Phosphorylation-dependent Manner Research Articles

Characterization of multiple binding sites on microtubule associated protein 2c recognized by dimeric and monomeric 14-3-3ζ.

Microtubule associated protein 2 (MAP2) interacts with the regulatory protein 14-3-3ζ in a cAMP-dependent protein kinase (PKA) phosphorylation dependent manner. Using selective phosphorylation, calorimetry, nuclear magnetic resonance, chemical crosslinking, and X-ray crystallography, we characterized interactions of 14-3-3ζ with various binding regions of MAP2c. Although PKA phosphorylation increases the affinity of MAP2c for 14-3-3ζ in the proline rich region and C-terminal domain, unphosphorylated MAP2c also binds the dimeric 14-3-3ζ via its microtubule binding domain and variable central domain. Monomerization of 14-3-3ζ leads to the loss of affinity for the unphosphorylated residues. In neuroblastoma cell extract, MAP2c is heavily phosphorylated by PKA and the proline kinase ERK2. Although 14-3-3ζ dimer or monomer do not interact with the residues phosphorylated by ERK2, ERK2 phosphorylation of MAP2c in the C-terminal domain reduces the binding of MAP2c to both oligomeric variants of 14-3-3ζ.

14-3-3 promotes sarcolemmal expression of cardiac CaV1.2 and nucleates isoproterenol-triggered channel superclustering

The L-type Ca2+ channel (CaV1.2) is essential for cardiac excitation-contraction coupling. To contribute to the inward Ca2+ flux that drives Ca2+-induced-Ca2+-release, CaV1.2 channels must be expressed on the sarcolemma; thus the regulatory mechanisms that tune CaV1.2 expression to meet contractile demand are an emerging area of research. A ubiquitously expressed protein called 14-3-3 has been proposed to affect Ca2+ channel trafficking in nonmyocytes; however, whether 14-3-3 has similar effects on CaV1.2 in cardiomyocytes is unknown. 14-3-3 preferentially binds phospho-serine/threonine residues to affect many cellular processes and is known to regulate cardiac ion channels including NaV1.5 and the human ether-à-go-go-related gene (hERG) potassium channel. Altered 14-3-3 expression and function have been implicated in cardiac pathologies including hypertrophy. Accordingly, we tested the hypothesis that 14-3-3 interacts with CaV1.2 in a phosphorylation-dependent manner and regulates cardiac CaV1.2 trafficking and recycling. Confocal imaging, proximity ligation assays, superresolution imaging, and coimmunoprecipitation revealed a population of 14-3-3 colocalized and closely associated with CaV1.2. The degree of 14-3-3/CaV1.2 colocalization increased upon stimulation of β-adrenergic receptors with isoproterenol. Notably, only the 14-3-3-associated CaV1.2 population displayed increased cluster size with isoproterenol, revealing a role for 14-3-3 as a nucleation factor that directs CaV1.2 superclustering. Isoproterenol-stimulated augmentation of sarcolemmal CaV1.2 expression, Ca2+ currents, and Ca2+ transients in ventricular myocytes were strengthened by 14-3-3 overexpression and attenuated by 14-3-3 inhibition. These data support a model where 14-3-3 interacts with CaV1.2 in a phosphorylation-dependent manner to promote enhanced trafficking/recycling, clustering, and activity during β-adrenergic stimulation.

Phosphorylation-dependent VaMYB4a regulates cold stress in grapevine by inhibiting VaPIF3 and activating VaCBF4.

Cold stress severely impacts the quality and yield of grapevine (Vitis L.). In this study, we extend our previous work to elucidate the role and regulatory mechanisms of Vitis amurensis MYB transcription factor 4a (VaMYB4a) in grapevine's response to cold stress. Our results identified VaMYB4a as a key positive regulator of cold stress. We demonstrated that VaMYB4a undergoes phosphorylation by V. amurensis CBL-interacting protein kinase 18 (VaCIPK18) under cold stress, a process that activates VaMYB4a transcriptional activity. Using ChIP-seq, we performed a comprehensive genomic search to identify downstream components that interact with VaMYB4a, leading to the discovery of a basic helix-loop-helix (bHLH) transcription factor, V. amurensis phytochrome-interacting factor 3 (VaPIF3). VaMYB4a attenuated the transcriptional activity of VaPIF3 through a phosphorylation-dependent interaction under cold conditions. Furthermore, VaPIF3, which interacts with and inhibits V. amurensis C-repeat binding factor 4 (VaCBF4, a known positive regulator of cold stress), has its activity attenuated by VaMYB4a, which mediates the modulation of this pathway. Notably, VaMYB4a also interacted with and promoted the expression of VaCBF4 in a phosphorylation-dependent manner. Our study shows that VaMYB4a positively modulates cold tolerance in plants by simultaneously downregulating VaPIF3 and upregulating VaCBF4. These findings provide a nuanced understanding of the transcriptional response in grapevine under cold stress and contribute to the broader field of plant stress physiology.

