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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 -
Advances in Protein Chemistry and... 2022Being phosphopeptide-binding hubs, 14-3-3 proteins coordinate multiple cellular processes in eukaryotes, including the regulation of apoptosis, cell cycle, ion channels... (Review)
Review
Being phosphopeptide-binding hubs, 14-3-3 proteins coordinate multiple cellular processes in eukaryotes, including the regulation of apoptosis, cell cycle, ion channels trafficking, transcription, signal transduction, and hormone biosynthesis. Forming constitutive α-helical dimers, 14-3-3 proteins predominantly recognize specifically phosphorylated Ser/Thr sites within their partners; this generally stabilizes phosphotarget conformation and affects its activity, intracellular distribution, dephosphorylation, degradation and interactions with other proteins. Not surprisingly, 14-3-3 complexes are involved in the development of a range of diseases and are considered promising drug targets. The wide interactome of 14-3-3 proteins encompasses hundreds of different phosphoproteins, for many of which the interaction is well-documented in vitro and in vivo but lack the structural data that would help better understand underlying regulatory mechanisms and develop new drugs. Despite obtaining structural information on 14-3-3 complexes is still lagging behind the research of 14-3-3 interactions on a proteome-wide scale, recent works provided some advances, including methodological improvements and accumulation of new interesting structural data, that are discussed in this review.
Topics: 14-3-3 Proteins; Phosphoproteins; Phosphorylation; Protein Conformation, alpha-Helical; Proteome
PubMed: 35534110
DOI: 10.1016/bs.apcsb.2021.12.004 -
Acta Biochimica Polonica Feb 2022Specific phosphorylation/dephosphorylation processes are fundamental for the transduction of external stimuli into physiological responses. A few of these processes...
Light-stimulated dephosphorylation of the BiP-like protein, SmicHSP75 (SBiP1) from Symbiodinium microadriaticum is inhibited by elevated but not low temperature and suggests regulation of the chaperone function.
Specific phosphorylation/dephosphorylation processes are fundamental for the transduction of external stimuli into physiological responses. A few of these processes appear to be modulated by light in cultured Symbiodinium microadriaticum since the BiP-like protein SmicHSP75 undergoes threonine dephosphorylation upon light stimuli. Several isoforms of the protein are encoded in the S. microadriaticum genome and thus, we identified and heterologously expressed a specific sequence corresponding to the previously identified SmicHSP75 isoform to obtain a highly specific antibody. We then determined by western blot analysis, that the detected light-stimulated changes in SmicHSP75 threonine phosphorylation were not due to changes in the protein expression and explored further the effect of lower than normal and higher stressful temperature, on the phosphorylation levels of the protein. Upon long-term (12 h) exposure of the cells to the low temperature of 21ºC under darkness, the protein was found significantly phosphorylated; however, light exposure for 30 min caused a dephosphorylation effect like the 26ºC control treatment. On the other hand, in cells exposed to 32ºC for 12 h under darkness, the highly Thr-phosphorylated SmicHSP75 was converted to a low-level phosphorylated protein. Likewise, short term (30 min) exposure to 32ºC under dark conditions caused dephosphorylation of the protein, similar to what was observed upon long-term exposure to 32ºC and upon light stimulation of cells under the normal temperature of 26ºC. These data suggested activation/inactivation of the chaperone function of SmicHSP75 by regulation of its Thr phosphorylation levels under heat stress conditions in Symbiodinium microadriaticum, independent of changes in protein expression.
