NH2 terminus of serum and glucocorticoid-regulated kinase 1 binds to phosphoinositides and is essential for isoform-specific physiological functions

2007 ◽  
Vol 292 (6) ◽  
pp. F1741-F1750 ◽  
Author(s):  
Alan C. Pao ◽  
James A. McCormick ◽  
Hongyan Li ◽  
John Siu ◽  
Cedric Govaerts ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Kinase Activity ◽  
Regulatory Protein ◽  
Forkhead Transcription Factor ◽  
Cellular Processes ◽  
Cellular Compartments ◽  
Stimulation Of

Serum and glucocorticoid regulated kinase 1 (SGK1) has been identified as a key regulatory protein that controls a diverse set of cellular processes including sodium (Na+) homeostasis, osmoregulation, cell survival, and cell proliferation. Two other SGK isoforms, SGK2 and SGK3, have been identified, which differ most markedly from SGK1 in their NH2-terminal domains. We found that SGK1 and SGK3 are potent stimulators of epithelial Na+ channel (ENaC)-dependent Na+ transport, while SGK2, which has a short NH2 terminus, is a weak stimulator of ENaC. Further characterization of the role of the SGK1 NH2 terminus revealed that its deletion does not affect in vitro kinase activity but profoundly limits the ability of SGK1 either to stimulate ENaC-dependent Na+ transport or inhibit Forkhead-dependent gene transcription. The NH2 terminus of SGK1, which shares sequence homology with the phosphoinositide 3-phosphate [PI( 3 )P] binding domain of SGK3, binds phosphoinositides in protein lipid overlay assays, interacting specifically with PI( 3 )P, PI( 4 )P, and PI( 5 )P, but not with PI( 3 , 4 , 5 )P3. Moreover, a point mutation that reduces phosphoinositide binding to the NH2 terminus also reduces SGK1 effects on Na+ transport and Forkhead activity. These data suggest that the NH2 terminus, although not required for PI 3-kinase-dependent modulation of SGK1 catalytic activity, is required for multiple SGK1 functions, including stimulation of ENaC and inhibition of the proapoptotic Forkhead transcription factor. Together, these observations support the idea that the NH2-terminal domain acts downstream of PI 3-kinase-dependent activation to target the kinase to specific cellular compartments and/or substrates, possibly through its interactions with a subset of phosphoinositides.

scholarly journals Characterization of the kinase activity of a WNK4 protein complex

2009 ◽  
Vol 297 (3) ◽  
pp. F685-F692 ◽  
Author(s):  
Robert Ahlstrom ◽  
Alan S. L. Yu
Keyword(s):  
Kinase Activity ◽  
Genetic Disorder ◽  
Signal Transduction Pathway ◽  
Kinase Domain ◽  
Full Length ◽  
Hek293 Cells ◽  
Pseudohypoaldosteronism Type Ii

Mutations in WNK4 protein kinase cause pseudohypoaldosteronism type II (PHAII), a genetic disorder that is characterized by renal NaCl and K+ retention leading to hypertension and hyperkalemia. Consistent with this, WNK4 is known to regulate several renal tubule transporters, including the NaCl cotransporter, NCC, and the K+ channel, ROMK, but the mechanisms are incompletely understood, and the role of the kinase activity in its actions is highly controversial. To assay WNK4 kinase activity, we have now succeeded in expressing and purifying full-length, enzymatically active WNK4 protein from HEK293 cells. We show that full-length wild-type WNK4 phosphorylates oxidative stress response kinase 1 (OSR1) and Ste20/SPS1-related proline/alanine-rich kinase (SPAK) in vitro. Introducing the PHAII-associated mutations, E559K, D561A, and Q562E, into our protein had no significant effect on this phosphorylation. We conclude that PHAII is unlikely to be caused by abnormal WNK4 kinase activity. We also made the intriguing observation that inactivating mutations of the WNK4 kinase domain did not completely abolish in vitro phosphorylation of OSR1/SPAK. Led by this, we identified a novel 40-kDa kinase that associates specifically with the COOH-terminal half of WNK4 and is able to phosphorylate both WNK4 and SPAK/OSR1. We suggest that this 40-kDa kinase functions in the WNK4 signal transduction pathway and may mediate some of the physiological actions attributed to WNK4.

scholarly journals Dissecting the roles of Haspin and VRK1 in Histone H3 threonine-3 phosphorylation during mitosis

