Protein Kinase Cε Modulates Insulin Receptor Localization and Trafficking in Mouse Embryonic Fibroblasts

We have previously shown that deletion of protein kinase C epsilon (PKCε) in mice results in protection against glucose intolerance caused by a high fat diet. This was in part due to reduced insulin uptake by hepatocytes and insulin clearance, which enhanced insulin availability. Here we employed mouse embryonic fibroblasts (MEFs) derived from wildtype (WT) and PKCε-deficient (PKCε^−/−) mice to examine this mechanistically. PKCε^−/− MEFs exhibited reduced insulin uptake which was associated with decreased insulin receptor phosphorylation, while downstream signalling through IRS-1 and Akt was unaffected. Cellular fractionation demonstrated that PKCε deletion changed the localization of the insulin receptor, a greater proportion of which co-fractionated with flotillin-1, a marker of membrane microdomains. Insulin stimulation resulted in redistribution of the receptor in WT cells, while this was markedly reduced in PKCε^−/− cells. These alterations in insulin receptor trafficking were associated with reduced expression of CEACAM1, a receptor substrate previously shown to modulate insulin clearance. Virally-mediated reconstitution of PKCε in MEFs increased CEACAM1 expression and partly restored the sensitivity of the receptor to insulin-stimulated redistribution. These data indicate that PKCε can affect insulin uptake in MEFs through promotion of receptor-mediated endocytosis, and that this may be mediated by regulation of CEACAM1 expression.     

An Examination of Focal Adhesion Formation and Tyrosine Phosphorylation in Fibroblasts Isolated from src¯, fyn¯, and yes¯ Mice

As cells adhere to extracellular matrix proteins, several focal adhesion proteins become tyrosine phosphorylated. One of the most prominent of these has been identified as the tyrosine kinase p125^FAK (focal adhesion kinase, FAK). An interaction between FAK and members of the Src family tyrosine kinases p59^fyn, pp60^v-src, and activated pp60^c-src (527F) has been demonstrated, raising the possibility that these kinases may regulate FAK activity. To explore the role of Src family kinases in focal adhesions and in the regulation of FAK activity, we isolated fibroblasts from transgenic mice that lack either pp60^c-src p59^fyn, or pp62^c-yes. These primary fibroblasts, and those of a control mouse, were passaged numerous times and resulted in spontaneously immortalized cell lines without the addition of transforming agents. After confirming the absence of the appropriate nonreceptor tyrosine kinases in the fyc¯, srn¯ and yes¯ fibroblasts, the ability of these fibroblasts to form focal adhesions and stress fibers was assessed by immunofluorescence microscopy and found to be comparable to that of normal fibroblasts. We investigated phosphotyrosine levels in response to adhesion to fibronectin and identified the pp60^src substrate p130 as the one major protein with reduced levels of tyrosine phosphorylation in the cells lacking p59^fyn and pp62^c-yes, and particularly in those lacking pp60^c-scr. We examined FAK phosphorylation and kinase activity and found that there were no significant differences between these cells.     

Serine 216 Phosphorylation of?Estrogen Receptor α in Neutrophils: Migration and Infiltration into the Mouse Uterus

Background

Whereas estrogen receptors are present in immune cells, it is not known if they are phosphorylated to regulate immune cell functions. Here we determined the phosphorylation status of estrogen receptor α (ERα) at residue serine 216 in mouse neutrophils and examined its　role in migration and infiltration. Serine 216 is the conserved phosphorylation site within the DNA binding domains found in the majority of nuclear receptors.

Methodology/Principal Findings

A phospho-peptide antibody specific to phosphorylated serine 216 and ERα KO mice were utilized in immunohistochemistry, double immuno-staining or Western blot to detect phosphorylation of ERα in peripheral blood as well as infiltrating neutrophils in the mouse uterus. Transwell assays were performed to examine migration of neutrophils. An anti-Ly6G antibody identified neutrophils. About 20% of neutrophils expressed phosphorylated ERα at serine 216 in peripheral white blood cells (WBC) from C3H/HeNCrIBR females. Phosphorylation was additively segregated between C3H/HeNCrIBR and C57BL/6 females. Only neutrophils that expressed phosphorylated ERα migrated in Transwell assays as well as infiltrated the mouse uterus during normal estrous cycles.

Conclusions/Significance

ERα was phosphorylated at serine 216 in about 20% of mouse peripheral blood neutrophils. Only those that express phosphorylated ERα migrate and infiltrate the mouse uterus. This phosphorylation was the first to be characterized in endogenous ERα found in normal tissues and cells. Phosphorylated ERα may have opened a novel research direction for biological roles of phosphorylation in ERα actions and can be developed as a drug target for treatment of immune-related diseases.     