Topics: Animals; Cold Temperature; Darkness; Dinoflagellida; Heat-Shock Proteins; Hot Temperature; Light; Molecular Chaperones; Phosphorylation; Temperature; Threonine
PubMed: 35148474
DOI: 10.18388/abp.2020_5788 -
Cellular Signalling Sep 2021Protein phosphatase 1(PP1) is a key regulator of cardiac function through dephosphorylating serine/threonine residues within target proteins to oppose the function of... (Review)
Review
Protein phosphatase 1(PP1) is a key regulator of cardiac function through dephosphorylating serine/threonine residues within target proteins to oppose the function of protein kinases. Studies from failing hearts of animal models and human patients have demonstrated significant increase of PP1 activity in myocardium, while elevated PP1 activity in transgenic mice leads to cardiac dysfunction, suggesting that PP1 might be a therapeutic target to ameliorate cardiac dysfunction in failing hearts. In fact, cardiac overexpression of inhibitor 1, the endogenous inhibitor of PP1, increases cardiac contractility and suppresses heart failure progression. However, this notion of PP1 inhibition for heart failure treatment has been challenged by recent studies on the isoform-specific roles of PP1 in the heart. PP1 is a holoenzyme composed of catalytic subunits (PP1α, PP1β, or PP1γ) and regulatory proteins that target them to distinct subcellular locations for functional specificity. This review will summarize how PP1 regulates phosphorylation of some of the key cardiac proteins involved in Ca handling and cardiac contraction, and the potential role of PP1 isoforms in controlling cardiac physiology and pathophysiology.
Topics: Animals; Heart; Humans; Mice; Myocardial Contraction; Myocardium; Phosphorylation; Protein Isoforms; Protein Phosphatase 1
PubMed: 34062239
DOI: 10.1016/j.cellsig.2021.110059 -
PeerJ 2023Golgin subfamily A member 3 (), a member of the golgin subfamily A, is highly expressed in mouse testis. The GOLGA3 protein, which contains eight phosphorylation sites,...
BACKGROUND
Golgin subfamily A member 3 (), a member of the golgin subfamily A, is highly expressed in mouse testis. The GOLGA3 protein, which contains eight phosphorylation sites, is involved in protein transport, cell apoptosis, Golgi localization, and spermatogenesis. Although it has been previously reported that nonsense mutations in cause multiple defects in spermatogenesis, the role of in the testis is yet to be clarified.
METHODS
Immunofluorescence co-localization in cells and protein dephosphorylation experiments were performed. mice were generated using cytosine base editors. Fertility tests as well as computer-assisted sperm analysis (CASA) were then performed to investigate sperm motility within caudal epididymis. Histological and immunofluorescence staining were used to analyze testis and epididymis phenotypes and TUNEL assays were used to measure germ cell apoptosis in spermatogenic tubules.
RESULTS
Immunofluorescence co-localization showed reduced Golgi localization of GOLGA3 with some protein scattered in the cytoplasm of HeLa cells .In addition, protein dephosphorylation experiments indicated a reduced band shift of the dephosphorylated GOLGA3, confirming S461 as the phosphorylation site. is an evolutionarily conserved gene and mice were successfully generated using cytosine base editors. These mice had normal fertility and spermatozoa, and did not differ significantly from wild-type mice in terms of spermatogenesis and apoptotic cells in tubules.
CONCLUSIONS
was found to be highly conserved in the testis, and GOLGA3 was shown to be involved in spermatogenesis, especially in apoptosis and Golgi complex-mediated effects. Infertility was also observed in KO male mice. Although GOLGA3showed reduced localization in the Golgi with some expression in the cytoplasm, this abnormal localization did not adversely affect fertility or spermatogenesis in male C57BL/6 mice. Therefore, mutation of the S461 GOLGA3 phosphorylation site did not affect mouse spermatogenesis.
Topics: Animals; Humans; Male; Mice; Golgi Matrix Proteins; HeLa Cells; Mice, Inbred C57BL; Mutation; Phosphorylation; Proteins; Semen; Sperm Motility; Spermatogenesis
PubMed: 37090114
DOI: 10.7717/peerj.15133 -
The Journal of Biological Chemistry Nov 2021Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome...
Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome assembly, alternative splicing, and mRNA export. Notably, serine-/arginine- (SR)-rich RBPs are densely phosphorylated compared with the remainder of the proteome. Previously, we showed that dephosphorylation of the splicing factor SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. However, the large-scale functional and structural impact of these modifications on RBPs remains unclear. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass-spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. We determined that phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high-molecular-weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine/arginine protein kinase 2 (SRPK2) in vitro decreased high-molecular-weight SRSF2 species formation. Furthermore, upon pharmacological inhibition of SRPKs in mammalian cells, we observed SRSF2 cytoplasmic mislocalization and increased formation of cytoplasmic granules as well as cytoplasmic tubular structures that associated with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.
