Добірка наукової літератури з теми "Phosphomimetic mutants"

Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration.

The cohesion of replicated sister chromatids promotes chromosome biorientation, gene regulation, DNA repair, and chromosome condensation. Cohesion is mediated by cohesin, which is deposited on chromosomes by a separate conserved loading complex composed of Scc2 and Scc4 in Saccharomyces cerevisiae. Although it is known to be required, the role of Scc2/Scc4 in cohesin deposition remains enigmatic. Scc2 is a phosphoprotein, although the functions of phosphorylation in deposition are unknown. We identified 11 phosphorylated residues in Scc2 by mass spectrometry. Mutants of SCC2 with substitutions that mimic constitutive phosphorylation retain normal Scc2–Scc4 interactions and chromatin association but exhibit decreased viability, sensitivity to genotoxic agents, and decreased stability of the Mcd1 cohesin subunit in mitotic cells. Cohesin association on chromosome arms, but not pericentromeric regions, is reduced in the phosphomimetic mutants but remains above a key threshold, as cohesion is only modestly perturbed. However, these scc2 phosphomimetic mutants exhibit dramatic chromosome condensation defects that are likely responsible for their high inviability. From these data, we conclude that normal Scc2 function requires modulation of its phosphorylation state and suggest that scc2 phosphomimetic mutants cause an increased incidence of abortive cohesin deposition events that result in compromised cohesin complex integrity and Mcd1 turnover.

ABSTRACT Although overactivation of RhoA is recognized as a common component of vascular disorders, the molecular mechanisms regulating RhoA activity in vascular smooth muscle cells (VSMC) are still unclear. We have previously shown that in VSMC, RhoA is phosphorylated on Ser188 by nitric oxide (NO)-stimulated cGMP-dependent kinase (PKG), which leads to RhoA-Rho kinase pathway inhibition. In this study, we showed that expression of phosphoresistant RhoA mutants prevented the stimulation of VSMC migration and adhesion induced by NO-PKG pathway activation. In contrast, under basal conditions, phosphomimetic RhoA mutants stimulated VSMC adhesion and migration through a signaling pathway requiring Rac1 and the Rho exchange factor Vav3. RhoA phosphorylation or phosphomimetic RhoA mutants induced Rac1 activation but did not activate Vav3. Indeed, phosphorylated RhoA or phosphomimetic mutants trapped guanine dissociation inhibitor α (GDIα), leading to the release of Rac1 and its translocation to the membrane, where it was then activated by the basal Vav3 nucleotide exchange activity. In vivo, RhoA phosphorylation induced by PKG activation in the aortas of rats treated with sildenafil induced dissociation of Rac1 from GDIα and activation of the Rac1 signaling pathway. These results suggest that the phosphorylation of RhoA represents a novel potent and physiological GDIα displacement factor that leads to Rac1 activation and regulation of Rac1-dependent VSMC functions.

Abstract An elevated level of nucleophosmin (NPM) is often found in actively proliferative cells including human tumors. To identify the regulatory role for NPM phosphorylation in proliferation and cell cycle control, a series of mutants targeting the consensus cyclin-dependent kinase (CKD) phosphorylation sites was created to mimic or abrogate either single-site or multi-site phosphorylation. Cells expressing the phosphomimetic NPM mutants showed enhanced proliferation and G2/M cell-cycle transition; whereas nonphosphorylatable mutants induced G2/M cell-cycle arrest. Simultaneous inactivation of two CKD phosphorylation sites at Ser10 and Ser70 (S10A/S70A, NPM-AA) induced phosphorylation of Cdk1 at Tyr15 (Cdc2Tyr15) and increased cytoplasmic accumulation of Cdc25C. Strikingly, stress-induced Cdk1Tyr15 and Cdc25C sequestration were completely suppressed by expression of a double phosphomimetic NPM mutant (S10E/S70E, NPM-EE). Further analysis revealed that phosphorylation of NPM at both Ser10 and Ser70 sites were required for proper interaction between Cdk1 and Cdc25C in mitotic cells. Moreover, the NPM-EE mutant directly bound to Cdc25C and prevented phosphorylation of Cdc25C at Ser216 during mitosis. Finally, NPM-EE overrided stress-induced G2/M arrest, increased peripheral-blood blasts and splenomegaly in a NOD/SCID xenograft model, and promoted leukemia development in Fanconi mouse hematopoietic stem/progenitor cells. Thus, these findings reveal a novel function of NPM on regulation of cell-cycle progression, in which Cdk1-dependent phosphorylation of NPM controls cell-cycle progression at G2/M transition through modulation of Cdc25C activity.

