AMP-activated Protein Kinase Signaling Activation by Resveratrol Modulates Amyloid-�� Peptide Metabolism

Alzheimer disease is an age-related neurodegenerative disorder characterized by amyloid-β (Aβ) peptide deposition into cerebral amyloid plaques. The natural polyphenol resveratrol promotes anti-aging pathways via the activation of several metabolic sensors, including the AMP-activated protein kinase (AMPK). Resveratrol also lowers Aβ levels in cell lines; however, the underlying mechanism responsible for this effect is largely unknown. Moreover, the bioavailability of resveratrol in the brain remains uncertain. Here we show that AMPK signaling controls Aβ metabolism and mediates the anti-amyloidogenic effect of resveratrol in non-neuronal and neuronal cells, including in mouse primary neurons. Resveratrol increased cytosolic calcium levels and promoted AMPK activation by the calcium/calmodulin-dependent protein kinase kinase-β. Direct pharmacological and genetic activation of AMPK lowered extracellular Aβ accumulation, whereas AMPK inhibition reduced the effect of resveratrol on Aβ levels. Furthermore, resveratrol inhibited the AMPK target mTOR (mammalian target of rapamycin) to trigger autophagy and lysosomal degradation of Aβ. Finally, orally administered resveratrol in mice was detected in the brain where it activated AMPK and reduced cerebral Aβ levels and deposition in the cortex. These data suggest that resveratrol and pharmacological activation of AMPK have therapeutic potential against Alzheimer disease.     

AMP-activated Protein Kinase Attenuates Nitric Oxide-induced ��-Cell Death

During the initial autoimmune response in type 1 diabetes, islets are exposed to a damaging mix of pro-inflammatory molecules that stimulate the production of nitric oxide by β-cells. Nitric oxide causes extensive but reversible cellular damage. In response to nitric oxide, the cell activates pathways for functional recovery and adaptation as well as pathways that direct β-cell death. The molecular events that dictate cellular fate following nitric oxide-induced damage are currently unknown. In this study, we provide evidence that AMPK plays a primary role controlling the response of β-cells to nitric oxide-induced damage. AMPK is transiently activated by nitric oxide in insulinoma cells and rat islets following IL-1 treatment or by the exogenous addition of nitric oxide. Active AMPK promotes the functional recovery of β-cell oxidative metabolism and abrogates the induction of pathways that mediate cell death such as caspase-3 activation following exposure to nitric oxide. Overall, these data show that nitric oxide activates AMPK and that active AMPK suppresses apoptotic signaling allowing the β-cell to recover from nitric oxide-mediated cellular stress.     

Sensing Domain Dynamics in Protein Kinase A-I�� Complexes by Solution X-ray Scattering

The catalytic (C) and regulatory (R) subunits of protein kinase A are exceptionally dynamic proteins. Interactions between the R- and C-subunits are regulated by cAMP binding to the two cyclic nucleotide-binding domains in the R-subunit. Mammalian cells express four different isoforms of the R-subunit (RIα, RIβ, RIIα, and RIIβ) that all interact with the C-subunit in different ways. Here, we investigate the dynamic behavior of protein complexes between RIα and C-subunits using small angle x-ray scattering. We show that a single point mutation in RIα, R333K (which alters the cAMP-binding properties of Domain B) results in a compact shape compared with the extended shape of the wild-type R·C complex. A double mutant complex that disrupts the interaction site between the C-subunit and Domain B in RIα, RIα_ABR333K·C(K285P), results in a broader P(r) curve that more closely resembles the P(r) profiles of wild-type complexes. These results together suggest that interactions between RIα Domain B and the C-subunit in the RIα·C complex involve large scale dynamics that can be disrupted by single point mutations in both proteins. In contrast to RIα·C complexes. Domain B in the RIIβ·C heterodimer is not dynamic and is critical for both inhibition and complex formation. Our study highlights the functional differences of domain dynamics between protein kinase A isoforms, providing a framework for elucidating the global organization of each holoenzyme and the cross-talk between the R- and C-subunits.     

AMP-activated Protein Kinase Mediates the Interferon-��-induced Decrease in Intestinal Epithelial Barrier Function

