Julianne J. Sando

Sequential Phosphorylation Mediates Receptor- and Kinase-induced Inhibition of TREK-1 Background Potassium Channels

Background potassium channels determine membrane potential and input resistance and serve as prominent effectors for modulatory regulation of cellular excitability. TREK-1 is a two-pore domain background K+ channel (KCNK2, K2P2.1) that is sensitive to a variety of physicochemical and humoral factors. In this work, we used a recombinant expression system to show that activation of Gαq-coupled receptors leads to inhibition of TREK-1 channels via protein kinase C (PKC), and we identified a critical phosphorylation site in a key regulatory domain that mediates inhibition of the channel. In HEK 293 cells co-expressing TREK-1 and either the thyrotropin-releasing hormone receptor (TRHR1) or the Orexin receptor (Orx1R), agonist stimulation induced robust channel inhibition that was suppressed by a bisindolylmaleimide PKC inhibitor but not by a protein kinase A blocker ((Rp)-cAMP-S). Channel inhibition by agonists or by direct activators of PKC (phorbol dibutyrate) and PKA (forskolin) was disrupted not only by alanine or aspartate mutations at an identified PKA site (Ser-333) in the C terminus, but also at a more proximal regulatory site in the cytoplasmic C terminus (Ser-300); S333A and S300A mutations enhanced basal TREK-1 current, whereas S333D and S300D substitutions mimicked phosphorylation and strongly diminished currents. When studied in combination, TREK-1 current density was enhanced in S300A/S333D but reduced in S300D/S333A mutant channels. Channel mutants were expressed and appropriately targeted to cell membranes. Together, these data support a sequential phosphorylation model in which receptor-induced kinase activation drives modification at Ser-333 that enables subsequent phosphorylation at Ser-300 to inhibit TREK-1 channel activity.

Lipid lateral heterogeneity in phosphatidylcholine/phosphatidylserine/diacylglycerol vesicles and its influence on protein kinase C activation

To test the hypothesis that the activation of protein kinase C (PKC) is influenced by lateral heterogeneities of the components of the lipid bilayer, the thermotropic phase behavior of dimyristoylphosphatidylcholine (DMPC)/dimyristoylphosphatidylserine (DMPS)/dioleoylglycerol (DO) vesicles was compared with the activation of PKC by this system. Differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to monitor the main transition (i.e., the gel-to-fluid phase transition) as a function of mole fraction DO (chi(DO)) in DMPC/DO, DMPS/DO, and [DMPC/DMPS (1:1, mol/mol)]/DO multilamellar vesicles (MLVs). In each case, when chi(DO) < or approximately 0.3, DO significantly broadened the main transition and shifted it to lower temperatures; but when chi(DO) > approximately 0.3, the main transition became highly cooperative, i.e., narrow, again. The coexistence of overlapping narrow and broad transitions was clearly evident in DSC thermograms from chi(DO) approximately 0.1 to chi(DO) approximately 0.3, with the more cooperative transition growing at the expense of the broader one as chi(DO) increased. FTIR spectroscopy, using analogs of DMPC and DMPS with perdeuterated acyl chains, showed that the melting profiles of all three lipid components in [DMPC/DMPS (1:1, mol/mol)]/DO MLVs virtually overlay when chi(DO) = 0.33, suggesting that a new type of phase, with a phospholipid/DO mole ratio near 2:1, is formed in this system. Collectively, the results are consistent with the coexistence of DO-poor and DO-rich domains throughout the compositions chi(DO) approximately 0.1 to chi(DO) approximately 0.3, even at temperatures above the main transition. Comparison of the phase behavior of the binary mixtures with that of the ternary mixtures suggests that DMPS/DO interactions may be more favorable than DMPC/DO interactions in the ternary system, especially in the gel state. PKC activity was measured using [DMPC/DMPS (1:1, mol/mol)]/DO MLVs as the lipid activator. At 35 degrees C (a temperature above the main transition of the lipids), PKC activity increased gradually with increasing chi(DO) from chi(DO) approximately 0.1 to chi(DO) approximately 0.4, and activity remained high at higher DO contents. In contrast, at 2 degrees C (a temperature below the main transition), PKC activity exhibited a maximum between chi(DO) approximately 0.1 and chi(DO) approximately 0.3, and at higher DO contents activity was essentially constant at 20-25% of the activity at the maximum. We infer from these results that the formation of DO-rich domains is related to PKC activation, and when the lipid is in the gel state, the coexistence of DO-poor and DO-rich phases also contributes to PKC activation.

