Phosphorylation-dependent Protein-protein Interactions Research Articles

Unveiling the mechanistic regulation of T148 phosphorylation-mediated neuroprotection by αA-crystallin.

α-crystallins (αA- and αB-) are chaperone proteins historically studied in the eye lens as crucial to maintaining lens transparency. More recently, both proteins have garnered increased attention for their protective and chaperone roles in the context of retinal neurodegeneration, and neurodegeneration in the central nervous system more broadly. Critically, for both αA- and αB-crystallin, post-translational modifications (PTMs) and in particular, phosphorylation, regulate these roles. S19, S45, and S59 phosphorylation on αB-crystallin as well as S122 and S/T148 phosphorylation on αA-crystallin have all been implicated in regulating their protective and chaperone abilities. T148 (S148 in rodents) phosphorylation is particularly intriguing, as it is decreased in diabetic retinopathy despite αA-crystallin's upregulation under these conditions. Using phosphomimetic (T148D) and non-phosphorylatable (T148A) αA-crystallin, our lab has shown T148 phosphorylation to be associated with higher protein solubility, greater chaperone function, phosphorylation-dependent protein-protein interactions, greater neuroprotective capabilities in retinal neurons, and regulation of glial function. However, little is known about the mechanistic regulation of T148 phosphorylation, and the involved kinase(s) have yet to be identified. Using the phosphosite-accurate kinase-substrate crosslinking assay (PhAXA) method in R28 retinal neurons and MIO-M1 glial cells, we have identified strong candidate kinases, with the top candidates being mTOR and PI3K-C2α. We also demonstrated significantly decreased mTOR expression in the peripheral retina of patients with diabetic retinopathy in comparison to nondiabetic patients, as well as reduced PI3K activity in an animal model of diabetes, both consistent with the involvement of these kinases. mTOR and PI3K were thus subjected to in-vitro validation by kinase and cell death assays, and T148 phosphorylation was assessed under basal and metabolic stress conditions. These findings shed light on the mechanisms underpinning T148 phosphorylation-mediated neuroprotection and will help further the pursuit of novel therapeutics targeting neurodegenerative retinal diseases.

A human kinase yeast array for the identification of kinases modulating phosphorylation‐dependent protein–protein interactions

Protein kinases play an important role in cellular signaling pathways and their dysregulation leads to multiple diseases, making kinases prime drug targets. While more than 500 human protein kinases are known to collectively mediate phosphorylation of over 290,000 S/T/Y sites, the activities have been characterized only for a minor, intensively studied subset. To systematically address this discrepancy, we developed a human kinase array in Saccharomyces cerevisiae as a simple readout tool to systematically assess kinase activities. For this array, we expressed 266 human kinases in four different S. cerevisiae strains and profiled ectopic growth as a proxy for kinase activity across 33 conditions. More than half of the kinases showed an activity‐dependent phenotype across many conditions and in more than one strain. We then employed the kinase array to identify the kinase(s) that can modulate protein–protein interactions (PPIs). Two characterized, phosphorylation‐dependent PPIs with unknown kinase–substrate relationships were analyzed in a phospho‐yeast two‐hybrid assay. CK2α1 and SGK2 kinases can abrogate the interaction between the spliceosomal proteins AAR2 and PRPF8, and NEK6 kinase was found to mediate the estrogen receptor (ERα) interaction with 14‐3‐3 proteins. The human kinase yeast array can thus be used for a variety of kinase activity‐dependent readouts.

Structural characterization of KKT4, an unconventional microtubule-binding kinetochore protein

SummaryThe kinetochore is the macromolecular machinery that drives chromosome segregation by interacting with spindle microtubules. Kinetoplastids (such as Trypanosoma brucei), a group of evolutionarily divergent eukaryotes, have a unique set of kinetochore proteins that lack any significant homology to canonical kinetochore components. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterize the structure and dynamics of KKT4. We show that its microtubule-binding domain consists of a coiled-coil structure followed by a positively charged disordered tail. The structure of the C-terminal BRCT domain of KKT4 reveals that it is likely a phosphorylation-dependent protein-protein interaction domain. The BRCT domain interacts with the N-terminal region of the KKT4 microtubule-binding domain and with a phosphopeptide derived from KKT8. Taken together, these results provide structural insights into the unconventional kinetoplastid kinetochore protein KKT4.

