An Amino-Terminal Polo Kinase Interaction Motif Acts in the Regulation of Centrosome Formation and Reveals a Novel Function for centrosomin (cnn) in Drosophila

The formation of the pericentriolar matrix (PCM) and a fully functional centrosome in syncytial Drosophila melanogaster embryos requires the rapid transport of Cnn during initiation of the centrosome replication cycle. We show a Cnn and Polo kinase interaction is apparently required during embryogenesis and involves the exon 1A-initiating coding exon, suggesting a subset of Cnn splice variants is regulated by Polo kinase. During PCM formation exon 1A Cnn-Long Form proteins likely bind Polo kinase before phosphorylation by Polo for Cnn transport to the centrosome. Loss of either of these interactions in a portion of the total Cnn protein pool is sufficient to remove native Cnn from the pool, thereby altering the normal localization dynamics of Cnn to the PCM. Additionally, Cnn-Short Form proteins are required for polar body formation, a process known to require Polo kinase after the completion of meiosis. Exon 1A Cnn-LF and Cnn-SF proteins, in conjunction with Polo kinase, are required at the completion of meiosis and for the formation of functional centrosomes during early embryogenesis.     

In vivo localisation of the mitotic POLO kinase shows a highly dynamic association with the mitotic apparatus during early embryogenesis in Drosophila

The gene polo encodes a highly conserved serine/threonine protein kinase that has been implicated in several functions during cell division. Polo-like kinases are important positive regulators of cell cycle progression and have also been implicated in the exit from mitosis through the activation of the anaphase-promoting complex. Several data indicate that Plks are required for centrosome function, bipolar spindle organisation and cytokinesis. The intracellular localisation of Plks reflects their multiple roles in cell division, however, in vivo studies that describe the distribution of this protein during different stages of mitosis have never been performed. In the present work, we report the in vivo distribution of a GFP-POLO fusion protein expressed in stable transformants and analysed during the early embryonic development of Drosophila melanogaster. The GFP-POLO protein can be detected in unfertilised oocytes associated with the centromeric region of chromosomes of the polar body and followed until the formation of mitotic domains in later development. Detailed analysis of the dynamic localisation of GFP-POLO during syncytial mitotic cycles shows the timing of localisation to the centrosomes, centromeres and midbody. The results also indicate that GFP-POLO is present in astral microtubules early in mitosis, accumulates around the nuclear envelope until nuclear envelop breakdown and at metaphase associates to spindle microtubules. These in vivo studies show a highly dynamic association of POLO with multiple compartments of the mitotic apparatus. Furthermore, the wide distribution of the GFP-POLO protein to all compartments of the mitotic apparatus provides a valuable tool for future studies on cell cycle during development.     

Biochemical and structural studies of the interaction of Cdc37 with Hsp90

The heat shock protein Hsp90 plays a key, but poorly understood role in the folding, assembly and activation of a large number of signal transduction molecules, in particular kinases and steroid hormone receptors. In carrying out these functions Hsp90 hydrolyses ATP as it cycles between ADP- and ATP-bound forms, and this ATPase activity is regulated by the transient association with a variety of co-chaperones. Cdc37 is one such co-chaperone protein that also has a role in client protein recognition, in that it is required for Hsp90-dependent folding and activation of a particular group of protein kinases. These include the cyclin-dependent kinases (Cdk) 4/6 and Cdk9, Raf-1, Akt and many others. Here, the biochemical details of the interaction of human Hsp90 beta and Cdc37 have been characterised. Small angle X-ray scattering (SAXS) was then used to study the solution structure of Hsp90 and its complexes with Cdc37. The results suggest a model for the interaction of Cdc37 with Hsp90, whereby a Cdc37 dimer binds the two N-terminal domain/linker regions in an Hsp90 dimer, fixing them in a single conformation that is presumably suitable for client protein recognition.     