Impaired epithelial barrier function plays a crucial role in the pathogenesis of inflammatory bowel disease. Elevated levels of the pro-inflammatory cytokine, interferon-γ (IFNγ), are believed to be prominently involved in the pathogenesis of Crohn disease. Treatment of T₈₄ intestinal epithelial cells with IFNγ severely impairs their barrier properties measured as transepithelial electrical resistance (TER) or permeability and reduces the expression of tight junction proteins such as occludin and zonula occludens-1 (ZO-1). However, little is known about the signaling events that are involved. The cellular energy sensor, AMP-activated protein kinase (AMPK), is activated in response to cellular stress, as occurs during inflammation. The aim of this study was to investigate a possible role for AMPK in mediating IFNγ-induced effects on the intestinal epithelial barrier. We found that IFNγ activates AMPK by phosphorylation, independent of intracellular energy levels. Inhibition of AMPK prevents, at least in part, the IFNγ-induced decrease in TER. Furthermore, AMPK knockdown prevented the increased epithelial permeability, the decreased TER, and the decrease in occludin and ZO-1 caused by IFNγ treatment of T₈₄ cells. However, AMPK activity alone was not sufficient to cause alterations in epithelial barrier function. These data show a novel role for AMPK, in concert with other signals induced by IFNγ, in mediating reduced epithelial barrier function in a cell model of chronic intestinal inflammation. These findings may implicate AMPK in the pathogenesis of chronic intestinal inflammatory conditions, such as inflammatory bowel disease.Inflammatory bowel disease (IBD)² consists of two major subgroups, ulcerative colitis and Crohn disease (CD). A complex cascade of genetic, immunological, and bacterial factors contributes to IBD pathogenesis (1). In the healthy intestine, the epithelial barrier separates the luminal bacterial microbiota and other aspects of the external environment from cells of the mucosal immune system. In CD in particular, an impaired epithelial barrier (2, 3) leads to increased exposure of the immune system to commensal bacteria. Along with possible genetic defects in bacterial sensing, this might contribute to a dysregulated immune response leading to further epithelial damage and active episodes of IBD (4). Epithelial barrier dysfunction in CD is characterized by alterations in intercellular tight junctions (5), as well as by an excessive loss of water and salt into the lumen. An important immunological marker in CD is the existence of excessively high levels of the pro-inflammatory cytokine, interferon gamma (IFNγ) (6).IFNγ treatment of intestinal epithelial cell monolayers severely compromises their barrier integrity. Most importantly from a functional perspective, IFNγ causes a decrease in transepithelial electrical resistance (TER) and increases epithelial permeability (7, 8). These defects closely resemble observations in CD, where there is a disruption of intercellular tight junctional complexes. This effect is due to disruption of the apical actin cytoskeleton in conjunction with decreased expression, as well as increased internalization, of important tight junction proteins such as occludin and zonula occludens-1 (ZO-1) (8–11). Conversely, induction of epithelial apoptosis by IFNγ is believed to contribute little to barrier dysfunction (12). IFNγ also induces further alterations in epithelial function that include reduced expression of various ion transporters and associated decreases in epithelial ion transport (13, 14). Despite the influence of IFNγ on a number of epithelial functions, relatively little is known about intracellular signaling mechanisms mediating its effects following receptor activation. Recent studies demonstrated the involvement of phosphatidylinositol 3′-kinase (PI3K) in mediating IFNγ-induced effects on epithelial barrier function (11, 15). However, this is unlikely to be the only regulatory pathway involved. Indeed, increased expression of receptors for tumor necrosis factor core family members, such as the tumor necrosis factor receptor and LIGHT (homologous to lymphotoxin, shows inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes), can also occur in response to IFNγ and lead to changes in intestinal barrier function (16–18).The effects of IFNγ in intestinal epithelial cells resemble, at least in part, those of the cellular energy sensor, AMP-activated protein kinase (AMPK). Upon activation, AMPK restores intracellular ATP levels by stimulating energy-producing pathways, such as glucose uptake (19) and glycolysis, while inhibiting energy-consuming pathways, such as the synthesis of fatty acids or triglycerides (20, 21). In the intestine, energy-consuming processes include epithelial ion transport, and, indeed, AMPK has been shown to decrease intestinal ATP-consuming ion transport as well as the synthesis of various proteins (22, 23). Moreover, it has previously been demonstrated that ion transport processes are suppressed in intestinal biopsies from IBD patients (24–26).AMPK is usually activated in response to cellular stress that depletes intracellular ATP and elevates the AMP:ATP ratio (27, 28). AMPK-activating conditions include oxidative stress (29), hypoxia (30), and hypoglycemia (31). Binding of AMP to AMPK causes an increase in activity of 5-fold or less (32). Further, binding of AMP to AMPK makes AMPK a better substrate for upstream kinase activation, resulting in phosphorylation of the catalytic α-subunit of AMPK on the Thr¹⁷² residue and subsequently in a 50- to 100-fold activation of the enzyme (32). A number of upstream kinases for AMPK have been identified, with LKB1 (33, 34) or calmodulin kinase II (35–37) being the most important and well studied. However, recent studies also indicate that PI3K can activate AMPK (38, 39).The goal of this study was to determine whether AMPK mediates IFNγ-induced alterations in intestinal epithelial barrier function. We found that IFNγ activates AMPK in intestinal epithelial cells and AMPK inhibition prevents, at least in part, IFNγ-induced barrier dysfunction. Our data indicate a novel role for the cellular energy sensor, AMPK, in the regulation of intestinal epithelial barrier properties in a cell model of chronic inflammation. These findings may have implications for barrier function in the setting of chronic inflammatory processes, such as IBD.     

