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International Journal of Molecular... Apr 2021More than 70% of eukaryotic proteins are regulated by phosphorylation. However, the mechanism of dephosphorylation that counteracts phosphorylation is less studied.... (Review)
Review
More than 70% of eukaryotic proteins are regulated by phosphorylation. However, the mechanism of dephosphorylation that counteracts phosphorylation is less studied. Phosphatases are classified into 104 distinct groups based on substrate-specific features and the sequence homologies in their catalytic domains. Among them, dual-specificity phosphatases (DUSPs) that dephosphorylate both phosphoserine/threonine and phosphotyrosine are important for cellular homeostasis. Ssu72 is a newly studied phosphatase with dual specificity that can dephosphorylate both phosphoserine/threonine and phosphotyrosine. It is important for cell-growth signaling, metabolism, and immune activation. Ssu72 was initially identified as a phosphatase for the Ser5 and Ser7 residues of the C-terminal domain of RNA polymerase II. It prefers the configuration of the serine-proline motif within its substrate and regulates Pin1, different from other phosphatases. It has recently been reported that Ssu72 can regulate sister chromatid cohesion and the separation of duplicated chromosomes during the cell cycle. Furthermore, Ssu72 appears to be involved in the regulation of T cell receptor signaling, telomere regulation, and even hepatocyte homeostasis in response to a variety of stress and damage signals. In this review, we aim to summarize various functions of the Ssu72 phosphatase, their implications in diseases, and potential therapeutic indications.
Topics: Animals; Chromatids; Chromosomes, Human; Humans; NIMA-Interacting Peptidylprolyl Isomerase; Phosphoprotein Phosphatases; Protein Domains; RNA Polymerase II; Receptors, Antigen, T-Cell; Signal Transduction
PubMed: 33917542
DOI: 10.3390/ijms22073791 -
Open Biology Jul 2023Mitotic exit requires the dephosphorylation of many proteins whose phosphorylation was needed for mitosis. Protein phosphatase 2A with its B55 regulatory subunit...
Mitotic exit requires the dephosphorylation of many proteins whose phosphorylation was needed for mitosis. Protein phosphatase 2A with its B55 regulatory subunit (PP2A-B55) promotes this transition. However, the events and substrates that it regulates are incompletely understood. We used proteomic approaches in to identify proteins that interact with and are dephosphorylated by PP2A-B55. Among several candidates, we identified emerin (otefin in ). Emerin resides in the inner nuclear membrane and interacts with the DNA-binding protein barrier-to-autointegration factor (BAF) via a LEM domain. We found that the phosphorylation of emerin at Ser50 and Ser54 near its LEM domain negatively regulates its association with BAF, lamin and additional emerin in mitosis. We show that dephosphorylation of emerin at these sites by PP2A-B55 determines the timing of nuclear envelope reformation. Genetic experiments indicate that this regulation is required during embryonic development. Phosphoregulation of the emerin-BAF complex formation by PP2A-B55 appears as a key event of mitotic exit that is likely conserved across species.
Topics: Animals; Drosophila; Nuclear Envelope; Protein Phosphatase 2; Proteomics; Mitosis
PubMed: 37463656
DOI: 10.1098/rsob.230104 -
The Biochemical Journal Jul 2015Activating mutations in the leucine rich repeat protein kinase 2 (LRRK2) gene are the most common cause of inherited Parkinson's disease (PD). LRRK2 is phosphorylated on...
Activating mutations in the leucine rich repeat protein kinase 2 (LRRK2) gene are the most common cause of inherited Parkinson's disease (PD). LRRK2 is phosphorylated on a cluster of phosphosites including Ser(910), Ser(935), Ser(955) and Ser(973), which are dephosphorylated in several PD-related LRRK2 mutants (N1437H, R1441C/G, Y1699C and I2020T) linking the regulation of these sites to PD. These serine residues are also dephosphorylated after kinase inhibition and lose 14-3-3 binding, which serves as a pharmacodynamic marker for inhibited LRRK2. Loss of 14-3-3 binding is well established, but the consequences of dephosphorylation are only now being uncovered. In the present study, we found that potent and selective inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(935) then ubiquitination and degradation of a significant fraction of LRRK2. GNE1023 treatment decreased the phosphorylation and stability of LRRK2 in expression systems and endogenous LRRK2 in A549 cells and in mouse dosing studies. We next established that LRRK2 is ubiquitinated through at least Lys(48) and Lys(63) ubiquitin linkages in response to inhibition. To investigate the link between dephosphorylation induced by inhibitor treatment and LRRK2 ubiquitination, we studied LRRK2 in conditions where it is dephosphorylated such as expression of PD mutants [R1441G, Y1699C and I2020T] or by blocking 14-3-3 binding to LRRK2 via difopein expression, and found LRRK2 is hyper-ubiquitinated. Calyculin A treatment prevents inhibitor and PD mutant induced dephosphorylation and reverts LRRK2 to a lesser ubiquitinated species, thus directly implicating phosphatase activity in LRRK2 ubiquitination. This dynamic dephosphorylation-ubiquitination cycle could explain detrimental loss-of-function phenotypes found in peripheral tissues of LRRK2 kinase inactive mutants, LRRK2 KO (knockout) animals and following LRRK2 inhibitor administration.