Production of and response to interleukin‐2 in peripheral blood lymphocytes of cancer patients

Interleukin‐2 (IL2) is essential for the expansion of antigen‐triggered lymphocytes and cytotoxic T‐cells, processes necessary for tumor control that are frequently depressed in malignancy. The authors measured certain aspects of IL2 function in cancer patients and controls and correlated the findings with the general immune response as indicated by the proliferative response to phytohemagglutinin (PHA) in peripheral blood lymphocytes (PBL). The major questions focused on the capacity of PBL to produce IL2, the correlation of this with the proliferative response to PHA, and whether exogenous IL2 could restore T‐cell responses and natural killer cell activity in immunodepressed cancer patients. IL2 production was measured by the 3H‐thymidine‐labeled CT6 assay on the supernatants of the PBL of cancer patients and normal controls after 24 hours of stimulation with PHA. There were 115 cancer patients (70 head and neck, 13 melanoma, 12 breast, 10 colorectal, and 6 other) and 52 controls. IL2 production was essentially normal in the head and neck cancer patients as a group, although their PHA response was depressed. The mean IL2 generated per 3 × 106 PBL over 24 hours were 129 μ/ml in the head and neck patients and 132 μ/ml in the breast patients, similar to the 129 μ/ml generated in the controls. There was modest but not significant depression in the melanoma (78 μ/ml) and colorectal cancer patients (81 μ/ml). Although subsets of patients showed depressed IL2 production, there was no significant correlation of IL2 production with the PHA response. Depressed IL2 production showed only limited correlation with depressed lymphocyte responses (r = −0.25), which suggested a dissociation of these functions. Of interest was the finding that indomethacin did augment IL2 production in both cancer patients and controls, suggesting that prostaglandin‐mediated regulation is involved. Addition of exogenous IL2 of recombinant origin (Biogen) produced significant augmentation in more than three fourths of the cancer patients and controls. Adding indomethacin further increased this response. Addition of IL2 also significantly increased natural killer activity in both groups. It was concluded that PBL in cancer patients generally have a normal capacity to generate IL2, and this capacity is not related to the proliferative response, which is frequently depressed in these patients. Exogenous IL2 can significantly augment lymphoproliferative and natural killer responses in cancer patients, suggesting that there is merit in exploring the potential therapeutic role of IL2 in these patients.

Phorbol 12-Myristate 13-Acetate Induces Protein Kinase Cη-specific Proliferative Response in Astrocytic Tumor Cells

Protein kinase C (PKC) activation has been implicated in cellular proliferation in neoplastic astrocytes. The roles for specific PKC isozymes in regulating this glial response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC-η expression in regulating cellular proliferation in two well characterized astrocytic tumor cell lines (U-1242 MG and U-251 MG) with different properties of growth in cell culture. Both cell lines expressed an array of conventional (α, βI, βII, and γ) and novel (θ and ε) PKC isozymes that can be activated by phorbol myristate acetate (PMA). Another novel PKC isozyme, PKC-η, was only expressed by U-251 MG cells. In contrast, PKC-δ was readily detected in U-1242 MG cells but was present only at low levels in U-251 MG cells. PMA (100 nm) treatment for 24 h increased cell proliferation by over 2-fold in the U-251 MG cells, whereas it decreased the mitogenic response in the U-1242 MG cells by over 90%. When PKC-η was stably transfected into U-1242 MG cells, PMA increased cell proliferation by 2.2-fold, similar to the response of U-251 MG cells. The cell proliferation induced by PMA in both the U-251 MG and U-1242-PKC-η cells was blocked by the PKC inhibitor bisindolylmaleimide (0.5 μm) and the MEK inhibitor, PD 98059 (50 μm). Transient transfection of wild type U-251 with PKC-η antisense oligonucleotide (1 μm) also blocked the PMA-induced increase in [3H]thymidine incorporation. The data demonstrate that two glioblastoma lines, with functionally distinct proliferative responses to PMA, express different novel PKC isozymes and that the differential expression of PKC-η plays a determining role in the different proliferative capacity.

Protein kinase C-η regulates resistance to UV- and γ-irradiation-induced apoptosis in glioblastoma cells by preventing caspase-9 activation