The interaction of PTP-BL PDZ domains with RIL: An enigmatic role for the RIL LIM domain

PDZ domains are protein-protein interaction modules that are crucial for the assembly of structural and signaling complexes. PDZ domains specifically bind short carboxyl-terminal peptides and occasionally internal sequences that structurally resemble peptide termini. Previously, using yeast two-hybrid methodology, we studied the interaction of two PDZ domains present in the large submembranous protein tyrosine phosphatase PTP-BL with the C-terminal half of the LIM domain-containing protein RIL. Deletion of the extreme RIL C-terminus did not eliminate binding, suggesting the presence of a PDZ binding site within the RIL LIM moiety. We have now performed experiments in mammalian cell lysates and found that the RIL C-terminus proper, but not the RIL LIM domain, can interact with PTP-BL, albeit very weakly. However, this interaction with PTP-BL PDZ domains is greatly enhanced when the combined RIL LIM domain and C-terminus is used, pointing to synergistic effects. NMR titration experiments and site-directed mutagenesis indicate that this result is not dependent on specific interactions that require surface exposed residues on the RIL LIM domain, suggesting a stabilizing role in the association with PTP-BL.     

The binding of actin to p38 MAPK and inhibiting its kinase activity<Emphasis Type="Italic">in vitro</Emphasis>

p38 MAP kinase mediates a signal pathway that is involved in many physiological and pathological processes such as inflammation, cellular stress, apoptosis, cell cycle and growth, ischemia/re-perfusion, and myocardium hypertrophy. To determine the molecular and regulative mechanism of p38 signal pathway, we usedin vitro binding methods to screen the proteins that interact with p38. Here we report two proteins from mouse macrophage RAW264.7 strain treated with lipopolysaccharide (LPS) or ultraviolet radiation (UV), binding directly to p38. One of them is β-actin identified by peptide mass spectrum and ProFound program. Actin can inhibit the autophosphorylation of p38 and the phosphorylation of ATF by p38. It suggests that the binding of actin to p38in vitro may represent a negative feedback to the kinase activity of p38, which leads to the regulation of p38 pathway and cellular function.     

A specific interaction between the NBD of the ABC-transporter HlyB and a C-terminal fragment of its transport substrate haemolysin A

A member of the family of RTX toxins, Escherichia coli haemolysin A, is secreted from Gram-negative bacteria. It carries a C-terminal secretion signal of approximately 50 residues, targeting the protein to the secretion or translocation complex, in which the ABC-transporter HlyB is a central element. We have purified the nucleotide-binding domain of HlyB (HlyB-NBD) and a C-terminal 23kDa fragment of HlyA plus the His-tag (HlyA1), which contains the secretion sequence. Employing surface plasmon resonance, we were able to demonstrate that the HlyB-NBD and HlyA1 interact with a K(D) of approximately 4 microM. No interaction was detected between the HlyA fragment and unrelated NBDs, OpuAA, involved in import of osmoprotectants, and human TAP1-NBD, involved in the export of antigenic peptides. Moreover, a truncated version of HlyA1, lacking the secretion signal, failed to interact with the HlyB-NBD. In addition, we showed that ATP accelerated the dissociation of the HlyB-NBD/HlyA1 complex. Taking these results together, we propose a model for an early stage of initiation of secretion in vivo, in which the NBD of HlyB, specifically recognizes the C terminus of the transport substrate, HlyA, and where secretion is initiated by subsequent displacement of HlyA from HlyB by ATP.     

The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein

Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H₂O₂). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin^−/− mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.     

A quantitative model for cyclin-dependent kinase control of the cell cycle: revisited

