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Annual Review of Pharmacology and... Jan 2023Phosphatases and kinases maintain an equilibrium of dephosphorylated and phosphorylated proteins, respectively, that are required for critical cellular functions.... (Review)
Review
Phosphatases and kinases maintain an equilibrium of dephosphorylated and phosphorylated proteins, respectively, that are required for critical cellular functions. Imbalance in this equilibrium or irregularity in their function causes unfavorable cellular effects that have been implicated in the development of numerous diseases. Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of protein substrates on tyrosine residues, and their involvement in cell signaling and diseases such as cancer and inflammatory and metabolic diseases has made them attractive therapeutic targets. However, PTPs have proved challenging in therapeutics development, garnering them the unfavorable reputation of being undruggable. Nonetheless, great strides have been made toward the inhibition of PTPs over the past decade. Here, we discuss the advancement in small-molecule inhibition for the PTP subfamily known as the mitogen-activated protein kinase (MAPK) phosphatases (MKPs). We review strategies and inhibitor discovery tools that have proven successful for small-molecule inhibition of the MKPs and discuss what the future of MKP inhibition potentially might yield.
Topics: Humans; Mitogen-Activated Protein Kinase Phosphatases; Neoplasms; Protein Tyrosine Phosphatases; Signal Transduction; Tyrosine Kinase Inhibitors
PubMed: 36662585
DOI: 10.1146/annurev-pharmtox-051921-121923 -
Cell Death and Differentiation Oct 2022The proper orientation of centrosome and spindle is essential for genome stability; however, the mechanism that governs these processes remains elusive. Here, we...
The proper orientation of centrosome and spindle is essential for genome stability; however, the mechanism that governs these processes remains elusive. Here, we demonstrated that polo-like kinase 1 (Plk1), a key mitotic kinase, phosphorylates residue Thr76 in VCP/p97 (an AAA-ATPase), at the centrosome from prophase to anaphase. This phosphorylation process recruits VCP to the centrosome and in this way, it regulates centrosome orientation. VCP exhibits strong co-localization with Eg5 (a mitotic kinesin motor), at the mitotic spindle, and the dephosphorylation of Thr76 in VCP is required for the enrichment of both VCP and Eg5 at the spindle, thus ensuring proper spindle architecture and chromosome segregation. We also showed that the phosphatase, PTEN, is responsible for the dephosphorylation of Thr76 in VCP; when PTEN was knocked down, the normal spread of VCP from the centrosome to the spindle was abolished. Cryo-EM structures of VCP and VCP, which represent dephosphorylated and phosphorylated states of VCP, respectively, revealed that the Thr76 phosphorylation modulates VCP by altering the inter-domain and inter-subunit interactions, and ultimately the nucleotide-binding pocket conformation. Interestingly, the tumor growth in nude mice implanted with VCP-reconstituted cancer cells was significantly slower when compared with those implanted with VCP-reconstituted cancer cells. Collectively, our findings demonstrate that the phosphorylation and dephosphorylation switch of VCP regulates the architecture of centrosome and spindle for faithful chromosome segregation.
Topics: Animals; Cell Cycle Proteins; Centrosome; HeLa Cells; Humans; Kinesins; Mice; Mice, Nude; Mitosis; Nucleotides; PTEN Phosphohydrolase; Phosphorylation; Spindle Apparatus; Valosin Containing Protein
PubMed: 35430615
DOI: 10.1038/s41418-022-01000-4 -
Cell Death & Disease Jun 2023The Raptor signaling pathway is a critical point of intervention in the invasion and progression of cancer. The non-receptor tyrosine kinase Src-mediated phosphorylation...
The Raptor signaling pathway is a critical point of intervention in the invasion and progression of cancer. The non-receptor tyrosine kinase Src-mediated phosphorylation of OTUB1-Y26 plays a critical role in Raptor stabilization, whereas cathepsin K inhibitor (odanacatib; ODN) and knockdown (siRNA) induce Raptor destabilization. However, the mechanisms involved in cathepsin K inhibition-induced OTUB1-Y26 phosphorylation in Raptor stabilization have not been yet elucidated. This study showed that cathepsin K inhibition activates SHP2, a tyrosine phosphatase, that dephosphorylates OTUB1 and destabilizes Raptor, whereas SHP2 deletion and pharmacological inhibition increase OTUB1-Y26 phosphorylation and Raptor expression. SHP2 deletion also led to the inhibition of ODN-induced mitochondrial ROS, fusion, and dysfunction. Furthermore, cathepsin K inhibition phosphorylated spleen tyrosine kinase (Syk) at Y525 and Y526, resulting in the SHP2-mediated dephosphorylation of OTUB1-Y26. Collectively, our findings identified Syk not only as an upstream tyrosine kinase required for SHP2 activation but also showed a critical mechanism that regulates ODN-induced Raptor downregulation and mitochondrial dysfunction. In conclusion, Syk/SHP2/Src/OTUB1 axis-mediated signaling can act as a therapeutic target in cancer management.