Both increased cell proliferation and apoptosis play important roles in the malignant growth of glioblastomas. We have demonstrated recently that the differential expression of protein kinase C (PKC)-eta increases the proliferative capacity of glioblastoma cells in culture; however, specific functions for this novel PKC isozyme in the regulation of apoptosis in these tumors has not been defined. In the present study of several glioblastoma cell lines, we investigated the role of PKC-eta in preventing UV- and gamma-irradiation-induced apoptosis and in caspase-dependent signaling pathways that mediate cell death. Exposure to UV or gamma irradiation killed 80% to 100% of PKC-eta-deficient nonneoplastic human astrocytes and U-1242 MG cells, but had little effect on the PKC-eta-expressing U-251 MG and U-373 MG cells. PKC-eta appears to mediate resistance to irradiation specifically such that when PKC-eta was stably expressed in U-1242 MG cells, more than 80% of these cells developed resistance to irradiation-induced apoptosis. Reducing PKC-eta expression by transient and stable expression of antisense PKC-eta in wild-type U-251 MG cells results in increased sensitivity to UV irradiation in a fashion similar to U-1242 MG cells and nonneoplastic astrocytes. Irradiation of PKC-eta-deficient glioblastoma cells resulted in the activation of caspase-9 and caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and a substantial increase in subdiploid DNA content that did not occur in PKC-eta-expressing tumor cells. A specific inhibitor (Ac-DEVD-CHO) of caspase-3 blocked apoptosis in PKC-eta-deficient U-1242 MG cells. The data demonstrate that resistance to UV and gamma irradiation in glioblastoma cell lines is modified significantly by PKC-eta expression and that PKC-eta appears to block the apoptotic cascade at caspase-9 activation.

Critical Role of Serine 465 in Isoflurane-induced Increase of Cell-surface Redistribution and Activity of Glutamate Transporter Type 3

Glutamate transporters (also called excitatory amino acid transporters, EAATs) bind extracellular glutamate and transport it to intracellular space to regulate glutamate neurotransmission and to maintain extracellular glutamate concentrations below neurotoxic levels. We previously showed that isoflurane, a commonly used anesthetic, enhanced the activity of EAAT3, a major neuronal EAAT. This effect required a protein kinase C (PKC) α-dependent EAAT3 redistribution to the plasma membrane. In this study, we prepared COS7 cells stably expressing EAAT3 with or without mutations of potential PKC phosphorylation sites in the putative intracellular domains. Here we report that mutation of threonine 5 or threonine 498 to alanine did not affect the isoflurane effects on EAAT3. However, the mutation of serine 465 to alanine abolished isoflurane-induced increase of EAAT3 activity and redistribution to the plasma membrane. The mutation of serine 465 to aspartic acid increased the expression of EAAT3 in the plasma membrane and also abolished the isoflurane effects on EAAT3. These results suggest an essential role of serine 465 in the isoflurane-increased EAAT3 activity and redistribution and a direct effect of PKC on EAAT3. Consistent with these results, isoflurane induced an increase in phosphorylation of wild type, T5A, and T498A EAAT3, and this increase was absent in S465A and S465D. Our current results, together with our previous data that showed the involvement of PKCα in the isoflurane effects on EAAT3, suggest that the phosphorylation of serine 465 in EAAT3 by PKCα mediates the increased EAAT3 activity and redistribution to plasma membrane after isoflurane exposure.

A Role for Protein Kinase CβI in the Regulation of Ca2+ Entry in Jurkat T Cells

T cell activation leading to cytokine production and cellular proliferation involves a regulated increase and subsequent decrease in the intracellular concentration of Ca2+([Ca2+] i ). While much is understood about agonist-induced increases in [Ca2+] i , less is known about down-regulation of this pathway. Understanding the mechanism of this down-regulation is critical to the prevention of cell death that can be the consequence of a sustained elevation in [Ca2+] i . Protein kinase C (PKC), activated by the diacylglycerol produced as a consequence of T cell receptor engagement, has long been presumed to be involved in this down-regulation, although the precise mechanism is not wholly clear. In this report we demonstrate that activation of PKC by phorbol esters slightly decreases the rate of Ca2+ efflux from the cytosol of Jurkat T cells following stimulation through the T cell receptor or stimulation in a receptor-independent manner by thapsigargin. On the other hand, phorbol ester treatment dramatically reduces the rate of Ca2+influx following stimulation. Phorbol ester treatment is without an effect on Ca2+ influx in a different T cell line, HSB. Down-regulation of PKCβI expression by 18-h phorbol ester treatment is associated with a loss of the response to acute phorbol ester treatment in Jurkat cells, suggesting that PKCβI may be the isozyme responsible for the effects on Ca2+ influx. Electroporation of an anti-PKCβI antibody, but not antibodies against PKCα or PKCγ, led to an increase in the rate of Ca2+ influx following stimulation. Taken together, these data suggest that PKCβI may be a component of the down-regulation of increases in [Ca2+] i associated with Jurkat T cell activation.