The eukaryotic cell division cycle encompasses an ordered series of events. Chromosomal DNA is replicated during S phase of the cell cycle before being distributed to daughter cells in mitosis. Both S phase and mitosis in turn consist of an intricately ordered sequence of molecular events. How cell cycle ordering is achieved, to promote healthy cell proliferation and avert insults on genomic integrity, has been a theme of Paul Nurse's research. To explain a key aspect of cell cycle ordering, sequential S phase and mitosis, Stern & Nurse proposed 'A quantitative model for cdc2 control of S phase and mitosis in fission yeast'. In this model, S phase and mitosis are ordered by their dependence on increasing levels of cyclin-dependent kinase (Cdk) activity. Alternative mechanisms for ordering have been proposed that rely on checkpoint controls or on sequential waves of cyclins with distinct substrate specificities. Here, we review these ideas in the light of experimental evidence that has meanwhile accumulated. Quantitative Cdk control emerges as the basis for cell cycle ordering, fine-tuned by cyclin specificity and checkpoints. We propose a molecular explanation for quantitative Cdk control, based on thresholds imposed by Cdk-counteracting phosphatases, and discuss its implications.     

Structure of the Saccharomyces cerevisiae Hrr25:Mam1 monopolin subcomplex reveals a novel kinase regulator

In budding yeast, the monopolin complex mediates sister kinetochore cross‐linking and co‐orientation in meiosis I. The CK1δ kinase Hrr25 is critical for sister kinetochore co‐orientation, but its roles are not well understood. Here, we present the structures of Hrr25 and its complex with the monopolin subunit Mam1. Hrr25 possesses a “central domain” that packs tightly against the kinase C‐lobe, adjacent to the binding site for Mam1. Together, the Hrr25 central domain and Mam1 form a novel, contiguous embellishment to the Hrr25 kinase domain that affects Hrr25 conformational dynamics and enzyme kinetics. Mam1 binds a hydrophobic surface on the Hrr25 N‐lobe that is conserved in CK1δ‐family kinases, suggesting a role for this surface in recruitment and/or regulation of these enzymes throughout eukaryotes. Finally, using purified proteins, we find that Hrr25 phosphorylates the kinetochore receptor for monopolin, Dsn1. Together with our new structural insights into the fully assembled monopolin complex, this finding suggests that tightly localized Hrr25 activity modulates monopolin complex–kinetochore interactions through phosphorylation of both kinetochore and monopolin complex components.     

Tetramerization and DNA ligase IV interaction of the DNA double-strand break repair protein XRCC4 are mutually exclusive

The XRCC4 protein is of critical importance for the repair of broken chromosomal DNA by non-homologous end joining (NHEJ). The absence of XRCC4 abolishes chromosomal NHEJ almost completely. One reason for this severe phenotype is that XRCC4 binds and modulates the stability and activity of the NHEJ-specific ligase, DNA ligase IV. XRCC4 in solution is in equilibrium between the dimeric and tetrameric forms. Previous structural studies have shown that the interface between dimers is located in the same region as that implicated in DNA ligase IV interaction. With the use of equilibrium sedimentation analysis, we show here that only the XRCC4 dimer can associate with DNA ligase IV, forming a monodisperse complex of 2:1 stoichiometry in solution. In addition, physical analysis of XRCC4/DNA ligase IV complex formation, combined with mutational analysis of XRCC4, indicates that tetramerization and DNA ligase IV binding are mutually exclusive. We propose that the putative function of the XRCC4 tetramer is distinct from its DNA ligase IV-associated function.     

The evolutionary rate of a protein is influenced by features of the interacting partners

Rates of protein evolution are thought to be influenced by features of protein-protein interaction (PPI). However, the most important features of interaction for determining the evolutionary rate are poorly understood. Here, we consider four categories for PPIs in Saccharomyces cerevisiae. Properties we consider are the extent to which proteins interact with proteins of the same function or different function (DF) and the extent to which these interactions involve connections in the dense part or sparse part (SP) of a PPI network. Our findings are that proteins with DF-SP interactions evolve at the slowest rate of all the proteins examined.     