Topics: Cathepsin K; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Signal Transduction; Protein-Tyrosine Kinases; Phosphorylation; Mitochondria
PubMed: 37330581
DOI: 10.1038/s41419-023-05884-z -
Frontiers in Immunology 2022Heart transplantation remains the optimal treatment option for patients with end-stage heart disease. Growing evidence demonstrates that purinergic signals mediated by... (Review)
Review
Heart transplantation remains the optimal treatment option for patients with end-stage heart disease. Growing evidence demonstrates that purinergic signals mediated by purine nucleotides and nucleosides play vital roles in heart transplantation, especially in the era of ischemia-reperfusion injury (IRI) and allograft rejection. Purinergic signaling consists of extracellular nucleotides and nucleosides, ecto-enzymes, and cell surface receptors; it participates in the regulation of many physiological and pathological processes. During transplantation, excess adenosine triphosphate (ATP) levels are released from damaged cells, and driver detrimental inflammatory responses largely purinergic P2 receptors. Ecto-nucleosidases sequentially dephosphorylate extracellular ATP to ADP, AMP, and finally adenosine. Adenosine exerts a cardioprotective effect by its anti-inflammatory, antiplatelet, and vasodilation properties. This review focused on the role of purinergic signaling in IRI and rejection after heart transplantation, as well as the clinical applications and prospects of purinergic signaling.
Topics: Adenosine; Adenosine Triphosphate; Heart Transplantation; Humans; Nucleosides; Nucleotides
PubMed: 35529844
DOI: 10.3389/fimmu.2022.826943 -
Autophagy May 2023Overexpression of PTP4A phosphatases are associated with advanced cancers, but their biological functions are far from fully understood due to limited knowledge about...
Overexpression of PTP4A phosphatases are associated with advanced cancers, but their biological functions are far from fully understood due to limited knowledge about their physiological substrates. VCP is implicated in lysophagy via collaboration with specific cofactors in the ELDR complex. However, how the ELDR complex assembly is regulated has not been determined. Moreover, the functional significance of the penultimate and conserved Tyr805 phosphorylation in VCP has not been established. Here, we use an unbiased substrate trapping and mass spectrometry approach and identify VCP/p97 as a substrate of PTP4A2. Biochemical studies show that PTP4A2 dephosphorylates VCP at Tyr805, enabling the association of VCP with its C-terminal cofactors UBXN6/UBXD1 and PLAA, which are components of the ELDR complex responsible for lysophagy, the autophagic clearance of damaged lysosomes. Functionally, PTP4A2 is required for cellular homeostasis by promoting lysophagy through facilitating ELDR-mediated K48-linked ubiquitin conjugate removal and autophagosome formation on the damaged lysosomes. Deletion of compromises the recovery of glycerol-injection induced acute kidney injury due to impaired lysophagy and sustained lysosomal damage. Taken together, our data establish PTP4A2 as a critical regulator of VCP and uncover an important role for PTP4A2 in maintaining lysosomal homeostasis through dephosphorylation of VCP at Tyr805. Our study suggests that PTP4A2 targeting could be a potential therapeutic approach to treat cancers and other degenerative diseases by modulating lysosomal homeostasis and macroautophagy/autophagy. AAA+: ATPases associated with diverse cellular activities; AKI: acute kidney injury; CBB: Coomassie Brilliant Blue; CRISPR: clustered regularly interspaced short palindromic repeats; ELDR: endo-lysosomal damage response; GFP: green fluorescent protein; GST: glutathione S-transferase; IHC: immunohistochemistry; IP: immunoprecipitation; LAMP1: lysosomal-associated membrane protein 1; LC-MS: liquid chromatography-mass spectrometry; LGALS3/Gal3: galectin 3; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; PLAA: phospholipase A2, activating protein; PTP4A2: protein tyrosine phosphatase 4a2; PUB: NGLY1/PNGase/UBA- or UBX-containing protein; PUL: PLAP, Ufd3, and Lub1; TFEB: transcription factor EB; UBXN6/UBXD1: UBX domain protein 6; UPS: ubiquitin-proteasome system; VCP/p97: valosin containing protein; VCPIP1: valosin containing protein interacting protein 1; YOD1: YOD1 deubiquitinase.