Identification of Phenazine-Based MEMO1 Small-Molecule Inhibitors: Virtual Screening, Fluorescence Polarization Validation, and Inhibition of Breast Cancer Migration.

Phosphorylation-dependent protein-protein interactions play a significant role in biological signaling pathways; therefore, small molecules that are capable of influencing these interactions can be valuable research tools and have potential as pharmaceutical agents. MEMO1 (mediator of ErbB2-cell driven motility) is a phosphotyrosine-binding protein that interacts with a variety of protein partners and has been found to be upregulated in breast cancer patients. Herein, we report the first small-molecule inhibitors of MEMO1 interactions identified through a virtual screening platform and validated in a competitive fluorescence polarization assay. Initial structure-activity relationships have been investigated for these phenazine-core inhibitors and the binding sites have been postulated using molecular dynamics simulations. The most potent biochemical inhibitor is capable of disrupting the large protein interface with a KI of 2.7 μm. In addition, the most promising phenazine core compounds slow the migration of breast cancer cell lines in a scratch assay.

Stafia-1: a STAT5a-Selective Inhibitor Developed via Docking-Based Screening of in Silico O-Phosphorylated Fragments.

We present a new approach for the identification of inhibitors of phosphorylation‐dependent protein–protein interaction domains, in which phenolic fragments are adapted by in silico O‐phosphorylation before docking‐based screening. From a database of 10 369 180 compounds, we identified 85 021 natural product‐derived phenolic fragments, which were virtually O‐phosphorylated and screened for in silico binding to the STAT3 SH2 domain. Nine screening hits were then synthesized, eight of which showed a degree of in vitro inhibition of STAT3. After analysis of its selectivity profile, the most potent inhibitor was then developed to Stafia‐1, the first small molecule shown to preferentially inhibit the STAT family member STAT5a over the close homologue STAT5b. A phosphonate prodrug based on Stafia‐1 inhibited STAT5a with selectivity over STAT5b in human leukemia cells, providing the first demonstration of selective in vitro and intracellular inhibition of STAT5a by a small‐molecule inhibitor.

Arabidopsis 14-3-3 epsilon members contribute to polarity of PIN auxin carrier and auxin transport-related development.

Eukaryotic 14-3-3 proteins have been implicated in the regulation of diverse biological processes by phosphorylation-dependent protein-protein interactions. The Arabidopsis genome encodes two groups of 14-3-3s, one of which - epsilon - is thought to fulfill conserved cellular functions. Here, we assessed the in vivo role of the ancestral 14-3-3 epsilon group members. Their simultaneous and conditional repression by RNA interference and artificial microRNA in seedlings led to altered distribution patterns of the phytohormone auxin and associated auxin transport-related phenotypes, such as agravitropic growth. Moreover, 14-3-3 epsilon members were required for pronounced polar distribution of PIN-FORMED auxin efflux carriers within the plasma membrane. Defects in defined post-Golgi trafficking processes proved causal for this phenotype and might be due to lack of direct 14-3-3 interactions with factors crucial for membrane trafficking. Taken together, our data demonstrate a fundamental role for the ancient 14-3-3 epsilon group members in regulating PIN polarity and plant development.

Phosphorylation of Capsaicinoid Derivatives Provides Highly Potent and Selective Inhibitors of the Transcription Factor STAT5b.