Phosphorylation of Serine 399 in LKB1 Protein Short Form by Protein Kinase Cζ Is Required for Its Nucleocytoplasmic Transport and Consequent AMP-activated Protein Kinase (AMPK) Activation

Two splice variants of LKB1 exist: LKB1 long form (LKB1_L) and LKB1 short form (LKB1_S). In a previous study, we demonstrated that phosphorylation of Ser-428/431 (in LKB1_L) by protein kinase Cζ (PKCζ) was essential for LKB1-mediated activation of AMP-activated protein kinase (AMPK) in response to oxidants or metformin. Paradoxically, LKB1_S also activates AMPK although it lacks Ser-428/431. Thus, we hypothesized that LKB1_S contained additional phosphorylation sites important in AMPK activation. Truncation analysis and site-directed mutagenesis were used to identify putative PKCζ phosphorylation sites in LKB1_S. Substitution of Ser-399 to alanine did not alter the activity of LKB1_S, but abolished peroxynitrite- and metformin-induced activation of AMPK. Furthermore, the phosphomimetic mutation (S399D) increased the phosphorylation of AMPK and its downstream target phospho-acetyl-coenzyme A carboxylase (ACC). PKCζ-dependent phosphorylation of Ser-399 triggered nucleocytoplasmic translocation of LKB1_S in response to metformin or peroxynitrite treatment. This effect was ablated by pharmacological and genetic inhibition of PKCζ, by inhibition of CRM1 activity and by substituting Ser-399 with alanine (S399A). Overexpression of PKCζ up-regulated metformin-mediated phosphorylation of both AMPK (Thr-172) and ACC (Ser-79), but the effect was ablated in the S399A mutant. We conclude that, similar to Ser-428/431 (in LKB1_L), Ser-399 (in LKB1_S) is a PKCζ-dependent phosphorylation site essential for nucleocytoplasmic export of LKB1_S and consequent AMPK activation.     

The PKARI�� Subunit of Protein Kinase A Modulates the Activation of p90RSK1 and Its Function

