Cell cycle phosphorylation of mitotic exit network (MEN) proteins

Phosphorylation of proteins is an important mechanism used to regulate most cellular processes. Recently, we completed an extensive phosphoproteomic analysis of the core proteins that constitute the Saccharomyces cerevisiae centrosome. Here, we present a study of phosphorylation sites found on the mitotic exit network (MEN) proteins, most of which are associated with the cytoplasmic face of the centrosome. We identified 55 sites on Bfa1, Cdc5, Cdc14 and Cdc15. Eight sites lie in cyclin-dependent kinase motifs (Cdk, S/T-P), and 22 sites are completely conserved within fungi. More than half of the sites were found in centrosomes from mitotic cells, possibly in preparation for their roles in mitotic exit. Finally, we report phosphorylation site information for other important cell cycle and regulatory proteins.Key words: in vivo phosphorylation, yeast centrosome, mitotic exit network (MEN), cell cycle, protein kinase, Cdk (cyclin-dependent kinase)/Cdc28, Plk1 (polo-like kinase)/Cdc5Reversible protein phosphorylation leads to changes in targeting, structure and stability of proteins and is used widely to modulate biochemical reactions in the cell. We are interested in phosphoregulation of centrosome duplication and function in the yeast Saccharomyces cerevisiae. Centrosomes nucleate microtubules and, upon duplication during the cell cycle, form the two poles of the bipolar mitotic spindle used to segregate replicated chromosomes into the two daughter cells. Timing and spatial cues are highly regulated to ensure that elongation of the mitotic spindle and separation of sister chromatids occur prior to progression into late telophase and initiation of mitotic exit. The mitotic exit network (MEN) regulates this timing through a complex signaling cascade activated at the centrosome that triggers the end of mitosis, ultimately through mitotic cyclin-dependent kinase (Cdk) inactivation (reviewed in ref. ¹).The major components of the MEN pathway (Fig. 1) are a Ras-like GTPase (Tem1), an activator (Lte1) with homology to nucleotide exchange factors, a GTPase-activating protein (GAP) complex (Bfa1/Bub2), several protein kinases [Cdc5 (Plk1 in humans), Cdc15 and Dbf2/Mob1] and Cdc14 phosphatase (reviewed in ref. ^2–5). Tem1 initiates the signal for the MEN pathway when switched to a GTP-active state. Prior to activation at anaphase, it is held at the centrosome in an inactive GDP-bound state by an inhibiting GAP complex, Bfa1/Bub2.⁶ The Bfa1/Bub2 complex and the inactive Tem1 are localized at the mother centrosome destined to move into the budded cell upon chromosome segregation, whereas the activator Lte1 is localized at the tip of the budded cell. These separate localizations ensure that Lte1 and Tem1 only interact in late anaphase, when the mitotic spindle elongates.^7,8 Lte1 has been thought to activate Tem1 as a nucleotide exchange factor, although more recent evidence suggests that it may instead affect Bfa1 localization.⁹ In addition, full activation of Tem1 is achieved through Cdc5 phosphorylation of the negative regulator Bfa1 ¹⁰ and potentially through phosphorylation of Lte1. GTP-bound Tem1 is then able to recruit Cdc15 to the centrosome, allowing for Dbf2 activation.³ The final step in the MEN pathway is release of Cdc14 from the nucleolus, which is at least partially due to phosphorylation by Dbf2¹¹ an leads to mitotic cyclin degradation and inactivation of the mitotic kinase.²Open in a separate windowFigure 1Schematic representation of the MEN proteins and pathway. MEN protein localization is shown within a metaphase cell when mitotic exit is inhibited and in a late anaphase cell when mitotic exit is initiated. Primary inhibition and activation events are described below the cells.Recently, we performed a large-scale analysis of phosphorylation sites on the 18 core yeast centrosomal proteins present in enriched centrosomal preparations.¹² In total, we mapped 297 sites on 17 of the 18 proteins and described their cell cycle regulation, levels of conservation and demonstrated defects in centrosome assembly and function resulting from mutating selected sites. MEN proteins were also identified in the centrosome preparations. This was expected, because Nud1, one of the 18 core centrosome components, is known to recruit several MEN proteins to the centrosome¹³ as part of its function in mitotic exit.^14,15 As phosphorylation is essential to several steps in the MEN pathway, beginning with recruitment of Bfa1/Bub2 by phosphorylated Nud1,¹⁵ we were interested in mapping in vivo phosphorylation sites on the MEN proteins associated with centrosomes and identifying when they occur during the cell cycle.We combined centrosome enrichment with mass spectrometry analysis to examine phosphorylation from asynchronously growing cells.¹² Centrosomes were also prepared from cells arrested in G₁ and mitosis¹² to monitor potentially cell cycle-regulated sites. We obtained significant coverage of a number of the MEN proteins, several of which have human homologs (​and33, column 1), of which eight sites lie within Cdk/Cdc28 motifs [S/T(P)], (23 Mob1 and Dbf2 are known phosphoproteins²⁴ for which we observed peptide coverage but no phosphorylation. Surprisingly, we did not detect phosphorylation on Bub2 despite the high peptide coverage; it is possible that the mitotic centrosome preparations (using a Cdc20 depletion protocol) affect the phosphorylation state of Bub2, as Bub2 is required for mitotic exit arrest in cdc20 mutants.²⁵ Additionally, specific phosphorylation sites have not been mapped on Bub2, suggesting that modifications on this protein may be difficult to observe by mass spectrometry. Lte1 does not localize to the centrosome, and we did not recover Lte1 peptides in our preparations. Many phosphorylation events on MEN proteins were observed in mitotic centrosomal preparations, most likely in preparation for their subsequent role in exit from mitosis (

