site. The training data is curated from scientific literature (PubMed) and includes 245 palmitoylated sites from 105 distinct proteins after redundancy elimination. The proper window length for a potential palmitoylated peptide is optimized as six. To evaluate the prediction performance of NBA-Palm, 3-fold cross-validation, 8-fold cross-validation and Jack-Knife validation have been carried out. Prediction accuracies reach 85.79% for 3-fold cross-validation, 86.72% for 8-fold cross-validation and 86.74% for Jack-Knife validation. Two more algorithms, RBF network and support vector machine (SVM), also have been employed and compared with NBA-Palm. CONCLUSION: Taken together, our analyses demonstrate that NBA-Palm is a useful computational program that provides insights for further experimentation. The accuracy of NBA-Palm is comparable with our previously described tool CSS-Palm. The NBA-Palm is freely accessible from: http://www.bioinfo.tsinghua.edu.cn/NBA-Palm.

Regulation of NDR1 activity by PLK1 ensures proper spindle orientation in mitosis.

Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids which depends on correct position of mitotic spindle relative to membrane cortex. Although recent work has identified the role of PLK1 in spindle orientation, the mechanisms underlying PLK1 signaling in spindle positioning and orientation have not been fully illustrated. Here, we identified a conserved signaling axis in which NDR1 kinase activity is regulated by PLK1 in mitosis. PLK1 phosphorylates NDR1 at three putative threonine residues (T7, T183 and T407) at mitotic entry, which elicits PLK1-dependent suppression of NDR1 activity and ensures correct spindle orientation in mitosis. Importantly, persistent expression of non-phosphorylatable NDR1 mutant perturbs spindle orientation. Mechanistically, PLK1-mediated phosphorylation protects the binding of Mob1 to NDR1 and subsequent NDR1 activation. These findings define a conserved signaling axis that integrates dynamic kinetochore-microtubule interaction and spindle orientation control to genomic stability maintenance.

Spatiotemporal dynamics of Aurora B-PLK1-MCAK signaling axis orchestrates kinetochore bi-orientation and faithful chromosome segregation.

Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. The microtubule depolymerase mitotic centromere-associated kinesin (MCAK) is a key regulator for an accurate kinetochore-microtubule attachment. However, the regulatory mechanism underlying precise MCAK depolymerase activity control during mitosis remains elusive. Here, we describe a novel pathway involving an Aurora B-PLK1 axis for regulation of MCAK activity in mitosis. Aurora B phosphorylates PLK1 on Thr210 to activate its kinase activity at the kinetochores during mitosis. Aurora B-orchestrated PLK1 kinase activity was examined in real-time mitosis using a fluorescence resonance energy transfer-based reporter and quantitative analysis of native PLK1 substrate phosphorylation. Active PLK1, in turn, phosphorylates MCAK at Ser715 which promotes its microtubule depolymerase activity essential for faithful chromosome segregation. Importantly, inhibition of PLK1 kinase activity or expression of a non-phosphorylatable MCAK mutant prevents correct kinetochore-microtubule attachment, resulting in abnormal anaphase with chromosome bridges. We reason that the Aurora B-PLK1 signaling at the kinetochore orchestrates MCAK activity, which is essential for timely correction of aberrant kinetochore attachment to ensure accurate chromosome segregation during mitosis.

The structure of the FANCM-MHF complex reveals physical features for functional assembly.

Fanconi anaemia is a rare genetic disease characterized by chromosomal instability and cancer susceptibility. The Fanconi anaemia complementation group protein M (FANCM) forms an evolutionarily conserved DNA-processing complex with MHF1/MHF2 (histone-fold-containing proteins), which is essential for DNA repair in response to genotoxic stress. Here we present the crystal structures of the MHF1-MHF2 complex alone and bound to a fragment of FANCM (FANCM(661-800), designated FANCM-F). The structures show that MHF1 and MHF2 form a compact tetramer to which FANCM-F binds through a 'dual-V' shaped structure. FANCM-F and (MHF1-MHF2)(2) cooperate to constitute a new DNA-binding site that is coupled to the canonical L1L2 region. Perturbation of the MHF-FANCM-F structural plasticity changes the localization of FANCM in vivo. The MHF-FANCM interaction and its subcellular localization are altered by a disease-associated mutant of FANCM. These findings reveal the molecular basis of MHF-FANCM recognition and provide mechanistic insights into the pathway leading to Fanconi anaemia.

GPS 2.1: enhanced prediction of kinase-specific phosphorylation sites with an algorithm of motif length selection.

As the most important post-translational modification of proteins, phosphorylation plays essential roles in all aspects of biological processes. Besides experimental approaches, computational prediction of phosphorylated proteins with their kinase-specific phosphorylation sites has also emerged as a popular strategy, for its low-cost, fast-speed and convenience. In this work, we developed a kinase-specific phosphorylation sites predictor of GPS 2.1 (Group-based Prediction System), with a novel but simple approach of motif length selection (MLS). By this approach, the robustness of the prediction system was greatly improved. All algorithms in GPS old versions were also reserved and integrated in GPS 2.1. The online service and local packages of GPS 2.1 were implemented in JAVA 1.5 (J2SE 5.0) and freely available for academic researches at: http://gps.biocuckoo.org.