Topics: Animals; Mice; Macroautophagy; Autophagy; Valosin Containing Protein; Fibroblasts; Proteins; Ubiquitin; Lysosomes; Protein Tyrosine Phosphatases; Immediate-Early Proteins
PubMed: 36300783
DOI: 10.1080/15548627.2022.2140558 -
Cellular & Molecular Biology Letters Jul 2023Sebaceous glands (SGs) synthesize and secret sebum to protect and moisturize the dermal system via the complicated endocrine modulation. Dysfunction of SG are usually...
BACKGROUND
Sebaceous glands (SGs) synthesize and secret sebum to protect and moisturize the dermal system via the complicated endocrine modulation. Dysfunction of SG are usually implicated in a number of dermal and inflammatory diseases. However, the molecular mechanism behind the differentiation, development and proliferation of SGs is far away to fully understand.
METHODS
Herein, the rat volar and mammary tissues with abundant SGs from female SD rats with (post-natal day (PND)-35) and without puberty onset (PND-25) were arrested, and conducted RNA sequencing. The protein complex of Neuropeptide Y receptor Y2 (NPY2R)/NPY5R/Nuclear factor of activated T cells 1 (NFATc1) was performed by immunoprecipitation, mass spectrum and gel filtration. Genome-wide occupancy of NFATc1 was measured by chromatin immunoprecipitation sequencing. Target proteins' expression and localization was detected by western blot and immunofluorescence.
RESULTS
NPY2R gene was significantly up-regulated in volar and mammary SGs of PND-25. A special protein complex of NPY2R/NPY5R/NFATc1 in PND-25. NFATc1 was dephosphorylated and activated, then localized into nucleus to exert as a transcription factor in volar SGs of PND-35. NFATc1 was especially binding at enhancer regions to facilitate the distal SG and sebum related genes' transcription. Dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) contributed to NFATc1 phosphorylation in PND-25, and inactivated of DYRK1A resulted in NFATc1 dephosphorylation and nuclear localization in PND-35.
CONCLUSIONS
Our findings unmask the new role of NPY2R/NFATc1/DYRK1A in pubertal SG, and are of benefit to advanced understanding the molecular mechanism of SGs' function after puberty, and provide some theoretical basis for the treatment of acne vulgaris from the perspective of hormone regulation.
Topics: Animals; Female; Rats; Acne Vulgaris; NFI Transcription Factors; Rats, Sprague-Dawley; Sebaceous Glands; Sebum; Dyrk Kinases
PubMed: 37501148
DOI: 10.1186/s11658-023-00467-4 -
Biomedicine & Pharmacotherapy =... May 2023Inositol monophosphatase (IMPase) is an enzyme with two homologs-IMPA1 and IMPA2-that is responsible for dephosphorylating myo-inositol monophosphate to generate... (Review)
Review
Inositol monophosphatase (IMPase) is an enzyme with two homologs-IMPA1 and IMPA2-that is responsible for dephosphorylating myo-inositol monophosphate to generate myo-inositol. IMPase has been extensively studied in neuropsychiatric diseases and is regarded as a susceptibility gene. Recently, emerging evidence has implied that IMPase is linked to cancer development and progression and correlates with patient survival outcomes. Interestingly, whether it acts as a tumor-promoter or tumor-suppressor is inconsistent among different research studies. In this review, we summarize the latest findings on IMPase in cancer, focusing on exploring the underlying mechanisms for its pro- and anticancer roles. In addition, we discuss the potential methods of IMPase regulation in cancer cells and the possible approaches for IMPase intervention in clinical practice.
Topics: Humans; 5'-Nucleotidase; Phosphoric Monoester Hydrolases; Inositol; Inositol Phosphates; Neoplasms
PubMed: 36841024
DOI: 10.1016/j.biopha.2023.114442 -
Frontiers in Physiology 2023The activity of the Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT) is finely tuned by phosphorylation networks involving serine/threonine kinases and... (Review)
Review
The activity of the Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT) is finely tuned by phosphorylation networks involving serine/threonine kinases and phosphatases. While much attention has been paid to the With-No-lysine (K) kinase (WNK)- STE20-related Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive kinase 1 (OSR1) signaling pathway, there remain many unanswered questions regarding phosphatase-mediated modulation of NCC and its interactors. The phosphatases shown to regulate NCC's activity, directly or indirectly, are protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), calcineurin (CN), and protein phosphatase 4 (PP4). PP1 has been suggested to directly dephosphorylate WNK4, SPAK, and NCC. This phosphatase increases its abundance and activity when extracellular K is increased, which leads to distinct inhibitory mechanisms towards NCC. Inhibitor-1 (I1), oppositely, inhibits PP1 when phosphorylated by protein kinase A (PKA). CN inhibitors, like tacrolimus and cyclosporin A, increase NCC phosphorylation, giving an explanation to the Familial Hyperkalemic Hypertension-like syndrome that affects some patients treated with these drugs. CN inhibitors can prevent high K-induced dephosphorylation of NCC. CN can also dephosphorylate and activate Kelch-like protein 3 (KLHL3), thus decreasing WNK abundance. PP2A and PP4 have been shown in models to regulate NCC or its upstream activators. However, no studies in native kidneys or tubules have been performed to test their physiological role in NCC regulation. This review focuses on these dephosphorylation mediators and the transduction mechanisms possibly involved in physiological states that require of the modulation of the dephosphorylation rate of NCC.