The transcription factor CREB regulates epithelial-mesenchymal transition of lens epithelial cells by phosphorylation-dependent and phosphorylation-independent mechanisms.

Epithelial mesenchymal transition (EMT) of lens epithelial cells (LECs) is one of the most important pathogenic mechanisms in lens fibrotic disorders, and the regulatory mechanisms of EMT have not been fully understood. Here, we demonstrate that the cAMP-response element binding protein (CREB) can regulate lens EMT in a phosphorylation-dependent and phosphorylation-independent manners with dual mechanisms. First, CREB-S133 phosphorylation is implicated in TGFβ-induced EMT of mouse LECs and also in injury-induced mouse anterior subcapsular cataract model. The interaction between CREB and p300 is necessary for CREB regulation of TGFβ-induced EMT, since inhibition of CREB-p300 interaction and p300 knockdown led to attenuated expression of mesenchymal genes. Second, S133A-CREB, a mutant mimicking constant dephosphorylation at S133, exhibits notable occupancy in the enhancers of mesenchymal genes and confers robust transcription activity on EMT genes. Introduction of R314A mutation in S133A-CREB, which abolishes the interaction between S133A-CREB and its co-activator, cAMP-regulated transcriptional co-activators led to substantial suppression of mesenchymal gene expression in mouse LECs. Taken together, our results showed that CREB regulates lens EMT in dual mechanisms and that the S133A-CREB acts as a novel transcription factor. Mechanistically, CREB interacts with p300 in a S133 phosphorylation-dependent manner to positively regulate lens EMT genes. In contrast, S133A-CREB interacts with cAMP-regulated transcriptional co-activators to confer a robust activation of lens EMT genes.

SNX9 family mediates βarrestin-independent GPCR endocytosis

Agonist-stimulated GPCR endocytosis typically occurs via the multi-faceted adaptor proteins known as βarrestins. However, endocytosis of several GPCRs occurs independently of β-arrestins, suggesting an additional mode of GPCR endocytosis, but the mechanisms remain unknown. Here we provide evidence that sorting nexin 9 (SNX9), a previously described endocytic remodeling protein, functions as a novel cargo adaptor that promotes agonist-stimulated GPCR endocytosis. We show that SNX9 and SNX18, but not β-arrestins, are necessary for endocytosis of the chemokine receptor CXCR4. SNX9 is recruited to CXCR4 at the plasma membrane and interacts directly with the carboxyl-terminal tail of the receptor in a phosphorylation-dependent manner. We also provide evidence that some receptors do not require SNX9 and SNX18 nor β-arrestins for endocytosis, suggesting additional modes for GPCR endocytosis. These results provide novel insights into the mechanisms regulating GPCR trafficking and broaden our overall understanding of GPCR regulation.

Formin-like 1β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes

We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.

Formin-like 1β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes.

We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.

