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Microtubule affinity‐regulating kinase 2 (MARK2) is a Ser/Thr protein kinase that regulates cell polarity and immune responses. Here, we report that Orf9b, one of the accessory proteins encoded in the SARS‐CoV‐2 genome, increases MARK2 activity via interaction with the autoinhibitory KAI domain. We found that co‐expression of Orf9b enhances the kinase activity of MARK2 in HEK293 cells. Orf9b does not bind to or enhance the activity of the mutant form of MARK2 lacking the KA1 domain. Orf9b lowers inhibitory phosphorylation of MARK2 at T595 while mutation experiments indicate that this site is dispensable for Orf9b‐mediated enhancement of MARK2 activity. Our results suggest that Orf9b enhances MARK2 activity by binding the autoinhibitory KA1 domain, which closely interacts with the kinase domain. [ABSTRACT FROM AUTHOR]

TG‐interacting factor 1 (TGIF1) contributes to the differentiation of murine white preadipocyte and human adipose tissue‐derived stem cells; however, its regulation is not well elucidated. Insulin is a component of the adipogenic cocktail that induces ERK signaling. TGIF1 phosphorylation and sustained stability in response to insulin were reduced through the use of specific MEK inhibitor U0126. Mutagenesis at T235 or T239 residue of TGIF1 in preadipocytes led to dephosphorylation of TGIF1. The reduced TGIF1 stability resulted in an increase in p27kip1 expression, a decrease in phosphorylated Rb expression and cellular proliferation, and a reduced accumulation of lipids compared to the TGIF1‐overexpressed cells. These findings highlight that insulin/ERK‐driven phosphorylation of the T235 or T239 residue at TGIF1 is crucial for adipocyte differentiation. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus DNA polymerase processivity factor UL44 is transported into the nucleus by importin (IMP) α/β through a classical nuclear localization signal (NLS), and this region is susceptible to cdc2‐mediated phosphorylation at position T427. Whilst phosphorylation within and close to the UL44 NLS regulates nuclear transport, the details remain elusive, due to the paucity of structural information regarding the role of negatively charged cargo phosphate groups. We addressed this issue by studying the effect of UL44 T427 phosphorylation on interaction with several IMPα isoforms by biochemical and structural approaches. Phosphorylation decreased UL44/IMPα affinity 10‐fold, and a comparative structural analysis of UL44 NLS phosphorylated and non‐phosphorylated peptides complexed with mouse IMPα2 revealed the structural rearrangements responsible for phosphorylation‐dependent inhibition of UL44 nuclear import. [ABSTRACT FROM AUTHOR]

The neuronal protein α‐synuclein is centrally involved in the neurodegeneration occurring in Parkinson's disease and related synucleinopathies. α‐Synuclein's membrane‐induced 3–11 helix conformation has a hydrophobic membrane‐embedded half and a hydrophilic cytosolic half. Here, we studied the significance of (a) the surprising hydrophobicity of amino‐acids at cytosol‐exposed helix position 8; (b) the absence of positively charged lysine/arginine from all cytosol‐exposed positions (1‐5‐8‐9). We found that (a) further increasing hydrophobicity or adding lysine, but not glutamate, at position 8 augments both membrane interaction and S129 phosphorylation; (b) adding lysines at cytosol‐exposed positions 1, 5, 8, or 9 has similar effects. Variants abundantly present in membranes by biochemical fractionation markedly colocalized with transferrin‐receptor (an endosomal marker) in immunofluorescence‐microscopy, indicating accumulation at vesicle membranes. Thus, we observed a striking correlation between membrane attraction and S129 phosphorylation, relevant for understanding α‐synuclein biology in health and disease. [ABSTRACT FROM AUTHOR]

E2F is the key transcription factor that determines the proliferative status of cells by regulating the G1/S phase of the cell cycle. In this study, we show that in rice (Oryza sativa), OsE2F2 is a phosphorylation target of MAP kinases. The MAP kinases OsMPK3, OsMPK4, and OsMPK6 interact with and phosphorylate OsE2F2. Next, we determined the serine and threonine residues that could play a role in the phosphorylation of OsE2F2. Subsequently, our study suggests a possible link between MAP kinase‐mediated OsE2F2 phosphorylation and its impact on DNA proliferation in the roots of rice seedlings. Finally, we found positive feedback regulation of OsMPK4 by OsE2F2. Therefore, our study hints at the potential impact of MAP kinase signaling on the cell cycle of rice plants. [ABSTRACT FROM AUTHOR]