Mass spectrometry analysis reveals that the three essential subunits of the cadherin–catenin complex are phosphorylated in vivo. Examination of phosphomimetic mutants in vitro suggests that phosphoregulation plays a key role in the assembly and disassembly of adherens junctions.

The disordered p53 transactivation domain (p53TAD) contains specific levels of transient helical secondary structure that are necessary for its binding to the negative regulators, mouse double minute 2 (Mdm2) and MdmX. The interactions of p53 with Mdm2 and MdmX are also modulated by posttranslational modifications (PTMs) of p53TAD including phosphorylation at S15, T18 and S20 that inhibits p53-Mdm2 binding. It is unclear whether the levels of transient secondary structure in p53TAD are changed by phosphorylation or other PTMs. We used phosphomimetic mutants to determine if adding a negative charge at positions 15 and 18 has any effect on the transient secondary structure of p53TAD and protein-protein binding. Using a combination of biophysical and structural methods, we investigated the effects of single and multisite phosphomimetics on the transient secondary structure of p53TAD and its interaction with Mdm2, MdmX, and the KIX domain. The phosphomimetics reduced Mdm2 and MdmX binding affinity by 3–5-fold, but resulted in minimal changes in transient secondary structure, suggesting that the destabilizing effect of phosphorylation on the p53TAD-Mdm2 interaction is primarily electrostatic. Phosphomimetics had no effect on the p53-KIX interaction, suggesting that increased binding of phosphorylated p53 to KIX may be influenced by decreased competition with its negative regulators.

Cytochrome c (Cc) underwent accelerated evolution from the stem of the anthropoid primates to humans. Of the 11 amino acid changes that occurred from horse Cc to human Cc, five were at Cc residues near the binding site of the Cc:CcO complex. Single-point mutants of horse and human Cc were made at each of these positions. The Cc:CcO dissociation constant KD of the horse mutants decreased in the order: T89E > native horse Cc > V11I Cc > Q12M > D50A > A83V > native human. The largest effect was observed for the mutants at residue 50, where the horse Cc D50A mutant decreased KD from 28.4 to 11.8 μM, and the human Cc A50D increased KD from 4.7 to 15.7 μM. To investigate the role of Cc phosphorylation in regulating the reaction with CcO, phosphomimetic human Cc mutants were prepared. The Cc T28E, S47E, and Y48E mutants increased the dissociation rate constant kd, decreased the formation rate constant kf, and increased the equilibrium dissociation constant KD of the Cc:CcO complex. These studies indicate that phosphorylation of these residues plays an important role in regulating mitochondrial electron transport and membrane potential ΔΨ.

Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the Togaviridae family, can cause a highly inflammatory and encephalitic disease upon infection. Although a category B select agent, no FDA-approved vaccines or therapeutics against VEEV currently exist. We previously demonstrated NF-κB activation and macromolecular reorganization of the IKK complex upon VEEV infection in vitro, with IKKβ inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKKβ and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKKβ kinase activity can directly phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5. Alanine substitution mutations at sites 204/5, 142, or 134/5 reduced VEEV replication by >30-100,000-fold corresponding to a severe decrease in negative-strand synthesis. Serial passaging rescued viral replication and negative-strand synthesis, and sequencing of revertant viruses revealed reversion to the wild-type TC-83 phosphorylation capable amino acid sequences at nsP3 sites 204/5, 142, and 135. Generation of phosphomimetic mutants using aspartic acid substitutions at site 204/5 resulted in rescue of both viral replication and negative-strand RNA production, whereas phosphomimetic mutant 134/5 rescued viral replication but failed to restore negative-strand RNA levels, and phosphomimetic mutant 142 did not rescue VEEV replication. Together, these data demonstrate that IKKβ can phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5, and suggest that phosphorylation is essential for negative-strand RNA synthesis at site 204/5, but may be important for infectious particle production at site 134/5.