Thematic Minireview Series: Complexities of Cellular Signaling Revealed by Simple Model Organisms*

All cells discriminate environmental signals and generate appropriate intracellular responses. Our understanding of these signal transduction mechanisms has benefitted from studies across the kingdoms of life, from fungi and fish to mice and men. This thematic minireview series examines lessons learned from three of the simplest (and best understood) eukaryotic model organisms. The first article focuses on the mating pheromone pathway in budding yeast Saccharomyces cerevisiae. The second describes stress-mediated signaling in the roundworm Caenorhabditis elegans. The third outlines some of the signaling pathways that dictate growth and development in the fruit fly Drosophila melanogaster. Each system has provided unique insights into hormone and neurotransmitter signaling mechanisms, in particular those mediated by the MAPKs. The advances described in these articles will continue to improve our understanding of human physiology and pharmacology.     

Coevolution is a short-distance force at the protein interaction level and correlates with the modular organization of protein networks

We investigated what roles coevolution plays in shaping yeast protein interaction network (PIN). We found that the extent of coevolution between two proteins decreases rapidly as their interacting distance on the PIN increases, suggesting coevolutionary constraint is a short-distance force at the molecular level. We also found that protein-protein interactions (PPIs) with strong coevolution tend to be enriched in interconnected clusters, whereas PPIs with weak coevolution are more frequently present at inter-cluster region. The findings indicate the close relationship between coevolution and modular organization of PINs, and may provide insights into evolution and modularity of cellular networks.     

Insights from the structural analysis of protein heterodimer interfaces

Protein heterodimer complexes are often involved in catalysis, regulation, assembly, immunity and inhibition. This involves the formation of stable interfaces between the interacting partners. Hence, it is of interest to describe heterodimer interfaces using known structural complexes. We use a non-redundant dataset of 192 heterodimer complex structures from the protein databank (PDB) to identify interface residues and describe their interfaces using amino-acids residue property preference. Analysis of the dataset shows that the heterodimer interfaces are often abundant in polar residues. The analysis also shows the presence of two classes of interfaces in heterodimer complexes. The first class of interfaces (class A) with more polar residues than core but less than surface is known. These interfaces are more hydrophobic than surfaces, where protein-protein binding is largely hydrophobic. The second class of interfaces (class B) with more polar residues than core and surface is shown. These interfaces are more polar than surfaces, where binding is mainly polar. Thus, these findings provide insights to the understanding of protein-protein interactions.     

The atomic resolution crystal structure of the YajL (ThiJ) protein from Escherichia coli: a close prokaryotic homologue of the Parkinsonism-associated protein DJ-1

The Escherichia coli protein YajL (ThiJ) is a member of the DJ-1 superfamily with close homologues in many prokaryotes. YajL also shares 40% sequence identity with human DJ-1, an oncogene and neuroprotective protein whose loss-of-function mutants are associated with certain types of familial, autosomal recessive Parkinsonism. We report the 1.1 angstroms resolution crystal structure of YajL in a crystal form with two molecules in the asymmetric unit. The structure of YajL is remarkably similar to that of human DJ-1 (0.9 angstroms C(alpha) RMSD) and both proteins adopt the same dimeric structure. The conserved cysteine residue located in the "nucleophile elbow" is oxidized to either cysteine sulfenic or sulfinic acid in the two molecules in the asymmetric unit, and a mechanism for this oxidation is proposed that may be valid for other proteins in the DJ-1 superfamily as well. Rosenfield difference matrix analysis of the refined anisotropic displacement parameters in the YajL structure reveals significant differences in the intramolecular flexibility of the two non-crystallographic symmetry-related molecules in the asymmetric unit. Lastly, a comparison of the crystal structures of the four different E.coli members of the DJ-1 superfamily indicates that the variable oligomerization in this superfamily is due to a combination of protein-specific insertions into the core fold that form specific interfaces while occluding others plus optimization of residues in the structurally invariant regions of the core fold that facilitate protein-protein interactions.     

