Pharmacological Activation Of Protein Kinase Research Articles

Constitutive protein kinase G activation exacerbates stress-induced cardiomyopathy.

Heart failure is associated with high morbidity and mortality, and new therapeutic targets are needed. Preclinical data suggest that pharmacological activation of protein kinase G (PKG) can reduce maladaptive ventricular remodelling and cardiac dysfunction in the stressed heart. However, clinical trial results have been mixed and the effects of long-term PKG activation in the heart are unknown. We characterized the cardiac phenotype of mice carrying a heterozygous knock-in mutation of PKG1 (Prkg1R177Q/+ ), which causes constitutive, cGMP-independent activation of the kinase. We examined isolated cardiac myocytes and intact mice, the latter after stress induced by surgical transaortic constriction or angiotensin II (Ang II) infusion. Cardiac myocytes from Prkg1R177Q/+ mice showed altered phosphorylation of sarcomeric proteins and reduced contractility in response to electrical stimulation, compared to cells from wild type mice. Under basal conditions, young PKG1R177Q/+ mice exhibited no obvious cardiac abnormalities, but aging animals developed mild increases in cardiac fibrosis. In response to angiotensin II infusion or fixed pressure overload induced by transaortic constriction, young PKGR177Q/+ mice exhibited excessive hypertrophic remodelling with increased fibrosis and myocyte apoptosis, leading to increased left ventricular dilation and dysfunction compared to wild type litter mates. Long-term PKG1 activation in mice may be harmful to the heart, especially in the presence of pressure overload and neurohumoral stress. This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Subunit-Specific Augmentation of AMPA Receptor Ubiquitination by Phorbol Ester.

Excitatory neurotransmission relies on the precise targeting of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors to the neuronal plasma membrane. Activity-dependent ubiquitination of AMPA receptor (AMPAR) subunits sorts internalised receptors to late endosomes for degradation, which ultimately determines the number of AMPARs on neuronal membrane. Our recent study has demonstrated a functional cross-talk between the phosphorylation and ubiquitination of the GluA1 subunit in mammalian central neurons. However, the existence of such a cross modulation for the GluA2 subunit remains unknown. Here, we have shown that bicuculline induced GluA2 ubiquitination on the same lysine residues (Lys-870 and Lys-882) in the C-terminal as those elicited by the AMPA treatment. Interestingly, bicuculline-induced ubiquitination was markedly enhanced by the phospho-mimetic GluA2 S880E mutant. Pharmacological activation of protein kinase C (PKC) by phorbol ester, which mediates the phosphorylation of GluA2 at Ser-880, augmented bicuculline-induced ubiquitination of GluA2 in cultured neurons. This effect was specific for the GluA2 subunit because phorbol ester did not alter the level of GluA1 ubiquitination. However, phorbol ester-induced enhancement of GluA2 ubiquitination did not require Ser-880 phosphorylation. This suggests that pseudo-phosphorylation of Ser-880 is sufficient but is not necessary for the augmentation of bicuculline-induced GluA2 ubiquitination. Collectively, these data provide the first demonstration of subunit-specific modulation of AMPAR ubiquitination by the PKC-dependent signalling pathway in mammalian central neurons.

An EPAC1/PDE1C-Signaling Axis Regulates Formation of Leading-Edge Protrusion in Polarized Human Arterial Vascular Smooth Muscle Cells.

Pharmacological activation of protein kinase A (PKA) reduces migration of arterial smooth muscle cells (ASMCs), including those isolated from human arteries (HASMCs). However, when individual migration-associated cellular events, including the polarization of cells in the direction of movement or rearrangements of the actin cytoskeleton, are studied in isolation, these individual events can be either promoted or inhibited in response to PKA activation. While pharmacological inhibition or deficiency of exchange protein activated by cAMP-1 (EPAC1) reduces the overall migration of ASMCs, the impact of EPAC1 inhibition or deficiency, or of its activation, on individual migration-related events has not been investigated. Herein, we report that EPAC1 facilitates the formation of leading-edge protrusions (LEPs) in HASMCs, a critical early event in the cell polarization that underpins their migration. Thus, RNAi-mediated silencing, or the selective pharmacological inhibition, of EPAC1 decreased the formation of LEPs by these cells. Furthermore, we show that the ability of EPAC1 to promote LEP formation by migrating HASMCs is regulated by a phosphodiesterase 1C (PDE1C)-regulated “pool” of intracellular HASMC cAMP but not by those regulated by the more abundant PDE3 or PDE4 activities. Overall, our data are consistent with a role for EPAC1 in regulating the formation of LEPs by polarized HASMCs and show that PDE1C-mediated cAMP hydrolysis controls this localized event.