Human developmental disorders encompass a wide range of debilitating physical conditions and intellectual disabilities. Perturbation of protein kinase signalling underlies the development of some of these disorders. For example, disrupted SRPK signalling is associated with intellectual disabilities, and the gene dosage of DYRKs can dictate the pathology of disorders including Down's syndrome. Here, we review the emerging roles of the CMGC kinase families SRPK, CLK, DYRK, and sub‐family HIPK during embryonic development and in developmental disorders. In particular, SRPK, CLK, and DYRK kinase families have key roles in developmental signalling and stem cell regulation, and can co‐ordinate neuronal development and function. Genetic studies in model organisms reveal critical phenotypes including embryonic lethality, sterility, musculoskeletal errors, and most notably, altered neurological behaviours arising from defects of the neuroectoderm and altered neuronal signalling. Further unpicking the mechanisms of specific kinases using human stem cell models of neuronal differentiation and function will improve our understanding of human developmental disorders and may provide avenues for therapeutic strategies. [ABSTRACT FROM AUTHOR]

Manganese (Mn) is essential for plants but is toxic when taken up in excess. To maintain Mn homeostasis, the root Mn transporter natural resistance associated macrophage protein 1 (NRAMP1) cycles from the plasma membrane to endosomes upon phosphorylation. To identify the kinase involved, a split‐luciferase screening was carried out between NRAMP1 and kinases of the CIPK family and identified CIPK23 as a partner of NRAMP1. The interaction was confirmed by split‐mCitrine bimolecular fluorescence complementation and co‐immunoprecipitation assays. In vitro phosphorylation assays pinpointed two CIPK23 target residues in NRAMP1, among which serine 20, important for endocytosis. Interestingly, Mn‐induced internalization of NRAMP1 was unaffected by cipk23 mutation suggesting a potential redundancy between CIPK23 and other kinase(s). How CIPK23 could regulate NRAMP1 in response to Mn availability is discussed. [ABSTRACT FROM AUTHOR]

IRE1 is a transmembrane signalling protein that activates the unfolded protein response under endoplasmic reticulum stress. IRE1 is endowed with kinase and endoribonuclease activities. The ribonuclease activity of IRE1 can switch substrate specificities to carry out atypical splicing of Xbp1 mRNA or trigger the degradation of specific mRNAs. The mechanisms regulating the distinct ribonuclease activities of IRE1 have yet to be fully understood. Here, we report the Bcl‐2 family protein Bid as a novel recruit of the IRE1 complex, which directly interacts with the cytoplasmic domain of IRE1. Bid binding to IRE1 leads to a decrease in IRE1 phosphorylation in a way that it can only perform Xbp1 splicing while mRNA degradation activity is repressed. The RNase outputs of IRE1 have been found to regulate the homeostatic–apoptotic switch. This study, thus, provides insight into IRE1‐mediated cell survival. [ABSTRACT FROM AUTHOR]

Rab GTPases comprise a large family of conserved GTPases that are critical regulators of the secretory and endocytic pathways. The human genome encodes ~ 65 Rabs that localize to discrete membrane compartments and, when in their GTP‐bound state, bind to effector proteins to carry out diverse functions. Activating mutations in LRRK2 kinase cause Parkinson's disease, and subsets of Rab GTPases are important LRRK2 substrates. LRRK2 phosphorylates a conserved threonine residue that is essential for Rab interaction with guanine nucleotide exchange factors, effectors, and GDI that recycles Rabs between membrane compartments. This brief review will highlight new findings related to LRRK2‐mediated phosphorylation of Rab GTPases and its consequences. Remarkably, Rab phosphorylation flips a switch on Rab effector selection with dominant consequences for cell pathophysiology. [ABSTRACT FROM AUTHOR]

Extracellular signal‐regulated kinase 7 (ERK7), also known as ERK8 and MAPK15, is an atypical member of the MAP kinase family. Compared with other MAP kinases, the biological roles of ERK7 remain poorly understood. Recent work, however, has revealed several novel functions for ERK7. These include a highly conserved essential role in ciliogenesis, the ability to control cell growth, metabolism and autophagy, as well as the maintenance of genomic integrity. ERK7 functions through phosphorylation‐dependent and ‐independent mechanisms and it is activated by cellular stressors, including DNA‐damaging agents, and nutrient deprivation. Here, we summarize recent developments in understanding ERK7 function, emphasizing its conserved roles in cellular and physiological regulation. [ABSTRACT FROM AUTHOR]

Ack1 is a proto‐oncogenic tyrosine kinase with homology to the tumour suppressor Mig6, an inhibitor of the epidermal growth factor receptor (EGFR). The residues critical for binding of Mig6 to EGFR are conserved within the Mig6 homology region (MHR) of Ack1. We tested whether intramolecular interactions between the Ack1 MHR and kinase domain (KD) are regulated by phosphorylation. We identified two Src phosphorylation sites within the MHR (Y859, Y860). Addition of Src‐phosphorylated MHR to the Ack1 KD enhanced enzymatic activity. Co‐expression of Src in cells led to increased Ack1 activity; mutation of Y859/Y860 blocked this increase. Collectively, the data suggest that phosphorylation of the Ack1 MHR regulates its kinase activity. Phosphorylation of Y859/Y860 occurs in cancers of the brain, breast, colon, and prostate, where genomic amplification or somatic mutations of Ack1 play a role in disease progression. Our findings suggest that MHR phosphorylation could contribute to Ack1 dysregulation in tumours. [ABSTRACT FROM AUTHOR]