Topics: HEK293 Cells; Humans; Nuclear Proteins; Phosphorylation; Protein Multimerization; Protein Serine-Threonine Kinases; Protein Stability; Ribonucleoprotein, U1 Small Nuclear; Serine-Arginine Splicing Factors
PubMed: 34673031
DOI: 10.1016/j.jbc.2021.101306 -
Methods in Molecular Biology (Clifton,... 2024Protein tyrosine phosphorylation and dephosphorylation are key regulatory mechanisms in eukaryotes. Protein tyrosine phosphorylation and dephosphorylation are catalyzed...
Protein tyrosine phosphorylation and dephosphorylation are key regulatory mechanisms in eukaryotes. Protein tyrosine phosphorylation and dephosphorylation are catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), respectively. The combinatorial action of both PTKs and PTPs is essential for properly maintaining cellular functions. In this unit, we discuss different novel methods to identify PTP substrates. PTPs depend on specific invariant residues that enable binding to tyrosine-phosphorylated substrates and aid catalytic activity. Identifying PTP substrates has paved the way to understanding their role in distinct intracellular signaling pathways. Due to their high specific activity, the interaction between PTPs and their substrates is transient; therefore, identifying the physiological substrates of PTPs has been challenging. To identify the physiological substrates of PTPs, various PTP mutants have been generated. These PTP mutants, named "substrate-trapping mutants," lack catalytic activity but bind tightly to their tyrosine-phosphorylated substrates. Identifying the substrates for the PTPs will provide critical insight into the function of physiological and pathophysiological signal transduction. In this chapter, we describe interaction assays used to identify the PTP substrates.
Topics: Protein Tyrosine Phosphatases; Signal Transduction; Phosphorylation; Protein-Tyrosine Kinases; Tyrosine
PubMed: 38147212
DOI: 10.1007/978-1-0716-3569-8_8 -
Frontiers in Immunology 2023Macrophages play a critical role in the regulation of inflammation and tissue homeostasis. In addition to their vital functions for cell survival and physiology,...
Macrophages play a critical role in the regulation of inflammation and tissue homeostasis. In addition to their vital functions for cell survival and physiology, mitochondria play a crucial role in innate immunity as a platform for the induction of inflammatory responses by regulating cell signaling and dynamics. Dynamin-related protein 1 (Drp1) plays a role in the induction of inflammatory responses and the subsequent development of various diseases. PGAM5 (phosphoglycerate mutase member 5) is a mitochondrial outer membrane phosphatase that dephosphorylates its substrate, Drp1. Previous studies showed that PGAM5 regulates the phosphorylation of Drp1 for the activation of NKT cells and T cells. However, it is not clear how PGAM5 regulates Drp1 activity for the induction of inflammation in macrophages. Here, we demonstrate that PGAM5 activity regulates the dephosphorylation of Drp1 in macrophages, leading to the induction of proinflammatory responses in macrophages. In TLR signaling, PGAM5 regulates the expression and production of inflammatory cytokines by regulating the activation of downstream signaling pathways, including the NF-κB and MAPK pathways. Upon LPS stimulation, PGAM5 interacts with Drp1 to form a complex, leading to the production of mtROS. Furthermore, PGAM5-Drp1 signaling promotes the polarization of macrophages toward a proinflammatory phenotype. Our study further demonstrates that PGAM5-Drp1 signaling promotes metabolic reprogramming by upregulating glycolysis and mitochondrial metabolism in macrophages. Altogether, PGAM5 signaling is a linker between alterations in Drp1-mediated mitochondrial dynamics and inflammatory responses in macrophages and may be a target for the treatment of inflammatory diseases.
Topics: Humans; Dynamins; Inflammation; Macrophages; Mitochondrial Proteins; Phosphoprotein Phosphatases; Signal Transduction; Animals
PubMed: 37771598
DOI: 10.3389/fimmu.2023.1243548 -
Frontiers in Molecular Biosciences 2020Several variant proteins are produced from , including two representative proteins produced via alternative splicing machinery. One protein is the canonical translation...