Pharmacological activation of protein kinase A improves memory loss and neuropathological changes in a mouse model of dementia of Alzheimer's type.

The study investigates the therapeutic potential of the protein kinase A (PKA) activator forskolin in cognitive deficits of mice. Streptozotocin (STZ) [3 mg/kg, intracerebroventricularly (i.c.v.)] was used to induce memory deficits in mice, whereas aged mice served as natural model of dementia. Forskolin (2.5, 5, and 10 mg/kg/day, oral) treatment was administered to i.c.v. STZ-treated and aged mice for 14 days. The Morris Water Maze test was used to evaluate learning and memory. Estimation of brain acetylcholinesterase (AChE) activity, brain glutathione, thiobarbituric acid-reactive species, brain myeloperoxidase levels, and histopathological studies were also performed. Both STZ i.c.v. and aging resulted in a marked decline in Morris Water Maze performance, reflecting impairment of learning and memory. STZ i.c.v.-treated mice and aged mice showed a marked accentuation of AChE activity, thiobarbituric acid-reactive species and myeloperoxidase levels along with a decrease in the glutathione level. Further, the stained micrographs of STZ-treated mice and aged mice indicated pathological changes, severe neutrophilic infiltration, and amyloid deposition. Forskolin treatment significantly attenuated STZ-induced and age-related memory deficits, and biochemical and histopathological alterations. The findings indicate that the PKA activator forskolin probably alleviated memory deficits by virtue of its anticholinesterase, antiamyloid, antioxidative, and anti-inflammatory effects. It is concluded that PKA could be explored as a potential therapeutic target in dementia.

Biological or pharmacological activation of protein kinase C alpha constrains hepatitis E virus replication

Although hepatitis E has emerged as a global health issue, there is limited knowledge of its infection biology and no FDA-approved medication is available. Aiming to investigate the role of protein kinases in hepatitis E virus (HEV) infection and to identify potential antiviral targets, we screened a library of pharmacological kinase inhibitors in a cell culture model, a subgenomic HEV replicon containing luciferase reporter. We identified protein kinase C alpha (PKCα) as an essential cell host factor restricting HEV replication. Both specific inhibitor and shRNA-mediated knockdown of PKCα enhanced HEV replication. Conversely, over-expression of the activated form of PKCα or treatment with its pharmacological activator strongly inhibited HEV replication. Interestingly, upon the stimulation by its activator, PKCα efficiently activates its downstream Activator Protein 1 (AP-1) pathway, leading to the induction of antiviral interferon-stimulated genes (ISGs). This process is independent of the JAK-STAT machinery and interferon production. However, PKCα induced HEV inhibition appears independent of the AP1 cascade. The discovery that activated PKCα restricts HEV replication reveals new insight of HEV-host interactions and provides new target for antiviral drug development.

Escitalopram Ameliorates Forskolin-Induced Tau Hyperphosphorylation in HEK239/tau441 Cells.

