Search Results (66)

The rabies virus (RABV) phosphoprotein (P protein) has multiple functions, including acting as the essential non-catalytic cofactor of the viral polymerase (L protein) for genome replication and transcription; the principal viral antagonist of the interferon (IFN)-mediated innate immune response; and the chaperone for the viral nucleoprotein (N protein). Although P protein is known to undergo phosphorylation by cellular kinases, the location and functions of the phosphorylation sites remains poorly defined. Here, we report the identification by mass-spectrometry (MS) of residues of P protein that are modified by phosphorylation in mammalian cells, including several novel sites. Analysis of P protein with phospho-mimetic and phospho-inhibitory mutations of three novel residues/clusters that were commonly identified by MS (Ser48, Ser183/187, Ser217/219/220) indicate that phosphorylation at each of these sites does not have a major influence on nuclear trafficking or antagonistic functions toward IFN signalling pathways. However, phosphorylation of Ser48 in the N-terminus of P protein impaired function in transcription/replication and in the formation of replication structures that contain complexes of P and N proteins, suggestive of altered interactions of these proteins. The crystal structure of P protein containing the S48E phospho-mimetic mutation indicates that Ser48 phosphorylation facilitates the binding of residues 41–52 of P protein into the RNA-binding groove of non-RNA-bound N protein (N⁰), primarily through the formation of a salt bridge with Arg434 of N protein. These data indicate that Ser48 modification regulates the cycling of P-N⁰ chaperone complexes that deliver N protein to RNA to enable transcription/replication, such that enhanced interaction due to S48E phospho-mimetic mutation reduces N protein delivery to the RNA, inhibiting subsequent transcription/replication processes. These data are, to our knowledge, the first to implicate phosphorylation of RABV P protein in conserved replication functions of the P gene. Full article

The immune response triggered when plant cell surface receptors recognize pathogen-associated molecular patterns (PAMPs) is known as PAMP-triggered immunity (PTI). Several studies have demonstrated that extracellular plant ferredoxin-like protein (PFLP) can enhance PTI signaling, thereby conferring resistance to bacterial diseases in various plants. The C-terminal casein kinase II (CK2) phosphorylation region of PFLP is essential for strengthening PTI. However, whether phosphorylation at this site directly enhances PTI signaling and consequently increases plant disease resistance remains unclear. To investigate this, site-directed mutagenesis was used to generate PFLPT90A, a non-phosphorylatable mutant, and PFLPT90D, a phospho-mimetic mutant, for functional analysis. Based on the experimental results, none of the recombinant proteins were able to enhance the hypersensitive response induced by the HrpN protein or increase resistance to the soft rot pathogen Pectobacterium carotovorum subsp. carotovorum ECC17. These findings suggest that phosphorylation at the T90 residue might be essential for PFLP-mediated enhancement of plant immune responses, implying that this post-translational modification is likely required for its disease resistance function in planta. To further explore the relationship between PFLP phosphorylation and endogenous CK2, the Arabidopsis insertion mutant cka2 and the complemented line CKA2R were analyzed under treatment with flg22_Pst from Pseudomonas syringae pv. tomato. The effects of PFLP on the hypersensitive response, rapid oxidative burst, callose deposition, and susceptibility to soft rot confirmed that CK2 is required for these immune responses. Furthermore, expression analysis of PTI-related genes FRK1 and WRKY22/29 in the mitogen-activated protein kinase (MAPK) signaling pathway demonstrated that CK2 is necessary for PFLP to enhance flg22_Pst-induced immune signaling. Taken together, these findings suggest that PFLP enhances A. thaliana resistance to bacterial soft rot primarily by promoting the MAPK signaling pathway triggered by PAMP recognition, with CK2-mediated phosphorylation being essential for its function. Full article