Previously, we showed that interactions between p90^RSK1 (RSK1) and the subunits of type I protein kinase A (PKA) regulate the activity of PKA and cellular distribution of active RSK1 (Chaturvedi, D., Poppleton, H. M., Stringfield, T., Barbier, A., and Patel, T. B. (2006) Mol. Cell Biol. 26, 4586–4600). Here we examined the role of the PKARIα subunit of PKA in regulating RSK1 activation and cell survival. In mouse lung fibroblasts, silencing of the PKARIα increased the phosphorylation and activation of RSK1, but not of RSK2 and RSK3, in the absence of any stimulation. Silencing of PKARIα also decreased the nuclear accumulation of active RSK1 and increased its cytoplasmic content. The increased activation of RSK1 in the absence of any agonist and changes in its subcellular redistribution resulted in increased phosphorylation of its cytoplasmic substrate BAD and increased cell survival. The activity of PKA and phosphorylation of BAD (Ser-155) were also enhanced when PKARIα was silenced, and this, in part, contributed to increased cell survival in unstimulated cells. Furthermore, we show that RSK1, PKA subunits, D-AKAP1, and protein phosphatase 2A catalytic subunit (PP2Ac) exist in a complex, and dissociation of RSK1 from D-AKAP1 by either silencing of PKARIα, depletion of D-AKAP1, or by using a peptide that competes with PKARIα for binding to AKAPs, decreased the amount of PP2Ac in the RSK1 complex. We also demonstrate that PP2Ac is one of the phosphatases that dephosphorylates RSK, but not ERK1/2. Thus, in unstimulated cells, the increased phosphorylation and activation of RSK1 after silencing of PKARIα or depletion of D-AKAP1 are due to decreased association of PP2Ac in the RSK1 complex.Cyclic AMP-dependent protein kinase (PKA)³ plays a pivotal role in manifesting an array of biological actions ranging from cell proliferation and tumorigenesis to increased inotropic and chronotropic effects in the heart as well as regulation of long term potentiation and memory. The PKA holoenzyme is a heterotetramer and consists of two catalytic (PKAc) subunits bound to a dimer of regulatory subunits. To date, four isoforms of the PKAc (PKAcα, PKAcβ, PKAcγ, and PKAcδ) and four isoforms of the regulatory subunits (RIα, RIβ, RIIα, and RIIβ) have been described (1). The various isoforms of PKA subunits are expressed differently in a tissue- and cell-specific manner (2). In addition to binding and inhibiting the activity of PKAc via their pseudo substrate region (3–6), the R subunits also interact with PKA-anchoring proteins (AKAPs) and facilitate the localization of PKA in specific subcellular compartments (7, 8). More than 50 AKAP family members have been described, and although most of these have a higher affinity for the RII subunits (9), certain AKAPs such as D-AKAP1 and D-AKAP2 preferentially bind the PKARIα subunit (10–12). Because the AKAPs also bind other signaling molecules such as phosphatases (PP2B) and kinases (protein kinase C), they act as scaffolds to organize and integrate specific signaling events within specific compartments in the cells (7, 8, 13, 14).We have shown that the PKARIα and PKAcα subunits of PKA interact with the inactive and active forms of p90^RSK1 (RSK1), respectively (15). Binding of inactive RSK1 to PKARIα decreases the interactions between PKARIα and PKAc, whereas the association of active RSK1 with PKAc increases interactions between PKARIα and PKAc such that larger amounts of cAMP are required to activate PKAc in the presence of active RSK1 (15). Moreover, the indirect (via subunits of PKA) interaction of RSK1 with AKAPs is required for the nuclear localization of active RSK1 (15), and disruption of the interactions of RSK1·PKA complex from AKAPs results in increased cytoplasmic distribution of active RSK1 with a concomitant increase in phosphorylation of its cytosolic substrates such as BAD and reduced cellular apoptosis (15). These findings show the functional and biological significance of RSK1·PKA·AKAP interactions.Besides inhibiting PKAc activity, the physiological role of PKARIα is underscored by the findings that mutations in the PKAR1A gene that result in haploinsufficiency of PKARIα are the underlying cause of Carney complex (CNC) (16, 17). CNC is an autosomal dominant multiple neoplasia syndrome in which myxomas of the skin, heart, and/or vicera are recurrent and also associated with high incidence of endocrine and ovarian tumors as well as Schwannomas (18–20). The majority of patients with the multiple neoplasia CNC syndrome harbor mutations in the PKAR1A gene (21) that result in PKARIα haploinsufficiency. Importantly, however, loss of heterozygosity or alterations in PKA activity may not contribute toward the tumorigenicity in either CNC patients or mouse model of CNC (21). This suggests that loss of function(s) of PKARIα other than inhibition of PKA activity is(are) involved in the enhanced tumorigenicity in CNC patients and in the murine CNC model.Because RSK1 regulates cell growth, survival, and tumorigenesis (22–27), and because its subcellular localization and ability to inhibit apoptosis is regulated by its interactions via PKARIα with AKAPs (15), we reasoned that in conditions such as CNC where PKARIα levels are decreased, the increase in tumorigenicity may emanate from aberrant regulation of the activity and/or subcellular localization of RSK1. Therefore, herein we have investigated whether PKARIα regulates the activation of RSK1 and its biological functions. Decreasing expression of PKARIα by small interfering RNA (siRNA) enhanced the activation of RSK1, but not RSK2 or RSK3, in the absence of an agonist such as EGF. This was accompanied by an increase in the cytoplasmic localization of the active RSK1 and enhanced cell survival in the absence of any growth factor. Silencing of PKARIα also increased PKAc activity and while part of the anti-apoptotic response could be attributed to an increase in PKAc activity, activation of RSK1 under basal conditions contributed significantly to cell survival. The elevation in RSK1 activity upon PKARIα silencing was not due to increased PKAc activity. Rather the activation of RSK1 in the absence of PKARIα was due to a decrease in PP2A in the RSK1 complex. These findings demonstrate a novel role for PKARIα in the regulation of RSK1 activation, a key enzyme that mediates the downstream actions of the ERK1/2 cascade.     

