scholarly journals A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs

2004 ◽  
Vol 384 (2) ◽  
pp. 391-400 ◽  
Author(s):  
Helen McNEILL ◽  
Axel KNEBEL ◽  
J. Simon C. ARTHUR ◽  
Ana CUENDA ◽  
Philip COHEN
Keyword(s):  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Glutathione S Transferase ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Extracellular Signal Regulated Kinase ◽  
Cell Extracts ◽  
Extracellular Signal ◽  
Sb 203580

A widely expressed protein containing UBA (ubiquitin-associated) and UBX (ubiquitin-like) domains was identified as a substrate of SAPKs (stress-activated protein kinases). Termed SAKS1 (SAPK substrate-1), it was phosphorylated efficiently at Ser200in vitro by SAPK3/p38γ, SAPK4/p38δ and JNK (c-Jun N-terminal kinase), but weakly by SAPK2a/p38α, SAPK2b/p38β2 or ERK (extracellular-signal-regulated kinase) 2. Ser200, situated immediately N-terminal to the UBX domain, became phosphorylated in HEK-293 (human embryonic kidney) cells in response to stressors. Phosphorylation was not prevented by SB 203580 (an inhibitor of SAPK2a/p38α and SAPK2b/p38β2) and/or PD 184352 (which inhibits the activation of ERK1 and ERK2), and was similar in fibroblasts lacking both SAPK3/p38γ and SAPK4/p38δ or JNK1 and JNK2. SAKS1 bound ubiquitin tetramers and VCP (valosin-containing protein) in vitro via the UBA and UBX domains respectively. The amount of VCP in cell extracts that bound to immobilized GST (glutathione S-transferase)–SAKS1 was enhanced by elevating the level of polyubiquitinated proteins, while SAKS1 and VCP in extracts were coimmunoprecipitated with an antibody raised against S5a, a component of the 19 S proteasomal subunit that binds polyubiquitinated proteins. PNGase (peptide N-glycanase) formed a 1:1 complex with VCP and, for this reason, also bound to immobilized GST–SAKS1. We suggest that SAKS1 may be an adaptor that directs VCP to polyubiquitinated proteins, and PNGase to misfolded glycoproteins, facilitating their destruction by the proteasome.

Hyperthermia with Magnetic Nanowires for Inactivating Living Cells

2008 ◽  
Vol 8 (5) ◽  
pp. 2323-2327 ◽  
Author(s):  
D. S. Choi ◽  
J. Park ◽  
S. Kim ◽  
D. H. Gracias ◽  
M. K. Cho ◽  
...  
Keyword(s):  
Cell Death ◽  
Magnetic Moments ◽  
Magnetic Hysteresis ◽  
Kidney Cells ◽  
Magnetic Nanowires ◽  
Human Embryonic Kidney ◽  
Lower Field ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells

We describe a method to induce hyperthermia in cells, in-vitro, by remotely heating Ni nanowires (NWs) with radio frequency (RF) electromagnetic fields. Ni NWs were internalized by human embryonic kidney cells (HEK-293). Only cells proximal to NWs or with internalized NWs changed shape on exposure to RF fields indicative of cell death. The cell death occurs as a result of hyperthermia, since the RF field remotely heats the NWs as a result of magnetic hysteresis. This is the first demonstration of hyperthermia induced by NWs; since the NWs have anisotropic and strong magnetic moments, our experiments suggest the possibility of performing hyperthermia at lower field strengths in order to minimize damage to untargeted cells in applications such as the treatment of cancer.

scholarly journals Functional interactions of the SPAK/OSR1 kinases with their upstream activator WNK1 and downstream substrate NKCC1

2006 ◽  
Vol 397 (1) ◽  
pp. 223-231 ◽  
Author(s):  
Alberto C. Vitari ◽  
Jacob Thastrup ◽  
Fatema H. Rafiqi ◽  
Maria Deak ◽  
Nick A. Morrice ◽  
...  
Keyword(s):  
Kidney Cells ◽  
Docking Site ◽  
Lower Blood Pressure ◽  
Peptide Substrate ◽  
Human Embryonic Kidney ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Functional Interactions ◽  
Cell Extracts ◽  
Lower Blood

The SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1) kinases interact and phosphorylate NKCC1 (Na+–K+–2Cl− co-transporter-1), leading to its activation. Recent studies indicated that SPAK and OSR1 are phosphorylated and activated by the WNK1 [with no K (lysine) protein kinase-1] and WNK4, genes mutated in humans affected by Gordon's hypertension syndrome. In the present study, we have identified three residues in NKCC1 (Thr175/Thr179/Thr184 in shark or Thr203/Thr207/Thr212 in human) that are phosphorylated by SPAK and OSR1, and have developed a peptide substrate, CATCHtide (cation chloride co-transporter peptide substrate), to assess SPAK and OSR1 activity. Exposure of HEK-293 (human embryonic kidney) cells to osmotic stress, which leads to phosphorylation and activation of NKCC1, increased phosphorylation of NKCC1 at the sites targeted by SPAK/OSR1. The residues on NKCC1, phosphorylated by SPAK/OSR1, are conserved in other cation co-transporters, such as the Na+–Cl− co-transporter, the target of thiazide drugs that lower blood pressure in humans with Gordon's syndrome. Furthermore, we characterize the properties of a 92-residue CCT (conserved C-terminal) domain on SPAK and OSR1 that interacts with an RFXV (Arg-Phe-Xaa-Val) motif present in the substrate NKCC1 and its activators WNK1/WNK4. A peptide containing the RFXV motif interacts with nanomolar affinity with the CCT domains of SPAK/OSR1 and can be utilized to affinity-purify SPAK and OSR1 from cell extracts. Mutation of the arginine, phenylalanine or valine residue within this peptide abolishes binding to SPAK/OSR1. We have identified specific residues within the CCT domain that are required for interaction with the RFXV motif and have demonstrated that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of CATCHtide which does not possess an RFXV motif. We establish that an intact CCT domain is required for WNK1 to efficiently phosphorylate and activate OSR1. These data establish that the CCT domain functions as a multipurpose docking site, enabling SPAK/OSR1 to interact with substrates (NKCC1) and activators (WNK1/WNK4).

scholarly journals Roles for TAB1 in regulating the IL-1-dependent phosphorylation of the TAB3 regulatory subunit and activity of the TAK1 complex

2008 ◽  
Vol 409 (3) ◽  
pp. 711-722 ◽  
Author(s):  
Heidi Mendoza ◽  
David G. Campbell ◽  
Kerry Burness ◽  
James Hastie ◽  
Natalia Ronkina ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Transforming Growth Factor ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Regulatory Subunit ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
P38α Mapk

The protein kinase TAK1 (transforming growth factor-β-activated kinase 1), which has been implicated in the activation of MAPK (mitogen-activated protein kinase) cascades and the production of inflammatory mediators by LPS (lipopolysaccharide), IL-1 (interleukin 1) and TNF (tumour necrosis factor), comprises the catalytic subunit complexed to the regulatory subunits, termed TAB (TAK1-binding subunit) 1 and either TAB2 or TAB3. We have previously identified a feedback-control mechanism by which p38α MAPK down-regulates TAK1 and showed that p38α MAPK phosphorylates TAB1 at Ser423 and Thr431. In the present study, we identified two IL-1-stimulated phosphorylation sites on TAB2 (Ser372 and Ser524) and three on TAB3 (Ser60, Thr404 and Ser506) in human IL-1R cells [HEK-293 (human embryonic kidney) cells that stably express the IL-1 receptor] and MEFs (mouse embryonic fibroblasts). Ser372 and Ser524 of TAB2 are not phosphorylated by pathways dependent on p38α/β MAPKs, ERK1/2 (extracellular-signal-regulated kinase 1/2) and JNK1/2 (c-Jun N-terminal kinase 1/2). In contrast, Ser60 and Thr404 of TAB3 appear to be phosphorylated directly by p38α MAPK, whereas Ser506 is phosphorylated by MAPKAP-K2/MAPKAP-K3 (MAPK-activated protein kinase 2 and 3), which are protein kinases activated by p38α MAPK. Studies using TAB1−/− MEFs indicate important roles for TAB1 in recruiting p38α MAPK to the TAK1 complex for the phosphorylation of TAB3 at Ser60 and Thr404 and in inhibiting the dephosphorylation of TAB3 at Ser506. TAB1 is also required to induce TAK1 catalytic activity, since neither IL-1 nor TNFα was able to stimulate detectable TAK1 activity in TAB1−/− MEFs. Surprisingly, the IL-1 and TNFα-stimulated activation of MAPK cascades and IκB (inhibitor of nuclear factor κB) kinases were similar in TAB1−/−, MEKK3−/− [MAPK/ERK (extracellular-signal-regulated kinase) kinase kinase 3] and wild-type MEFs, suggesting that another MAP3K (MAPK kinase kinase) may mediate the IL-1/TNFα-induced activation of these signalling pathways in TAB1−/− and MEKK3−/− MEFs.

scholarly journals Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor

2008 ◽  
Vol 414 (3) ◽  
pp. 441-452 ◽  
Author(s):  
Huihui Kong ◽  
Peter P. Jones ◽  
Andrea Koop ◽  
Lin Zhang ◽  
Henry J. Duff ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Cardiac Arrhythmias ◽  
Single Channel ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Activation Threshold ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
293 Cells ◽  
High Concentrations