PubMed: 36875042
DOI: 10.3389/fphys.2023.1100522 -
Bio-protocol Sep 2022Disturbance of the dynamic balance between protein tyrosine phosphorylation and dephosphorylation, modulated by protein tyrosine kinases (PTKs) and protein tyrosine...
Disturbance of the dynamic balance between protein tyrosine phosphorylation and dephosphorylation, modulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), is known to be crucial for the development of many human diseases. The discovery of agents that restore this balance has been the subject of many drug research efforts, most of which have focused on tyrosine kinase inhibitors (TKIs), resulting in the development of more than 50 FDA-approved TKIs during the past two decades. More recently, accumulating evidence has suggested that members of the PTP superfamily are also promising drug targets, and efforts to discover tyrosine phosphatase inhibitors (TPIs) have increased dramatically. Here, we provide protocols for determining the potency of TPIs in vitro. We focus on the use of fluorescence-based substrates, which exhibit a dramatic increase in fluorescence emission when dephosphorylated by the PTP, and thus allow setting up highly sensitive and miniaturized phosphatase activity assays using 384-well or 1536-well microplates and a continuous (kinetic) assay format. The protocols cover PTP specific activity assays, Michaelis-Menten kinetics, dose-response inhibition assays, and dose-response data analysis for determining IC values. Potential pitfalls are also discussed. While advanced instrumentation is utilized for compound spotting and liquid dispensing, all the assays can be adapted to existing equipment in most laboratories. Assays are described for selected PTP drug targets, including SHP2 ( ), PTP1B ( ), STEP ( ), and VHR ( ). However, all protocols are applicable to members of the PTP enzyme family in general. Graphical abstract.
PubMed: 36248604
DOI: 10.21769/BioProtoc.4510 -
Heliyon May 2023Connexin 43 (Cx43), the predominant gap junction protein in hearts, is modified by specific (de)phosphorylation events under physiological and pathological states to...
BACKGROUND
Connexin 43 (Cx43), the predominant gap junction protein in hearts, is modified by specific (de)phosphorylation events under physiological and pathological states to affect myocardium function and structure. Previously we found that deficiency in Cx43 S282 phosphorylation could impair intercellular communication and contribute to cardiomyocyte apoptosis by activating p38 mitogen-activated protein kinase (p38 MAPK)/factor-associated suicide (Fas)/Fas-associating protein with a novel death domain (FADD) pathway, which is involved in myocardium injury in ischemia/reperfusion () heart. In addition, mutant at Cx43 S282 substituted with alanine heterozygous mice (S282A) exhibited different degrees of ventricular arrhythmias and only some underwent myocardium apoptosis. In this study, we aimed to investigate the role of Cx43 pS282 in different cardiac pathological phenotypes.
METHODS
We examined cardiac function, structure, and relevant protein expression in S282A mice (aged 2, 10 and 30 weeks) by electrocardiograph, echocardiography, histological staining, and co-immunoprecipitation followed by Western blot. Intraperitoneal isoprenaline injection and I/R surgery were applied in S282A mice as external stimulus. 2,3,5-triphenyltetrazolium chloride staining was used for myocardium infarction evaluation.
RESULTS
Adult S282A mice (aged 10 and 30 weeks) still exhibited spontaneous arrhythmia. Unlike neonatal stage (aged around 2 weeks), no apoptosis-related manifestations and the activation of p38 MAPK-Fas-FADD apoptotic pathway were observed in adult S282A hearts. S282A neonatal mice with cardiomyocytes apoptosis exhibited more than 60% dephosphorylation at Cx43 S282 than WT mice, while less than 40% S282 dephosphorylation were found in adult S282A mice. In addition, although S282A mice displayed normal cardiac function, they were highly susceptible to isoproterenol-induced ECG alternans and prone to cardiac injury and deaths upon attack
CONCLUSIONS
These results reinforce that Cx43 S282 dephosphorylation acts as a susceptibility factor in regulating cardiomyocyte survival and cardiac electrical homeostasis in basal conditions and contributes to myocardium injury in the setting of . Cx43 S282 phosphorylation was competent to induce spontaneous arrhythmias, cardiomyocyte apoptosis and deaths based on the degree of S282 dephosphorylation.
PubMed: 37215881
DOI: 10.1016/j.heliyon.2023.e15879