Neurotrophins Induce Neuregulin Release through Protein Kinase C�� Activation

Proper, graded communication between different cell types is essential for normal development and function. In the nervous system, heart, and for some cancer cells, part of this communication requires signaling by soluble and membrane-bound factors produced by the NRG1 gene. We have previously shown that glial-derived neurotrophic factors activate a rapid, localized release of soluble neuregulin from neuronal axons that can, in turn promote proper axoglial development (Esper, R. M., and Loeb, J. A. (2004) J. Neurosci. 24, 6218–6227). Here we elucidate the mechanism of this localized, regulated release by implicating the delta isoform of protein kinase C (PKC). Blocking the PKC delta isoform with either rottlerin, a selective antagonist, or small interference RNA blocks the regulated release of neuregulin from both transfected cells and primary neuronal cultures. PKC activation also leads to the rapid phosphorylation of the pro-NRG1 cytoplasmic tail on serine residues adjacent to the membrane-spanning segment, that, when mutated markedly reduce the rate of NRG1 activity release. These findings implicate this specific PKC isoform as an important factor for the cleavage and neurotrophin-regulated release of soluble NRG1 forms that have important effects in nervous system development and disease.The neuregulins (NRGs)² are a family of growth and differentiation factors with a broad range of functions during development and in the adult. NRGs are necessary for glial and cardiac development and participate in a wide range of biologic processes ranging from proper formation of peripheral nerves and the neuromuscular junction to tumor growth (2–9). The NRGs have also been implicated as both potential mediators and therapeutic targets for a number of human diseases including cancer, schizophrenia, and multiple sclerosis (10–12). NRGs function as mediators of cell-to-cell communication through a multitude of alternatively spliced isoforms arising from at least four distinct genes that bind to and activate members of the epidermal growth factor receptor family HER-2/3/4 (ErbB-2/3/4) (13–19).Although all known isoforms of the NRG1 gene have an epidermal growth factor-like domain sufficient to bind to and activate its receptors (20), products of this gene are divided into three classes based on structurally and functionally different N-terminal regions (21) The type I and II forms have a unique N-terminal, heparin-binding Ig-like domain (22–26). This Ig-like domain potentiates the biological activities of soluble NRG1 forms and leads to their highly selective tissue distributions through its affinity for specific cell-surface heparan sulfates (12, 20, 27, 28). These forms are first expressed as transmembrane precursors (pro-NRG1) that undergo proteolytic cleavage to release their soluble ectodomains. The type III NRG1 forms, on the other hand, are not typically released from cells, because their N-terminal domain consists of a cysteine-rich domain that can serve as a membrane tether making this form ideal for juxtacrine signaling. This form has been strongly implicated to be important peripheral nerve myelination (29–31).While many of the biological functions of type I/II NRG1 forms are less clear, their ability to be released from axons in the peripheral and central nervous systems in a regulated manner provides the potential for long range cell-cell communication not possible from membrane-bound forms. Studies examining the regulation of type I NRG1 release from neuronal axons have implicated protein kinase C (PKC) as a mediator of NRG1 release from pro-NRG1 in transfected cell lines (32). Subsequent studies in intact neurons found that PKC activation was sufficient to release NRG1 from sensory and motor neuron axons and that NRG1 could also be released by Schwann cell-derived neurotrophic factors, such as BDNF and GDNF (1). Recently, the β-secretase protease BACE1 has been suggested to cleave these NRG1 forms so that when it is knocked out in mice, deficits similar to those seen in NRG1 knockouts are seen (33, 34). These findings suggest that reciprocal communication between NRG1s and neurotrophins could be an important mechanisms for local axoglial communication that is critical for normal peripheral nerve development. Consistently, PKC has been implicated as a key mediator for the electrically mediated release of NRG1 from cultured cerebellar granule cells and pontine nucleus neurons (35).The PKC family consists of 10 serine/threonine kinases isoforms (α, βI, βII, γ, δ, ϵ, ζ, θ, λ, and η) each with a unique cellular distribution, target specificity, mechanism of activation, and function (36). One of these functions promotes the cleavage and release of soluble signaling proteins that are initially synthesized as membrane-spanning precursors. In addition to NRG1, other proteins released upon PKC activation include epidermal growth factor, transforming growth factor-α, amyloid precursor protein, l-selectin, and interleukins (1, 37–43). We hypothesize that neurotrophic factors induce the cleavage and release of NRG1 from pro-NRG1 through PKC activation. This hypothesis seems reasonable, because neurotrophin binding to the Trk family of neurotrophin receptor tyrosine kinases, but not the low affinity neurotrophin receptor p75 (44), activates phospholipase Cγ-mediated conversion of membrane-bound phosphatidylinositol bisphosphate to inositol triphosphate and diacylglycerol, which in turn, can activate PKC (45–48). Although this can be achieved using phorbol 12-myristate 13-acetate (PMA), a diacylglycerol analog sufficient to activate most PKC isozymes (48), the exact PKC isoform and mechanism by which this occurs is not known. Here, we demonstrate NRG1 is released from cells through direct activation of the PKCδ isoform using siRNA and PKC isoform-specific inhibitors in transfected Chinese hamster ovary (CHO) cells, PC12, and primary neuronal cultures. We further demonstrate that PKC activation induces rapid phosphorylation of the cytoplasmic tail of pro-NRG1 on specific serine residues that are required for efficient NRG1 activity release. These findings provide mechanistic insights into how highly localized, reciprocal signaling occurs along neuronal axons, which has important implications for normal development and disease.     

Mechanism of Activation and Functional Role of Protein Kinase C�� in Human Platelets