Topics: 14-3-3 Proteins; Amino Acid Substitution; Animals; Enzyme Inhibitors; HEK293 Cells; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Marine Toxins; Mice; Mutation, Missense; Oxazoles; Phosphorylation; Protein Serine-Threonine Kinases; Ubiquitination
PubMed: 25939886
DOI: 10.1042/BJ20141305 -
Open Biology May 2017Ubiquitin-like domain-containing C-terminal domain phosphatase 1 (UBLCP1), an FCP/SCP phosphatase family member, was identified as the first proteasome phosphatase....
Ubiquitin-like domain-containing C-terminal domain phosphatase 1 (UBLCP1), an FCP/SCP phosphatase family member, was identified as the first proteasome phosphatase. UBLCP1 binds to proteasome subunit Rpn1 and dephosphorylates the proteasome However, it is still unclear which proteasome subunit(s) are the substrate(s) of UBLCP1 and the precise mechanism for proteasome regulation remains elusive. Here, we show that UBLCP1 selectively binds to the 19S regulatory particle (RP) through its interaction with Rpn1, but not the 20S core particle (CP) or the 26S proteasome holoenzyme. In the RP, UBLCP1 dephosphorylates the subunit Rpt1, impairs its ATPase activity, and consequently disrupts the 26S proteasome assembly, yet it has no effects on the RP assembly from precursor complexes. The Rpn1-binding and phosphatase activities of UBLCP1 are essential for its function on Rpt1 dephosphorylation and proteasome activity both and Our study establishes the essential role of the UBLCP1/Rpn1/Rpt1 complex in regulating proteasome assembly.
Topics: Adenosine Triphosphatases; HEK293 Cells; HeLa Cells; Humans; Nuclear Proteins; Phosphoprotein Phosphatases; Phosphorylation; Proteasome Endopeptidase Complex; Protein Binding; Protein Subunits
PubMed: 28539385
DOI: 10.1098/rsob.170042 -
Cancer Medicine Aug 2016Dual-specificity phosphatase-1 (DUSP1/MKP1), as a member of the threonine-tyrosine dual-specificity phosphatase family, was first found in cultured murine cells. The... (Review)
Review
Dual-specificity phosphatase-1 (DUSP1/MKP1), as a member of the threonine-tyrosine dual-specificity phosphatase family, was first found in cultured murine cells. The molecular mechanisms of DUSP1-mediated extracellular signal-regulated protein kinases (ERKs) dephosphorylation have been subsequently identified by studies using gene knockout mice and gene silencing technology. As a protein phosphatase, DUSP1 also downregulates p38 MAPKs and JNKs signaling through directly dephosphorylating threonine and tyrosine. It has been detected that DUSP1 is involved in various functions, including proliferation, differentiation, and apoptosis in normal cells. In various human cancers, abnormal expression of DUSP1 was observed which was associated with prognosis of tumor patients. Further studies have revealed its role in tumorigenesis and tumor progression. Besides, DUSP1 has been found to play a role in tumor chemotherapy, immunotherapy, and biotherapy. In this review, we will focus on the function and mechanism of DUSP1 in tumor cells and tumor treatment.