Design approaches for inhibitors of protein-protein interactions are rare, but highly sought after. Here, we report that O-phosphorylation of simple derivatives of the natural products dihydrocapsaicin and N-vanillylnonanamide leads to inhibitors of the SH2 domain of the transcription factor STAT5b. The most potent molecule is obtained from dihydrocapsaicin in only three synthetic steps. It has submicromolar affinity for the SH2 domain of STAT5b (Ki = 0.34 μM), while displaying 35-fold selectivity over the highly homologous STAT5a (Ki = 13.0 μM). The corresponding pivaloyloxymethyl ester inhibits STAT5b with selectivity over STAT5a in human tumor cells. Importantly, it inhibits cell viability and induces apoptosis in human tumor cells in a STAT5-dependent manner. Our data validate O-phosphorylation of appropriately preselected natural products or natural product derivatives as a semirational design approach for small molecules that selectively inhibit phosphorylation-dependent protein-protein interaction domains in cultured human tumor cells.

Phosphorylation‐dependent Association of PDE3A1 with SERCA2 and its Regulation of SERCA2 Activity in Human Myocardium

PDE3 regulates cAMP‐mediated signaling in the heart. Studies in mice showed that PDE3A is associated with SERCA2, PLB and AKAP18 in a signaling complex in the SR that regulates inotropic responses. Immunohistochemical studies demonstrate that PDE3A is co‐localized in Z‐bands of human cardiomyocytes with desmin, SERCA2, PLB and AKAP18. In SR fractions, cAMP or rPKAc increased PLB phosphorylation and SERCA2 activity; this was potentiated specifically by PDE3 inhibition. During S6 chromatography of solubilized SR membranes, PDE3 activity was recovered in high‐ and low‐molecular‐weight (HMW, LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, while LMW contained PDE3A1, PDE3A2 and PDE3A3; PDE3A1 was the principal PKA‐phosphorylated isoform. Phosphorylation by rPKAc increased cAMP‐hydrolytic activity, shifted PDE3A from LMW to HMW peaks and increased the co‐IP of PDE3A with SERCA2 and AKAP18. Phosphorylation of rPDE3A by rPKAc increased co‐IP of rSERCA2 and rAKAP18. S292/S293, a site unique to PDE3A1, was identified by alanine substitutions as the principal site regulating this interaction. Phosphorylation of PDE3A1 at this site thus promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates contractility by modulating phosphorylation‐dependent protein‐protein interactions, PLB phosphorylation and SERCA2 activity. Agents capable of blocking this interaction and increasing myocardial contractility may constitute a novel therapeutic approach for heart failure.

Regulation of Sarcoplasmic Reticulum Ca2+ ATPase 2 (SERCA2) Activity by Phosphodiesterase 3A (PDE3A) in Human Myocardium: PHOSPHORYLATION-DEPENDENT INTERACTION OF PDE3A1 WITH SERCA2

Cyclic nucleotide phosphodiesterase 3A (PDE3) regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects and that murine PDE3A1 associates with sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2), phospholamban (PLB), and AKAP18 in a multiprotein signalosome in human sarcoplasmic reticulum (SR). Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB, and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, whereas LMW peaks contained PDE3A1, PDE3A2, and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immunoprecipitation of SERCA2, cav3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of recombinant human PDE3A isoforms by recombinant PKA catalytic subunit increased co-immunoprecipitation with rSERCA2 and rat rAKAP18 (recombinant AKAP18). Deletion of the recombinant human PDE3A1/PDE3A2 N terminus blocked interactions with recombinant SERCA2. Serine-to-alanine substitutions identified Ser-292/Ser-293, a site unique to human PDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of human PDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation, and SERCA2 activity.

Construction of phosphorylation interaction networks by text mining of full-length articles using the eFIP system.