To investigate the effect of escitalopram (a widely used and highly efficacious antidepressant from the SSRI class) on tau hyperphosphorylation, HEK293/tau441 cells were pretreated with 4 μM of forskolin for 2 h. Then we treated the cells with different doses of escitalopram (0, 5, 10, 20, 40, 80 μM) for 22 h. We measured the phosphorylation level of tau by Western blotting. It was shown that escitalopram could protect tau from hyperphosphorylation induced by pharmacological activation of protein kinase A (PKA) at a dose of 20, 40, and 80 μM in vitro. Interestingly, the same dose of escitalopram could also increase the level of serine-9-phosphorylated GSK-3β (inactive form) and the phosphorylation level of Akt at Ser473 (active form) with no significant change in the level of total GSK-3β and Akt. Unexpectedly, 5-hydroxytryptamine 1A receptor (5-HT1A) agonist 8-OH-DPAT did not decrease forskolin-induced tau hyperphosphorylation. Our results suggest that escitalopram can ameliorate forskolin-induced tau hyperphosphorylation, which is not through the typical 5-HT1A pathway, and Akt/GSK-3β signaling pathway is involved. These findings may support an effective role of antidepressants in the prevention of dementia associated with depression in patients.

α2 noradrenergic receptor suppressed CaMKII signaling in spinal dorsal horn of mice with inflammatory pain

Intrathecal application of α2 noradrenergic receptor agonists effectively alleviates the pathological pain induced by peripheral tissue injury. However, the spinal antinociceptive mechanisms of α2 noradrenergic receptors remain to be characterized. The present study performed immunohistochemistry and western blot to elucidate the signaling pathway initiated by α2 noradrenergic receptors in spinal dorsal horn of mice, and identified calcium/calmodulin-dependent protein kinase II (CaMKII) as an important target for noradrenergic suppression of inflammatory pain. Our data showed that intraplantar injection of Complete Freund's Adjuvant (CFA) substantially enhanced CaMKII autophosphorylation at Threonine 286, which could be abolished by intrathecal administration of α2 noradrenergic receptor agonist clonidine. Gi protein-coupled α2 noradrenergic receptor might inhibit cAMP-dependent protein kinase (PKA) to disturb CaMKII signaling. We found that pharmacological activation of PKA in intact mice also enhanced spinal CaMKII autophosphorylation level, which was completely antagonized by clonidine. Moreover, direct PKA inhibition in CFA-injected mice mimicked the suppressive effect of α2 noradrenergic receptors on CaMKII. PKA inhibition has been shown to downregulate CaMKII by enhancing protein phosphatase activity. Consistent with this notion, spinal treatment with protein phosphatase inhibitor okadaic acid ruled out clonidine-mediated CaMKII dephosphorylation in CFA-injected mice. Through PKA/protein phosphatase/CaMKII pathway, clonidine noticeably decreased CFA-evoked phosphorylation of N-methyl-d-aspartate subtype glutamate receptor GluN1 and GluN2B subunit as well as α-amino-3-hydroxy-5-methylisoxazole-4-propionic Acid subtype glutamate receptor GluA1 subunit. These data suggested that interference with CaMKII signaling might represent an important mechanism underlying noradrenergic suppression of inflammatory pain.

Arc/Arg3.1 Translation Is Controlled by Convergent N-Methyl-D-aspartate and Gs-coupled Receptor Signaling Pathways

Arc/Arg3.1 is an immediate early gene whose expression is necessary for the late-phase of long-term potentiation (LTP) and memory consolidation. Whereas pathways regulating Arc transcription have been extensively investigated, less is known about the role of post-transcriptional mechanisms in Arc expression. Fluorescence microscopy experiments in cultured hippocampal neurons revealed that Arc protein level was dramatically increased by activation of the cAMP-dependent protein kinase (PKA) pathway, which is implicated in long-term memory. A PKA-dependent increase in Arc protein level was observed after pharmacological or synaptic activation of N-methyl-D-aspartate (NMDA) receptors, which play a critical role in both LTP induction and learning. Arc protein was also up-regulated by activation of PKA through G(s)-coupled dopamine and beta-adrenergic receptors, which regulate the late-phase of LTP and memory. When agonists for the NMDA and G(s)-coupled receptors were co-applied, they had an additive effect on Arc protein expression. Interestingly, G(s)-coupled receptor stimulation was ineffective in the presence of an NMDA receptor antagonist, suggesting calcium influx through the NMDA receptor plays a gating role in this pathway. Stimulation of the cAMP/PKA pathway did not affect Arc mRNA level or protein stability, identifying translational efficacy as the main determinant of Arc protein expression level. It is concluded that efficient Arc translation requires NMDA receptor activity, whereas a further enhancement can be achieved with activation of G(s)-coupled receptors. These experiments have, therefore, revealed remarkable similarities in the signaling pathways that control Arc expression and those that regulate LTP, learning, and memory.