Topics: Animals; Cell Transformation, Neoplastic; Combined Modality Therapy; Disease Progression; Dual Specificity Phosphatase 1; Gene Expression Regulation, Neoplastic; Humans; Immunotherapy; Molecular Targeted Therapy; Neoplasms; Signal Transduction
PubMed: 27227569
DOI: 10.1002/cam4.772 -
Clinical and Experimental Nephrology Oct 2019Potassium (K) intake is intrinsically linked to blood pressure. High-K intake decreases hypertension and associated lower mortality. On the other hand, hyperkalemia... (Review)
Review
INTRODUCTION
Potassium (K) intake is intrinsically linked to blood pressure. High-K intake decreases hypertension and associated lower mortality. On the other hand, hyperkalemia causes sudden death with fatal cardiac arrhythmia and is also related to higher mortality. Renal sodium (Na)-chloride (Cl) cotransporter (NCC), expressed in the distal convoluted tubule, is a key molecule in regulating urinary K excretion. K intake affects the activity of the NCC, which is related to salt-sensitive hypertension. A K-restrictive diet activates NCC, and K loading suppresses NCC. Hyperpolarization caused by decreased extracellular K concentration ([K]) increases K and Cl efflux, leading to the activation of Cl-sensitive with-no-lysine (WNK) kinases and their downstream molecules, including STE20/SPS1-related proline/alanine-rich kinase (SPAK) and NCC.
RESULTS
We investigated the role of the ClC-K2 Cl channel and its β-subunit, barttin, using barttin hypomorphic (Bsnd) mice and found that these mice did not show low-K-induced NCC activation and salt-sensitive hypertension. Additionally, we discovered that the suppression of NCC by K loading was regulated by another mechanism, whereby tacrolimus (a calcineurin [CaN] inhibitor) inhibited high-K-induced NCC dephosphorylation and urinary K excretion. The K loading and the tacrolimus treatment did not alter the expression of WNK4 and SPAK. The depolarization induced by increased [K] activated CaN, which dephosphorylates NCC.
CONCLUSIONS
We concluded that there were two independent molecular mechanisms controlling NCC activation and K excretion. This review summarizes the clinical importance of K intake and explains how NCC phosphorylation is regulated by different molecular mechanisms between the low- and the high-K condition.
Topics: Animals; Blood Pressure; Humans; Potassium; Potassium, Dietary; Sodium-Potassium-Chloride Symporters
PubMed: 31317362
DOI: 10.1007/s10157-019-01766-x -
Cell Communication and Signaling : CCS Jun 2021Invadopodia are actin-based cell-membrane protrusions associated with the extracellular matrix degradation accompanying cancer invasion. The elucidation of the molecular...
BACKGROUND
Invadopodia are actin-based cell-membrane protrusions associated with the extracellular matrix degradation accompanying cancer invasion. The elucidation of the molecular mechanisms leading to invadopodia formation and activity is central for the prevention of tumor spreading and growth. Protein tyrosine kinases such as Src are known to regulate invadopodia assembly, little is however known on the role of protein tyrosine phosphatases in this process. Among these enzymes, we have selected the tyrosine phosphatase Shp1 to investigate its potential role in invadopodia assembly, due to its involvement in cancer development.
METHODS
Co-immunoprecipitation and immunofluorescence studies were employed to identify novel substrate/s of Shp1AQ controlling invadopodia activity. The phosphorylation level of cortactin, the Shp1 substrate identified in this study, was assessed by immunoprecipitation, in vitro phosphatase and western blot assays. Short interference RNA and a catalytically-dead mutant of Shp1 expressed in A375MM melanoma cells were used to evaluate the role of the specific Shp1-mediated dephosphorylation of cortactin. The anti-invasive proprieties of glycerophosphoinositol, that directly binds and regulates Shp1, were investigated by extracellular matrix degradation assays and in vivo mouse model of metastasis.
RESULTS
The data show that Shp1 was recruited to invadopodia and promoted the dephosphorylation of cortactin at tyrosine 421, leading to an attenuated capacity of melanoma cancer cells to degrade the extracellular matrix. Controls included the use of short interference RNA and catalytically-dead mutant that prevented the dephosphorylation of cortactin and hence the decrease the extracellular matrix degradation by melanoma cells. In addition, the phosphoinositide metabolite glycerophosphoinositol facilitated the localization of Shp1 at invadopodia hence promoting cortactin dephosphorylation. This impaired invadopodia function and tumor dissemination both in vitro and in an in vivo model of melanomas.
CONCLUSION
The main finding here reported is that cortactin is a specific substrate of the tyrosine phosphatase Shp1 and that its phosphorylation/dephosphorylation affects invadopodia formation and, as a consequence, the ability of melanoma cells to invade the extracellular matrix. Shp1 can thus be considered as a regulator of melanoma cell invasiveness and a potential target for antimetastatic drugs. Video abstract.