Autophagy is a highly conserved quality control process that maintains cellular health by eliminating deleterious cargoes. Compared with the extensive studies in yeast and mammalian models, the molecular details and significance of post‐translational modifications (PTMs) in the autophagy process in plants remain less well defined. In this review, we discuss recent progress in our understanding of phosphorylation, one of the most extensively studied PTMs, in the regulation of autophagosome biogenesis and autophagic degradation in plants. Based on the plant mass spectrometric database, we summarize the experimentally verified phosphorylation sites of the core autophagy machinery in plants. Furthermore, we put forward several approaches to test the roles of phosphorylation in the regulation of plant autophagy. [ABSTRACT FROM AUTHOR]

Timing by the circadian clock of Neurospora is associated with hyperphosphorylation of frequency (FRQ), which depends on anchoring casein kinase 1a (CK1a) to FRQ. It is not known how CK1a is anchored so that approximately 100 sites in FRQ can be targeted. Here, we identified two regions in CK1a, p1 and p2, that are required for anchoring to FRQ. Mutation of p1 or p2 impairs progressive hyperphosphorylation of FRQ. A p1‐mutated strain is viable but its circadian clock is non‐functional, whereas a p2‐mutated strain is non‐viable. Our data suggest that p1 and potentially also p2 in CK1a provide an interface for interaction with FRQ. Anchoring via p1‐p2 leaves the active site of CK1a accessible for phosphorylation of FRQ at multiple sites. [ABSTRACT FROM AUTHOR]

Bone morphogenetic proteins (BMPs) are essential regulators of skeletal homeostasis, and BMP9 is the most potently osteogenic among them. Here, we found that BMP9 and BMP2 rapidly induced early growth response 1 (EGR1) protein expression in osteoblasts through MEK/ERK pathway-dependent transcriptional activation. Knock-down of EGR1 using siRNA significantly inhibited BMP9-induced matrix mineralization and osteogenic marker gene expression in osteoblasts. Knock-down of EGR1 significantly reduced SMAD1/5 phosphorylation and inhibited the expression of their transcriptional targets in osteoblasts stimulated by BMP9. In contrast, forced EGR1 overexpression in osteoblasts enhanced BMP9-mediated osteoblast differentiation and SMAD1/5 phosphorylation. An intracellular association between EGR1 and SMAD1/5 was identified using immunoprecipitation assays. These results indicated that EGR1 plays an important role in BMP9-stimulated osteoblast differentiation by enhancing SMAD1/5 phosphorylation. [ABSTRACT FROM AUTHOR]

Hippo signalling plays key role in tissue growth and homeostasis, and its dysregulation is implicated in various human diseases. Expanded (Ex) is an important upstream activator of Hippo signalling; however, how Ex activates Hippo signalling is still poorly understood. Here, we demonstrate that Ex forms a homodimer via C-terminal interaction, and that the ExC2 region (912-1164 aa) is sufficient and essential for Ex dimerization. Functional analysis shows that ExC2 is required for Ex to promote the phosphorylation and inactivation of Yki in Drosophila cells. Further in vivo analysis shows that ExC2 is important for Ex to control Drosophila tissue growth. Our study, thus, uncovers a novel mechanism whereby Ex homodimerization mediates its full activation to promote Hippo signalling in growth control. [ABSTRACT FROM AUTHOR]

N-acetylglucosamine (GlcNAc) is an important structural component of the cell wall chitin, N-glycans, glycolipids, and GPI-anchors in eukaryotes. GlcNAc kinase phosphorylates GlcNAc into GlcNAc-6-phosphate, a precursor of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) that serves as a substrate for glycan synthesis. Although GlcNAc kinase is found widely in organisms ranging from microorganisms to mammals, it has never been found in the model yeast Saccharomyces cerevisiae. Here, we demonstrate the presence of GlcNAc metabolism for UDP-GlcNAc biosynthesis in S. cerevisiae through Ngk1, a GlcNAc kinase we discovered previously. The overexpression or deletion of Ngk1 in the presence of GlcNAc affected the amount of both UDP-GlcNAc and chitin, suggesting that GlcNAc metabolism via Ngk1 promotes UDP-GlcNAc synthesis. Our data suggest that the Ngk1-mediated GlcNAc metabolism compensates for the hexosamine pathway, a known pathway for UDP-GlcNAc synthesis., (© 2024 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The phosphomimetic mutation S82D in the cancer‐associated, FAD‐dependent human NADP(H):quinone oxidoreductase 1 (hNQO1) causes a decrease in flavin‐adenine dinucleotide‐binding affinity and intracellular stability. We test in this work whether the evolutionarily recent neutral mutation R80H in the vicinity of S82 may alter the strong functional effects of S82 phosphorylation through electrostatic interactions. We show using biophysical and bioinformatic analyses that the reverse mutation H80R prevents the effects of S82D phosphorylation on hNQO1 by modulating the local stability. Consistently, in rat NQO1 (rNQO1) which contains R80, the effects of phosphorylation were milder, resembling the behaviour found in hNQO1 when this residue was humanized in rNQO1 (by the R80H mutation). Thus, apparently neutral and evolutionarily divergent mutations may determine the functional response of mammalian orthologues towards phosphorylation. [ABSTRACT FROM AUTHOR]