Neural plasticity maintained high by activation of cyclic AMP-dependent protein kinase: An age-independent, general mechanism in cat striate cortex

Adult cats lack ocular dominance plasticity, showing little change in the ocular dominance distribution following monocular deprivation. Ocular dominance plasticity is also lost in kitten visual cortex that has been continuously infused with either catecholaminergic neurotoxin, β-adrenoreceptor blocker, or inhibitor of cyclic AMP-dependent protein kinase (protein kinase A). Complementarily, in adult cats we showed earlier that pharmacological activation of protein kinase A, albeit partially, restored ocular dominance plasticity. In the present study, we first asked whether, mediated by protein kinase A activation, the same molecular mechanisms could restore ocular dominance plasticity to kitten cortex that once lost the expression of plasticity due to prior pharmacological treatments. Concurrently with monocular deprivation, two kinds of cyclic AMP-related drugs (cholera toxin A-subunit or dibutyryl cyclic AMP) were directly infused in two types of aplastic kitten cortex pretreated with either 6-hydroxydopamine or propranolol. The combined treatment resulted in clear ocular dominance shift to the non-deprived eye, indicating that cortical plasticity was fully restored to aplastic kitten cortex. Next, to directly prove the sensitivity difference in protein kinase A activation between the immature and mature cortex, we compared the thus-obtained data in kittens with the published data derived from adult cats under the comparable experimental paradigm. The extent of ocular dominance changes following monocular deprivation was compared at different drug concentrations in the two preparations: the shifted ocular dominance distribution in aplastic kitten cortex infused with dibutyryl cyclic AMP at the lowest concentration tested and the W-shaped distribution in similarly treated adult cortex at a thousandfold-higher drug concentration that induced nearly maximal changes. We conclude that, irrespective of the animal’s age, activation of protein kinase A cascades is a general mechanism to maintain ocular dominance plasticity high, their sensitivity being substantially higher in the immature than mature cortex.

Phosphorylation by Protein Kinase A Inhibits Nuclear Import of 5-Lipoxygenase

The enzyme 5-lipoxygenase initiates the synthesis of leukotrienes from arachidonic acid. Protein kinase A phosphorylates 5-lipoxygenase on Ser(523), and this reduces its activity. We report here that phosphorylation of Ser(523) also shifts the subcellular distribution of 5-lipoxygenase from the nucleus to the cytoplasm. Phosphorylation and redistribution of 5-lipoxygenase could be produced by overexpression of the protein kinase A catalytic subunit alpha, by pharmacological activators of protein kinase A, and by prostaglandin E(2). Mimicking phosphorylation by replacing Ser(523) with glutamic acid caused cytoplasmic localization; replacement of Ser(523) with alanine prevented phosphorylation and redistribution in response to protein kinase A activation. Because Ser(523) is positioned within the nuclear localization sequence-518 of 5-lipoxygenase, the ability of protein kinase A to phosphorylate and alter the localization of green fluorescent protein fused to the nuclear localization sequence-518 peptide was also tested. Site-directed replacement of Ser(523) with glutamic acid within the peptide impaired nuclear accumulation; overexpression of the protein kinase A catalytic subunit alpha and pharmacological activation of protein kinase caused phosphorylation of the fusion protein at Ser(523), and the phosphorylated protein was found chiefly in the cytoplasm. Taken together, these results indicate that phosphorylation of Ser(523) inhibits the nuclear import function of a nuclear localization sequence, resulting in the accumulation of 5-lipoxygenase enzyme in the cytoplasm. As cytoplasmic localization can be associated with reduced leukotriene synthetic capacity, phosphorylation of Ser(523) serves to inhibit leukotriene production by both impairing catalytic activity and by placing the enzyme in a site that is unfavorable for action.