USF2 and TFEB compete in regulating lysosomal and autophagy genes

Autophagy, a highly conserved self-digestion process crucial for cellular homeostasis, is triggered by various environmental signals, including nutrient scarcity. The regulation of lysosomal and autophagy-related processes is pivotal to maintaining cellular homeostasis and basal metabolism. The consequences of disrupting or diminishing lysosomal and autophagy systems have been investigated; however, information on the implications of hyperactivating lysosomal and autophagy genes on homeostasis is limited. Here, we present a mechanism of transcriptional repression involving upstream stimulatory factor 2 (USF2), which inhibits lysosomal and autophagy genes under nutrient-rich conditions. We find that USF2, together with HDAC1, binds to the CLEAR motif within lysosomal genes, thereby diminishing histone H3K27 acetylation, restricting chromatin accessibility, and downregulating lysosomal gene expression. Under starvation, USF2 competes with transcription factor EB (TFEB), a master transcriptional activator of lysosomal and autophagy genes, to bind to target gene promoters in a phosphorylation-dependent manner. The GSK3β-mediated phosphorylation of the USF2 S155 site governs USF2 DNA-binding activity, which is involved in lysosomal gene repression. These findings have potential applications in the treatment of protein aggregation-associated diseases, including α1-antitrypsin deficiency. Notably, USF2 repression is a promising therapeutic strategy for lysosomal and autophagy-related diseases.

Protein-Sequence-Based Search of Nonreceptor ITAM-Like Regions to Identify Cytosolic Syk-Recruiting Proteins.

The recruitment of the protein spleen tyrosine kinase (Syk) to membrane-bound immune receptors is an essential step in initiating an immune response mediated through the activated receptors. Syk recognizes intracellular phosphorylated regions of membrane receptors known as immunoreceptor tyrosine-based activation motifs (ITAMs) defined by a sequence with two tyrosine (Y) amino acids separated by a certain spacing of six to eight residues: YXX(I/L)X6-8YXX(I/L). Syk with doubly phosphorylated ITAM is high-affinity and negatively regulated when Syk itself becomes phosphorylated. While the role of Syk in immune signaling is well characterized, recent information affords new functionality to Syk related to cytoplasmic processes, including the clearance of stress granules and P-bodies, both formed by liquid-liquid phase separation. Little to nothing is known about the molecular interactions involving Syk in these cytoplasmic processes. Given the essential role of receptor ITAMs in recruiting and localizing Syk for immune signaling, we explore here the possibility of a similar localization mechanism occurring for cytoplasmic processes by searching sequences of proteins related to Syk cytoplasmic function for regions that resemble receptor ITAMs. Protein sequence databases were generated from a Syk-dependent phosphoproteome and from genes related to P-bodies. A search of these databases for ITAM-like sequences yielded 102 unique hits, and 33 of these were synthesized and tested experimentally for binding to Syk tandem SH2 domains. The equilibrium dissociation constants were 0.1-50 μM for 28 peptides, and binding was negatively regulated by phosphorylation for many peptides. These results identify cytoplasmic proteins with potential for regulating the localization of Syk in a phosphorylation-dependent manner to nonmembrane cellular regions.

Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

Sister chromatid cohesion is mediated by the cohesin complex. In mitotic prophase cohesin is removed from chromosome arms in a Wapl- and phosphorylation-dependent manner. Sgo1-PP2A protects pericentromeric cohesion by dephosphorylation of cohesin and its associated Wapl antagonist sororin. However, Sgo1-PP2A relocates to inner kinetochores well before sister chromatids are separated by separase, leaving pericentromeric regions unprotected. Why deprotected cohesin is not removed by Wapl remains enigmatic. By reconstituting Wapl-dependent cohesin removal from chromatin in vitro, we discovered a requirement for Nek2a and Cdk1/2-cyclin A2. These kinases phosphorylate cohesin-bound Pds5b, thereby converting it from a sororin- to a Wapl-interactor. Replacement of endogenous Pds5b by a phosphorylation mimetic variant causes premature sister chromatid separation (PCS). Conversely, phosphorylation-resistant Pds5b impairs chromosome arm separation in prometaphase-arrested cells and suppresses PCS in the absence of Sgo1. Early mitotic degradation of Nek2a and cyclin A2 may therefore explain why only separase, but not Wapl, can trigger anaphase.

PIN1 is a novel interaction partner and a negative upstream regulator of the transcription factor NFIB.

NFIB is a transcription factor of the Nuclear Factor One (NFI) family that is essential for embryonic development. Post-translational control of NFIB or its upstream regulators have not been well characterized. Here, we show that PIN1 binds NFIB in a phosphorylation-dependent manner, via its WW domain. PIN1 interacts with the well-conserved N-terminal domains of all NFIs. Moreover, PIN1 attenuates the transcriptional activity of NFIB; this attenuation requires substrate binding by PIN1 but not its isomerase activity. Paradoxically, we found stabilization of NFIB by PIN1. We propose that PIN1 represses NFIB function not by regulating its abundance but by inducing a conformational change. These results identify NFIB as a novel PIN1 target and posit a role for PIN1 in post-translational regulation of NFIB and other NFIs.