The timing of cytokinesis relative to other mitotic events in the fission yeast Schizosaccharomyces pombe is controlled by the septation initiation network (SIN). During a mitotic checkpoint, the SIN is inhibited by the E3 ubiquitin ligase Dma1 to prevent chromosome mis‐segregation. Dma1 dynamically localizes to spindle pole bodies (SPBs) and the contractile ring (CR) during mitosis, though its role at the CR is unknown. Here, we examined whether Dma1 phosphorylation affects its localization or function. We found that preventing Dma1 phosphorylation by substituting the six phosphosites with alanines diminished its CR localization but did not affect its mitotic checkpoint function. These studies reinforce the conclusion that Dma1 localization to the SPB is key to its role in the mitotic checkpoint. [ABSTRACT FROM AUTHOR]

The NLRP3 inflammasome, a critical component of the innate immune system, induces caspase‐1 activation and interleukin‐1β maturation and drives cell fate toward pyroptosis. However, the mechanism of NLRP3 inflammasome activation still remains elusive. Here we provide evidence that AKT regulates NLRP3 inflammasome activation. Upon NLRP3 activation, AKT activity is inhibited by second stimulus‐induced reactive oxygen species. In contrast, AKT activation leads to NLRP3 inhibition and improved mitochondrial fitness. Mechanistically, AKT induces the phosphorylation of the DDX3X (DEAD‐box helicase 3, X‐linked), a recently identified NLRP3 inflammasome component, and impairs the interaction between DDX3X and NLRP3. Furthermore, an AKT agonist reduces NLRP3‐dependent inflammation in two in vivo models of LPS‐induced sepsis and Alum‐induced peritonitis. Altogether, our study highlights an important role of AKT in controlling NLRP3 inflammasome activation. [ABSTRACT FROM AUTHOR]

The ribosome is subjected to post‐translational modifications, including phosphorylation, that affect its biological activity. Among ribosomal elements, the P‐proteins undergo phosphorylation within the C terminus, the element which interacts with trGTPases or ribosome‐inactivating proteins (RIPs); however, the role of phosphorylation has never been elucidated. Here, we probed the function of phosphorylation on the interaction of P‐proteins with RIPs using the ribosomal P1‐P2 dimer. We determined the kinetic parameters of the interaction with the toxins using biolayer interferometry and microscale thermophoresis. The results present the first mechanistic insight into the function of P‐protein phosphorylation, showing that introduction of a negative charge into the C terminus of P1‐P2 proteins promotes α‐helix formation and decreases the affinity of the P‐proteins for the RIPs. [ABSTRACT FROM AUTHOR]

Previous studies have suggested that phosphorylation of translation elongation factor 1A (eEF1A) can alter its function, and large‐scale phospho‐proteomic analyses in Saccharomyces cerevisiae have identified 14 eEF1A residues phosphorylated under various conditions. Here, a series of eEF1A mutations at these proposed sites were created and the effects on eEF1A activity were analyzed. The eEF1A‐S53D and eEF1A‐T430D phosphomimetic mutant strains were inviable, while corresponding alanine mutants survived but displayed defects in growth and protein synthesis. The activity of an eEF1A‐S289D mutant was significantly reduced in the absence of the guanine nucleotide exchange factor eEF1Bα and could be restored by an exchange‐deficient form of the protein, suggesting that eEF1Bα promotes eEF1A activity by a mechanism other than nucleotide exchange. Our data show that several of the phosphorylation sites identified by high‐throughput analysis are critical for eEF1A function. [ABSTRACT FROM AUTHOR]

Activated and promoter‐bound heat‐shock transcription factor 1 (HSF1) induces RNA polymerase II recruitment upon heat shock, and this is facilitated by the core Mediator in Drosophila and yeast. Another Mediator module, CDK8 kinase module (CKM), consisting of four subunits including MED12 and CDK8, plays a negative or positive role in the regulation of transcription; however, its involvement in HSF1‐mediated transcription remains unclear. We herein demonstrated that HSF1 interacted with MED12 and recruited MED12 and CDK8 to the HSP70 promoter during heat shock in mammalian cells. The kinase activity of CDK8 (and its paralog CDK19) promoted HSP70 expression partly by phosphorylating HSF1‐S326 and maintained proteostasis capacity. These results indicate an important role for CKM in the protection of cells against proteotoxic stress. [ABSTRACT FROM AUTHOR]