Converting Repulsion to Attraction with CICR

Growth cones guide neurites to their targets by monitoring chemoattractive and chemorepellant cues. Many cues elicit localized increases in cytosolic free calcium concentration ([Ca 2+ ] c ), but, curiously, both attractive and repulsive diffusible cues can increase [Ca 2+ ] c on the side of the growth cone facing the source of the gradient so that the growth cone turns toward (attraction) or away from (repulsion) the side with greater [Ca 2+ ] c . Ooashi et al . used focal laser-induced photolysis (FLIP) of caged Ca 2+ to transiently increase [Ca 2+ ] c in the growth cones of dorsal root ganglion (DRG) neurons grown on different substrata. Neurons grown on L1 or N-cadherin turned toward, whereas neurons grown on laminin turned away from, the side on which [Ca 2+ ] c was greater. Neurons grown on L1 and N-cadherin had more adenosine 3′,5′-monophosphate (cAMP) and showed increased cAMP binding to the regulatory subunits of cAMP-dependent protein kinase (PKA) than did neurons grown on laminin. Inhibition of cAMP signaling converted Ca 2+ -mediated attraction to repulsion, whereas pharmacological activation of PKA converted Ca 2+ -mediated repulsion to attraction. PKA phosphorylates ryanodine receptors (RyRs), facilitating calcium-induced calcium release (CICR), and pharmacological analysis combined with calcium imaging in conjunction with FLIP showed PKA-dependent activation of RyR-mediated CICR in growth cones on L1 or N-cadherin. Analysis of calcium signals and of the turning behavior of DRG neurons from mice lacking the type 3 RyR isoform (RyR3) implicated RyR3 in PKA-dependent CICR and attractive turning. Experiments with calcium buffers or with different amounts of the Ca 2+ -caging compound indicated that the source of the cytosolic Ca 2+ signal--rather than its amplitude--determined turning behavior. Thus, the authors conclude that a Ca 2+ signal that triggers CICR stimulates attractive turning, whereas a Ca 2+ signal without CICR elicits repulsion. N. Ooashi, A. Futatsugi, F. Yoshihara, K. Mikoshiba, H. Kamiguchi, Cell adhesion molecules regulate Ca 2+ -mediated steering of growth cones via cyclic AMP and ryanodine receptor type 3. J. Cell Biol. 170 , 1159-1167 (2005). [Abstract] [Full Text]

Intracellular cholesterol mobilization involved in the ABCA1/apolipoprotein-mediated assembly of high density lipoprotein in fibroblasts

Differential regulation has been suggested for cellular cholesterol and phospholipid release mediated by apolipoprotein A-I (apoA-I)/ABCA1. We investigated various factors involved in cholesterol mobilization related to this pathway. ApoA-I induced a rapid decrease of the cellular cholesterol compartment that is in equilibrium with the ACAT-accessible pool in cells that generate cholesterol-rich HDL. Pharmacological and genetic inactivation of ACAT enhanced the apoA-I-mediated cholesterol release through upregulation of ABCA1 and through cholesterol enrichment in the HDL generated. Pharmacological activation of protein kinase C (PKC) also decreased the ACAT-accessible cholesterol pool, not only in the cells that produce cholesterol-rich HDL by apoA-I (i.e., human fibroblast WI-38 cells) but also in the cells that generate cholesterol-poor HDL (mouse fibroblast L929 cells). In L929 cells, the PKC activation caused an increase in apoA-I-mediated cholesterol release without detectable change in phospholipid release and in ABCA1 expression. These results indicate that apoA-I mobilizes intracellular cholesterol for the ABCA1-mediated release from the compartment that is under the control of ACAT. The cholesterol mobilization process is presumably related to PKC activation by apoA-I.

