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Autophagy Aug 2023Macroautophagy/autophagy is an important process responsible for protein turnover and cell survival in amino acid-deprived conditions, especially for leucine (Leu). With...
Macroautophagy/autophagy is an important process responsible for protein turnover and cell survival in amino acid-deprived conditions, especially for leucine (Leu). With the dramatic advances in mass spectrometry, many new post-translational modifications (PTMs) have been identified. However, whether these PTMs regulate autophagy remains unclear. Here we found global lysine crotonylation levels are significantly upregulated during Leu deprivation-induced autophagy. A comprehensive crotonylome profiling showed that YWHA/14-3-3 proteins are significantly enriched in the Leu regulated-crotonylome. The inhibition of YWHAE/14-3-3ε crotonylation by mutating two crotonylated sites to arginine, K73R K78R, significantly attenuates autophagy induced by Leu deprivation. Molecular dynamics suggest that YWHAE K73 and K78 crotonylations decrease protein conformation and thermodynamic stability. Moreover, we found crotonylation of YWHAE releases PPM1B to dephosphorylate ULK1 and consequently activate autophagy. Decrotonylation of YWHAE is mediated by HDAC7 whose activity is inhibited significantly by Leu deprivation. Taken together, our finding reveals a critical role of YWHAE crotonylation in Leu deprivation-induced autophagy.
Topics: Leucine; 14-3-3 Proteins; Autophagy; Mass Spectrometry; Protein Processing, Post-Translational
PubMed: 36628438
DOI: 10.1080/15548627.2023.2166276 -
Cell Death & Disease Nov 2021Cervical cancer is the leading cause of cancer-related deaths in women, and treatment for cervical cancer is very limited. Emerging evidence suggests that targeting...
Cervical cancer is the leading cause of cancer-related deaths in women, and treatment for cervical cancer is very limited. Emerging evidence suggests that targeting ferroptosis is a promising way to treat cancer. Here, we investigated the role of ferroptosis in cervical cancer, with a focus on the Cdc25A/PKM2/ErbB2 axis. Cervical cancer cells were treated with sorafenib to induce ferroptosis. Cellular MDA/ROS/GSH/iron detection assays were used to measure ferroptosis. MTT assays were performed to assess cell viability. qRT-PCR, western blot, and immunostaining assays were performed to measure the levels of proteins. Autophagy was monitored by fluorescence microscopy. Nuclear and cytosolic fractions were isolated to examine the location of PKM2 modifications. Co-IP experiments were conducted to determine the Cdc25A/PKM2 interaction. ChIP assays were performed to measure the binding affinity between H3K9Ac and the ErbB3 promoter, and a dual luciferase assay was performed to examine the transcriptional activity of ErbB2. A nude mouse xenograft model was used to examine the effects of the Cdc25A/ErbB2 axis on tumour growth in vivo. Cdc25A was elevated in human cervical cancer tissues but was reduced during sorafenib-induced ferroptosis of cervical cancer cells. Overexpression of Cdc25A inhibited sorafenib-induced ferroptosis by dephosphorylating nuclear PKM2 and suppressing autophagy. Cdc25A regulated autophagy-induced ferroptosis by increasing ErbB2 levels via the PKM2-pH3T11-H3K9Ac pathway. Cdc25A increased the resistance of cervical cancer to sorafenib, while knockdown of ErbB2 blocked these effects. Cdc25A suppressed autophagy-dependent ferroptosis in cervical cancer cells by upregulating ErbB2 levels through the dephosphorylation of PKM2. These studies revealed that Cdc25A/PKM2/ErbB2 pathway-regulated ferroptosis could serve as a therapeutic target in cervical cancer.