Heart failure is associated with dysregulation in cellular Ca²⁺ that could involve sarcolemmal L-type Ca²⁺ currents (LTCCs). Building on previous observations showing that recombinant Ca_V1.2 channels are upregulated by phosphorylated calmodulin (CaM) variants, the cellular mechanism(s) underlying this posttranslational modification was investigated in cultured cardiomyocytes. Whole-cell LTCCs decreased by ≈75% after silencing the gene coding for casein kinase 2 (CK2), a constitutively active kinase in cardiomyocytes, or after its pharmacological inhibition. The overexpression of the dominant negative phosphoresistant single, double T79A/S81A, or triple T79A/S81A/S101A CaM variants resulted in a similar inhibition. In contrast, the overexpression of CaM WT or its double T79D/S81D and triple T79D/S81D/S101D phosphomimetic variants curtailed the downregulation of LTCCs caused by CK2 partial knockdown, suggesting that CK2 is responsible for the posttranslational modification of these CaM target residues. Catecholamines, triggering the protein kinase A (PKA) cascade, partially rescued LTCCs treated with siRNA without or after the overexpression of either CaM WT or stimulating CaM phosphomimetic variants. More importantly, they thwarted the negative impact of the phosphoresistant CaM variants, altogether arguing that CK2 and PKA are acting in synergy to regulate the activity of LTCCs. We conclude that CK2-mediated phosphorylation processes exacerbate the Ca²⁺ load associated with heart failure. Full article

Cytochrome c (Cytc) is a multifunctional protein, essential for respiration and intrinsic apoptosis. Post-translational modifications of Cytc have been linked to physiological and pathophysiologic conditions, including cancer. Cytc tyrosine 67 (Y67) is a conserved residue that is important to the structure and function of Cytc. We here report the phosphorylation of Y67 of Cytc purified from bovine heart mapped by mass spectrometry. We characterized the functional effects of Y67 Cytc modification using in vitro and cell culture models. Y67 was mutated to the phosphomimetic glutamate (Y67E) and to phenylalanyl (Y67F) as a control. The phosphomimetic Y67E Cytc inhibited cytochrome c oxidase (COX) activity, redirecting energy metabolism toward glycolysis, and decreased the pro-apoptotic capabilities of Cytc. The phosphomimetic Y67E Cytc showed a significantly impaired rate of superoxide scavenging and a reduced rate of oxidation by hydrogen peroxide, suggesting a lower ability to transfer electrons and scavenge reactive oxygen species (ROS). Phosphomimetic Y67E replacement led to an almost complete loss of cardiolipin peroxidase activity, pointing to a central role of Y67 for this catalytic function of Cytc. In intact cells, phosphomimetic replacement leads to a reduction in cell respiration, mitochondrial membrane potential, and ROS levels. We propose that Y67 phosphorylation is cardioprotective and promotes cell survival. Full article

It was previously reported that tyrosine 97 (Y97) of cytochrome c is phosphorylated in cow heart tissue under physiological conditions. Y97 phosphorylation was shown to partially inhibit respiration in vitro in the reaction with purified cytochrome c oxidase. Here, we use phosphomimetic Y97E Cytc to further characterize the functional effects of this modification both in vitro and in cell culture models. In vitro, phosphomimetic Y97E Cytc showed lower activity in the reaction with purified cow heart cytochrome c oxidase (COX), decreased caspase-3 activity, and reduced rate of reduction. Additionally, the phosphomimetic Y97E Cytc tended to be resistant to heme degradation and showed an increased rate of oxidation. Intact mouse Cytc double knockout fibroblasts were transfected with plasmids coding for phosphomimetic Y97E Cytc and other variants. Compared to cells expressing wild-type Cytc, the cells expressing phosphomimetic Y97E Cytc showed reduced respiration, mitochondrial membrane potential, and reactive oxygen species production, and protection from apoptosis. In an oxygen–glucose deprivation/reoxygenation cell culture model of ischemia/reperfusion injury, mitochondrial membrane potential and reactive oxygen species production were decreased. These data show that Cytc phosphorylation controls the overall flux through the electron transport chain by maintaining optimal intermediate ΔΨm potentials for efficient ATP production while minimizing reactive oxygen species production, thus protecting the cell from apoptosis. Full article