The lysosome is a cellular signalling hub at the point of convergence of endocytic and autophagic pathways, where the contents are degraded and recycled. Pleckstrin homology domain‐containing family member 1 (PLEKHM1) acts as an adaptor to facilitate the fusion of endocytic and autophagic vesicles with the lysosome. However, it is unclear how PLEKHM1 function at the lysosome is controlled. Herein, we show that PLEKHM1 coprecipitates with, and is directly phosphorylated by, mTOR. Using a phosphospecific antibody against Ser432/S435 of PLEKHM1, we show that the same motif is a direct target for ERK2‐mediated phosphorylation in a growth factor‐dependent manner. This dual regulation of PLEKHM1 at a highly conserved region points to a convergence of both growth factor‐ and amino acid‐sensing pathways, placing PLEKHM1 at a critical juncture of cellular metabolism. [ABSTRACT FROM AUTHOR]

Mutations in PINK1 (PTEN‐induced putative kinase 1) are associated with autosomal recessive early‐onset Parkinson's disease. Full‐length PINK1 (PINK1‐l) has been extensively studied in mitophagy; however, the functions of the short form of PINK1 (PINK1‐s) remain poorly understood. Here, we report that PINK1‐s is recruited to ribosome fractions after short‐term inhibition of proteasomes. The expression of PINK1‐s greatly inhibits protein synthesis even without proteasomal stress. Mechanistically, PINK1‐s phosphorylates the translation elongation factor eEF1A1 during proteasome inhibition. The expression of the phosphorylation mimic mutation eEF1A1S396E rescues protein synthesis defects and cell viability caused by PINK1 knockout. These findings implicate an important role for PINK1‐s in protecting cells against proteasome stress through inhibiting protein synthesis. [ABSTRACT FROM AUTHOR]

Integrin‐mediated adhesion regulates cellular responses to changes in the mechanical and biochemical properties of the extracellular matrix. Cell–matrix adhesion regulates caveolar endocytosis, dependent on caveolin 1 (Cav1) Tyr14 phosphorylation (pY14Cav1), to control anchorage‐dependent signaling. We find that cell–matrix adhesion regulates pY14Cav1 levels in mouse fibroblasts. Biochemical fractionation reveals endogenous pY14Cav1 to be present in caveolae and focal adhesions (FA). Adhesion does not affect caveolar pY14Cav1, supporting its regulation at FA, in which PF‐228‐mediated inhibition of focal adhesion kinase (FAK) disrupts. Cell adhesion on 2D polyacrylamide matrices of increasing stiffness stimulates Cav1 phosphorylation, which is comparable to the phosphorylation of FAK. Inhibition of FAK across varying stiffnesses shows it regulates pY14Cav1 more prominently at higher stiffness. Taken together, these studies reveal the presence of FAK–pY14Cav1 crosstalk at FA, which is regulated by cell–matrix adhesion. [ABSTRACT FROM AUTHOR]

The self‐renewal efficiency of mouse embryonic stem cells (ESCs) is determined by the concentration of the transcription factor NANOG. While NANOG binds thousands of sites in chromatin, the regulatory systems that control DNA binding are poorly characterised. Here, we show that NANOG is phosphorylated by casein kinase I, and identify target residues. Phosphomimetic substitutions at phosphorylation sites within the homeodomain (S130 and S131) have site‐specific functional effects. Phosphomimetic substitution of S130 abolishes DNA binding by NANOG and eliminates LIF‐independent self‐renewal. In contrast, phosphomimetic substitution of S131 enhances LIF‐independent self‐renewal, without influencing DNA binding. Modelling the DNA–homeodomain complex explains the disparate effects of these phosphomimetic substitutions. These results indicate how phosphorylation may influence NANOG homeodomain interactions that underpin ESC self‐renewal. [ABSTRACT FROM AUTHOR]

'Phosphorylation by CIPK23 regulates the high-affinity Mn transporter NRAMP1 in Arabidopsis'. B Cover illustration b The cover image refers to Kosuth I et al i . [Extracted from the article]

The transcription factor B‐MYB is an important regulator of cell cycle‐related processes that is activated by step‐wise phosphorylation of multiple sites by cyclin‐dependent kinases (CDKs) and conformational changes induced by the peptidylprolyl cis/trans isomerase Pin1. Here, we show that a conserved amino acid sequence around Ser‐577 in the C‐terminal part of B‐MYB is able to interact with the B‐MYB DNA‐binding domain. Phosphorylation of Ser‐577 disrupts this interaction and is regulated by the interplay of CDKs and the phosphatase CDC14B. Deletion of sequences surrounding Ser‐577 hyperactivates the transactivation potential of B‐MYB, decreases its proteolytic stability, and causes cell cycle defects. Overall, we show for the first time that B‐MYB can undergo an intramolecular interaction that is controlled by the phosphorylation state of Ser‐577. [ABSTRACT FROM AUTHOR]

The uL10 protein is the main constituent of the ribosomal P‐stalk, anchoring the whole stalk to the ribosome through interactions with rRNA. The P‐stalk is the core of the GTPase‐associated center (GAC), a critical element for ribosome biogenesis and ribosome translational activity. All P‐stalk proteins (uL10, P1, and P2) undergo phosphorylation within their C termini. Here, we show that uL10 has multiple phosphorylation sites, mapped also within the N‐terminal rRNA‐binding domain. Our results reveal that the introduction of a negative charge within the N terminus of uL10 impairs its association with the ribosome. These findings demonstrate that uL10 N‐terminal phosphorylation has regulatory potential governing the uL10 interaction with the ribosome and may control the activity of GAC. [ABSTRACT FROM AUTHOR]