Topics: Animals; Autophagy; Carrier Proteins; Cell Line, Tumor; Female; Ferroptosis; Gene Expression Regulation, Neoplastic; Humans; Male; Membrane Proteins; Mice, Nude; Phosphorylation; RNA, Messenger; Receptor, ErbB-2; Signal Transduction; Sorafenib; Thyroid Hormones; Up-Regulation; Uterine Cervical Neoplasms; cdc25 Phosphatases; Thyroid Hormone-Binding Proteins; Mice
PubMed: 34743185
DOI: 10.1038/s41419-021-04342-y -
International Journal of Molecular... Nov 2021Mitogen-activated protein kinase (MAPK) signaling pathways are highly conserved regulators of eukaryotic cell function. These enzymes regulate many biological processes,... (Review)
Review
Mitogen-activated protein kinase (MAPK) signaling pathways are highly conserved regulators of eukaryotic cell function. These enzymes regulate many biological processes, including the cell cycle, apoptosis, differentiation, protein biosynthesis, and oncogenesis; therefore, tight control of the activity of MAPK is critical. Kinases and phosphatases are well established as MAPK activators and inhibitors, respectively. Kinases phosphorylate MAPKs, initiating and controlling the amplitude of the activation. In contrast, MAPK phosphatases (MKPs) dephosphorylate MAPKs, downregulating and controlling the duration of the signal. In addition, within the past decade, pseudoenzymes of these two families, pseudokinases and pseudophosphatases, have emerged as bona fide signaling regulators. This review discusses the role of pseudophosphatases in MAPK signaling, highlighting the function of phosphoserine/threonine/tyrosine-interacting protein (STYX) and TAK1-binding protein (TAB 1) in regulating MAPKs. Finally, a new paradigm is considered for this well-studied cellular pathway, and signal transduction pathways in general.
Topics: Humans; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Phosphatases; Phosphorylation
PubMed: 34830476
DOI: 10.3390/ijms222212595 -
International Journal of Molecular... Jul 2019Extensive research over several decades in plant light signaling mediated by photoreceptors has identified the molecular mechanisms for how phytochromes regulate... (Review)
Review
Extensive research over several decades in plant light signaling mediated by photoreceptors has identified the molecular mechanisms for how phytochromes regulate photomorphogenic development, which includes degradation of phytochrome-interacting factors (PIFs) and inactivation of COP1-SPA complexes with the accumulation of master transcription factors for photomorphogenesis, such as HY5. However, the initial biochemical mechanism for the function of phytochromes has not been fully elucidated. Plant phytochromes have long been known as phosphoproteins, and a few protein phosphatases that directly interact with and dephosphorylate phytochromes have been identified. However, there is no report thus far of a protein kinase that acts on phytochromes. On the other hand, plant phytochromes have been suggested as autophosphorylating serine/threonine protein kinases, proposing that the kinase activity might be important for their functions. Indeed, the autophosphorylation of phytochromes has been reported to play an important role in the regulation of plant light signaling. More recently, evidence that phytochromes function as protein kinases in plant light signaling has been provided using phytochrome mutants displaying reduced kinase activities. In this review, we highlight recent advances in the reversible phosphorylation of phytochromes and their functions as protein kinases in plant light signaling.
Topics: Enzyme Activation; Light Signal Transduction; Phosphorylation; Phytochrome; Plant Physiological Phenomena; Plant Proteins; Plants; Protein Binding; Protein Interaction Domains and Motifs; Protein Kinases
PubMed: 31337079
DOI: 10.3390/ijms20143450 -
The Biochemical Journal Mar 2021Adhesive structures between cells and with the surrounding matrix are essential for the development of multicellular organisms. In addition to providing mechanical... (Review)
Review
Adhesive structures between cells and with the surrounding matrix are essential for the development of multicellular organisms. In addition to providing mechanical integrity, they are key signalling centres providing feedback on the extracellular environment to the cell interior, and vice versa. During development, mitosis and repair, cell adhesions must undergo extensive remodelling. Post-translational modifications of proteins within these complexes serve as switches for activity. Tyrosine phosphorylation is an important modification in cell adhesion that is dynamically regulated by the protein tyrosine phosphatases (PTPs) and protein tyrosine kinases. Several PTPs are implicated in the assembly and maintenance of cell adhesions, however, their signalling functions remain poorly defined. The PTPs can act by directly dephosphorylating adhesive complex components or function as scaffolds. In this review, we will focus on human PTPs and discuss their individual roles in major adhesion complexes, as well as Hippo signalling. We have collated PTP interactome and cell adhesome datasets, which reveal extensive connections between PTPs and cell adhesions that are relatively unexplored. Finally, we reflect on the dysregulation of PTPs and cell adhesions in disease.
Topics: Animals; Cell Adhesion; Cell Adhesion Molecules; Humans; Protein Tyrosine Phosphatases
PubMed: 33710332
DOI: 10.1042/BCJ20200511 -
Cell Research Dec 2023Combination therapy with PD-1 blockade and IL-2 substantially improves anti-tumor efficacy comparing to monotherapy. The underlying mechanisms responsible for the...