Background: FAT10 is a member of the ubiquitin-like modifier family. Similar to ubiquitin, FAT10 has a distinct enzyme cascade consisting of E1-activating, E2-conjugating, and possibly several E3-ligating enzymes, which will covalently link FAT10 to substrate proteins in order to target them directly for proteasomal degradation. FAT10 was reported to be phosphorylated by IKKβ during infection with influenza A virus. Methods: To assess the difference between the FAT10-dependent degradation of phosphorylated FAT10 and the non-phosphorylated FAT10 wild type (FAT10 WT), a mutated FAT10 that mimicked phosphorylation (FAT10 D) was constructed by replacing several serine residues and one threonine residue with aspartic or glutamic acid. The FAT10 degradation or conjugation was compared between the phospho-mimetic FAT10 and the wild-type FAT10 with respect to the dependence of the E3 ligase TRIM25, the UBL-UBA protein NUB1L, and the proteasomal ubiquitin receptor RPN10. Results: The phospho-mimetic FAT10 was more efficiently conjugated to substrate proteins as compared to the wild-type FAT10, particularly if TRIM25 was co-expressed. Additionally, the phospho-mimetic FAT10 was not bound by NUB1L. However, this did not affect FAT10 D or FAT10 WT degradation. No differences were found in the binding affinity of phospho-mimetic FAT10 to RPN10. Conclusions: In brief, the phospho-mimetic FAT10 shows enhanced conjugation efficiency, but phosphorylation does not alter its degradation by the proteasome. This reveals that phosphorylation may fine-tune FAT10’s interactions with specific interaction partners without disrupting its core function of proteasomal degradation. Full article

Dicer, a cytoplasmic type III RNase, is essential for the maturation of microRNAs (miRNAs) and is implicated in cancer progression and chemoresistance. Our previous research demonstrated that phosphorylation of Dicer at S1016 alters miRNA maturation and glutamine metabolism, contributing to gemcitabine (GEM) resistance in pancreatic ductal adenocarcinoma (PDAC). In this study, we focused on the role of Dicer phosphorylation at S1728/S1852 in GEM-resistant PDAC cells. Using shRNA to knock down Dicer in GEM-resistant PANC-1 (PANC-1 GR) cells, we examined cell viability through MTT and clonogenic assays. We also expressed phosphomimetic Dicer 2E (S1728E/S1852E) and phosphomutant Dicer 2A (S1728A/S1852A) to evaluate their effects on GEM resistance and metabolism. Our results show that phosphorylation at S1728/S1852 promotes GEM resistance by reprogramming glutamine metabolism. Specifically, phosphomimetic Dicer 2E increased intracellular glutamine, driving pyrimidine synthesis and raising dCTP levels, which compete with gemcitabine’s metabolites. This metabolic shift enhanced drug resistance. In contrast, phosphomutant Dicer 2A reduced GEM resistance. These findings highlight the importance of Dicer phosphorylation in regulating metabolism and drug sensitivity, offering insights into potential therapeutic strategies for overcoming GEM resistance in pancreatic cancer. Full article

Cell death-inducing p53-target protein 1 (CDIP1) is a proapoptotic protein that is normally expressed at low levels and is upregulated by genotoxic and endoplasmic reticulum stresses. CDIP1 has been reported to be localized to endosomes and to interact with several proteins, including B-cell receptor-associated protein 31 (BAP31) and apoptosis-linked gene 2 (ALG-2). However, the cellular and molecular mechanisms underlying CDIP1 expression-induced apoptosis remain unclear. In this study, we first demonstrated that CDIP1 was upregulated after treatment with the anticancer drug adriamycin in human breast cancer MCF-7 cells but was degraded rapidly in the lysosomal pathway. We also demonstrated that treatment with the cyclin-dependent kinase 5 (CDK5) inhibitor roscovitine led to an increase in the electrophoretic mobility of CDIP1. In addition, a phosphomimetic mutation at Ser-32 in CDIP1 resulted in an increase in CDIP1 expression-induced apoptosis. We also found that CDIP1 expression led to the induction of autophagy prior to apoptosis. Treatment of cells expressing CDIP1 with SAR405, an inhibitor of the class III phosphatidylinositol 3-kinase VPS34, caused a reduction in autophagy and promoted apoptosis. Therefore, autophagy is thought to be a defense mechanism against CDIP1 expression-induced apoptosis. Full article

The supramolecular striatin-interacting phosphatases and kinases (STRIPAK) complex is highly conserved in eukaryotes and controls diverse developmental processes in fungi. STRIPAK is genetically and physically linked to the Hippo-related septation initiation network (SIN), which signals through a chain of three kinases, including the terminal nuclear Dbf2-related (NDR) family kinase DBF2. Here, we provide evidence for the function of DBF2 during sexual development and vegetative growth of the homothallic ascomycetous model fungus Sordaria macrospora. Using mutants with a deleted dbf2 gene and complemented strains carrying different variants of dbf2, we demonstrate that dbf2 is essential for fruiting body formation, as well as septum formation of vegetative hyphae. Furthermore, we constructed dbf2 mutants carrying phospho-mimetic and phospho-deficient codons for two conserved phosphorylation sites. Growth tests of the phosphorylation mutants showed that coordinated phosphorylation is crucial for controlling vegetative growth rates and maintaining proper septum distances. Finally, we investigated the function of DBF2 by overexpressing the dbf2 gene. The corresponding transformants showed disturbed cytokinesis during ascospore formation. Thus, regulated phosphorylation of DBF2 and precise expression of the dbf2 gene are essential for accurate septation in vegetative hyphae and coordinated cell division during septation and sexual spore formation. Full article

