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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 -
Cold Spring Harbor Perspectives in... Apr 2020Germline pathogenic phosphatase and tensin homolog () mutations cause hamartoma tumor syndrome (PHTS), characterized by various benign and malignant tumors of the... (Review)
Review
Germline pathogenic phosphatase and tensin homolog () mutations cause hamartoma tumor syndrome (PHTS), characterized by various benign and malignant tumors of the thyroid, breast, endometrium, and other organs. Patients with PHTS may present with other clinical features such as macrocephaly, intestinal polyposis, cognitive changes, and pathognomonic skin changes. Clinically, deregulation of PTEN function is implicated in other human diseases in addition to many types of human cancer. PTEN is an important phosphatase that counteracts one of the most critical cancer pathways: the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathways. Although PTEN can dephosphorylate lipids and proteins, it also has functions independent of phosphatase activity in normal and pathological states. It is positively and negatively regulated at the transcriptional level as well as posttranslationally by phosphorylation, ubiquitylation, oxidation, and acetylation. Although most of its tumor-suppressor activity is likely to be caused by lipid dephosphorylation at the plasma membrane, PTEN also resides in the cytoplasm and nucleus, and its subcellular distribution is under strict control. In this review, we highlight our current knowledge of PTEN function and recent discoveries in understanding PTEN function regulation and how this can be exploited therapeutically for cancer treatment.
Topics: Animals; Disease Management; Genetic Predisposition to Disease; Germ-Line Mutation; Hamartoma Syndrome, Multiple; Humans; Neoplasms; PTEN Phosphohydrolase; Phosphatidylinositol 3-Kinases; Signal Transduction
PubMed: 31570378
DOI: 10.1101/cshperspect.a036087 -
Molecular Cell Dec 2022Chai et al. reveal that the eukaryotic-like effector protein PtpB from Mycobacterium tuberculosis (MTB) dephosphorylates phospholipid membrane proteins, which prevents...
Chai et al. reveal that the eukaryotic-like effector protein PtpB from Mycobacterium tuberculosis (MTB) dephosphorylates phospholipid membrane proteins, which prevents membrane localization of cleaved gasdermin D, inhibiting pyroptosis and cytokine release by infected macrophages to enable MTB immune evasion.
Topics: Mycobacterium tuberculosis; Pyroptosis; Ubiquitin; Macrophages; Cytokines
PubMed: 36525954
DOI: 10.1016/j.molcel.2022.11.020 -
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 -
A noncanonical function of SKP1 regulates the switch between autophagy and unconventional secretion.Science Advances Oct 2023Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides...
Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient-poor conditions, SKP1 is phosphorylated on Thr, allowing its interaction with V subunits of the vacuolar ATPase (V-ATPase). This event, in turn, promotes V-ATPase assembly to acidify late endosomes and enhance endolysosomal degradation. Under nutrient-rich conditions, SUMOylation of phosphorylated SKP1 allows its binding to and dephosphorylation by the PPM1B phosphatase. Dephosphorylated SKP1 interacts with SEC22B to promote unconventional secretion of the content of less acidified hybrid endosomal/autophagic compartments. Collectively, our study implicates SKP1 phosphorylation as a switch between autophagy and unconventional secretion in a manner dependent on cellular nutrient status.
Topics: Autophagy; Cell Membrane; Endosomes; Lysosomes; Vacuolar Proton-Translocating ATPases; Humans
PubMed: 37831778
DOI: 10.1126/sciadv.adh1134 -
The Journal of Clinical Investigation Nov 2023Consumption of low dietary potassium, common with ultraprocessed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the with no (K) lysine...
Consumption of low dietary potassium, common with ultraprocessed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the with no (K) lysine kinase/STE20/SPS1-related proline-alanine-rich protein kinase (WNK/SPAK) pathway to induce salt retention and elevate blood pressure (BP). However, it remains unclear how high-potassium "DASH-like" diets (dietary approaches to stop hypertension) inactivate the cotransporter and whether this decreases BP. A transcriptomics screen identified Ppp1Ca, encoding PP1A, as a potassium-upregulated gene, and its negative regulator Ppp1r1a, as a potassium-suppressed gene in the kidney. PP1A directly binds to and dephosphorylates NCC when extracellular potassium is elevated. Using mice genetically engineered to constitutively activate the NCC-regulatory kinase SPAK and thereby eliminate the effects of the WNK/SPAK kinase cascade, we confirmed that PP1A dephosphorylated NCC directly in a potassium-regulated manner. Prior adaptation to a high-potassium diet was required to maximally dephosphorylate NCC and lower BP in constitutively active SPAK mice, and this was associated with potassium-dependent suppression of Ppp1r1a and dephosphorylation of its cognate protein, inhibitory subunit 1 (I1). In conclusion, potassium-dependent activation of PP1A and inhibition of I1 drove NCC dephosphorylation, providing a mechanism to explain how high dietary K+ lowers BP. Shifting signaling of PP1A in favor of activation of WNK/SPAK may provide an improved therapeutic approach for treating salt-sensitive hypertension.
