Protein kinase C phosphorylation regulates membrane insertion of GABAA receptor subtypes that mediate tonic inhibition

Tonic inhibition in the brain is mediated largely by specialized populations of extrasynaptic receptors, γ-aminobutyric acid receptors (GABA(A)Rs). In the dentate gyrus region of the hippocampus, tonic inhibition is mediated primarily by GABA(A)R subtypes assembled from α4β2/3 with or without the δ subunit. Although the gating of these receptors is subject to dynamic modulation by agents such as anesthetics, barbiturates, and neurosteroids, the cellular mechanisms neurons use to regulate their accumulation on the neuronal plasma membrane remain to be determined. Using immunoprecipitation coupled with metabolic labeling, we demonstrate that the α4 subunit is phosphorylated at Ser(443) by protein kinase C (PKC) in expression systems and hippocampal slices. In addition, the β3 subunit is phosphorylated on serine residues 408/409 by PKC activity, whereas the δ subunit did not appear to be a PKC substrate. We further demonstrate that the PKC-dependent increase of the cell surface expression of α4 subunit-containing GABA(A)Rs is dependent on Ser(443). Mechanistically, phosphorylation of Ser(443) acts to increase the stability of the α4 subunit within the endoplasmic reticulum, thereby increasing the rate of receptor insertion into the plasma membrane. Finally, we show that phosphorylation of Ser(443) increases the activity of α4 subunit-containing GABA(A)Rs by preventing current run-down. These results suggest that PKC-dependent phosphorylation of the α4 subunit plays a significant role in enhancing the cell surface stability and activity of GABA(A)R subtypes that mediate tonic inhibition.     

HER2/ErbB2-induced Breast Cancer Cell Migration and Invasion Require p120 Catenin Activation of Rac1 and Cdc42

Breast cancers that overexpress the receptor tyrosine kinase ErbB2/HER2/Neu result in poor patient outcome because of extensive metastatic progression. Herein, we delineate a molecular mechanism that may govern this malignant phenotype. ErbB2 induction of migration requires activation of the small GTPases Rac1 and Cdc42. The ability of ErbB2 to activate these small GTPases necessitated expression of p120 catenin, which is itself up-regulated by signaling through ErbB2 and the tyrosine kinase Src. Silencing p120 in ErbB2-dependent breast cancer cell lines dramatically inhibited migration and invasion as well as activation of Rac1 and Cdc42. In contrast, overexpression of constitutively active mutants of these GTPases reversed the effects of p120 silencing. Lastly, ectopic expression of p120 promoted migration and invasion and potentiated metastatic progression of a weakly metastatic, ErbB2-dependent breast cancer cell line. These results suggest that p120 acts as an obligate intermediate between ErbB2 and Rac1/Cdc42 to modulate the metastatic potential of breast cancer cells.     

Hyperphosphorylation of extracellular regulated kinase 2 (ERK2) and inhibition of JNK2 phosphorylation are associated with increased S-phase during transformation of Syrian hamster embryo cells by Malachite Green

Malachite Green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by MG. In this study, we have studied the mitogen activated protein (MAP) kinase signal transduction pathway in preneoplastic cells induced by MG. Western blots of MG induced preneoplastic cells showed no phosphorylation of ERK1, an increased phosphoactive ERK2 associated with a decreased expression of phosphoactive JNK2. However, total forms of ERKs, JNKs and p38 Kinases showed similar levels of expression in control and preneoplastic SHE cells. Indirect immunofluorescence studies have shown a distinct nuclear localisation of phosphoactive ERKs in MG induced preneoplastic cells. Flow cytometric analysis showed an increase of S-phase cells in preneoplastic cells compared to control SHE cells. The present study indicates that hyperphosphorylation of ERK2, decreased JNK2 phosphorylation and an increase in S-phase cells seems to be the early changes associated with the MG induced malignant transformation of SHE cells in primary culture.     