Transmitter- and Activity-Dependent Inhibition of Neuronal Firing

Neuronal excitability depends upon the activation of voltage-gated Na + channels. Various neuromodulators reduce Na + channel availability (and thus neuronal excitability) through the activation of heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPCR)-mediated signaling pathways that lead to channel phosphorylation. Carr et al . used a combination of electrophysiological and pharmacological analysis to show that the reduction in Na + channel availability produced by GPCR activation closely resembled slow inactivation--a slow, voltage-dependent state that occurs in response to prolonged depolarization and during repetitive firing. The authors investigated the kinetics and voltage dependence of changes in Na + channel availability in mouse prefrontal cortex pyramidal neurons in response to treatment with the 5-HT 2a/c (5-hydroxytryptamine) receptor agonist 2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI). The effects of receptor stimulation were voltage-dependent, and DOI accelerated the development and extent of slow inactivation without affecting the time constant of recovery. Moreover, the effects of DOI, like slow inactivation, were inhibited by increases in the extracellular Na + concentration. Similarly, direct pharmacological activation of protein kinase A and protein kinase C promoted a slow inactivation-like state. The voltage dependence of phosphorylation-dependent channel inactivation occurred after phosphorylation, and mutational analysis indicated that channel phosphorylation was not required for depolarization-dependent development of slow inactivation. DOI accelerated the elevation of threshold that occurs during sustained firing (when the interspike membrane potential remains somewhat depolarized) and led to early failure of action potential generation. Thus, GPCR stimulation, by promoting a slow inactivation-like state, can selectively modulate sustained neuronal activity to produce a novel form of neuronal plasticity. D. B. Carr, M. Day, A. R. Cantrell, J. Held, T. Scheuer, W. A. Catterall, D. J. Surmeier, Transmitter modulation of slow, activity-dependent alterations in sodium channel availability endows neurons with a novel form of cellular plasticity. Neuron 39 , 793-806 (2003). [Online Journal]

NF-kappaB is involved in the regulation of CD154 (CD40 ligand) expression in primary human T cells.

Cognate interactions between CD154 (CD40 ligand, CD40L) on activated T cells and its receptor CD40 on various antigen-presenting cells are involved in thymus-dependent humoral immune responses and multiple other cell-mediated immune responses. We have studied the regulation of CD154 expression in human T cells after activation with anti-CD3 and anti-CD28 antibodies or after pharmacological activation of protein kinase C with phorbol 12-myristate 13-acetate, and the calcium ionophore ionomycin. Under these conditions, transcription of the CD154 gene was rapidly induced without requiring de novo protein synthesis. Pharmacological inhibitors of NF-kappaB activation down-regulated CD154 mRNA and protein levels. Cyclosporin A, an inhibitor of NF-AT activation, acted similarly, and the effects of both inhibitors were additive. A potential NF-kappaB binding site is present within the CD154 promoter at positions -1190 to - 1181. In electrophoretic mobility shift assays, this sequence was specifically bound by NF-kappaB present in nuclear extracts from activated T cells. Furthermore, in transient co-transfection of Jurkat T cells, p65 activated the transcription of a reporter construct containing a multimer of this NF-kappaB binding site. These observations demonstrate a role of NF-kappaB transcription factors in the regulation of CD40L expression in activated primary human T cells.

Inhibition of CaM kinase II activation and force maintenance by KN-93 in arterial smooth muscle.

Ca(+)/calmodulin-dependent protein kinase II (CaM kinase II) has been implicated in the regulation of smooth muscle contractility. The goals of this study were to determine: 1) to what extent CaM kinase II is activated by contractile stimuli in intact arterial smooth muscle, and 2) the effect of a CaM kinase II inhibitor (KN-93) on CaM kinase II activation, phosphorylation of myosin regulatory light chains (MLC(20)), and force. Both histamine (1 microM) and KCl depolarization activated CaM kinase II with a time course preceding maximal force development, and suprabasal CaM kinase II activation was sustained during tonic contractions. CaM kinase II activation was inhibited by KN-93 pretreatment (IC(50) approximately 1 microM). KN-93 inhibited histamine-induced tonic force maintenance, whereas early force development and MLC(20) phosphorylation responses during the entire time course were unaffected. Both force development and maintenance in response to KCl were inhibited by KN-93. Rapid increases in KCl-induced MLC(20) phosphorylation were also inhibited by KN-93, whereas steady-state MLC(20) phosphorylation responses were unaffected. In contrast, phorbol 12,13-dibutyrate (PDBu) did not activate CaM kinase II and PDBu-stimulated force development was unaffected by KN-93. Thus KN-93 appears to target a step(s) essential for force maintenance in response to physiological stimuli, suggesting a role for CaM kinase II in regulating tonic contractile responses in arterial smooth muscle. Pharmacological activation of protein kinase C bypasses the KN-93 sensitive step.