As the final stage of leaf development, leaf senescence is affected by a variety of internal and external signals including age and environmental stresses. Although significant progress has been made in elucidating the mechanisms of age-dependent leaf senescence, it is not clear how stress conditions induce a similar process. Here, we report the roles of a stress-responsive and senescence-induced gene, ERD7 (EARLY RESPONSIVE TO DEHYDRATION 7), in regulating both age-dependent and stress-induced leaf senescence in Arabidopsis. The results showed that the leaves of erd7 mutant exhibited a significant delay in both age-dependent and stress-induced senescence, while transgenic plants overexpressing the gene exhibited an obvious accelerated leaf senescence. Furthermore, based on the results of LC-MS/MS and PRM quantitative analyses, we selected two phosphorylation sites, Thr-225 and Ser-262, which have a higher abundance during senescence, and demonstrated that they play a key role in the function of ERD7 in regulating senescence. Transgenic plants overexpressing the phospho-mimetic mutant of the activation segment residues ERD7^T225D and ERD7^T262D exhibited a significantly early senescence, while the inactivation segment ERD7^T225A and ERD7^T262A displayed a delayed senescence. Moreover, we found that ERD7 regulates ROS accumulation by enhancing the expression of AtrbohD and AtrbohF, which is dependent on the critical residues, i.e., Thr-225 and Ser-262. Our findings suggest that ERD7 is a positive regulator of senescence, which might function as a crosstalk hub between age-dependent and stress-induced leaf senescence. Full article

Activity-regulated cytoskeleton-associated protein (Arc) plays essential roles in diverse forms of synaptic plasticity, including long-term potentiation (LTP), long-term depression (LTD), and homeostatic plasticity. In addition, it assembles into virus-like particles that may deliver mRNAs and/or other cargo between neurons and neighboring cells. Considering this broad range of activities, it is not surprising that Arc is subject to regulation by multiple types of post-translational modification, including phosphorylation, palmitoylation, SUMOylation, ubiquitylation, and acetylation. Here we explore the potential regulatory role of Arc phosphorylation by protein kinase C (PKC), which occurs on serines 84 and 90 within an α-helical segment in the N-terminal domain. To mimic the effect of PKC phosphorylation, we mutated the two serines to negatively charged glutamic acid. A consequence of introducing these phosphomimetic mutations is the almost complete inhibition of Arc palmitoylation, which occurs on nearby cysteines and contributes to synaptic weakening. The mutations also inhibit the binding of nucleic acids and destabilize high-order Arc oligomers. Thus, PKC phosphorylation of Arc may limit the full expression of LTD and may suppress the interneuronal transport of mRNAs. Full article

The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has shown promise in preclinical models of myocardial infarction, but graft myocardium exhibits incomplete host–graft electromechanical integration and a propensity for pro-arrhythmic behavior. Perhaps contributing to this situation, hPSC-CM grafts show low expression of connexin 43 (Cx43), the major gap junction (GJ) protein, in ventricular myocardia. We hypothesized that Cx43 expression and function could be rescued by engineering Cx43 in hPSC-CMs with a series of phosphatase-resistant mutations at three casein kinase 1 phosphorylation sites (Cx43-S3E) that have been previously reported to stabilize Cx43 GJs and reduce arrhythmias in transgenic mice. However, contrary to our predictions, transgenic Cx43-S3E hPSC-CMs exhibited reduced Cx43 expression relative to wild-type cells, both at baseline and following ischemic challenge. Cx43-S3E hPSC-CMs showed correspondingly slower conduction velocities, increased automaticity, and differential expression of other connexin isoforms and various genes involved in cardiac excitation–contraction coupling. Cx43-S3E hPSC-CMs also had phosphorylation marks associated with Cx43 GJ internalization, a finding that may account for their impaired GJ localization. Taken collectively, our data indicate that the Cx43-S3E mutation behaves differently in hPSC-CMs than in adult mouse ventricular myocytes and that multiple biological factors likely need to be addressed synchronously to ensure proper Cx43 expression, localization, and function. Full article