Protein phosphorylation regulates a large variety of biological processes in all living cells. In pathogenic bacteria, the study of serine, threonine, and tyrosine (Ser/Thr/Tyr) phosphorylation has shed light on the course of infectious diseases, from adherence to host cells to pathogen virulence, replication, and persistence. Mass spectrometry (MS)‐based phosphoproteomics has provided global maps of Ser/Thr/Tyr phosphosites in bacterial pathogens. Despite recent developments, a quantitative and dynamic view of phosphorylation events that occur during bacterial pathogenesis is currently lacking. Temporal, spatial, and subpopulation resolution of phosphorylation data is required to identify key regulatory nodes underlying bacterial pathogenesis. Herein, we discuss how technological improvements in sample handling, MS instrumentation, data processing, and machine learning should improve bacterial phosphoproteomic datasets and the information extracted from them. Such information is expected to significantly extend the current knowledge of Ser/Thr/Tyr phosphorylation in pathogenic bacteria and should ultimately contribute to the design of novel strategies to combat bacterial infections. [ABSTRACT FROM AUTHOR]

Receptor‐interacting serine/threonine‐protein kinase 3 (RIPK3) is a central protein in necroptosis with great potential as a target for treating necroptosis‐associated diseases, such as Crohn's disease. However, blockade of RIPK3 kinase activity leads to unexpected RIPK3‐initiated apoptosis. Herein, we found that PP2, a known SRC inhibitor, inhibits TNF‐α‐induced necroptosis without initiating apoptosis. Further investigation showed that PP2 acts as an inhibitor of not only SRC but also RIPK3. PP2 does not disturb the integrity of the RIPK1–RIPK3–mixed lineage kinase domain‐like pseudokinase (MLKL) necroptosome or the autophosphorylation of RIPK3 at T231/S232 but disrupts RIPK3 oligomerization, thereby impairing the phosphorylation and oligomerization of MLKL. These results demonstrate the essential role of RIPK3 oligomerization in necroptosis and suggest a potential RIPK3 oligomerization‐targeting strategy for therapeutic development. [ABSTRACT FROM AUTHOR]

Membrane traffic between secretory and endosomal compartments is vesicle‐mediated and must be tightly balanced to maintain a physiological compartment size. Vesicle formation is initiated by guanine nucleotide exchange factors (GEFs) that activate the ARF family of small GTPases. Regulatory mechanisms, including reversible phosphorylation, allow ARF‐GEFs to support vesicle formation only at the right time and place in response to cellular needs. Here, we review current knowledge of how the Golgi‐specific brefeldin A‐resistance factor 1 (GBF1)/brefeldin A‐inhibited guanine nucleotide exchange protein (BIG) family of ARF‐GEFs is influenced by phosphorylation and use predictive paradigms to propose new regulatory paradigms. We describe a conserved cluster of phosphorylation sites within the N‐terminal domains of the GBF1/BIG ARF‐GEFs and suggest that these sites may respond to homeostatic signals related to cell growth and division. In the C‐terminal region, GBF1 shows phosphorylation sites clustered differently as compared with the similar configuration found in both BIG1 and BIG2. Despite this similarity, BIG1 and BIG2 phosphorylation patterns are divergent in other domains. The different clustering of phosphorylation sites suggests that the nonconserved sites may represent distinct regulatory nodes and specify the function of GBF1, BIG1, and BIG2. [ABSTRACT FROM AUTHOR]

Activation of the nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) transcription factor, a central player in immune response regulation, is based on phosphorylation of inhibitor of kappaB alpha (IκBα) by the Inhibitor of kappaB kinase (IKK) that triggers IκBα degradation. Although inhibitor of kappaB beta (IκBβ) is structurally similar to IκBα, its precise characteristics remain undefined. Herein, we report that the molecular interactivity of IκBβ with the kinase‐active region of IKK subunit 2 (IKK2), as well as its phosphorylation status, differs markedly from those of IκBα. A mass spectrometry analysis revealed that IκBβ phosphorylation sites are distributed in its C‐terminal region, whereas IκBα phosphorylation sites are located in the N‐terminal region. Furthermore, IKK2 phosphorylation sites in IκBβ are found in a region distinct from typical degradation signals, such as phosphodegron and proline/glutamic acid/serine/threonine‐rich sequence (PEST) motifs. Mutation of the IκBβ phosphorylation sites enhances its resistance to homeostatic proteasomal degradation. These findings contribute a novel concept in NF‐κB/IKK signalling research. [ABSTRACT FROM AUTHOR]