The novel class of protein kinase C (nPKC) isoform η is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCη using pharmacological and gene knock-out approaches. nPKCη was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y₁ receptor antagonist, or YM-254890, a G_q blocker, abolished 2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to activate nPKCη in platelets isolated from P2Y₁ and G_q knock-out mice. However, pretreatment of platelets with P2Y₁₂ receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCη phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCη was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin α_IIbβ₃ mediated outside-in signaling. Moreover, in the presence of SC-57101, a α_IIbβ₃ receptor antagonist, nPKCη dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cγ, a catalytic subunit of serine/threonine phosphatase, α_IIbβ₃ failed to dephosphorylate nPKCη. Thus, we conclude that ADP activates nPKCη via P2Y₁ receptor and is subsequently dephosphorylated by PP1γ phosphatase activated by α_IIbβ₃ integrin. In addition, pretreatment of platelets with η-RACK antagonistic peptides, a specific inhibitor of nPKCη, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCη positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.Platelets are the key cellular components in maintaining hemostasis (1). Vascular injury exposes subendothelial collagen that activates platelets to change shape, secrete contents of granules, generate thromboxane, and finally aggregate via activated α_IIbβ₃ integrin, to prevent further bleeding (2, 3). ADP is a physiological agonist of platelets secreted from dense granules and is involved in feedback activation of platelets and hemostatic plug stabilization (4). It activates two distinct G-protein-coupled receptors (GPCRs) on platelets, P2Y₁ and P2Y₁₂, which couple to G_q and G_i, respectively (5–8). G_q activates phospholipase Cβ (PLCβ), which leads to diacyl glycerol (DAG)² generation and calcium mobilization (9, 10). On the other hand, G_i is involved in inhibition of cAMP levels and PI 3-kinase activation (4, 6). Synergistic activation of G_q and G_i proteins leads to the activation of the fibrinogen receptor integrin α_IIbβ₃. Fibrinogen bound to activated integrin α_IIbβ₃ further initiates feed back signaling (outside-in signaling) in platelets that contributes to the formation of a stable platelet plug (11).Protein kinase Cs (PKCs) are serine/threonine kinases known to regulate various platelet functional responses such as dense granule secretion and integrin α_IIbβ₃ activation (12, 13). Based on their structure and cofactor requirements, PKCs are divided in to three classes: classical (cofactors: DAG, Ca²⁺), novel (cofactors: DAG) and atypical (cofactors: PIP₃) PKC isoforms (14). All the members of the novel class of PKC isoforms (nPKC), viz. nPKC isoforms δ, θ, η, and ε, are expressed in platelets (15), and they require DAG for activation. Among all the nPKCs, PKCδ (15, 16) and PKCθ (17–19) are fairly studied in platelets. Whereas nPKCδ is reported to regulate protease-activated receptor (PAR)-mediated dense granule secretion (15, 20), nPKCθ is activated by outside-in signaling and contributes to platelet spreading on fibrinogen (18). On the other hand, the mechanism of activation and functional role of nPKCη is not addressed as yet.PKCs are cytoplasmic enzymes. The enzyme activity of PKCs is modulated via three mechanisms (14, 21): 1) cofactor binding: upon cell stimulus, cytoplasmic PKCs mobilize to membrane, bind cofactors such as DAG, Ca²⁺, or PIP3, release autoinhibition, and attain an active conformation exposing catalytic domain of the enzyme. 2) phosphorylations: 3-phosphoinositide-dependent kinase 1 (PDK1) on the membrane phosphorylates conserved threonine residues on activation loop of catalytic domain; this is followed by autophosphorylations of serine/threonine residues on turn motif and hydrophobic region. These series of phosphorylations maintain an active conformation of the enzyme. 3) RACK binding: PKCs in active conformation bind receptors for activated C kinases (RACKs) and are lead to various subcellular locations to access the substrates (22, 23). Although various leading laboratories have elucidated the activation of PKCs, the mechanism of down-regulation of PKCs is not completely understood.The premise of dynamic cell signaling, which involves protein phosphorylations by kinases and dephosphorylations by phosphatases has gained immense attention over recent years. PP1, PP2A, PP2B, PHLPP are a few of the serine/threonine phosphatases reported to date. Among them PP1 and PP2 phosphatases are known to regulate various platelet functional responses (24, 25). Furthermore, PP1c, is the catalytic unit of PP1 known to constitutively associate with α_IIb and is activated upon integrin engagement with fibrinogen and subsequent outside-in signaling (26). Among various PP1 isoforms, recently PP1γ is shown to positively regulate platelet functional responses (27). Thus, in this study we investigated if the above-mentioned phosphatases are involved in down-regulation of nPKCη. Furthermore, reports from other cell systems suggest that nPKCη regulates ERK/JNK pathways (28). In platelets ERK is known to regulate agonist induced thromboxane generation (29, 30). Thus, we also investigated if nPKCη regulates ERK phosphorylation and thereby agonist-induced platelet functional responses.In this study, we evaluated the activation of nPKCη downstream of ADP receptors and its inactivation by an integrin-associated phosphatase PP1γ. We also studied if nPKCη regulates functional responses in platelets and found that this isoform regulates ADP-induced thromboxane generation, but not fibrinogen receptor activation in platelets.     

TWEAK-Independent Fn14 Self-Association and NF-κB Activation Is Mediated by the C-Terminal Region of the Fn14 Cytoplasmic Domain

The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TWEAK) is a pro-inflammatory and pro-angiogenic cytokine implicated in physiological tissue regeneration and wound repair. TWEAK binds to a 102-amino acid type I transmembrane cell surface receptor named fibroblast growth factor-inducible 14 (Fn14). TWEAK:Fn14 engagement activates several intracellular signaling cascades, including the NF-κB pathway, and sustained Fn14 signaling has been implicated in the pathogenesis of chronic inflammatory diseases and cancer. Although several groups are developing TWEAK- or Fn14-targeted agents for therapeutic use, much more basic science research is required before we fully understand the TWEAK/Fn14 signaling axis. For example, we and others have proposed that TWEAK-independent Fn14 signaling may occur in cells when Fn14 levels are highly elevated, but this idea has never been tested directly. In this report, we first demonstrate TWEAK-independent Fn14 signaling by showing that an Fn14 deletion mutant that is unable to bind TWEAK can activate the NF-κB pathway in transfected cells. We then show that ectopically-expressed, cell surface-localized Fn14 can self-associate into Fn14 dimers, and we show that Fn14 self-association is mediated by an 18-aa region within the Fn14 cytoplasmic domain. Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions. Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122. These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.     