Protein phosphorylation is a reversible post-translational modification where a protein kinase adds a phosphate group to a protein, potentially regulating its function, localization and/or activity. Phosphorylation can affect protein–protein interactions (PPIs), abolishing interaction with previous binding partners or enabling new interactions. Extracting phosphorylation information coupled with PPI information from the scientific literature will facilitate the creation of phosphorylation interaction networks of kinases, substrates and interacting partners, toward knowledge discovery of functional outcomes of protein phosphorylation. Increasingly, PPI databases are interested in capturing the phosphorylation state of interacting partners. We have previously developed the eFIP (Extracting Functional Impact of Phosphorylation) text mining system, which identifies phosphorylated proteins and phosphorylation-dependent PPIs. In this work, we present several enhancements for the eFIP system: (i) text mining for full-length articles from the PubMed Central open-access collection; (ii) the integration of the RLIMS-P 2.0 system for the extraction of phosphorylation events with kinase, substrate and site information; (iii) the extension of the PPI module with new trigger words/phrases describing interactions and (iv) the addition of the iSimp tool for sentence simplification to aid in the matching of syntactic patterns. We enhance the website functionality to: (i) support searches based on protein roles (kinases, substrates, interacting partners) or using keywords; (ii) link protein entities to their corresponding UniProt identifiers if mapped and (iii) support visual exploration of phosphorylation interaction networks using Cytoscape. The evaluation of eFIP on full-length articles achieved 92.4% precision, 76.5% recall and 83.7% F-measure on 100 article sections. To demonstrate eFIP for knowledge extraction and discovery, we constructed phosphorylation-dependent interaction networks involving 14-3-3 proteins identified from cancer-related versus diabetes-related articles. Comparison of the phosphorylation interaction network of kinases, phosphoproteins and interactants obtained from eFIP searches, along with enrichment analysis of the protein set, revealed several shared interactions, highlighting common pathways discussed in the context of both diseases.Database URL: http://proteininformationresource.org/efip

Analysis of phosphorylation-dependent protein-protein interactions of histone h3.

Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. Differential pull-down experiments with H3-tails synthesized from l- and d-amino acids uncovered that histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) are part of the protein network, which interacts with the unmodified H3-tail. An additional H3-derived bait containing the nonhydrolyzable phospho-serine mimic phosphonomethylen-alanine (Pma) at S10 recruited several isoforms of the 14-3-3 family and blocked the recruitment of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide new insights into the many functions of H3S10 phosphorylation. In addition, the outlined methodology is generally applicable for studying specific binding partners of unmodified histone tails.

The bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system: regulation by protein phosphorylation and phosphorylation-dependent protein-protein interactions.

The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.

The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells

Cell signaling, one of key processes in both normal cellular function and disease, is coordinated by numerous interactions between membrane proteins that change in response to stimuli. We present a split ubiquitin-based method for detection of integral membrane protein-protein interactions (PPIs) in human cells, termed mammalian-membrane two-hybrid assay (MaMTH). We show that this technology detects stimulus (hormone or agonist)-dependent and phosphorylation-dependent PPIs. MaMTH can detect changes in PPIs conferred by mutations such as those in oncogenic ErbB receptor variants or by treatment with drugs such as the tyrosine kinase inhibitor erlotinib. Using MaMTH as a screening assay, we identified CRKII as an interactor of oncogenic EGFR(L858R) and showed that CRKII promotes persistent activation of aberrant signaling in non-small cell lung cancer cells. MaMTH is a powerful tool for investigating the dynamic interactomes of human integral membrane proteins.

Examining post‐translational modification‐mediated protein–protein interactions using a chemical proteomics approach

Post-translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM-dependent protein-protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein-protein interactions. We have recently developed CLASPI (cross-linking-assisted and stable isotope labeling in cell culture-based protein identification), a chemical proteomics approach to examine protein-protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation-dependent protein-protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine-9 (H3K9Me₃)-dependent protein-protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation-dependent protein-protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine-3 (H3T3-Phos), a mitotic histone "mark" appearing exclusively during cell division. Our approach identified survivin, the only known H3T3-Phos-binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation "mark". Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3-Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me₃). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein-protein interactions mediated by PTMs.