Chronic morphine treatment induces sex- and synapse-specific cellular tolerance on thalamo-cortical mu opioid receptor signaling.

How cellular adaptations give rise to opioid analgesic tolerance to opioids like morphine is not well understood. For one, pain is a complex phenomenon comprising both sensory and affective components, largely mediated through separate circuits. Glutamatergic projections from the medial thalamus (MThal) to the anterior cingulate cortex (ACC) are implicated in processing of affective pain, a relatively understudied component of the pain experience. The goal of this study was to determine the effects of chronic morphine exposure on mu-opioid receptor (MOR) signaling on MThal-ACC synaptic transmission within the excitatory and feedforward inhibitory pathways. Using whole cell patch-clamp electrophysiology and optogenetics to selectively target these projections, we measured morphine-mediated inhibition of optically evoked postsynaptic currents in ACC layer V pyramidal neurons in drug-naïve and chronically morphine-treated mice. We found that morphine perfusion inhibited the excitatory and feedforward inhibitory pathways similarly in females but caused greater inhibition of the inhibitory pathway in males. Chronic morphine treatment robustly attenuated morphine presynaptic inhibition within the inhibitory pathway in males, but not females, and mildly attenuated presynaptic inhibition within the excitatory pathway in both sexes. These effects were not observed in MOR phosphorylation-deficient mice. This study indicates that chronic morphine treatment induces cellular tolerance to morphine within a thalamo-cortical circuit relevant to pain and opioid analgesia. Furthermore, it suggests this tolerance may be driven by MOR phosphorylation. Overall, these findings improve our understanding of how chronic opioid exposure alters cellular signaling in ways that may contribute to opioid analgesic tolerance.NEW & NOTEWORTHY Opioid signaling within the anterior cingulate cortex (ACC) is important for opioid modulation of affective pain. Glutamatergic medial thalamus (MThal) neurons synapse in the ACC and opioids, acting through mu opioid receptors (MORs), acutely inhibit synaptic transmission from MThal synapses. However, the effect of chronic opioid exposure on MThal-ACC synaptic transmission is not known. Here, we demonstrate that chronic morphine treatment induces cellular tolerance at these synapses in a sex-specific and phosphorylation-dependent manner.

Zbtb11 interacts with Otx2 and patterns the anterior neuroectoderm in Xenopus.

The zinc finger and BTB domain-containing 11 gene (zbtb11) is expressed in the Xenopus anterior neuroectoderm, but the molecular nature of the Zbtb11 protein during embryonic development remains to be elucidated. Here, we show the role of Zbtb11 in anterior patterning of the neuroectoderm and the cooperative action with the transcription factor Otx2. Both overexpression and knockdown of zbtb11 caused similar phenotypes: expanded expression of the posterior gene gbx2 in the neural plate, and later microcephaly with reduced eyes, suggesting that a proper level of zbtb11 expression is necessary for normal patterning of the neuroectoderm, including eye formation. Co-immunoprecipitation assays showed that Zbtb11 formed a complex with itself and with a phosphomimetic and repressive form of Otx2, suggesting that Zbtb11 forms a dimer or oligomer and interacts with Otx2 in a phosphorylation-dependent manner. Reporter analysis further showed that Zbtb11 enhanced the activity of the phosphomimetic Otx2 to repress a silencer element of the posterior gene meis3. These data suggest that Zbtb11 coordinates with phosphorylated Otx2 to specify the anterior neuroectoderm by repressing posterior genes.

Deubiquitinating enzyme mutagenesis screens identify a USP43-dependent HIF-1 transcriptional response

The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1α isoform, and while USP43 does not alter HIF-1α stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.

EIF4E integrates into stress response

In this issue, Diamond etal.1 and Kim etal.2 report that depletion of eIF4E leads to translational upregulation of GCN4, a key player in the integrated stress response, in an eIF2α phosphorylation-independent manner, suggesting a new mode of translational adaptation.