Evidence for mechanistic alterations of Ca2+ homeostasis in Type 2 diabetes mellitus.

Altered cytosolic Ca2+ is implicated in the aetiology of many diseases including diabetes but there are few studies on the mechanism(s) of the altered Ca2+ regulation. Using human lymphocytes, we studied cytosolic calcium (Cai) and various Ca2+ transport mechanisms in subjects with Type 2 diabetes mellitus and control subjects. Ca2+-specific fluorescent probes (Fura-2 and Fluo-3) were used to monitor the Ca2+ signals. Thapsigargin, a potent and specific inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), was used to study Ca2+- store dependent Ca2+ fluxes. Significant (P < 0.05) elevation of basal Cai levels was observed in lymphocytes from diabetic subjects. Cai levels were positively correlated with fasting, plasma glucose and HbAlc. There was also a significant (P < 0.05) reduction in plasma membrane calcium (PMCA) ATPase activity in diabetic subjects compared to controls. Cells from Type 2 diabetics exhibited an increased Ca2+ influx (as measured both by Fluo-3 fliorescence and C45a assays) as a consequence of of thapsigargin-mediated Ca2+ store depletion. Upon addition of Mn2+ (a surrogate of Ca2+), the fura-2 fluorescence decayed in an exponential fashion and the rate and extent of this decline was steeper and greater in cells from type 2 diabetic patients. There was also a significant (P < 0.05) difference in the Na+/Ca2+ exchange activity in Type 2 diabetic patients, both under resting conditions and after challenging the cells with thapsigargin, when the internal store Ca2+ sequestration was circumvented. Pharmacological activation of protein kinase C (PKC) in cells from patients resulted in only partial inhibition of Ca2+ entry. We conclude that cellular Ca2+ accumulation in cells from Type 2 diabetes results from (a) reduction in PMCA ATPase activity, (b) modulation of Na+/Ca2+ exchange and (3) increased Ca2+ influx across the plasma membrane.

Degradation of ZAP-70 Following Antigenic Stimulation in Human T Lymphocytes: Role of Calpain Proteolytic Pathway

T cell activation by the specific Ag results in dramatic changes of the T cell phenotype that include a rapid and profound down-regulation and degradation of triggered TCRs. In this work, we investigated the fate of the TCR-associated ZAP-70 kinase in Ag-stimulated T cells. T cells stimulated by peptide-pulsed APCs undergo an Ag dose-dependent decrease of the total cellular content of ZAP-70, as detected by FACS analysis and confocal microscopy on fixed and permeabilized T cell-APC conjugates and by Western blot on total cell lysates. The time course of ZAP-70 consumption overlaps with that of zeta-chain degradation, indicating that ZAP-70 is degraded in parallel with TCR internalization and degradation. Pharmacological activation of protein kinase C (PKC) does not induce ZAP-70 degradation, which, on the contrary, requires activation of protein tyrosine kinases. Two lines of evidence indicate that the Ca2+-dependent cysteine protease calpain plays a major role in initiating ZAP-70 degradation: 1) treatment of T cells with cell-permeating inhibitors of calpain markedly reduces ZAP-70 degradation; 2) ZAP-70 is cleaved in vitro by calpain. Our results show that, in the course of T cell-APC cognate interaction, ZAP-70 is rapidly degraded via a calpain-dependent mechanism.