AbstractThe pigment epithelium–derived factor (PEDF) belongs to the family of noninhibitory serpins. Although originally identified in the eye, PEDF is widely expressed in other body regions including the plasma. This factor can act either as a neurotrophic or as an antiangiogenic factor, and we previously showed that the 2 effects of PEDF are regulated through phosphorylation by PKA and CK2. Here, we studied the interplay between the PKA and CK2 phosphorylation of PEDF, and found that a PEDF mutant mimicking the CK2-phosphorylated PEDF cannot be phosphorylated by PKA, while the mutant mimicking the PKA-phosphorylated PEDF is a good CK2 substrate. Using triple mutants that mimic the PKA- and CK2-phosphorylated and nonphosphorylated PEDF, we found that PEDF can induce several distinct cellular activities dependent on its phosphorylation. The mutant mimicking the accumulative PKA plus CK2 phosphorylation exhibited the strongest antiangiogenic and neurotrophic activities, while the mutants mimicking the individual phosphorylation site mutants had either a reduced activity or only one of these activities. Thus, differential phosphorylation induces variable effects of PEDF, and therefore contributes to the complexity of PEDF action. It is likely that the triple phosphomimetic mutant can be used to generate effective antiangiogenic or neurotrophic drugs.

AbstractBcl2's antiapoptotic function is regulated by phosphorylation. Bcl2 also regulates cell cycle progression, but the molecular mechanism is unclear. Bcl2 is functionally expressed in mitochondria where it can act as an antioxidant that may regulate intracellular reactive oxygen species (ROS). Since ROS have been reported to act as second messengers in cell signaling, we tested whether Bcl2 phosphorylation regulates ROS and cell cycle progression. G1 → S transition and ROS levels were measured in cells expressing either the gain of function phosphomimetic Bcl2 mutants S70E and T69E/S70E/S87E (EEE) or the nonphosphorylatable and survival-deficient mutants S70A and T69A/S70A/S87A (AAA). Expression of S70E and EEE but not the A-containing Bcl2 mutants retards G1 → S transition by 35% to 50% and significantly slows cell growth in association with reduced levels of intracellular ROS. In addition to expression of the phosphomimetic Bcl2 mutants, either interleukin-3 withdrawal or treatment of cells with the antioxidant pyrrolidine dithiocarbamate (PDTC) also reduces intracellular ROS levels in association with up-regulation of p27 and accumulation of cells in G0/G1. Retardation of G1 → S transition can be overridden by directly adding H2O2 to the cells in a mechanism that involves down-regulation of p27 and activation of Cdk2. Thus, Bcl2 may regulate G1 → S transition by a novel signaling mechanism that couples regulation of intracellular ROS with p27 and Cdk2. Furthermore, phosphorylation of Bcl2 may functionally link its antiapoptotic, cell cycle retardation, and antioxidant properties.