Evidence against the use of surrogates for biomonitoring of Neotropical floodplains

1. Community concordance measures the level of association between the compositional patterns shown by two groups of organisms. If strong community concordance occurs, one group could be used as a surrogate for another in conservation planning and biodiversity monitoring. In this study, we evaluated the variability in the strength of community concordance, the likely mechanisms underlying community concordance and the degree to which one community can predict another in a set of Neotropical floodplain lakes (Upper Paraná River floodplain, Brazil). 2. We used a data set including six aquatic communities: fish, macrophytes, benthic macroinvertebrates, zooplankton, phytoplankton and periphyton. We used Mantel and PROTEST approaches to evaluate the levels of community concordance in up to four sampling periods. Also, we used partial Mantel test and information about biotic interactions to investigate reasons for observed patterns of concordance. Finally, we used co‐correspondence analysis to evaluate the performance of one taxonomic group in predicting the structures of other communities. 3. The levels of community concordance varied over time for almost all cross‐taxa comparisons. Concordance between phytoplankton and periphyton probably resulted from similar responses to environmental gradients, whereas other patterns of concordance were likely generated by interactions among groups. However, the levels of predictability were low, and no particular taxonomic group significantly predicted all other groups. 4. The low and temporally variable levels of community concordance cast doubts on the use of surrogate groups for biodiversity management in Neotropical floodplains.     

Allele Specific Expression of the Transthyretin Gene in Swedish Patients with Hereditary Transthyretin Amyloidosis (ATTR V30M) Is Similar between the Two Alleles

Background

Hereditary transthyretin (TTR) amyloidosis (ATTR) is an autosomal dominant disease characterized by extracellular deposits of amyloid fibrils composed of misfolded TTR. The differences in penetrance and age at onset are vast, both between and within populations, with a generally late onset for Swedish carriers. In a recent study the entire TTR gene including the 3′ UTR in Swedish, French and Japanese ATTR patients was sequenced. The study disclosed a SNP in the V30M TTR 3′ UTR of the Swedish ATTR population that was not present in either the French or the Japanese populations (rs62093482-C>T). This SNP could create a new binding site for miRNA, which would increase degradation of the mutated TTR’s mRNA thus decrease variant TTR formation and thereby delay the onset of the disease. The aim of the present study was to disclose differences in allele specific TTR expression among Swedish V30M patients, and to see if selected miRNA had any effect upon the expression.

Methodology/Principal Findings

Allele-specific expression was measured on nine liver biopsies from Swedish ATTR patients using SNaPshot Multiplex assay. Luciferase activity was measured on cell lines transfected with constructs containing the TTR 3′ UTR. Allele-specific expression measured on liver biopsies from Swedish ATTR patients showed no difference in expression between the two alleles. Neither was there any difference in expression between cell lines co-transfected with two constructs with or without the TTR 3′ UTR SNP regardless of added miRNA.