Activation-dependent degradation of protein kinase Cη

Prolonged activation of protein kinase Cs (PKCs) by long-term treatment of cells with phorbol ester tumor promoters down-regulates the expression of many PKCs. To investigate the molecular mechanisms involved in the down-regulation of PKCη, we expressed the novel PKCs η and θ and various mutant forms in baby hamster kidney cells. Upon overexpression, constitutively active PKCη, but not wild type or kinase-dead PKCη, underwent rapid degradation to generate several lower molecular weight polypeptides. When co-expressed with active kinases, kinase-dead PKCη with a pseudosubstrate site mutation designed to give an active conformation was down-regulated while the wild type PKCη was not. These results suggest requirements for kinase activity and an active conformation for down-regulation of PKCη. Treatment with the proteasome inhibitors N-Ac-Leu-Leu-norleucinal and lactacystin led to accumulation of PKCη proteolytic products and potentially ubiquitinated forms. While wild type PKCη localizes mostly to the detergent-soluble fraction of the cell, a significant portion of full-length constitutively active PKCη and of kinase-dead, active conformation PKCη were found in the detergent-insoluble fraction. Several proteolytic fragments of constitutively active PKCη also were found in the detergent insoluble fraction. These full-length and proteolytic fragments of PKCη in the detergent-insoluble fraction accumulated further in the presence of proteasome inhibitors. These data suggest that active conformation PKCη accumulates in the detergent-insoluble compartment, is degraded by proteolysis in the presence of kinase activity, and that the cleavage products undergo further degradation via ubiquitin-mediated degradation in the proteasome.

Regulation of ghrelin structure and membrane binding by phosphorylation.

The peptide hormone ghrelin requires Ser-3 acylation for receptor binding, orexigenic and anti-inflammatory effects. Functions of desacylghrelin are less well understood. In vitro kinase assays reveal that the evolutionarily conserved Ser-18 in the basic C-terminus is an excellent substrate for protein kinase C. Circular dichroism reveals that desacylghrelin is approximately 12% helical in aqueous solution and approximately 50% helical in trifluoroethanol. Ser-18-phosphorylation, Ser-18-Ala substitution, or Ser-3-acylation reduces the helical character in trifluoroethanol to approximately 24%. Both ghrelin and desacylghrelin bind to phosphatidylcholine:phosphatidylserine sucrose-loaded vesicles in a phosphatidylserine-dependent manner. Phosphoghrelin and phosphodesacylghrelin show greatly diminished phosphatidylserine-dependent binding. These results are consistent with binding of ghrelin and desacylghrelin to acidic lipids via the basic face of an amphipathic helix with Ser-18 phosphorylation disrupting both helical character and membrane binding.

Inhibitory Role of Ser-425 of the α1 2.2 Subunit in the Enhancement of Cav 2.2 Currents by Phorbol-12-myristate, 13-Acetate

Voltage-gated calcium channels (Cav) 2.2 currents are potentiated by phorbol-12-myristate, 13-acetate (PMA), whereas Cav 2.3 currents are increased by both PMA and acetyl-β-methylcholine (MCh). MCh-selective sites were identified in the α1 2.3 subunit, whereas the identified PMA sites responded to both PMA and MCh (Kamatchi, G. L., Franke, R., Lynch, C., III, and Sando, J. J. (2004) J. Biol. Chem. 279, 4102–4109; Fang, H., Franke, R., Patanavanich, S., Lalvani, A., Powell, N. K., Sando, J. J., and Kamatchi, G. L. (2005) J. Biol. Chem. 280, 23559–23565). The hypothesis that PMA sites in the α1 2.2 subunit are homologous to the PMA-responsive sites in α1 2.3 subunit was tested with Ser/Thr → Ala mutations in the α1 2.2 subunit. WT α1 2.2 or mutants were expressed in Xenopus oocytes in combination with β1b and α2/δ subunits. Inward current (IBa) was recorded using Ba2+ as the charge carrier. T422A, S1757A, S2108A, or S2132A decreased the PMA response. In contrast, S425A increased the response to PMA, and thus, it was considered an inhibitory site. Replacement of each of the identified stimulatory Ser/Thr sites with Asp increased the basal current and decreased the PMA-induced enhancement, consistent with regulation by phosphorylation at these sites. Multiple mutant combinations showed (i) greater inhibition than that caused by the single Ala mutations; (ii) that enhancement observed when Thr-422 and Ser-2108 are available may be inhibited by the presence of Ser-425; and (iii) that the combination of Thr-422, Ser-2108, and either Ser-1757 or Ser-2132 can provide a greater response to PMA when Ser-425 is replaced with Ala. The homologous sites inα1 2.2 andα1 2.3 subunits seem to be functionally different. The existence of an inhibitory phosphorylation site in the I-II linker seems to be unique to the α1 2.2 subunit.