Selon le paradigme structure-fonction, la fonction d'une protéine dépend de sa structure, et une connaissance approfondie de cette structure révèle des mécanismes fonctionnels essentiels. Cependant, ce paradigme est remis en question par les protéines intrinsèquement désordonnées (IDPs), qui, bien qu'elles n'aient pas de structure stable, restent fonctionnelles. Le facteur d'initiation de la traduction eucaryotique 4B (eIF4B) est une IDP clé dans la régulation de l'initiation de la traduction chez les eucaryotes. Cofacteur essentiel de l'hélicase eIF4A, eIF4B est crucial pour la traduction des ARNm dotés de longues régions 5' non traduites structurées. Il possède plusieurs domaines fonctionnels, dont le domaine structuré du motif de reconnaissance de l'ARN (RRM) en N-terminal, la région désordonnée DRYG, riche en aspartate, arginine, tyrosine et glycine, et la région désordonnée riche en arginine (ARM) en C-terminal. Les domaines RRM et ARM sont connus pour se lier à l'ARN, tandis que la région DRYG est essentielle à l'auto-association de l'eIF4B. La surexpression d'eIF4B dans les cellules cancéreuses pourrait influencer la formation des granules de stress, et son activité est régulée par la phosphorylation, en particulier aux sites Ser406 et Ser422.Malgré l'importance de l'eIF4B, seul son domaine RRM structuré a été caractérisé au niveau atomique. Les détails moléculaires de sa région intrinsèquement désordonnée (IDR) restent inconnus, car ces protéines sont difficiles à étudier en raison de leur hétérogénéité conformationnelle et de leur comportement dynamique. Pendant ma thèse, j'ai poursuivi quatre objectifs :(i) caractériser structurellement l'IDR de l'eIF4B à l'état monomérique ;(ii) explorer la dynamique conformationnelle et les interactions de l'IDR au niveau moléculaire lors de l'oligomérisation et de la condensation à l'échelle mésoscopique ;(iii) étudier les dynamiques conformationnelles de l'IDR lors des interactions avec l'ARN ;(iv) analyser l'impact des mutations phosphomimétiques de l'eIF4B sur l'auto-association, sur la séparation de phase et sur les interactions ARN-protéine.Grâce au transfert d'énergie par résonance de Förster à molécule unique (smFRET), j'ai étudié l'IDR de l'eIF4B en tant que monomère, révélant un comportement conformationnel non uniforme et une flexibilité variable selon les régions. La région DRYG, bien que désordonnée, est étonnamment compacte, tandis que la région C-terminale (CTR) est plus étendue et flexible. Ces caractéristiques sont largement dictées par la composition spécifique de chaque sous-région. SmFRET a aussi permis d'analyser l'oligomérisation de l'eIF4B et les changements conformationnels associés. L'augmentation de la concentration protéique au-delà d'un certain seuil a conduit à une séparation de phase d'eIF4B. Ces analyses ont permis de cartographier un paysage d'auto-association complexe d'eIF4B, passant des monomères aux oligomères et aux gouttelettes condensées. Les mutations phosphomimétiques S406E et S422E n'affectent que peu l'oligomérisation d'eIF4B, mais réduisent sa tendance à la séparation des phases.Enfin, une combinaison d'expériences smFRET et RMN a permis d'étudier les interactions eIF4B-ARN. Il en ressort que la liaison concerne principalement la région 332 à 457, qui se compacte lors de la liaison à l'ARN. Cette interaction dépend de la force ionique, suggérant que les interactions électrostatiques en sont le moteur principal, tandis que la spécificité pour les ARN riches en guanosine indique des interactions π-π supplémentaires. Notamment, les mutations phosphomimétiques Ser406 et Ser422 affectent significativement l'affinité de liaison entre l'eIF4B et l'ARN. En résumé, ce travail offre une compréhension approfondie du comportement conformationnel de l'eIF4B et des mécanismes de ses interactions, fournissant des éclaircissements sur la manière dont une protéine sans structure stable peut remplir des fonctions essentielles
The structure-function paradigm defines that protein function is determined by its structure, and detailed structural knowledge provides critical insights into its functional mechanisms. This paradigm has been challenged with the intrinsically disordered proteins (IDPs) that lack a stable structure, yet they are functional under physiological conditions. Eukaryotic translation initiation factor 4B (eIF4B) is an IDP involved in the regulation of translation initiation in eukaryotes. As an essential co-factor of RNA helicase eIF4A, eIF4B is particularly important for translation of mRNAs with long and structured 5' untranslated regions. It contains several defined functional domains/regions, including the structured N-terminal RNA recognition motif (RRM) domain, the disordered DRYG region, enriched with aspartate, arginine, tyrosine and glycine and the disordered C-terminal arginine-rich motif (ARM) region. While the RRM and ARM domains mediate RNA binding, the DRYG region is essential for eIF4B self-association. eIF4B is overexpressed in cancer cells, and may influence stress granule formation. The cellular activity of eIF4B is regulated by phosphorylation, notably at Ser406 and Ser422 residues.Despite its importance, only the well-structured RRM domain has been characterized at atomic level. The molecular details of its large intrinsically disordered region (IDR) are still unknown, as proteins of this nature are difficult to characterize due to their conformational heterogeneity and dynamic behavior. During my PhD work I had four objectives:i) structural characterization of eIF4B IDR in its monomeric state;ii) characterization of conformational dynamics and interactions of eIF4B IDR on the molecular level upon oligomerization and upon condensation on the mesoscopic scale;iii) investigation of conformational dynamics of eIF4B IDR upon RNA interactions;iv) analysis of the impact of key phosphomimetic mutations on eIF4B protein - protein interactions, eIF4B condensation and eIF4B - RNA interactions.Using single-molecule Förster resonance energy Transfer spectroscopy (smFRET) I studied the eIF4B IDR as a monomer, demonstrating its non-uniform conformational behavior and flexibility, with different regions showing varying degrees of compactness and dynamics. Although the DRYG region is disordered, it is surprisingly compact, whereas the CTR is more expanded and flexible. These characteristics are largely dictated by the specific sequence composition of each subregion. smFRET also enabled probing eIF4B oligomerization behavior and associated protein conformational changes. Increasing the protein concentration above certain thresholds lead to eIF4B phase separation, which was studied by dedicated phase separation assays. Altogether, these experiments enabled mapping of the self-association landscape of eIF4B, which represents a complex transition from monomers to oligomers to condensed droplets. Interestingly, phosphomimetic mutations, such as S406E and S422E minimally affect eIF4B oligomerization, but considerably reduce the phase separation propensity.Finally, I used a combination of smFRET and NMR experiments to investigate eIF4B - RNA interactions, confirming that binding primarily involves the 332 to 457 region (overlapping with previously identified ARM region), which undergoes compaction upon RNA binding. The binding is ionic strength dependent, suggesting that electrostatic interactions are the main driving force, while sequence specificity towards guanosine-containing RNAs, indicates additional π-π stacking interactions. Importantly, the Ser406 and Ser422 phosphomimetic mutations within the RNA binding region significantly affect eIF4B-RNA binding affinity.Altogether, this work provides a detailed molecular understanding of conformational behavior of eIF4B and mechanisms of its interactions