Topics: Animals; Cell Line, Tumor; Cortactin; Extracellular Matrix; Humans; Inositol Phosphates; Lung Neoplasms; Melanoma; Mice, Inbred BALB C; Mice, Nude; Models, Biological; Neoplasm Invasiveness; Phosphorylation; Protein Binding; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Pseudopodia; Substrate Specificity; Mice
PubMed: 34088320
DOI: 10.1186/s12964-021-00747-6 -
International Journal of Biological... 2023Numerous mitochondrial abnormalities are reported to result from excessive inflammation during endotoxemia. Prohibitin 2 (PHB2) and phosphoglycerate mutase 5 (Pgam5)...
Numerous mitochondrial abnormalities are reported to result from excessive inflammation during endotoxemia. Prohibitin 2 (PHB2) and phosphoglycerate mutase 5 (Pgam5) have been associated with altered mitochondrial homeostasis in several cardiovascular diseases; however, their role in endotoxemia-related myocardial dysfunction has not been explored. Our experiments were aimed to evaluate the potential contribution of Pgam5 and PHB2 to endotoxemia-induced mitochondrial dysfunction in cardiomyocytes, with a focus on two endogenous protective programs that sustain mitochondrial integrity, namely mitophagy and the mitochondrial unfolded protein response (UPR). We found that PHB2 transgenic mice are resistant to endotoxemia-mediated myocardial depression and mitochondrial damage. Our assays indicated that PHB2 overexpression activates mitophagy and the UPR, which maintains mitochondrial metabolism, prevents oxidative stress injury, and enhances cardiomyocyte viability. Molecular analyses further showed that Pgam5 binds to and dephosphorylates PHB2, resulting in cytosolic translocation of mitochondrial PHB2. Silencing of Pgam5 or transfection of a phosphorylated PHB2 mutant in mouse HL-1 cardiomyocytes prevented the loss of mitochondrially-localized PHB2 and activated mitophagy and UPR in the presence of LPS. Notably, cardiomyocyte-specific deletion of Pgam5 attenuated LPS-mediated myocardial dysfunction and preserved cardiomyocyte viability. These findings suggest that Pgam5/PHB2 signaling and mitophagy/UPR are potential targets for the treatment of endotoxemia-related cardiac dysfunction.
Topics: Animals; Mice; Endotoxemia; Lipopolysaccharides; Mitophagy; Phosphoprotein Phosphatases; Prohibitins; Unfolded Protein Response
PubMed: 37781037
DOI: 10.7150/ijbs.85767 -
The FEBS Journal Jul 2016Glucan phosphatases are a recently discovered class of enzymes that dephosphorylate starch and glycogen, thereby regulating energy metabolism. Plant genomes encode two... (Review)
Review
Glucan phosphatases are a recently discovered class of enzymes that dephosphorylate starch and glycogen, thereby regulating energy metabolism. Plant genomes encode two glucan phosphatases, called Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), that regulate starch metabolism by selectively dephosphorylating glucose moieties within starch glucan chains. Recently, the structures of both SEX4 and LSF2 were determined, with and without phosphoglucan products bound, revealing the mechanism for their unique activities. This review explores the structural and enzymatic features of the plant glucan phosphatases, and outlines how they are uniquely adapted to perform their cellular functions. We outline the physical mechanisms used by SEX4 and LSF2 to interact with starch glucans: SEX4 binds glucan chains via a continuous glucan-binding platform comprising its dual-specificity phosphatase domain and carbohydrate-binding module, while LSF2 utilizes surface binding sites. SEX4 and LSF2 both contain a unique network of aromatic residues in their catalytic dual-specificity phosphatase domains that serve as glucan engagement platforms and are unique to the glucan phosphatases. We also discuss the phosphoglucan substrate specificities inherent to SEX4 and LSF2, and outline structural features within the active site that govern glucan orientation. This review defines the structural mechanism of the plant glucan phosphatases with respect to phosphatases, starch metabolism and protein-glucan interaction, thereby providing a framework for their application in both agricultural and industrial settings.
Topics: Arabidopsis Proteins; Dual-Specificity Phosphatases; Glucans; Plant Proteins; Protein Binding; Starch
PubMed: 26934589
DOI: 10.1111/febs.13703 -
Cell Apr 2020Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class...
Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates.
Topics: Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Enzyme Activators; G1 Phase; Humans; Multiprotein Complexes; Phenothiazines; Phosphorylation; Protein Phosphatase 2; Protein Subunits; Trans-Activators; Transcription Factors
PubMed: 32315619
DOI: 10.1016/j.cell.2020.03.051