2021 ◽  
Author(s):  
Tyrell N Cartwright ◽  
Rebecca J Harris ◽  
Stephanie K Meyer ◽  
Nikolaus A. Watson ◽  
Cheryl Tan ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Neuromuscular Disease ◽  
Kinase Activity ◽  
Histone H3 ◽  
Direct Role ◽  
Dividing Cells ◽  
Histone Kinase ◽  
Mitosis And Meiosis

Protein kinases that phosphorylate histones are ideally-placed to influence the behavior of chromosomes during cell division. Indeed, a number of conserved histone phosphorylation events occur prominently during mitosis and meiosis in most eukaryotes, including on histone H3 at threonine-3 (H3T3ph). At least two kinases, Haspin and VRK1 (NHK-1/ballchen in Drosophila), have been proposed to carry out this modification. Phosphorylation of H3 by Haspin has defined roles in mitosis, but the significance of VRK1 activity towards histones in dividing cells has been unclear. Here, using in vitro kinase assays, KiPIK screening, RNA interference, and CRISPR/Cas9 approaches, we were unable to substantiate a direct role for VRK1, or its homologue VRK2, in the phosphorylation of threonine-3 or serine-10 of Histone H3 in mitosis, although loss of VRK1 did slow cell proliferation. We conclude that the role of VRK1, and its more recently identified association with neuromuscular disease in humans, is unlikely to involve mitotic histone kinase activity. In contrast, Haspin is required to generate H3T3ph during mitosis.

scholarly journals Endothelin-1 Couples βPix to p66Shc: Role of βPix in Cell Proliferation through FOXO3a Phosphorylation and p27kip1 Down-Regulation Independently of Akt

2008 ◽  
Vol 19 (6) ◽  
pp. 2609-2619 ◽  
Author(s):  
Ahmed Chahdi ◽  
Andrey Sorokin
Keyword(s):  
Cell Proliferation ◽  
Direct Evidence ◽  
Mesangial Cells ◽  
Second Phase ◽  
Forkhead Transcription Factor ◽  
Down Regulation ◽  
Human Mesangial Cells ◽  
Endothelin 1 ◽  
Stimulation Of

The phosphorylation of forkhead transcription factor FOXO3a by Akt is critical regulator of cell proliferation induced by serum. We show that endothelin-1 (ET-1) stimulation of primary human mesangial cells (HMCs) induces βPix and p66Shc up-regulation, resulting in the formation of the βPix/p66Shc complex. In transformed HMCs, ET-1 induces a biphasic phosphorylation of p66Shc and FOXO3a. The second phase leads to p27kip1 down-regulation independently of Akt. Depletion of βPix blocks the second phase of p66Shc and FOXO3a phosphorylation and prevents p27kip1 down-regulation induced by ET-1. Depletion of either βPix or p66Shc inhibits ET-1–induced cell proliferation. The expression of β1Pix induces FOXO3a phosphorylation through activation of Rac1, ERK1/2, and p66Shc. Using either p66Shc- or Akt-depleted cells; we show that β1Pix-induced FOXO3a phosphorylation requires p66Shc but not Akt. β1Pix-induced p27kip1 down-regulation was blocked by U0126 but not by wortmannin. Endogenous βPix and FOXO3a are constitutively associated with endogenous p66Shc. FOXO3a and p66Shc binding requires β1Pix homodimerization. Expression of β1Pix homodimerization deficient mutant abrogates β1Pix-induced p27kip1 down-regulation and cell proliferation. Our results identify p66Shc and FOXO3a as novel partners of β1Pix and represent the first direct evidence of β1Pix in cell proliferation via Erk/p66Shc-dependent and Akt-independent mechanisms.

scholarly journals Identification and characterization of XPC-binding domain of hHR23B.

1997 ◽  
Vol 17 (12) ◽  
pp. 6915-6923 ◽  
Author(s):  
C Masutani ◽  
M Araki ◽  
K Sugasawa ◽  
P J van der Spek ◽  
A Yamada ◽  
...  
Keyword(s):  
Hydrophobic Interactions ◽  
Excision Repair ◽  
Binding Domain ◽  
Cell Extracts ◽  
Nucleotide Excision ◽  
Stimulation Of