Phosphorylation of ��6-Tubulin by Protein Kinase C�� Activates Motility of Human Breast Cells

Engineered overexpression of protein kinase Cα (PKCα) was previously shown to endow nonmotile MCF-10A human breast cells with aggressive motility. A traceable mutant of PKCα (Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364–2370) revealed that α6-tubulin is phosphorylated in cells expressing traceable PKCα and in vitro by wild type PKCα. Gain-of-function, single site mutations (Ser → Asp) were constructed at each PKC consensus site in α6-tubulin (Ser¹⁵⁸, Ser¹⁶⁵, Ser²⁴¹, and Thr³³⁷) to simulate phosphorylation. Following expression of each construct in MCF-10A cells, motility assays identified Ser¹⁶⁵ as the only site in α6-tubulin whose pseudophosphorylation reproduced the motile behavior engendered by PKCα. Expression of a phosphorylation-resistant mutant (S165N-α6-tubulin) resulted in suppression of MCF-10A cell motility stimulated either by expression of PKCα or by treatment with PKCα-selective activator diacylglycerol-lactone. MCF-10A cells treated with diacylglycerol-lactone showed strong phosphorylation of endogenous α-tubulin that could be blocked when S165N-α6-tubulin was expressed. The S165N mutant also inhibited intrinsically motile human breast tumor cells that express high endogenous PKCα levels (MDA-MB-231 cells) or lack PKCα and other conventional isoforms (MDA-MB-468 cells). Comparison of Myc-tagged wild type α6-tubulin and S165N-α6-tubulin expressed in MDA-MB-468 cells demonstrated that Ser¹⁶⁵ is also a major site of phosphorylation for endogenously active, nonconventional PKC isoforms. PKC-stimulated motility of MCF-10A cells was nocodazole-sensitive, thereby implicating microtubule elongation in the mechanism. These findings support a model in which PKC phosphorylates α-tubulin at Ser¹⁶⁵, leading to microtubule elongation and motility.     

A Biologically Active Sequence of the Laminin ��2 Large Globular 1 Domain Promotes Cell Adhesion through Syndecan-1 by Inducing Phosphorylation and Membrane Localization of Protein Kinase C��

Laminin-2 promotes basement membrane assembly and peripheral myelinogenesis; however, a receptor-binding motif within laminin-2 and the downstream signaling pathways for motif-mediated cell adhesion have not been fully established. The human laminin-2 α2 chain cDNAs cloned from human keratinocytes and fibroblasts correspond to the laminin α2 chain variant sequence from the human brain. Individually expressed recombinant large globular (LG) 1 protein promotes cell adhesion and has heparin binding activities. Studies with synthetic peptides delineate the DLTIDDSYWYRI motif (Ln2-P3) within the LG1 as a major site for both heparin and cell binding. Cell adhesion to LG1 and Ln2-P3 is inhibited by treatment of heparitinase I and chondroitinase ABC. Syndecan-1 from PC12 cells binds to LG1 and Ln2-P3 and colocalizes with both molecules. Suppression of syndecan-1 with RNA interference inhibits cell adhesion to LG1 and Ln2-P3. The binding of syndecan-1 with LG1 and Ln2-P3 induces the recruitment of protein kinase Cδ (PKCδ) into the membrane and stimulates its tyrosine phosphorylation. A decrease in PKCδ activity significantly reduces cell adhesion to LG1 and Ln2-P3. Taken together, these results indicate that the Ln2-P3 motif and LG1 domain, containing the motif, within the human laminin-2 α2 chain are major ligands for syndecan-1, which mediates cell adhesion through the PKCδ signaling pathway.     