Adapter Protein Shc Regulates Janus Kinase 3 Phosphorylation

Although constitutive activation of Janus kinase 3 (Jak3) leads to different cancers, the mechanism of trans-molecular regulation of Jak3 activation is not known. Previously we reported that Jak3 interactions with adapter protein p52ShcA (Shc) facilitate mucosal homeostasis. In this study, we characterize the structural determinants that regulate the interactions between Jak3 and Shc and demonstrate the trans-molecular mechanism of regulation of Jak3 activation by Shc. We show that Jak3 autophosphorylation was the rate-limiting step during Jak3 trans-phosphorylation of Shc where Jak3 directly phosphorylated two tyrosine residues in Src homology 2 (SH2) domain and one tyrosine residue each in calponin homology 1 (CH1) domain and phosphotyrosine interaction domain (PID) of Shc. Direct interactions between mutants of Jak3 and Shc showed that although FERM domain of Jak3 was sufficient for binding to Shc, CH1 and PID domains of Shc were responsible for binding to Jak3. Functionally Jak3 was autophosphorylated under IL-2 stimulation in epithelial cells. However, Shc recruited tyrosine phosphatases SHP2 and PTP1B to Jak3 and thereby dephosphorylated Jak3. Thus we not only characterize Jak3 interaction with Shc, but also demonstrate the molecular mechanism of intracellular regulation of Jak3 activation where Jak3 interactions with Shc acted as regulators of Jak3 dephosphorylation through direct interactions of Shc with both Jak3 and tyrosine phosphatases.     

Long Term Expression of Drosophila melanogaster Nucleoside Kinase in Thymidine Kinase 2-deficient Mice with No Lethal Effects Caused by Nucleotide Pool Imbalances

Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2^−/−) mice extended the life span of Tk2^−/− mice from 3 weeks to at least 20 months. The Dm-dNK^+/−Tk2^−/− mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK^+/−Tk2^−/− mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK^+/− mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues.     

Protein Kinase C,Focal Adhesions and the Regulation of Cell Migration

Cell adhesion to extracellular matrix is a complex process involving protrusive activity driven by the actin cytoskeleton, engagement of specific receptors, followed by signaling and cytoskeletal organization. Thereafter, contractile and endocytic/recycling activities may facilitate migration and adhesion turnover. Focal adhesions, or focal contacts, are widespread organelles at the cell-matrix interface. They arise as a result of receptor interactions with matrix ligands, together with clustering. Recent analysis shows that focal adhesions contain a very large number of protein components in their intracellular compartment. Among these are tyrosine kinases, which have received a great deal of attention, whereas the serine/threonine kinase protein kinase C has received much less. Here the status of protein kinase C in focal adhesions and cell migration is reviewed, together with discussion of its roles and potential substrates.     

Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity.     

Sphingosine 1-Phosphate Receptor 4 Uses HER2 (ERBB2) to Regulate Extracellular Signal Regulated Kinase-1/2 in MDA-MB-453 Breast Cancer Cells

We demonstrate here that the bioactive lipid sphingosine 1-phosphate (S1P) uses sphingosine 1-phosphate receptor 4 (S1P₄) and human epidermal growth factor receptor 2 (HER2) to stimulate the extracellular signal regulated protein kinase 1/2 (ERK-1/2) pathway in MDA-MB-453 cells. This was based on several lines of evidence. First, the S1P stimulation of ERK-1/2 was abolished by JTE013, which we show here is an S1P_2/4 antagonist and reduced by siRNA knockdown of S1P₄. Second, the S1P-stimulated activation of ERK-1/2 was almost completely abolished by a HER2 inhibitor (ErbB2 inhibitor II) and reduced by siRNA knockdown of HER2 expression. Third, phyto-S1P, which is an S1P₄ agonist, stimulated ERK-1/2 activation in an S1P₄- and HER2-dependent manner. Fourth, FTY720 phosphate, which is an agonist at S1P_1,3,4,5 but not S1P₂ stimulated activation of ERK-1/2. Fifth, S1P stimulated the tyrosine phosphorylation of HER2, which was reduced by JTE013. HER2 which is an orphan receptor tyrosine kinase is the preferred dimerization partner of the EGF receptor. However, EGF-stimulated activation of ERK-1/2 was not affected by siRNA knockdown of HER2 or by ErbB2 (epidermal growth factor receptor 2 (or HER2)) inhibitor II in MDA-MB-453 cells. Moreover, S1P-stimulated activation of ERK-1/2 does not require an EGF receptor. Thus, S1P and EGF function in a mutually exclusive manner. In conclusion, the magnitude of the signaling gain on the ERK-1/2 pathway produced in response to S1P can be increased by HER2 in MDA-MB-453 cells. The linkage of S1P with an oncogene suggests that S1P and specifically S1P₄ may have an important role in breast cancer progression.     