Conclusions/Significance

The SNP found in the 3′ UTR of the TTR gene has no effect on degrading the variant allele’s expression and thus has no impact on the diminished penetrance of the trait in the Swedish population. However, the 3′ UTR SNP is unique for patients descending from the Swedish founder, and this SNP could be utilized to identify ATTR patients of Swedish descent.     

Statistical discovery of site inter-dependencies in sub-molecular hierarchical protein structuring

Background

Much progress has been made in understanding the 3D structure of proteins using methods such as NMR and X-ray crystallography. The resulting 3D structures are extremely informative, but do not always reveal which sites and residues within the structure are of special importance. Recently, there are indications that multiple-residue, sub-domain structural relationships within the larger 3D consensus structure of a protein can be inferred from the analysis of the multiple sequence alignment data of a protein family. These intra-dependent clusters of associated sites are used to indicate hierarchical inter-residue relationships within the 3D structure. To reveal the patterns of associations among individual amino acids or sub-domain components within the structure, we apply a k-modes attribute (aligned site) clustering algorithm to the ubiquitin and transthyretin families in order to discover associations among groups of sites within the multiple sequence alignment. We then observe what these associations imply within the 3D structure of these two protein families.

Results

The k-modes site clustering algorithm we developed maximizes the intra-group interdependencies based on a normalized mutual information measure. The clusters formed correspond to sub-structural components or binding and interface locations. Applying this data-directed method to the ubiquitin and transthyretin protein family multiple sequence alignments as a test bed, we located numerous interesting associations of interdependent sites. These clusters were then arranged into cluster tree diagrams which revealed four structural sub-domains within the single domain structure of ubiquitin and a single large sub-domain within transthyretin associated with the interface among transthyretin monomers. In addition, several clusters of mutually interdependent sites were discovered for each protein family, each of which appear to play an important role in the molecular structure and/or function.

Conclusions

Our results demonstrate that the method we present here using a k- modes site clustering algorithm based on interdependency evaluation among sites obtained from a sequence alignment of homologous proteins can provide significant insights into the complex, hierarchical inter-residue structural relationships within the 3D structure of a protein family.

     

The G protein-coupled receptor GPR4 suppresses ERK activation in a ligand-independent manner

The lysophospholipids, lysophosphatidic acid, sphingosine-1-phosphate, and sphingosylphosphorylcholine (SPC), are bioactive lipid molecules that regulate diverse biological processes. Although the specific G protein-coupled receptors for lysophosphatidic acid and sphingosine-1-phosphate have been well-characterized, much less is known of the SPC receptors. It has been reported that ovarian cancer G protein-coupled receptor 1 (OGR1) is a high affinity receptor for SPC, and its closely related homologue GPR4 is a high affinity receptor for SPC with low affinity for lysophosphatidylcholine (LPC). However, in a functional assay to examine the specificity of ligand binding, we found that neither SPC nor LPC, or other related lysophospholipids, induced internalization of GPR4 from the plasma membrane. In agreement, these lysolipids also did not induce translocation of beta-arrestin2-GFP from the cytosol to the plasma membrane in GPR4 expressing cells. However, when these cells were cotransfected with G protein-coupled receptor kinase 2, in the absence of added ligands, beta-arrestin2-GFP accumulated in cytoplasmic vesicles, reminiscent of vesicular labeling usually observed after agonist stimulation of GPCRs. In addition, neither SPC nor LPC stimulated the binding of GTPgammaS to membranes prepared from GPR4 expressing cells and did not activate ERK1/2. Surprisingly, enforced expression of GPR4 inhibited activation of ERK1/2 induced by several stimuli, including SPC, sphingosine-1-phosphate, and even EGF. Collectively, our results suggest that SPC and LPC are not the ligands for GPR4 and that this receptor may constitutively inhibit ERK1/2 activation.     