Caffeine has long been used as a pharmacological probe for studying RyR (ryanodine receptor)-mediated Ca2+ release and cardiac arrhythmias. However, the precise mechanism by which caffeine activates RyRs is elusive. In the present study, we investigated the effects of caffeine on spontaneous Ca2+ release and on the response of single RyR2 (cardiac RyR) channels to luminal or cytosolic Ca2+. We found that HEK-293 cells (human embryonic kidney cells) expressing RyR2 displayed partial or ‘quantal’ Ca2+ release in response to repetitive additions of submaximal concentrations of caffeine. This quantal Ca2+ release was abolished by ryanodine. Monitoring of endoplasmic reticulum luminal Ca2+ revealed that caffeine reduced the luminal Ca2+ threshold at which spontaneous Ca2+ release occurs. Interestingly, spontaneous Ca2+ release in the form of Ca2+ oscillations persisted in the presence of 10 mM caffeine, and was diminished by ryanodine, demonstrating that unlike ryanodine, caffeine, even at high concentrations, does not hold the channel open. At the single-channel level, caffeine markedly reduced the threshold for luminal Ca2+ activation, but had little effect on the threshold for cytosolic Ca2+ activation, indicating that the major action of caffeine is to reduce the luminal, but not the cytosolic, Ca2+ activation threshold. Furthermore, as with caffeine, the clinically relevant, pro-arrhythmic methylxanthines aminophylline and theophylline potentiated luminal Ca2+ activation of RyR2, and increased the propensity for spontaneous Ca2+ release, mimicking the effects of disease-linked RyR2 mutations. Collectively, our results demonstrate that caffeine triggers Ca2+ release by reducing the threshold for luminal Ca2+ activation of RyR2, and suggest that disease-linked RyR2 mutations and RyR2-interacting pro-arrhythmic agents may share the same arrhythmogenic mechanism.

scholarly journals Induction of Sertoli cells from human fibroblasts by NR5A1 and GATA4

2018 ◽  
Author(s):  
Jianlin Liang ◽  
Nan Wang ◽  
Jing He ◽  
Jian Du ◽  
Yahui Guo ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Lipid Droplets ◽  
Human Fibroblasts ◽  
Seminiferous Tubules ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Human Embryonic Kidney Cells ◽  
Reprogramming Factors ◽  
Spermatogonia Cells

AbstractSertoli cells are essential nurse cells in the testis that regulate the process of spermatogenesis and establish the immune-privileged environment of the blood-testis-barrier (BTB). The induction of human Sertoli cells from fibroblasts could provide cellular sources for fertility and transplantation treatments. Here, we report the in vitro reprogramming of human fibroblasts to Sertoli cells and characterize these human induced Sertoli cells (hiSCs). Initially, five transcriptional factors (NR5A1, GATA4, WT1, SOX9 and DMRT1) and a gene reporter carrying the AMH promoter were utilized to obtain the hiSCs. We further reduce the number of reprogramming factors to two, i.e., NR5A1 and GATA4, and show that these hiSCs have transcriptome profiles that are similar to those of primary human Sertoli cells. Consistent with the known cellular properties of Sertoli cells, hiSCs attract endothelial cells and exhibit high number of lipid droplets in the cytoplasm. More importantly, hiSCs can sustain the viability of spermatogonia cells harvested from mouse seminiferous tubules. In addition, hiSCs suppress the production of IL-2 and proliferation of human T lymphocytes. When hiSCs were cotransplanted with human embryonic kidney cells, these xenotransplanted human cells survived longer in mice with normal immune systems. hiSCs also allow us to determine a gene associated with Sertoli-only syndrome (SCO), CX43, is indeed important in regulating the maturation of Sertoli cells.

scholarly journals Evaluation of the effect of montelukast on cadmium induced toxicity in human embryonic kidney cells (HEK-293)

2019 ◽  
Vol 24 (4) ◽  
pp. 125-137
Author(s):  
Farnoosh Kaviani ◽  
Seyedeh Missagh Jalali ◽  
Elham Hoveizi ◽  
Javad Jamshidian ◽  
Masoomeh Ahmadizadeh ◽  
...  
Keyword(s):  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells

Molecular insight to in vitro biocompatibility of phytofabricated copper oxide nanoparticles with human embryonic kidney cells

Nanomedicine ◽  
2018 ◽  
Vol 13 (19) ◽  
pp. 2415-2433 ◽  
Author(s):  
Puja Kumari ◽  
Pritam Kumar Panda ◽  
Ealisha Jha ◽  
Nandini Pramanik ◽  
Kumari Nisha ◽  
...  
Keyword(s):  
Copper Oxide ◽  
Copper Oxide Nanoparticles ◽  
Oxide Nanoparticles ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Human Embryonic Kidney Cells ◽  
In Vitro Biocompatibility
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