The Spleen Tyrosine Kinase (Syk) Regulates Alzheimer Amyloid-β Production and Tau Hyperphosphorylation

We have previously shown that the L-type calcium channel (LCC) antagonist nilvadipine reduces brain amyloid-β (Aβ) accumulation by affecting both Aβ production and Aβ clearance across the blood-brain barrier (BBB). Nilvadipine consists of a mixture of two enantiomers, (+)-nilvadipine and (−)-nilvadipine, in equal proportion. (+)-Nilvadipine is the active enantiomer responsible for the inhibition of LCC, whereas (−)-nilvadipine is considered inactive. Both nilvadipine enantiomers inhibit Aβ production and improve the clearance of Aβ across the BBB showing that these effects are not related to LCC inhibition. In addition, treatment of P301S mutant human Tau transgenic mice (transgenic Tau P301S) with (−)-nilvadipine reduces Tau hyperphosphorylation at several Alzheimer disease (AD) pertinent epitopes. A search for the mechanism of action of (−)-nilvadipine revealed that this compound inhibits the spleen tyrosine kinase (Syk). We further validated Syk as a target-regulating Aβ by showing that pharmacological inhibition of Syk or down-regulation of Syk expression reduces Aβ production and increases the clearance of Aβ across the BBB mimicking (−)-nilvadipine effects. Moreover, treatment of transgenic mice overexpressing Aβ and transgenic Tau P301S mice with a selective Syk inhibitor respectively decreased brain Aβ accumulation and Tau hyperphosphorylation at multiple AD relevant epitopes. We show that Syk inhibition induces an increased phosphorylation of the inhibitory Ser-9 residue of glycogen synthase kinase-3β, a primary Tau kinase involved in Tau phosphorylation, by activating protein kinase A, providing a mechanism explaining the reduction of Tau phosphorylation at GSK3β-dependent epitopes following Syk inhibition. Altogether our data highlight Syk as a promising target for preventing both Aβ accumulation and Tau hyperphosphorylation in AD.     

Functional Modulation of the Glutamate Transporter Variant GLT1b by the PDZ Domain Protein PICK1

The dominant glutamate transporter isoform in the mammalian brain, GLT1, exists as at least three splice variants, GLT1a, GLT1b, and GLT1c. GLT1b interacts with the scaffold protein PICK1 (protein interacting with kinase C1), which is implicated in glutamatergic neurotransmission via its regulatory effect on trafficking of AMPA-type glutamate receptors. The 11 extreme C-terminal residues specific for the GLT1b variant are essential for its specific interaction with the PICK1 PDZ domain, but a functional consequence of this interaction has remained unresolved. To identify a functional effect of PICK1 on GLT1a or GLT1b separately, we employed the Xenopus laevis expression system. GLT1a and GLT1b displayed similar electrophysiological properties and EC₅₀ for glutamate. Co-expressed PICK1 localized efficiently to the plasma membrane and resulted in a 5-fold enhancement of the leak current in GLT1b-expressing oocytes with only a minor effect on [³H]glutamate uptake. Three different GLT1 substrates all caused a slow TBOA-sensitive decay in the membrane current upon prolonged application, which provides support for the leak current being mediated by GLT1b itself. Leak and glutamate-evoked currents in GLT1a-expressing oocytes were unaffected by PICK1 co-expression. PKC activation down-regulated GLT1a and GLT1b activity to a similar extent, which was not affected by co-expression of PICK1. In conclusion, PICK1 may not only affect glutamatergic neurotransmission by its regulatory effect on glutamate receptors but may also affect neuronal excitability via an increased GLT1b-mediated leak current. This may be particularly relevant in pathological conditions such as amyotrophic lateral sclerosis and cerebral hypoxia, which are associated with neuronal GLT1b up-regulation.