High-yield expression in Escherichia coli, purification, and characterization of properly folded major peanut allergen Ara h 2

Allergic reactions to peanuts are a serious health problem because of their high prevalence, associated with potential severity, and chronicity. One of the three major allergens in peanut, Ara h 2, is a member of the conglutin family of seed storage proteins. Ara h 2 shows high sequence homology to proteins of the 2S albumin family. Presently, only very few structural data from allergenic proteins of this family exist. For a detailed understanding of the molecular mechanisms of food-induced allergies and for the development of therapeutic strategies knowledge of the high-resolution three-dimensional structure of allergenic proteins is essential. We report a method for the efficient large-scale preparation of properly folded Ara h 2 for structural studies and report CD-spectroscopic data. In contrast to other allergenic 2S albumins, Ara h 2 exists as a single continuous polypeptide chain in peanut seeds, and thus heterologous expression in Escherichia coli was possible. Ara h 2 was expressed as Trx-His-tag fusion protein in E. coli Origami (DE3), a modified E. coli strain with oxidizing cytoplasm which allows the formation of disulfide bridges. It could be shown that recombinant Ara h 2, thus overexpressed and purified, and the allergen isolated from peanuts are identical as judged from immunoblotting, analytical HPLC, and circular dichroism spectra.     

Changing concepts in plant hormone action

Summary A plant hormone is not, in the classic animal sense, a chemical synthesized in one organ, transported to a second organ to exert a chemical action to control a physiological event. Any phytohormone can be synthesized everywhere and can influence different growth and development processes at different places. The concept of physiological activity under hormonal control cannot be dissociated from changes in concentrations at the site of action, from spatial differences and changes in the tissue's sensitivity to the compound, from its transport and its metabolism, from balances and interactions with the other phytohormones, or in their metabolic relationships, and in their signaling pathways as well. Secondary messengers are also involved. Hormonal involvement in physiological processes can appear through several distinct manifestations (as environmental sensors, homeostatic regulators and spatio-temporal synchronizers, resource allocators, biotime adjusters, etc.), dependent on or integrated with the primary biochemical pathways. The time has also passed for the hypothesized ‘specific’ developmental hormones, rhizocaline, canlocaline, and florigen: root, stem, and flower formation result from a sequential control of specific events at the right places through a coordinated control by electrical signals, the known phytohormones and nonspecific molecules of primary and secondary metabolism, and involve both cytoplasmic and apoplastic compartments. These contemporary views are examined in this review.     

An easy and reliable method for establishment and maintenance of leaf and root cell cultures ofArabidopsis thaliana

Cell suspension cultures are useful for a wide range of biochemical and physiological studies, yet their production can be technically demanding and often unreliable. Here we describe a protocol for producing Arabidopsis cell suspension cultures that is reliable and easy to use.     

Synthetic lipopeptide analogs of bacterial lipoprotein are potent polyclonal activators for murine B lymphocytes

The lipoprotein from the outer membrane of Escherichia coli and other Enterobacteriaceae is a potent polyclonal activator for B lymphocytes. To determine the molecular structure responsible for the biologic activity of lipoprotein, a well-defined series of analogs of its N-terminal part was synthesized: S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-cysteine, -cysteine methyl ester, -cysteinyl-serine, -cysteinyl-seryl-serine, -cysteinyl-seryl-seryl-asparagine, and -cysteinyl-seryl-seryl-asparaginyl-alanine. All compounds were tested for mitogenic activity toward spleen cells from BALB/c, LPS-non-responder C3H/HeJ, and congenitally athymic C3H/Tif/Bom/nu/nu mice, measuring the incorporation of [3H]thymidine into DNA. Lymphocyte activation was confirmed by determination of the incorporation of [3H]uridine into RNA and [3H]leucine into protein. The synthetic lipopeptides were also investigated for their ability to stimulate B lymphocytes into immunoglobulin secretion, as shown by a hemolytic plaque assay. Throughout our studies, the compounds carrying two to five amino acids exhibited strong stimulation activity toward B lymphocytes comparable to native lipoprotein. In contrast, products containing only one amino acid, cysteine or cysteine methyl ester, were only marginally active, indicating that to obtain full biologic activity the presence of the hydrophilic dipeptide structure is necessary. All compounds exhibited only a marginal effect on thymocytes. Thus, a series of defined synthetic fragments of a bacterial outer membrane component exhibits a pronounced mitogenic and polyclonally stimulating activity towards B lymphocytes. The substances will be valuable tools for more detailed investigations on the molecular mechanisms of B cell activation.