Topics: Animals; Mice; Blood Pressure; Solute Carrier Family 12, Member 3; Protein Serine-Threonine Kinases; Sodium Chloride; Potassium, Dietary; Kidney; Hypertension; Potassium; Phosphorylation
PubMed: 37676724
DOI: 10.1172/JCI158498 -
Antioxidants (Basel, Switzerland) Feb 2021Phosphatase and tensin homolog deleted on chromosome 10 () is known as a tumor suppressor gene that is frequently mutated in numerous human cancers and inherited... (Review)
Review
Phosphatase and tensin homolog deleted on chromosome 10 () is known as a tumor suppressor gene that is frequently mutated in numerous human cancers and inherited syndromes. PTEN functions as a negative regulator of PI3K/Akt signaling pathway by dephosphorylating phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3) to phosphatidylinositol (4, 5)-bisphosphate (PIP2), which leads to the inhibition of cell growth, proliferation, cell survival, and protein synthesis. PTEN contains a cysteine residue in the active site that can be oxidized by peroxides, forming an intramolecular disulfide bond between Cys and Cys. Redox regulation of PTEN by reactive oxygen species (ROS) plays a crucial role in cellular signaling. Peroxiredoxins (Prxs) are a superfamily of peroxidase that catalyzes reduction of peroxides and maintains redox homeostasis. Mammalian Prxs have 6 isoforms (I-VI) and can scavenge cellular peroxides. It has been demonstrated that Prx I can preserve and promote the tumor-suppressive function of PTEN by preventing oxidation of PTEN under benign oxidative stress via direct interaction. Also, Prx II-deficient cells increased PTEN oxidation and insulin sensitivity. Furthermore, Prx III has been shown to protect PTEN from oxidation induced by 15s-HpETE and 12s-HpETE, these are potent inflammatory and pro-oxidant mediators. Understanding the tight connection between PTEN and Prxs is important for providing novel therapies. Herein, we summarized recent studies focusing on the relationship of Prxs and the redox regulation of PTEN.
PubMed: 33669370
DOI: 10.3390/antiox10020302 -
Nature Communications Aug 2022The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation, supporting anabolic reactions and...
The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation, supporting anabolic reactions and inhibiting catabolic pathways like autophagy. Its hyperactivation is a frequent event in cancer promoting tumor cell proliferation. Several intracellular membrane-associated mTORC1 pools have been identified, linking its function to distinct subcellular localizations. Here, we characterize the N-terminal kinase-like protein SCYL1 as a Golgi-localized target through which mTORC1 controls organelle distribution and extracellular vesicle secretion in breast cancer cells. Under growth conditions, SCYL1 is phosphorylated by mTORC1 on Ser754, supporting Golgi localization. Upon mTORC1 inhibition, Ser754 dephosphorylation leads to SCYL1 displacement to endosomes. Peripheral, dephosphorylated SCYL1 causes Golgi enlargement, redistribution of early and late endosomes and increased extracellular vesicle release. Thus, the mTORC1-controlled phosphorylation status of SCYL1 is an important determinant regulating subcellular distribution and function of endolysosomal compartments. It may also explain the pathophysiology underlying human genetic diseases such as CALFAN syndrome, which is caused by loss-of-function of SCYL1.
Topics: Adaptor Proteins, Vesicular Transport; DNA-Binding Proteins; Golgi Apparatus; Humans; Intracellular Membranes; Lysosomes; Mechanistic Target of Rapamycin Complex 1; Phosphorylation
PubMed: 35948564
DOI: 10.1038/s41467-022-32487-7 -
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 Journal of Clinical Investigation Nov 2023Dual-specificity phosphatase 8 (DUSP8) is a MAPK phosphatase that dephosphorylates and inactivates the kinase JNK. DUSP8 is highly expressed in T cells; however, the in...
Dual-specificity phosphatase 8 (DUSP8) is a MAPK phosphatase that dephosphorylates and inactivates the kinase JNK. DUSP8 is highly expressed in T cells; however, the in vivo role of DUSP8 in T cells remains unclear. Using T cell-specific Dusp8 conditional KO (T-Dusp8 cKO) mice, mass spectrometry analysis, ChIP-Seq, and immune analysis, we found that DUSP8 interacted with Pur-α, stimulated interleukin-9 (IL-9) gene expression, and promoted Th9 differentiation. Mechanistically, DUSP8 dephosphorylated the transcriptional repressor Pur-α upon TGF-β signaling, leading to the nuclear export of Pur-α and subsequent IL-9 transcriptional activation. Furthermore, Il-9 mRNA levels were induced in Pur-α-deficient T cells. In addition, T-Dusp8-cKO mice displayed reduction of IL-9 and Th9-mediated immune responses in the allergic asthma model. Reduction of Il-9 mRNA levels in T cells and allergic responses of T-Dusp8-cKO mice was reversed by Pur-α knockout. Remarkably, DUSP8 protein levels and the DUSP8-Pur-α interaction were indeed increased in the cytoplasm of T cells from people with asthma and patients with atopic dermatitis. Collectively, DUSP8 induces TGF-β-stimulated IL-9 transcription and Th9-induced allergic responses by inhibiting the nuclear translocation of the transcriptional repressor Pur-α. DUSP8 may be a T-cell biomarker and therapeutic target for asthma and atopic dermatitis.
Topics: Animals; Humans; Mice; Active Transport, Cell Nucleus; Asthma; Dermatitis, Atopic; Dual-Specificity Phosphatases; Hypersensitivity; Inflammation; Interleukin-9; RNA, Messenger; Transcription Factors; Transforming Growth Factor beta
PubMed: 37909329
DOI: 10.1172/JCI166269