hHR23B was originally isolated as a component of a protein complex that specifically complements nucleotide excision repair (NER) defects of xeroderma pigmentosum group C cell extracts in vitro and was identified as one of two human homologs of the Saccharomyces cerevisiae NER gene product Rad23. Recombinant hHR23B has previously been shown to significantly stimulate the NER activity of recombinant human XPC protein (rhXPC). In this study we identify and functionally characterize the XPC-binding domain of hHR23B protein. We prepared various internal as well as terminal deletion products of hHR23B protein in a His-tagged form and examined their binding with rhXPC by using nickel-chelating Sepharose. We demonstrate that a domain covering 56 amino acids of hHR23B is required for binding to rhXPC as well as for stimulation of in vitro NER reactions. Interestingly, a small polypeptide corresponding to the XPC-binding domain is sufficient to exert stimulation of XPC NER activity. Comparison with known crystal structures and analysis with secondary structure programs provided strong indications that the binding domain has a predominantly amphipathic alpha-helical character, consistent with evidence that the affinity with XPC is based on hydrophobic interactions. Our work shows that binding to XPC alone is required and sufficient for the role of hHR23B in in vitro NER but does not rule out the possibility that the protein has additional functions in vivo.

Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†

2021 ◽  
Author(s):  
Cecilia Valencia ◽  
Felipe Alonso Pérez ◽  
Carola Matus ◽  
Ricardo Felmer ◽  
María Elena Arias
Keyword(s):  
Protein Synthesis ◽  
Kinase Activity ◽  
Cyclin B1 ◽  
Protein Synthesis Inhibitors ◽  
Vitro Fertilization ◽  
New Findings ◽  
Bovine Oocytes ◽  
Dependent Pathway

Abstract The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P < 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P < 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P < 0.05) and its activity at 4 and 1 hpa, respectively (P < 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P < 0.05); however, its kinase activity decreased at 6 hpf (P < 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P < 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.

scholarly journals KLF7/VPS35 axis contributes to hepatocellular carcinoma progression through CCDC85C-activated β-catenin pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yarong Guo ◽  
Bao Chai ◽  
Junmei Jia ◽  
Mudan Yang ◽  
Yanjun Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Luciferase Reporter ◽  
Aberrant Expression ◽  
Proliferation And Invasion ◽  
Hcc Cells

Abstract Objective Dysregulation of KLF7 participates in the development of various cancers, but it is unclear whether there is a link between HCC and aberrant expression of KLF7. The aim of this study was to investigate the role of KLF7 in proliferation and migration of hepatocellular carcinoma (HCC) cells. Methods CCK8, colony growth, transwell, cell cycle analysis and apoptosis detection were performed to explore the effect of KLF7, VPS35 and Ccdc85c on cell function in vitro. Xenografted tumor growth was used to assess in vivo role of KLF7. Chip-qPCR and luciferase reporter assays were applied to check whether KLF7 regulated VPS35 at transcriptional manner. Co-IP assay was performed to detect the interaction between VPS35 and Ccdc85c. Immunohistochemical staining and qRT-PCR analysis were performed in human HCC sampels to study the clinical significance of KLF7, VPS35 and β-catenin. Results Firstly, KLF7 was highly expressed in human HCC samples and correlated with patients’ differentiation and metastasis status. KLF7 overexpression contributed to cell proliferation and invasion of HCC cells in vitro and in vivo. KLF7 transcriptional activation of VPS35 was necessary for HCC tumor growth and metastasis. Further, co-IP studies revealed that VPS35 could interact with Ccdc85c in HCC cells. Rescue assay confirmed that overexpression of VPS35 and knockdown of Ccdc85c abolished the VPS35-medicated promotion effect on cell proliferation and invasion. Finally, KLF7/VPS35 axis regulated Ccdc85c, which involved in activation of β-catenin signaling pathway, confirmed using β-catenin inhibitor, GK974. Functional studies suggested that downregulation of Ccdc85c partly reversed the capacity of cell proliferation and invasion in HCC cells, which was regulated by VPS35 upregulation. Lastly, there was a positive correlation among KLF7, VPS35 and active-β-catenin in human HCC patients. Conclusion We demonstrated that KLF7/VPS35 axis promoted HCC cell progression by activating Ccdc85c-medicated β-catenin pathway. Targeting this signal axis might be a potential treatment strategy for HCC.

scholarly journals Characterization of a Dye-Decolorizing Peroxidase from Irpex lacteus Expressed in Escherichia coli: An Enzyme with Wide Substrate Specificity Able to Transform Lignosulfonates

2021 ◽  
Vol 7 (5) ◽  
pp. 325
Author(s):  
Laura Isabel de de Eugenio ◽  
Rosa Peces-Pérez ◽  
Dolores Linde ◽  
Alicia Prieto ◽  
Jorge Barriuso ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Veratryl Alcohol ◽  
Dye Decolorization ◽  
Irpex Lacteus ◽  
Type D ◽  
Lignin Peroxidases