Subunit interaction slows the unfolding of the N-terminal domain of creatine kinase in urea

Fluorescence emission intensity changes with two different excitation wavelengths were used to measure the unfolding rate constants of different domains of muscle type creatine kinase (CK-MM) according to the heterogeneity of aromatic amino acid distributions in the crystal structure of CK-MM. The results were compared with those of brain type creatine kinase (CK-BB) and dithio-bis(succinimidyl propionate) cross-linked CK-MM. CK-BB differed greatly in its distribution of aromatic amino acids in each domain and the unfolding process of cross-linked CK-MM was not accompanied by the dissociation of the dimer. The N-terminal domain of CK-MM was shown to be well protected by subunit interaction during the unfolding of CK-MM in 4 M urea. Dissociating the CK dimer in high urea concentration (6 M) eliminated the subunit protection. Subunit interactions are also important in preserving secondary structure and forming contracted conformation at low urea concentration.     

Borealin: a novel chromosomal passenger required for stability of the bipolar mitotic spindle

The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Here, we describe Borealin, a novel member of the complex. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin; and Borealin binds Survivin and INCENP in vitro. A second complex contains Aurora B and INCENP, but no Borealin or Survivin. Depletion of Borealin by RNA interference delays mitotic progression and results in kinetochore-spindle misattachments and an increase in bipolar spindles associated with ectopic asters. The extra poles, which apparently form after chromosomes achieve a bipolar orientation, severely disrupt the partitioning of chromosomes in anaphase. Borealin depletion has little effect on histone H3 serine10 phosphorylation. These results implicate the chromosomal passenger holocomplex in the maintenance of spindle integrity and suggest that histone H3 serine10 phosphorylation is performed by an Aurora B-INCENP subcomplex.     

Involvement of the rolled/MAP kinase gene in Drosophila mitosis: interaction between genes for the MAP kinase cascade and abnormal spindle

We have found that mutations that lead to loss of rolled/MAP kinase function result in a reduced mitotic index in the larval central nervous system, consistent with an interphase block to cell cycle progression, associated with a low frequency of cells showing chromosome over-condensation in mitosis and abnormal anaphase figures. In contrast to wild-type tissue, such rolled mutants do not show a significant increase in accumulation of mitotic cells when treated with colchicine. We have studied double mutant combinations between mutations affecting the activity of rolled/MAP kinase and several genes that are essential to the establishment of a bipolar spindle during progression through mitosis, and find no interactions with mutations in polo, mgr,or aurora. However, partial loss-of-function mutations in rolled enhance the abnormal spindle (asp) phenotype, whereas gain-of function mutations in rolled or in the gene encoding its activating kinase Dsor1, act as suppressors. We discuss these findings in relation to the proposed role of MAP kinase in mediating the spindle integrity checkpoint. Received: 27 October 1997 / Accepted: 18 December 1997     

The protein Dredd is an essential component of the c-Jun N-terminal kinase pathway in the Drosophila immune response

The Drosophila immune deficiency (IMD) pathway mobilizes c-Jun N-terminal kinase (JNK), caspase, and nuclear factor-κB (NF-κB) modules to counter infection with gram-negative bacteria. Dredd is an essential caspase in the IMD pathway, and it is widely established that NF-κB activation depends on Dredd. More recent cell culture studies suggested a role for Dredd in the activation of dJNK (Drosophila JNK). However, there are no epistatic or mechanistic data on the involvement of Dredd in dJNK activation. More importantly, there is no in vivo evidence to demonstrate a physiological requirement for Dredd in the IMD/dJNK pathway. We performed a comprehensive analysis of the role of Dredd in the IMD/dJNK pathway, and we demonstrated that Dredd is essential for the activation of IMD/dJNK in cell culture. We positioned Dredd activity at an early point of the IMD/dJNK pathway and uncovered a series of interactions between Dredd and additional proximal IMD pathway molecules. Mechanistically, we showed that the caspase activity inhibitor p35 blocked dJNK activation and the induction of dJNK-dependent genes in cell culture and in vivo. Most importantly, we demonstrated that dredd mutant flies are completely inhibited in their ability to activate dJNK or express dJNK-responsive target genes after bacterial infection in vivo. In conclusion, we established Dredd as an essential component of the IMD pathway required for the full activation of IMD/dJNK in cell culture and in vivo. Our data enhance our appreciation of Dredd-dependent IMD signal transduction events.