Combination therapy with PD-1 blockade and IL-2 substantially improves anti-tumor efficacy comparing to monotherapy. The underlying mechanisms responsible for the synergistic effects of the combination therapy remain enigmatic. Here we show that PD-1 ligation results in BATF-dependent transcriptional induction of the membrane-associated E3 ubiquitin ligase MARCH5, which mediates K27-linked polyubiquitination and lysosomal degradation of the common cytokine receptor γ chain (γ). PD-1 ligation also activates SHP2, which dephosphorylates γ, leading to impairment of γ family cytokine-triggered signaling. Conversely, PD-1 blockade restores γ level and activity, thereby sensitizing CD8 T cells to IL-2. We also identified Pitavastatin Calcium as an inhibitor of MARCH5, which combined with PD-1 blockade and IL-2 significantly improves the efficacy of anti-tumor immunotherapy in mice. Our findings uncover the mechanisms by which PD-1 signaling antagonizes γ family cytokine-triggered immune activation and demonstrate that the underlying mechanisms can be exploited for increased efficacy of combination immunotherapy of cancer.
Topics: Animals; Mice; CD8-Positive T-Lymphocytes; Immune Checkpoint Inhibitors; Interleukin Receptor Common gamma Subunit; Interleukin-2; Neoplasms; Programmed Cell Death 1 Receptor; Ubiquitination; Ubiquitin-Protein Ligases; Mitochondrial Proteins; Membrane Proteins
PubMed: 37932447
DOI: 10.1038/s41422-023-00890-4 -
Molekuliarnaia Biologiia 2021Small SCP phosphatases CTDSP1, CTDSP2, and CTDSPL specifically dephosphorylate serine and threonine residues in protein molecules. The enzymes are involved in regulating... (Review)
Review
Small SCP phosphatases CTDSP1, CTDSP2, and CTDSPL specifically dephosphorylate serine and threonine residues in protein molecules. The enzymes are involved in regulating activity of RNA polymerase II at the transition from transcription initiation to elongation, regulating expression of neuron-specific genes, and activating the key cell-cycle protein pRb at the G1/S boundary. In addition, the substrates of SCP phosphatases include SMAD transcription modulators; AKT1 protein kinase, which regulates the cell cycle, apoptosis, and angiogenesis; the TWIST1 and c-MYC transcription factors; Ras family proteins, which are involved in signaling pathways regulating the cell growth and apoptosis; CDCA3, which is associated with cell division; the cyclin-dependent kinase inhibitor p21; and the promyelocytic leukemia protein (PML), which is involved in regulation of the tumor suppressors p53, PTEN, and mTOR. Dysfunction or inactivation of SCP phosphatases leads to various diseases, including cancer. Recently the increase in interest to SCP phosphatases is due to their their tumor growth-inhibiting properties or role in the development of malignant tumors of various etiology and localization. The review discusses the properties of SCP phosphatases and their role in oncogenesis. Understanding the functions of SCP phosphatases and their regulatory mechanisms can be useful in searching for efficient targets for tumor therapy.
Topics: Carcinogenesis; Cell Cycle; Cell Cycle Proteins; Cell Transformation, Neoplastic; Humans; Neoplasms; Phosphoprotein Phosphatases
PubMed: 34432772
DOI: 10.31857/S0026898421040091 -
Cell Reports May 2023The molecular and pathogenic mechanisms of esophageal squamous cell carcinoma (ESCC) development are still unclear, which hinders the development of effective...
The molecular and pathogenic mechanisms of esophageal squamous cell carcinoma (ESCC) development are still unclear, which hinders the development of effective treatments. In this study, we report that DUSP4 is highly expressed in human ESCC and is negatively correlated with patient prognosis. Knockdown of DUSP4 suppresses cell proliferation and patient-derived xenograft (PDX)-derived organoid (PDXO) growth and inhibits cell-derived xenograft (CDX) development. Mechanistically, DUSP4 directly binds to heat shock protein isoform β (HSP90β) and promotes the ATPase activity of HSP90β by dephosphorylating HSP90β on T214 and Y216. These dephosphorylation sites are critical for the stability of JAK1/2-STAT3 signaling and p-STAT3 (Y705) nucleus translocation. In vivo, Dusp4 knockout in mice significantly inhibits 4-nitrochinoline-oxide-induced esophageal tumorigenesis. Moreover, DUSP4 lentivirus or treatment with HSP90β inhibitor (NVP-BEP800) significantly impedes PDX tumor growth and inactivates the JAK1/2-STAT3 signaling pathway. These data provide insight into the role of the DUSP4-HSP90β-JAK1/2-STAT3 axis in ESCC progression and describe a strategy for ESCC treatment.