A dye-decolorizing peroxidase (DyP) from Irpex lacteus was cloned and heterologously expressed as inclusion bodies in Escherichia coli. The protein was purified in one chromatographic step after its in vitro activation. It was active on ABTS, 2,6-dimethoxyphenol (DMP), and anthraquinoid and azo dyes as reported for other fungal DyPs, but it was also able to oxidize Mn2+ (as manganese peroxidases and versatile peroxidases) and veratryl alcohol (VA) (as lignin peroxidases and versatile peroxidases). This corroborated that I. lacteus DyPs are the only enzymes able to oxidize high redox potential dyes, VA and Mn+2. Phylogenetic analysis grouped this enzyme with other type D-DyPs from basidiomycetes. In addition to its interest for dye decolorization, the results of the transformation of softwood and hardwood lignosulfonates suggest a putative biological role of this enzyme in the degradation of phenolic lignin.

scholarly journals Mass Spectrometry-Based Proteomic Characterization of Cutaneous Melanoma Ectosomes Reveals the Presence of Cancer-Related Molecules

2020 ◽  
Vol 21 (8) ◽  
pp. 2934 ◽  
Author(s):  
Magdalena Surman ◽  
Sylwia Kędracka-Krok ◽  
Dorota Hoja-Łukowicz ◽  
Urszula Jankowska ◽  
Anna Drożdż ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Content ◽  
Cell Lines ◽  
Cutaneous Melanoma ◽  
Epithelial Mesenchymal Transition ◽  
Mesenchymal Transition ◽  
Starting Point ◽  
Novel Biomarkers

Cutaneous melanoma (CM) is an aggressive type of skin cancer for which effective biomarkers are still needed. Recently, the protein content of extracellular vesicles (ectosomes and exosomes) became increasingly investigated in terms of its functional role in CM and as a source of novel biomarkers; however, the data concerning the proteome of CM-derived ectosomes is very limited. We used the shotgun nanoLC–MS/MS approach to the profile protein content of ectosomes from primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cell lines. Additionally, the effect exerted by CM ectosomes on recipient cells was assessed in terms of cell proliferation (Alamar Blue assay) and migratory properties (wound healing assay). All cell lines secreted heterogeneous populations of ectosomes enriched in the common set of proteins. A total of 1507 unique proteins were identified, with many of them involved in cancer cell proliferation, migration, escape from apoptosis, epithelial–mesenchymal transition and angiogenesis. Isolated ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of different cancer-promoting molecules. Taken together, these results confirm the significant role of ectosomes in several biological processes leading to CM development and progression, and might be used as a starting point for further studies exploring their diagnostic and prognostic potential.

scholarly journals The Role of Interleukin-23 in the Early Development of Emphysema in HIV1+Smokers

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Igor Z. Barjaktarevic ◽  
Ronald G. Crystal ◽  
Robert J. Kaner
Keyword(s):  
Tissue Destruction ◽  
Epithelial Lining Fluid ◽  
Protein Levels ◽  
Knowing That ◽  
Lung Epithelial ◽  
Interleukin 23 ◽  
Metalloproteinase 9 ◽  
Stimulation Of

Rationale.Matrix metalloproteinase-9 (MMP-9) expression is upregulated in alveolar macrophages (AM) of HIV1+smokers who develop emphysema. Knowing that lung epithelial lining fluid (ELF) of HIV1+smokers contains increased levels of inflammatory cytokines compared to HIV1−smokers, we hypothesized that upregulation of lung cytokines in HIV1+smokers may be functionally related to increased MMP-9 expression.Methods.Cytokine arrays evaluated cytokine protein levels in ELF obtained from 5 groups of individuals: HIV1−healthy nonsmokers, HIV1−healthy smokers, HIV1−smokers with low diffusing capacity (DLCO), HIV1+nonsmokers, and HIV1+smokers with lowDLCO.Results. Increased levels of the Th17 related cytokine IL-23 were found in HIV1−smokers with lowDLCOand HIV1+smokers and nonsmokers. Relative IL-23 gene expression was increased in AM of HIV1+individuals, with greater expression in AM of HIV1+smokers with lowDLCO. Infection with HIV1in vitroinduced IL-23 expression in normal AM. IL-23 stimulation of AM/lymphocyte coculturesin vitroinduced upregulation of MMP-9. Lung T lymphocytes express receptor IL-23R and interact with AM in order to upregulate MMP-9.Conclusion. This mechanism may contribute to the increased tissue destruction in the lungs of HIV1+smokers and suggests that Th17 related inflammation may play a role.

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