E-cadherin Mediates Aggregation-dependent Survival of Prostate and Mammary Epithelial Cells through the Retinoblastoma Cell Cycle Control Pathway

E-cadherin and the retinoblastoma tumor suppressor (Rb) are traditionally associated with diverse regulatory aspects of cell growth and differentiation. However, we have discovered new evidence, which suggests that these proteins are functionally linked in a physiologic pathway required for cell survival and programmed cell death. Pharmacological activation of protein kinase C (PKC) or inducible overexpression and activation of the alpha isozyme of PKC (PKCalpha) resulted in approximately 60% apoptosis of mammary and prostate epithelial cells. Interestingly, the surviving cells had undergone dramatic aggregation concurrent with increased E-cadherin expression. When aggregation was inhibited by the addition of an E-cadherin-blocking antibody, apoptosis increased synergistically. We hypothesized that survival of the aggregated population was associated with contact-inhibited growth and that apoptosis might result from aberrant growth regulatory signals in non-aggregated, cycling cells. This hypothesis was confirmed by experiments that demonstrated that E-cadherin-dependent aggregation resulted in Rb-mediated G1 arrest and survival. Immunoblot analysis and flow cytometry revealed that hypophosphorylated Rb was present in non-aggregated, S phase cultures concurrent with synergistic cell death. We have also determined that the loss of membrane E-cadherin and subsequent hypophosphorylation of Rb in luminal epithelial cells preceded apoptosis induced by castration. These findings provide compelling evidence that suggests that E-cadherin-mediated aggregation results in Rb activation and G1 arrest that is critical for survival of prostate and mammary epithelial cells. These data also indicate that Rb can initiate a fatal growth signal conflict in non-aggregated, cycling cells when the protein is hypophosphorylated as these epithelial cells enter S phase.

Dephosphorylation of the catenins p120 and p100 in endothelial cells in response to inflammatory stimuli

Inflammatory mediators such as histamine and thrombin increase the tight-junction permeability of endothelial cells. Tight-junction permeability may be independently controlled, but is dependent on the adherens junction, where adhesion is achieved through homotypic interaction of cadherins, which in turn are associated with cytoplasmic proteins, the catenins. p120, also termed p120cas/p120ctn, and its splice variant, p100, are catenins. p120, originally discovered as a substrate of the tyrosine kinase Src, is also a target for a protein kinase C-stimulated pathway in epithelial cells, causing its serine/threonine dephosphorylation. The present study shows that pharmacological activation of protein kinase C stimulated a similar pathway in endothelial cells. Activation of receptors for agents such as histamine (H1), thrombin and lysophosphatidic acid in the endothelial cells also caused serine/threonine dephosphorylation of p120 and p100, suggesting physiological relevance. However, protein kinase C inhibitors, although blocking the effect of pharmacological activation of protein kinase C, did not block the effects due to receptor activation. Calcium mobilization and the myosin-light-chain-kinase pathway do not participate in p120/p100 signalling. In conclusion, endothelial cells possess protein kinase C-dependent and -independent pathways regulating p120/p100 serine/threonine phosphorylation. These data describe a new connection between inflammatory agents, receptor-stimulated signalling and pathways potentially influencing intercellular adhesion in endothelial cells.

Dephosphorylation of the catenins p120 and p100 in endothelial cells in response to inflammatory stimuli

Inflammatory mediators such as histamine and thrombin increase the tight-junction permeability of endothelial cells. Tight-junction permeability may be independently controlled, but is dependent on the adherens junction, where adhesion is achieved through homotypic interaction of cadherins, which in turn are associated with cytoplasmic proteins, the catenins. p120, also termed p120(cas)/p120(ctn), and its splice variant, p100, are catenins. p120, originally discovered as a substrate of the tyrosine kinase Src, is also a target for a protein kinase C-stimulated pathway in epithelial cells, causing its serine/threonine dephosphorylation. The present study shows that pharmacological activation of protein kinase C stimulated a similar pathway in endothelial cells. Activation of receptors for agents such as histamine (H1), thrombin and lysophosphatidic acid in the endothelial cells also caused serine/threonine dephosphorylation of p120 and p100, suggesting physiological relevance. However, protein kinase C inhibitors, although blocking the effect of pharmacological activation of protein kinase C, did not block the effects due to receptor activation. Calcium mobilization and the myosin-light-chain-kinase pathway do not participate in p120/p100 signalling. In conclusion, endothelial cells possess protein kinase C-dependent and -independent pathways regulating p120/p100 serine/threonine phosphorylation. These data describe a new connection between inflammatory agents, receptor-stimulated signalling and pathways potentially influencing intercellular adhesion in endothelial cells.