Topics: Animals; Humans; Mice; Cell Line, Tumor; Cell Proliferation; Dual-Specificity Phosphatases; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Gene Expression Regulation, Neoplastic; Heterografts; Mitogen-Activated Protein Kinase Phosphatases; Signal Transduction
PubMed: 37141098
DOI: 10.1016/j.celrep.2023.112445 -
The Journal of Neuroscience : the... Apr 2022Deactivation of G-protein-coupled receptors (GPCRs) involves multiple phosphorylations followed by arrestin binding, which uncouples the GPCR from G-protein activation....
Deactivation of G-protein-coupled receptors (GPCRs) involves multiple phosphorylations followed by arrestin binding, which uncouples the GPCR from G-protein activation. Some GPCRs, such as rhodopsin, are reused many times. Arrestin dissociation and GPCR dephosphorylation are key steps in the recycling process. evidence suggests that visual arrestin (ARR1) binding to light-activated, phosphorylated rhodopsin hinders dephosphorylation. Whether ARR1 binding also affects rhodopsin dephosphorylation is not known. We investigated this using both male and female mice lacking ARR1. Mice were exposed to bright light and placed in darkness for different periods of time, and differently phosphorylated species of rhodopsin were assayed by isoelectric focusing. For WT mice, rhodopsin dephosphorylation was nearly complete by 1 h in darkness. Surprisingly, we observed that, in the KO rods, rhodopsin remained phosphorylated even after 3 h. Delayed dephosphorylation in KO rods cannot be explained by cell stress induced by persistent signaling, since it is not prevented by the removal of transducin, the visual G-protein, nor can it be explained by downregulation of protein phosphatase 2A, the putative rhodopsin phosphatase. We further show that cone arrestin (ARR4), which binds light-activated, phosphorylated rhodopsin poorly, had little effect in enhancing rhodopsin dephosphorylation, whereas mice expressing binding-competent mutant ARR1-3A showed a similar time course of rhodopsin dephosphorylation as WT. Together, these results reveal a novel role of ARR1 in facilitating rhodopsin dephosphorylation G-protein-coupled receptors (GPCRs) are transmembrane proteins used by cells to receive and respond to a broad range of extracellular signals that include neurotransmitters, hormones, odorants, and light (photons). GPCR signaling is terminated by two sequential steps: phosphorylation and arrestin binding. Both steps must be reversed when GPCRs are recycled and reused. Dephosphorylation, which is required for recycling, is an understudied process. Using rhodopsin as a prototypical GPCR, we discovered that arrestin facilitated rhodopsin dephosphorylation in living mice.
Topics: Animals; Arrestin; Female; GTP-Binding Proteins; Male; Mice; Phosphorylation; Retinal Rod Photoreceptor Cells; Rhodopsin
PubMed: 35332081
DOI: 10.1523/JNEUROSCI.0141-22.2022 -
Frontiers in Immunology 2023NLRP3 is a prototypical sensor protein connecting cellular stress to pro-inflammatory signaling. A complex array of regulatory steps is required to switch NLRP3 from an... (Review)
Review
NLRP3 is a prototypical sensor protein connecting cellular stress to pro-inflammatory signaling. A complex array of regulatory steps is required to switch NLRP3 from an inactive state into a primed entity that is poised to assemble an inflammasome. Accumulating evidence suggests that post-translational mechanisms are critical. In particular, phosphorylation/dephosphorylation and ubiquitylation/deubiquitylation reactions have been reported to regulate NLRP3. Taken individually, several post-translational modifications appear to be essential. However, it remains difficult to understand how they may be coordinated, whether there is a unique sequence of regulatory steps accounting for the functional maturation of NLRP3, or whether the sequence is subject to variations depending on cell type, the stimulus, and other parameters such as the cellular context. This review will focus on the regulation of the NLRP3 inflammasome by phosphorylation and dephosphorylation, and on kinases and phosphatases that have been reported to modulate NLRP3 activity. The aim is to try to integrate the current understanding and highlight potential gaps for further studies.
Topics: Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Phosphorylation; Protein Processing, Post-Translational; Proteins
PubMed: 38022631
DOI: 10.3389/fimmu.2023.1281607