Gravin Is a Transitory Effector of Polo-like Kinase 1 during Cell Division

The mitogenic and second-messenger signals that promote cell proliferation often proceed through multienzyme complexes. The kinase-anchoring protein Gravin integrates cAMP and calcium/phospholipid signals at the plasma membrane by sequestering protein kinases A and C with G protein-coupled receptors. In this report we define a role for Gravin as a temporal organizer of phosphorylation-dependent protein-protein interactions during mitosis. Mass spectrometry, molecular, and cellular approaches show that CDK1/Cyclin B1 phosphorylates Gravin on threonine 766 to prime the recruitment of the polo-like kinase Plk1 at defined phases of mitosis. Fluorescent live-cell imaging reveals that cells depleted of Gravin exhibit mitotic defects that include protracted prometaphase and misalignment of chromosomes. Moreover, a Gravin T766A phosphosite mutant that is unable to interact with Plk1 negatively impacts cell proliferation. In situ detection of phospho-T766 Gravin in biopsy sections of human glioblastomas suggests that this phosphorylation event might identify malignant neoplasms.

Phosphorylation-Dependent Protein-Protein Interaction Modules As Potential Molecular Targets for Cancer Therapy

Protein phosphorylation is a key event in signal transduction pathways. When upstream signals are stimulated, protein kinases are activated and phosphorylate their substrates, modulating their localization, conformation, and activity. In some cases, phosphorylated substrates become recognizable to other proteins-such interactions transduce and propel the signal onward. Certain domains specifically recognize phosphorylated residues of proteins, regulating cell growth and differentiation. Because the proteins that contain these domains also mediate diseases that are caused by dysregulated signal transduction, small molecules that inhibit such motifs are attractive candidates for the treatment of diseases, such as cancer. In this review, we summarize the domains that recognize phosphorylated proteins, particularly serine- and threonine-phosphorylated sequences in target proteins. In addition, we introduce a high-throughput screen that we developed to identify small-molecule inhibitors of phosphorylation-dependent protein-protein interactions. An example is presented, and the potential uses of this system are discussed.

Cleavage of the BRCT tandem domains of nibrin by the 657del5 mutation affects the DNA damage response less than the Arg215Trp mutation

The Nijmegen breakage syndrome (NBS) is a genetic disorder caused by mutations in NBN gene and characterized by chromosomal instability and hypersensitivity to ionizing radiations (IR). The N-terminus of nibrin (NBN) contains a tandem breast cancer 1 (BRCA1) carboxy-terminal (BRCT) domain that represents one of the major mediators of phosphorylation-dependent protein-protein interactions in processes related to cell cycle checkpoint and DNA repair functions. Patients with NBS compound heterozygous for the 657del5 hypomorphic mutation and for the Arg215Trp missense mutation (corresponding to the 643C>T gene mutation) display a clinical phenotype more severe than that of patients homozygous for the 657del5 mutation. Here, we show that both the 657del5 and Arg215Trp mutations, occurring within the tandem BRCT domains of NBN, although not altering the assembly of the MRE11/RAD50/NBN (MRN) complex, affect the MRE11 IR-induced nuclear foci (IRIF) formation and the DNA double-strand break (DSB) signaling via the phosphorylation of both ataxia-telangiectasia-mutated (ATM) kinase and ATM downstream targets (e.g., SMC1 and p53). Remarkably, data obtained indicate that the cleavage of the BRCT tandem domains of NBN by the 657del5 mutation affects the DNA damage response less than the Arg215Trp mutation. Indeed, the 70-kDa NBN fragment, arising from the 657del5 mutation, maintains the capability to interact with MRE11 and γ-H2AX and to form IRIF. Altogether, the role of the tandem BRCT domains of NBN in the localization of the MRN complex at the DNA DSB and in the activation of the damage response is highlighted.