ELONGATED HYPOCOTYL 5 (HY5) is a transcription factor that coordinates nitrogen uptake in the roots with carbon fixation in the shoot in Arabidopsis thaliana. HY5 is regulated by phosphorylation, but much about this process remains unknown. Two receptors involved in nitrogen responses may be involved: the CLAVATA1 (CLV1) receptor, which is involved in regulating lateral root initiation in response to nitrogen, and XYLEM INTERMIXED WITH PHLOEM (XIP1), a receptor that is involved in activating nitrogen uptake. In this study, epistasis analysis determined that XIP1 and HY5 most likely function in interconnected pathways involving sucrose. Once identified, hy5 clv1 double mutants will be used in an additional epistasis test to determine how HY5 interacts with CLV1 and XIP1. In addition, this study has focused on using phosphomimetics to analyze two phosphorylation sites, S23 and S36. I have completed the first step of site-directed mutagenesis by successfully piecing together the gene with the substituted amino acid. In the future, lateral root growth assays of the resulting mutants can be employed in order to determine whether the change had phenotypic effects. This study reveals information about the signaling pathways that regulate HY5, a gene involved in the vital process of nitrogen uptake.

Signal transduction plays a crucial role in the intricate functioning of the pituitary gland. Dopamine receptor type 2 (DRD2) signaling is representative. The actin-binding protein filamin A (FLNA) is essential for the expression and signaling of dopamine receptor type 2 (DRD2) in GH- and PRL-secreting pituitary tumors (PitNETs). FLNA acts, facilitating DRD2 signal transduction and influencing tumor responsiveness to dopaminergic drugs and somatostatin receptor ligands. Remarkably, when FLNA is phosphorylated at Ser2152 (P-FLNA), its role transitions from being a scaffold that facilitates SSTR2 signal transduction to becoming a signal termination protein that impairs SSTR2’s antitumoral effects in GH-secreting PitNETs. Activation of the cAMP pathway and stimulation of DRD2 agonists impact P-FLNA levels. Overexpression of a phosphomimetic (S2152D) FLNA mutant prevents DRD2’s antiproliferative effects, emphasizing the role of P-FLNA in DRD2 signaling. These include the phosphorylation of Janus Kinase (Jak) 2 and Signal Transduction and Activator of Transcription (STAT) 5. Once phosphorylated, these proteins modulate the activity of specific genes. For instance, they enhance the expression of tyrosine hydroxylase, which stimulates dopamine production, and activate the beta-casein gene, promoting milk protein synthesis. In lower vertebrates, the pituitary gland exhibits signal transduction mechanisms related to the glucagon-like peptide-1 (GLP-1) system.