Effects of Aging and Fatigue on Human Skeletal Muscle Cellular Contractile Function

Aging is associated with a reduction in skeletal muscle mass and function, known as sarcopenia. While reductions in contractile force are typically explained by loss in muscle cellular cross sectional area (CSA) loss of contractile power during high velocity contractions exceed reductions in force and cannot be solely explained by loss of muscle size and are observed at the single fiber within the same myosin heavy chain isoform (MHC). These phenomenon may be explained by myosin binding protein C (MyBP-C), which is known to modulate contractile force and velocity in a phosphorylation-dependent manner in both cardiac and skeletal muscle. Few studies have investigated the inherent contractile performance of single fibers between old and young adults, and none have explored the effects of altered MyBP-C phosphorylation on single fiber function. Therefore, the PURPOSE of this study was to investigate the effects of age and fatigue-induced MyBP-C phosphorylation on single fiber contractile performance. We HYPOTHESIZED that isometric tension will be enhanced in older adults but velocity and power will be reduced, and fatigue will enhance velocity and power in single fibers. To test this hypothesis, we recruited 4 young (18-35) and 3 older (65-80) adult males to perform a single bout of unilateral knee extensions at 30% maximal voluntary isometric contraction (MVIC) until task failure. Immediately following, a bilateral biopsy of the vastus lateralis (VL) muscle was performed on the fatigued and non-fatigued control limbs. Tissue was either flash frozen for western blot or prepared for mechanical analysis of single fiber tension (Tmax), velocity (Vmax), and power (Pmax) under optimal intracellular metabolic conditions. Our analyses were limited to MHC IIA due to differences in fiber type distribution between the groups. Our RESULTS showed that neither age nor fatigue had an effect on contractile performance in MHC IIA fibers. However, independent analysis of fatigue within age groups suggest trends towards an increase in velocity (p=0.055) in young (MHC IIA) and reduction in tension (p=0.085) in old (MHC I) adult fibers. We CONCLUDE that fatigue may modulate contractile function in the single fiber in an age- and fiber-type dependent manner. Future studies will be aimed at collecting multiple fiber types and investigating the role of biological sex. Wu Tsai Human Performance Alliance. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Probing the Immunoreceptor Tyrosine-Based Inhibition Motif Interaction Protein Partners with Proteomics.

Phosphorylation of tyrosine is the basic mode of protein function and signal transduction in organisms. This process is regulated by protein tyrosine kinases (PTKs) and protein tyrosinases (PTPs). Immunoreceptor tyrosine-based inhibition motif (ITIM) has been considered as regulating the PTP activity through the interaction with the partner proteins in the cell signal pathway. The ITIM sequences need to be phosphorylated first to active the downstream signaling proteins. To explore potential regulatory mechanisms, the ITIM sequences of two transmembrane immunoglobulin proteins, myelin P0 protein-related protein (PZR) and programmed death 1 (PD-1), were analyzed to investigate their interaction with proteins involved in regulatory pathways. We discovered that phosphorylated ITIM sequences can selectively interact with the tyrosine phosphatase SHP2. Specifically, PZR-N-ITIM (pY) may be critical in the interaction between the ITIM and SH2 domains of SHP2, while PD1-C-ITSM (pY) may play a key role in the interaction between the ITIM and SH2 domains of SHP2. Quite a few proteins were identified containing the SH2 domain, exhibiting phosphorylation-mediated interaction with PZR-ITIM. In this study, 14 proteins with SH2 structural domains were identified by GO analysis on 339 proteins associated to the affinity pull-down of PZR-N-ITIM (pY). Through the SH2 domains, these proteins may interact with PZR-ITIM in a phosphorylation-dependent manner.

Multifaceted regulation of siderophore synthesis by multiple regulatory systems in Shewanella oneidensis