The endothelin-1 (ET1) peptide has a pathological role in the activation of proliferation, survival and invasiveness pathways in different cancers. ET1’s effects rely on its activation by the endothelin-converting enzyme-1 (ECE1), which is expressed as four isoforms, differing only in their cytoplasmic N-terminuses. We already demonstrated in colorectal cancer, glioblastoma, and preliminarily lung cancer, that the isoform ECE1c heightens aggressiveness by promoting cancer stem cell traits. This is achieved through a non-canonical ET1-independent mechanism of enhancement of ECE1c’s stability upon CK2-dependent phosphorylation at S18 and S20. Here, a K6 residue is presumably responsible for ECE1c ubiquitination as its mutation to R impairs proteasomal degradation. However, how phosphorylation enhances ECE1c’s stability and how this translates into aggressiveness are still open questions. In this brief report, by swapping residues to either phospho-mimetic or phospho-resistant amino acids, we propose that the N-terminus may also be phosphorylated at Y5 and/or T9 by an unknown kinase(s). In addition, N-terminus phosphorylation may lead to a blockage of K6 ubiquitination, increasing ECE1c’s stability and presumably activating the Wnt/β-catenin signaling pathway. Thus, a novel CK2/ECE1c partnership may be emerging to promote aggressiveness and thus become a biomarker of poor prognosis and a potential therapeutic target for several cancers. Full article

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells by binding to the angiotensin-converting enzyme 2 (hACE2) receptor. This process is aided by the transmembrane protease serine 2 (TMPRSS2), which enhances entry efficiency and infectiousness by cleaving the SARS-CoV-2 surface glycoprotein (Spike). The cleavage primes the Spike protein, promoting membrane fusion instead of receptor-mediated endocytosis. Despite the pivotal role played by TMPRSS2, our understanding of its non-protease distinct domains remains limited. In this report, we present evidence indicating the potential phosphorylation of a minimum of six tyrosine residues within the cytosolic tail (CT) of TMPRSS2. Via the use of TMPRSS2 CT phospho-mimetic mutants, we observed a reduction in TMPRSS2 protease activity, accompanied by a decrease in SARS-CoV-2 pseudovirus transduction, which was found to occur mainly via the endosomal pathway. We expanded our investigation beyond TMPRSS2 CT and discovered the involvement of other non-protease domains in regulating infection. Our co-immunoprecipitation experiments demonstrated a strong interaction between TMPRSS2 and Spike. We revealed a 21 amino acid long TMPRSS2-Spike-binding region (TSBR) within the TMPRSS2 scavenger receptor cysteine-rich (SRCR) domain that contributes to this interaction. Our study sheds light on novel functionalities associated with TMPRSS2’s cytosolic tail and SRCR region. Both of these regions have the capability to regulate SARS-CoV-2 entry pathways. These findings contribute to a deeper understanding of the complex interplay between viral entry and host factors, opening new avenues for potential therapeutic interventions. Full article

Retinal neurons that form ribbon-style synapses operate over a wide dynamic range, continuously relaying visual information to their downstream targets. The remarkable signaling abilities of these neurons are supported by specialized presynaptic machinery, one component of which is syntaxin3B. Syntaxin3B is an essential t-SNARE protein of photoreceptors and bipolar cells that is required for neurotransmitter release. It has a light-regulated phosphorylation site in its N-terminal domain at T14 that has been proposed to modulate membrane fusion. However, a direct test of the latter has been lacking. Using a well-controlled in vitro fusion assay, we found that a phosphomimetic T14 syntaxin3B mutation leads to a small but significant enhancement of SNARE-mediated membrane fusion following the formation of the t-SNARE complex. While the addition of Munc18a had only a minimal effect on membrane fusion mediated by SNARE complexes containing wild-type syntaxin3B, a more significant enhancement was observed in the presence of Munc18a when the SNARE complexes contained a syntaxin3B T14 phosphomimetic mutant. Finally, we showed that the retinal-specific complexins (Cpx III and Cpx IV) inhibited membrane fusion mediated by syntaxin3B-containing SNARE complexes in a dose-dependent manner. Collectively, our results establish that membrane fusion mediated by syntaxin3B-containing SNARE complexes is regulated by the T14 residue of syntaxin3B, Munc18a, and Cpxs III and IV. Full article