Several variant proteins are produced from , including two representative proteins produced via alternative splicing machinery. One protein is the canonical translation eukaryotic elongation factor eEF1Bδ1, and the other is the heat shock-responsive transcription factor eEF1BδL. eEF1Bδ1 is phosphorylated by cyclin-dependent kinase 1 (CDK1), but the machinery controlling eEF1BδL phosphorylation and dephosphorylation has not been clarified. In this study, we found that both proteins were dephosphorylated under heat shock and proteotoxic stress, and this dephosphorylation was inhibited by okadaic acid. Using proteins with mutations at putative phosphorylated residues, we revealed that eEF1Bδ1 and eEF1BδL are phosphorylated at S133 and S499, respectively, and these residues are both CDK1 phosphorylation sites. The eEF1BδL S499A mutant more strongly activated promoter-driven reporter than the wild-type protein and S499D mutant. Furthermore, protein phosphatase 1 (PP1) was co-immunoprecipitated with eEF1Bδ1 and eEF1BδL, and PP1 dephosphorylated both proteins . Thus, this study clarified the role of phosphorylation/dephosphorylation in the functional regulation of eEF1BδL during heat shock.
PubMed: 33521052
DOI: 10.3389/fmolb.2020.598578 -
Drug Metabolism and Disposition: the... Mar 2023Tenofovir (TFV; prescribed as TFV disoproxil fumarate and TFV alafenamide prodrugs) is currently used for HIV prevention and treatment. TFV must be phosphorylated twice...
Tenofovir (TFV; prescribed as TFV disoproxil fumarate and TFV alafenamide prodrugs) is currently used for HIV prevention and treatment. TFV must be phosphorylated twice into TFV-diphosphate (TFV-DP) to become pharmacologically active. Previously, we reported heterogeneity in TFV-DP distribution in colorectal tissue (a putative site of HIV infection) sections collected from research participants receiving a TFV-containing enema. This observed heterogeneity is likely multifactorial. Of note, TFV-DP is structurally similar to ATP. It is known that nucleotidases such as nucleoside triphosphate diphosphohydrolases (NTPDases) dephosphorylate ATP. Thus, it was hypothesized that NTPDase-mediated dephosphorylation plays a role in regulating TFV-DP levels in colorectal tissue. To test this hypothesis, recombinant NTPDase proteins (NTPDase 1, 3, 4, 5, 6, and 8) were incubated, individually, with TFV-DP to determine their abilities to dephosphorylate TFV-DP in vitro. Following incubations, TFV-DP dephosphorylation was determined using both malachite green phosphate assays and ultrahigh-performance liquid chromatography tandem mass spectrometry. From these, NTPDase 1 exhibited the highest activity toward TFV-DP. Further, enzyme kinetic analysis revealed Michaelis-Menten kinetics for NTPDase 1-mediated TFV-DP dephosphorylation. Next, immunoblot analyses were conducted to confirm the expression of NTPDase 1 protein in human colorectal tissue. Liquid chromatography coupled to mass spectrometry proteomics analysis was used to measure the relative abundance of NTPDases in human colorectal tissue among healthy adult individuals ( = 4). These analyses confirmed the high abundance of NTPDase 1 in human colorectal tissue. Taken together, results suggest that NTPDase 1 may contribute to the regulation of TFV-DP levels. The above data provide important insights into the dephosphorylation of TFV-DP. SIGNIFICANCE STATEMENT: Nucleoside triphosphate diphosphohydrolases (NTPDases) that are involved in enzymatic ATP dephosphorylation may contribute to tenofovir-diphosphate (TFV-DP) dephosphorylation, leading to its inactivation. In this study, the NTPDases responsible for TFV-DP dephosphorylation in vitro and their expression in human colorectal tissue were investigated. Through this work, it was demonstrated that NTPDase 1 has the highest activity toward TFV-DP dephosphorylation, and it was abundant in human colorectal tissue. Importantly, these studies will increase our understanding of TFV-DP disposition.
Topics: Adult; Humans; HIV Infections; Nucleosides; Anti-HIV Agents; Diphosphates; Kinetics; Tenofovir; Nucleotides; Colorectal Neoplasms; Adenosine Triphosphate
PubMed: 36396461
DOI: 10.1124/dmd.122.000855