Novel Role of Phosphorylation-Dependent Interaction between FtsZ and FipA in Mycobacterial Cell Division

The bacterial divisome is a multiprotein complex. Specific protein-protein interactions specify whether cell division occurs optimally, or whether division is arrested. Little is known about these protein-protein interactions and their regulation in mycobacteria. We have investigated the interrelationship between the products of the Mycobacterium tuberculosis gene cluster Rv0014c-Rv0019c, namely PknA (encoded by Rv0014c) and FtsZ-interacting protein A, FipA (encoded by Rv0019c) and the products of the division cell wall (dcw) cluster, namely FtsZ and FtsQ. M. smegmatis strains depleted in components of the two gene clusters have been complemented with orthologs of the respective genes of M. tuberculosis. Here we identify FipA as an interacting partner of FtsZ and FtsQ and establish that PknA-dependent phosphorylation of FipA on T77 and FtsZ on T343 is required for cell division under oxidative stress. A fipA knockout strain of M. smegmatis is less capable of withstanding oxidative stress than the wild type and showed elongation of cells due to a defect in septum formation. Localization of FtsQ, FtsZ and FipA at mid-cell was also compromised. Growth and survival defects under oxidative stress could be functionally complemented by fipA of M. tuberculosis but not its T77A mutant. Merodiploid strains of M. smegmatis expressing the FtsZ(T343A) showed inhibition of FtsZ-FipA interaction and Z ring formation under oxidative stress. Knockdown of FipA led to elongation of M. tuberculosis cells grown in macrophages and reduced intramacrophage growth. These data reveal a novel role of phosphorylation-dependent protein-protein interactions involving FipA, in the sustenance of mycobacterial cell division under oxidative stress.

Regulation of Dauer Formation by O-GlcNAcylation in Caenorhabditis elegans

Modification of proteins at serine or threonine residues with N-acetylglucosamine, termed O-GlcNAcylation, plays an important role in most eukaryotic cells. To understand the molecular mechanism by which O-GlcNAcylation regulates the entry of Caenorhabditis elegans into the non-aging dauer state, we performed proteomic studies using two mutant strains: the O-GlcNAc transferase-deficient ogt-1(ok430) strain and the O-GlcNAcase-defective oga-1(ok1207) strain. In the presence of the dauer pheromone daumone, ogt-1 showed suppression of dauer formation, whereas oga-1 exhibited enhancement of dauer formation. Consistent with these findings, treatment of wild-type N2 worms with low concentrations of daumone and the O-GlcNAcase inhibitor O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) enhanced dauer formation, which was dependent on intact O-GlcNAcylation metabolism. We also found that the treatment of daumone enhanced O-GlcNAcylation in vivo. Seven proteins, identified by coupled two-dimensional electrophoresis/liquid chromatography-mass spectroscopy (LC-MS) analysis, were differentially expressed in oga-1(ok1207) worms compared with wild-type N2 worms. The identities of these proteins suggest that O- GlcNAcylation influences stress resistance, protein folding, and mitochondrial function. Using O-GlcNAc labeling with fluorescent dye combined with two-dimensional electrophoresis/LC-MS analysis, we also identified five proteins that were differentially O-GlcNAcylated during dauer formation. Analysis of these candidate O-GlcNAcylated proteins suggests that O-GlcNAcylation may regulate cytoskeleton modifications and protein turnover during dauer formation.

Plant 14-3-3 proteins catch up with their mammalian orthologs

Members of the eukaryotic 14-3-3 family are highly conserved proteins that have been implicated in the modulation of distinct biological processes by phosphorylation-dependent protein-protein interactions. In plants, 14-3-3 mediated regulation of house-keeping proteins such as nitrate reductase and the plasma membrane localized H(+)-ATPase has been intensely studied. Recent proteome-wide approaches have indicated that the plant 14-3-3 interactome is comparable in size and functional complexity to its animal counterpart and, furthermore, shifted the focus of attention to signal mediators. In this regard, in vivo analyses of certain signaling proteins, such as BRASSINAZOLE-RESISTANT 1, a transcription factor controlling brassinosteroid responsive gene expression, verified an essential role for 14-3-3s in hormonal signal transduction processes.