Glucagon‐like peptide 1 receptor (GLP‐1R) belongs to the family B of G protein‐coupled receptors (GPCRs) and has antidiabetic and cardioprotective effects. Classical GLP‐1R at the plasma membrane undergoes desensitization and internalization and is recycled back to the plasma membrane under the control of GLP‐1 in islet β‐cells. However, the subcellular localization of GLP‐1R in the vascular system remains unclear. Here, we find that GLP‐1R is localized in the nucleus of rat aortic smooth muscle cells (RASMCs) and in the tunica media. We identify a functional nuclear localization signal (NLS; 412‐442aa) at the C‐terminal region of GLP‐1R. Nuclear import of GLP‐1R is mediated by an importin‐α‐dependent pathway and regulated by phosphorylation of Ser416 in the NLS. Upon leaving the nucleus, GLP‐1R promotes cell proliferation in RASMCs. These findings may provide insights into the cardiovascular functions of GLP‐1R. [ABSTRACT FROM AUTHOR]

Dual‐specificity tyrosine‐regulated kinases (DYRKs) are evolutionarily conserved from yeast to mammals. Accumulating studies have revealed that DYRKs have important roles in regulation of the cell cycle and survival. DYRK2, a member of the class II DYRK family protein, is a key regulator of p53, and phosphorylates it at Ser46 to induce apoptosis in response to DNA damage. Moreover, recent studies have uncovered that DYRK2 regulates G1/S transition, epithelial‐mesenchymal‐transition, and stemness in human cancer cells. DYRK2 also appears to have roles in tissue development in lower eukaryotes. Thus, the elucidation of mechanisms for DYRK2 during mammalian tissue development will promote the understanding of cell differentiation, tissue homeostasis, and congenital diseases as well as cancer. In this review, we discuss the roles of DYRK2 in tumor cells. Moreover, we focus on DYRK2‐dependent developmental mechanisms in several species including fly (Drosophila), worm (Caenorhabditis elegans), zebrafish (Danio rerio), and mammals. [ABSTRACT FROM AUTHOR]

Although signal-transducing adaptor protein-2 (STAP-2) acts in certain immune responses, its role in B cell receptor (BCR)-mediated signals remains unknown. In this study, we have revealed that BCR-mediated signals, cytokine production and antibody production were increased in STAP-2 knockout (KO) mice compared with wild-type (WT) mice. Phosphorylation of tyrosine-protein kinase LYN Y508 was reduced in STAP-2 KO B cells after BCR stimulation. Mechanistic analysis revealed that STAP-2 directly binds to LYN, dependently of STAP-2 Y250 phosphorylation by LYN. Furthermore, phosphorylation of STAP-2 enhanced interactions between LYN and tyrosine-protein kinase CSK, resulting in enhanced CSK-mediated LYN Y508 phosphorylation. These results suggest that STAP-2 is crucial for controlling BCR-mediated signals and antibody production by enhanced CSK-mediated feedback regulation of LYN., (© 2023 Federation of European Biochemical Societies.)

The inter‐organelle transport of lipids must be regulated to ensure appropriate lipid composition of each organelle. In mammalian cells, ceramide synthesised in the endoplasmic reticulum (ER) is transported to the trans‐Golgi regions, where ceramide is converted to sphingomyelin (SM) with the concomitant production of diacylglycerol. Ceramide transport protein (CERT) transports ceramide from the ER to the trans‐Golgi regions at the ER–Golgi membrane contact sites (MCS). The function of CERT is down‐regulated by multisite phosphorylation of a serine‐repeat motif (SRM) and up‐regulated by phosphorylation of serine 315 in CERT. Multisite phosphorylation of the SRM is primed by protein kinase D, which is activated by diacylglycerol. The function of CERT is regulated by a phosphorylation‐dependent feedback mechanism in response to cellular requirements of SM. CERT‐dependent ceramide transport is also affected by the pool of phosphatidylinositol (PtdIns)‐4‐phosphate (PtdIns(4)P) in the trans‐Golgi regions, while the PtdIns(4)P pool is regulated by PtdIns‐4‐kinases and oxysterol‐binding protein. The ER‐Golgi MCS may serve as inter‐organelle communication zones, in which many factors work in concert to serve as an extensive rheostat of SM, diacylglycerol, cholesterol and PtdIns(4)P. [ABSTRACT FROM AUTHOR]

Connivance of cellular factors during virus‐host cell membrane fusion is poorly understood. We have recently shown that cellular villin plays an important role during membrane fusion of reconstituted Sendai virosomes with hepatocytes. Here, we employed villin‐null Chinese Hamster Ovary (CHO) cells, where villin expression led to an increased fusion with virosomes, which was further enhanced due to tyrosine phosphorylation in the presence of c‐src. However, the villin RRI mutant, lacking actin‐severing function, failed to augment membrane fusion. Furthermore, quantitative mass spectrometry and detailed analysis revealed Tyr499 to be the key phosphorylation site of villin responsible for the enhancement of virosome‐CHO cell fusion. Overall, our results demonstrate a critical role for villin and its cell‐type dependent phosphorylation in regulating membrane fusion. [ABSTRACT FROM AUTHOR]