The Tyrosine Phosphorylation and Cytoskeletal Translocation of Phospholipase Cγ1 in Bovine Adrenal Medullary Chromaffin Cells

The tyrosine phosphatase inhibitor BpV(phen) stimulated a concentration-dependent increase of phospholipase C (PLC) activity in bovine adrenal medullary chromaffin cells. This response was accompanied by an increase in PLCgamma1 tyrosine phosphorylation and its cytosketetal translocation. Insulin, at high concentrations, stimulated PLC activity to a similar extent as BpV(phen), a response that was also accompanied by an increase in PLCgamma1 translocation but not its tyrosine phosphorylation. BpV(phen) strongly enhanced the insulin-stimulated increase in PLC activity and caused a small rise in PLCgamma1 translocation above that seen with insulin alone. Despite the synergistic rise in activity PLCgamma1 tyrosine phosphorylation did not increase beyond that seen with BpV(phen) alone. These results indicate that PLCgamma1 activation in chromaffin cells may be more closely associated with its cytoskeletal translocation than its tyrosine phosphorylation although other factors may also be important for activation of enzyme activity.     

Phosphorylation of C3a Receptor at Multiple Sites Mediates Desensitization, β-Arrestin-2 Recruitment and Inhibition of NF-κB Activity in Mast Cells

Background

Phosphorylation of G protein coupled receptors (GPCRs) by G protein coupled receptor kinases (GRKs) and the subsequent recruitment of β-arrestins are important for their desensitization. Using shRNA-mediated gene silencing strategy, we have recently shown that GRK2, GRK3 and β-arrestin-2 promote C3a receptor (C3aR) desensitization in human mast cells. We also demonstrated that β-arrestin-2 provides an inhibitory signal for NF-κB activation. C3aR possesses ten potential phosphorylation sites within its carboxyl terminus but their role on desensitization, β-arrestin recruitment and NF-κB activation has not been determined.

Methodology/Principal Findings

We utilized a site directed mutagenesis approach in transfected HEK293 cells to determine the role of receptor phosphorylation on β-arrestin-2 recruitment and RBL-2H3 cells for functional studies. We found that although Ala substitution of Ser475/479, Thr480/481 residues resulted in 58±3.8% decrease in agonist-induced C3aR phosphorylation there was no change in β-arrestin-2 binding or receptor desensitization. By contrast, Ala substitution of Thr463, Ser465, Thr466 and Ser470 led to 40±1.3% decrease in agonist-induced receptor phosphorylation but this was associated with 74±2.4% decreases in β-arrestin-2 binding, significantly reduced desensitization and enhanced NF-κB activation. Combined mutation of these Ser/Thr residues along with Ser459 (mutant MT7), resulted in complete loss of receptor phosphorylation and β-arrestin-2 binding. RBL-2H3 cells expressing MT7 responded to C3a for greater Ca²⁺ mobilization, degranulation and NF-κB activation when compared to the wild-type receptor. Interestingly, co-expression of MT7 with a constitutively active mutant of β-arrestin (R169E) inhibited C3a-induced degranulation by 28±2.4% and blocked NF-κB activation by 80±2.4%.

Conclusion/Significance

This study demonstrates that although C3a causes phosphorylation of its receptor at multiple sites, Ser459, Thr463, Ser465, Thr466 and Ser470 participate in C3aR desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity. Furthermore, β-arrestin-2 inhibits C3a-induced NF-κB activation via receptor desensitization-dependent and independent pathways.     

Inhibition of Wnt and PI3K Signaling Modulates GSK-3β Activity and Induces Morphological Changes in Cortical Neurons: Role of Tau Phosphorylation

Glycogen synthase kinase GSK-3β has been identified as one of the major candidates mediating tau hyperphosphorylation at the same sites as those present in tau protein in brain from Alzheimer′s disease (AD) patients. However, the signal transduction pathways involved in the abnormal activation of GSK-3β, have not been completely elucidated. GSK-3β activity is repressed by the canonical Wnt signaling pathway, but it is also modulated through the PI3K/Akt route. Recent studies have suggested that Wnt signaling might be involved in the pathophysiology of AD. On the other hand, modulators of the PI3K pathway might be reduced during aging leading to a sustained activation of GSK-3β, which in turn would increase the risk of tau hyperphosphorylation. The role of Wnt and PI3K signaling inhibition on the extent of tau phosphorylation and neuronal morphology has not been completely elucidated. Thus, in the present investigation we analyzed the effects of different negative modulators of the Wnt and the PI3K pathways on GSK-3β activation and phosphorylation of tau at the PHF-1 epitope in cortical cultured neurons and hippocampal slices from adult rat brain. Changes in the microtubule network were also studied. We found that a variety of Wnt and PI3K inhibitors, significantly increased tau phosphorylation at the PHF-1 site, induced the disarrangement of the microtubule network and the accumulation of tau within cell bodies. These changes correlated with alterations in neuronal morphology. Special issue article in honor of Dr. Ricardo Tapia.     