Siderophore-dependent iron uptake is a mechanism by which microorganisms scavenge and utilize iron for their survival, growth, and many specialized activities, such as pathogenicity. The siderophore biosynthetic system PubABC in Shewanella can synthesize a series of distinct siderophores, yet how it is regulated in response to iron availability remains largely unexplored. Here, by whole genome screening we identify TCS components histidine kinase (HK) BarA and response regulator (RR) SsoR as positive regulators of siderophore biosynthesis. While BarA partners with UvrY to mediate expression of pubABC post-transcriptionally via the Csr regulatory cascade, SsoR is an atypical orphan RR of the OmpR/PhoB subfamily that activates transcription in a phosphorylation-independent manner. By combining structural analysis and molecular dynamics simulations, we observe conformational changes in OmpR/PhoB-like RRs that illustrate the impact of phosphorylation on dynamic properties, and that SsoR is locked in the ‘phosphorylated’ state found in phosphorylation-dependent counterparts of the same subfamily. Furthermore, we show that iron homeostasis global regulator Fur, in addition to mediating transcription of its own regulon, acts as the sensor of iron starvation to increase SsoR production when needed. Overall, this study delineates an intricate, multi-tiered transcriptional and post-transcriptional regulatory network that governs siderophore biosynthesis.

Phosphorylation of ELYS promotes its interaction with VAPB at decondensing chromosomes during mitosis.

ELYS is a nucleoporin that localizes to the nuclear side of the nuclear pore complex (NPC) in interphase cells. In mitosis, it serves as an assembly platform that interacts with chromatin and then with nucleoporin subcomplexes to initiate post-mitotic NPC assembly. Here we identify ELYS as a major binding partner of the membrane protein VAPB during mitosis. In mitosis, ELYS becomes phosphorylated at many sites, including a predicted FFAT (two phenylalanines in an acidic tract) motif, which mediates interaction with the MSP (major sperm protein)-domain of VAPB. Binding assays using recombinant proteins or cell lysates and co-immunoprecipitation experiments show that VAPB binds the FFAT motif of ELYS in a phosphorylation-dependent manner. In anaphase, the two proteins co-localize to the non-core region of the newly forming nuclear envelope. Depletion of VAPB results in prolonged mitosis, slow progression from meta- to anaphase and in chromosome segregation defects. Together, our results suggest a role of VAPB in mitosis upon recruitment to or release from ELYS at the non-core region of the chromatin in a phosphorylation-dependent manner.

Characterization of the transcriptionally active form of dephosphorylated DctD complexed with dephospho-IIAGlc.

Bacterial enhancer-binding proteins (bEBPs) acquire a transcriptionally active state via phosphorylation. However, transcriptional activation by the dephosphorylated form of bEBP has been observed in DctD, which belongs to Group I bEBP. The formation of a complex between dephosphorylated DctD (d-DctD) and dephosphorylated IIAGlc (d-IIAGlc) is a prerequisite for the transcriptional activity of d-DctD. In the present study, characteristics of the transcriptionally active complex composed of d-IIAGlc and phosphorylation-deficient DctD (DctDD57Q) of Vibrio vulnificus were investigated in its multimeric conformation and DNA-binding ability. DctDD57Q formed a homodimer that could not bind to the DNA. In contrast, when DctDD57Q formed a complex with d-IIAGlc in a 1:1 molar ratio, it produced two conformations: dimer and dodecamer of the complex. Only the dodecameric complex exhibited ATP-hydrolyzing activity and DNA-binding affinity. For successful DNA-binding and transcriptional activation by the dodecameric d-IIAGlc/DctDD57Q complex, extended upstream activator sequences were required, which encompass the nucleotide sequences homologous to the known DctD-binding site and additional nucleotides downstream. This is the first report to demonstrate the molecular characteristics of a dephosphorylated bEBP complexed with another protein to form a transcriptionally active dodecameric complex, which has an affinity for a specific DNA-binding sequence.IMPORTANCEResponse regulators belonging to the bacterial two-component regulatory system activate the transcription initiation of their regulons when they are phosphorylated by cognate sensor kinases and oligomerized to the appropriate multimeric states. Recently, it has been shown that a dephosphorylated response regulator, DctD, could activate transcription in a phosphorylation-independent manner in Vibrio vulnificus. The dephosphorylated DctD activated transcription as efficiently as phosphorylated DctD when it formed a complex with dephosphorylated form of IIAGlc, a component of the glucose-phosphotransferase system. Functional mimicry of this complex with the typical form of transcriptionally active phosphorylated DctD led us to study the molecular characteristics of this heterodimeric complex. Through systematic analyses, it was surprisingly determined that a multimer constituted with 12 complexes gained the ability to hydrolyze ATP and recognize specific upstream activator sequences containing a typical inverted-repeat sequence flanked by distinct nucleotides.