The Paip2 protein is a factor regulating mRNA translation and stability in the cytoplasm. It has also been found in the nuclei of several cell types in Drosophila. Here, we aim to elucidate the functions of Paip2 in the cell nucleus. We find that nuclear Paip2 is a component of an ~300‐kDa protein complex. Paip2 interacts with mRNA capping factor and factors of RNA polymerase II (Pol II) transcription initiation and early elongation. Paip2 functionally cooperates with the Cbp80 subunit of the cap‐binding complex, with both proteins ensuring proper Pol II C‐terminal domain (CTD) Ser5 phosphorylation at the promoter. Thus, Paip2 is a novel player at the stage of mRNA capping and early Pol II elongation. [ABSTRACT FROM AUTHOR]

During bacterial conjugation, plasmid DNA is transferred from cell to cell. In Agrobacterium fabrum, conjugation is regulated by the phytochrome photoreceptors Agp1 and Agp2. Both contribute equally to this regulation. Agp1 and Agp2 are histidine kinases, but, for Agp2, we found no autophosphorylation activity. A clear autophosphorylation signal, however, was obtained with mutants in which the phosphoaccepting Asp of the C‐terminal response regulator domain is replaced. Thus, the Agp2 histidine kinase differs from the classical transphosphorylation pattern. We performed size exclusion, photoconversion, dark reversion, autophosphorylation, chromophore assembly kinetics and fluorescence resonance energy transfer measurements on mixed Agp1/Agp2 samples. These assays pointed to an interaction between both proteins. This could partially explain the coaction of both phytochromes in the cell. [ABSTRACT FROM AUTHOR]

Baicalin is one of the main flavonoids of the dried root of Scutellaria baicalensis Georgi and is reported to exert beneficial effects on the regulation of glucose/lipid metabolism. However, understanding its specific target and unique mechanism for improving glucose utilization is a challenge. In this paper, target fishing with a baicalin probe reveals that baicalin interacts with AKT. An immunofluorescence assay further demonstrates the colocalization of baicalin with AKT in the cytoplasm. A competitive test and virtual docking show that baicalin might bind to the pleckstrin homology domain of AKT. This specific binding hampers AKT membrane translocation, activates the phosphorylation of AKT on Ser473, induces the downstream glycogen synthase kinase 3β activation, and affects glycogen synthesis. [ABSTRACT FROM AUTHOR]

G2/M checkpoints ensure the proper timing of cell mitosis. We previously reported that p38 mitogen‐activated protein kinase (MAPK) activation is essential for stress‐induced G2 arrest in the U‐2OS osteosarcoma cell line, but the molecular mechanism was obscure. Here, using the T7 phage display system, we find p38 directly binds to human polycomb protein 2 (HPC2), and arsenate‐induced G2 arrest in U‐2OS cell is p38‐ and phosphorylation of HPC2‐dependent. Phosphorylation of HPC2 at threonine 495 is required for recruiting Ring1 and Rb family proteins to form the polycomb repressive complex (PRC), and PRC is required for arsenate‐induced downregulation of CDC2 expression. Thus, p38 MAPK regulates cell cycle progression through phosphorylation of HPC2 to mediate transcriptional repression, providing a mechanistic link for arsenate‐induced transcriptional silencing. [ABSTRACT FROM AUTHOR]

Almost 100 years after the first descriptions of proteins conjugated to carbohydrates (mucins), several studies suggested that glycoproteins were not restricted to the serum, extracellular matrix, cell surface, or endomembrane system. In the 1980s, key data emerged demonstrating that intracellular proteins were modified by monosaccharides of O‐linked β‐N‐acetylglucosamine (O‐GlcNAc). Subsequently, this modification was identified on thousands of proteins that regulate cellular processes as diverse as protein aggregation, localization, post‐translational modifications, activity, and interactions. In this Review, we will highlight critical discoveries that shaped our understanding of the molecular events underpinning the impact of O‐GlcNAc on protein function, the role that O‐GlcNAc plays in maintaining cellular homeostasis, and our understanding of the mechanisms that regulate O‐GlcNAc‐cycling. [ABSTRACT FROM AUTHOR]

The MEKK1 kinase is a key regulator of stress signaling in Arabidopsis; however, little is known about the regulation of its kinase activity. Here, we found that recombinant MEKK1, expressed in both mammalian HEK293 cells and Escherichia coli, shows a mobility shift in SDS‐PAGE, and immunoblotting detected phosphorylation of serine, threonine, and tyrosine residues. N‐terminal deletions, site‐directed mutagenesis, and protein phosphatase treatment revealed that the mobility shift results from autophosphorylation of the kinase domain. We identified the tyrosine autophosphorylation sites in the N‐terminal region of MEKK1. Tyrosine to phenylalanine mutations decrease phosphorylation of the substrate MKK1, suggesting the important role of this residue in the regulation of MEKK1 kinase activity. The present study is the first to show that plant MAPKKKs are regulated by tyrosine phosphorylation. [ABSTRACT FROM AUTHOR]