Presenilin-1/γ-Secretase Controls Glutamate Release,Tyrosine Phosphorylation,and Surface Expression of N-Methyl-d-aspartate Receptor (NMDAR) Subunit GluN2B

Abnormally high concentrations of extracellular glutamate in the brain may cause neuronal damage via excitotoxicity. Thus, tight regulation of glutamate release is critical to neuronal function and survival. Excitotoxicity is caused mainly by overactivation of the extrasynaptic NMDA receptor (NMDAR) and results in specific cellular changes, including calcium-induced activation of calpain proteases. Here, we report that presenilin-1 (PS1) null mouse cortical neuronal cultures have increased amounts of calpain-dependent spectrin breakdown products (SBDPs) compared with WT cultures. NMDAR antagonists blocked accumulation of SBDPs, suggesting abnormal activation of this receptor in PS1 null cultures. Importantly, an increase in SBDPs was detected in cultures of at least 7 days in vitro but not in younger cultures. Conditioned medium from PS1 null neuronal cultures at 8 days in vitro contained higher levels of glutamate than medium from WT cultures and stimulated production of SBDPs when added to WT cultures. Use of glutamate reuptake inhibitors indicated that accumulation of this neurotransmitter in the media of PS1 null cultures was due to increased rates of release. PS1 null neurons showed decreased cell surface expression and phosphorylation of the GluN2B subunit of NMDAR, indicating decreased amounts of extrasynaptic NMDAR in the absence of PS1. Inhibition of γ-secretase activity in WT neurons caused changes similar to those observed in PS1 null neurons. Together, these data indicate that the PS1/γ-secretase system regulates release of glutamate, tyrosine phosphorylation, and surface expression of GluN2B-containing NMDARs.     

Tyrosine Kinase Phosphorylation of GABAA Receptor α1, β2 and γ2 Subunits Following Chronic Intermittent Ethanol (CIE) Exposure of Cultured Cortical Neurons of Mice

There is evidence that many of the GABA_A receptor subunits contain consensus sequence for tyrosine kinase, and phosphorylation may play a key role in ethanol’s regulation of GABA_A receptors. Recently, we investigated the effect of chronic exposure of ethanol (CE) on tyrosine kinase phosphorylation and reported that there was an up-regulation in tyrosine kinase phosphorylation of the β₂- and γ₂- subunits and no effect on α₁-subunit of the GABA_A receptor in the cultured cortical neurons of mice. In the present study, we have further investigated the effect of chronic intermittent administration of ethanol (CIE) on tyrosine kinase phosphorylation of the GABA_A receptor subunits (α₁, β₂, and γ₂) in the mouse cultured cortical neurons by immunoprecipitation and Western blot techniques. We observed that there was an up-regulation in the tyrosine kinase phosphorylation of the GABA_A receptor β₂- and γ₂-subunits following CIE exposure, and no effect on α₁-subunit in the cultured cortical neurons of mice. These CIE changes, unlike CE, were not reverted back to the control level following ethanol withdrawal even after 7 days. Acute exposure of ethanol did not cause any change in the tyrosine kinase regulation of the GABA_A receptor subunits. In conclusion, the CIE exposure, unlike chronic/acute ethanol exposure, regulates the tyrosine kinase phosphorylation of the selective population of GABA_A receptors in a long lasting manner.     

Stimulation of duodenal epithelial HCO3− transport in the guinea pig and cat by luminal prostaglandin E2

Segments of guinea pig or cat duodenum distal to the Brunner gland containing area and devoid of bile or pancreatic secretions were cannulated in situ. The unbuffered luminal solution was gassed with 100% O₂ or N₂ and HCO₃⁻ transport titrated at pH 7.40 or 8.00 with solutions containing HCl. Cat duodenum transported HCO₃⁻ at a greater rate (∼17μeq, cm⁻¹, h⁻¹) than did jejunum in the same animals (∼5μeq, cm⁻¹, h⁻¹) and also developed a greater transmucosal electrical potential difference. Luminal application of PGE₂ (1 – 12 μM) in cat duodenum increased HCO₃⁻ transport and the potential difference. HCO₃⁻ transport by guinea pig duodenum (∼27 μeq, cm⁻¹, h⁻¹) was increased by luminal PGE₂ only in animals where transport had been inhibited by pretreatment with aspirin (30 mg/kg intravenously). Exposure of the cat duodenal lumen to HCl (1 – 25 mM, 5 min) stimulated HCO₃⁻ transport and continuous exposure of duodenum in the guinea pig to acid discharged from the stomach may increase endogenous prostaglandin concentrations, resulting in an apparent lack of effect of exogenous prostaglandins. The present results and previous similar findings in amphibians in vitro suggest that surface epithelial transport of HCO₃⁻ protects duodenal mucosa against acid.