Protein Kinase C�� Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B

Activation of the 5-hydroxytryptamine receptor 2B (5-HT_2B), a G_q/11 protein-coupled receptor, results in proliferation of various cell types. The 5-HT_2B receptor is also expressed on the pacemaker cells of the gastrointestinal tract, the interstitial cells of Cajal (ICC), where activation triggers ICC proliferation. The goal of this study was to characterize the mitogenic signal transduction cascade activated by the 5-HT_2B receptor. All of the experiments were performed on mouse small intestine primary cell cultures. Activation of the 5-HT_2B receptor by its agonist BW723C86 induced proliferation of ICC. Inhibition of phosphatidylinositol 3-kinase by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 decreased base-line proliferation but had no effect on 5-HT_2B receptor-mediated proliferation. Proliferation of ICC through the 5-HT_2B receptor was inhibited by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and by the inositol 1,4,5-trisphosphate receptor inhibitor Xestospongin C. Calphostin C, the α, β, γ, and μ protein kinase C (PKC) inhibitor Gö6976, and the α, β, γ, δ, and ζ PKC inhibitor Gö6983 inhibited 5-HT_2B receptor-mediated proliferation, indicating the involvement of PKC α, β, or γ. Of all the PKC isoforms blocked by Gö6976, PKCγ and μ mRNAs were found by single-cell PCR to be expressed in ICC. 5-HT_2B receptor activation in primary cell cultures obtained from PKCγ^−/− mice did not result in a proliferative response, further indicating the requirement for PKCγ in the proliferative response to 5-HT_2B receptor activation. The data demonstrate that the 5-HT_2B receptor-induced proliferative response of ICC is through phospholipase C, [Ca²⁺]_i, and PKCγ, implicating this PKC isoform in the regulation of cellular proliferation.Tight control of cell proliferation is essential to maintain organ size and function. Proliferation needs to be tightly regulated to maintain a critical mass of a particular cell type while preventing dysplasia or malignancy. Cell proliferation is regulated by a complex interaction between extrinsic and intrinsic factors. Extrinsic factors usually signal through cell surface receptors such as various growth factor receptors. 5-Hydroxytryptamine (5-HT,² serotonin) is well established as a neurotransmitter and a paracrine factor with over 90% of 5-HT produced by the gastrointestinal tract (1, 2). There is now substantial evidence that, together with these established functions, 5-HT is involved in the control of cell proliferation through various 5-HT receptors, in particular the 5-hydroxytryptamine receptor 2B (5-HT_2B (3–9)). The 5-HT_2B receptor is G_q/11 protein-coupled. Activation of the 5-HT_2B receptor regulates cardiac function, smooth muscle contractility, vascular physiology, and mood control. Recently it was demonstrated that activation of the 5-HT_2B receptor also induces proliferation of neurons, retinal cells (3, 4), hepatocytes (5), osteoblasts (8), and interstitial cells of Cajal (ICC) (9). ICC express the 5-HT_2B receptor, and activation by 5-HT induces proliferation of ICC (9). ICC are specialized, mesoderm-derived mesenchymal cells in the gastrointestinal tract. Their best known function is the generation of slow waves (10), but they also conduct and amplify neuronal signals (11, 12), release carbon monoxide to set the intestinal smooth muscle membrane potential gradient (13), and act as mechanosensors (14, 15). Loss of ICC has been associated with pathological conditions such as gastroparesis (16–18), infantile pyloric stenosis (19, 20), pseudo-obstruction (21, 22), and slow transit constipation (23), whereas increased proliferation of ICC or their precursors is associated with gastrointestinal stromal tumors (24).The mechanisms by which activation of the 5-HT_2B receptor results in increased proliferation are not well understood. In cultured cardiomyocytes, stimulation of the 5-HT_2B receptor activated both phosphatidylinositol 3-kinase (PI3′-K)/Akt and ERK1/2/mitogen-activated protein kinase (MAPK) signaling pathways to protect cardiomyocytes from apoptosis (25). On the other hand, the 5-HT2 subfamily of receptors are also known to couple to phospholipase C (PLC) (26–28).The objective of this study was to utilize the known expression of the 5-HT_2B receptor on ICC to determine whether proliferation through the 5-HT_2B receptor required PI3′-K or PLC. This study demonstrates that proliferation mediated by the 5-HT_2B receptor requires PLC, intracellular calcium release, and the ERK/MAPK signaling pathway and identifies the PKC isoform activated by the 5-HT_2B receptor in ICC as PKCγ.     

Mechanism Underlying IκB Kinase Activation Mediated by the Linear Ubiquitin Chain Assembly Complex

The linear ubiquitin chain assembly complex (LUBAC) ligase, consisting of HOIL-1L, HOIP, and SHARPIN, specifically generates linear polyubiquitin chains. LUBAC-mediated linear polyubiquitination has been implicated in NF-κB activation. NEMO, a component of the IκB kinase (IKK) complex, is a substrate of LUBAC, but the precise molecular mechanism underlying linear chain-mediated NF-κB activation has not been fully elucidated. Here, we demonstrate that linearly polyubiquitinated NEMO activates IKK more potently than unanchored linear chains. In mutational analyses based on the crystal structure of the complex between the HOIP NZF1 and NEMO CC2-LZ domains, which are involved in the HOIP-NEMO interaction, NEMO mutations that impaired linear ubiquitin recognition activity and prevented recognition by LUBAC synergistically suppressed signal-induced NF-κB activation. HOIP NZF1 bound to NEMO and ubiquitin simultaneously, and HOIP NZF1 mutants defective in interaction with either NEMO or ubiquitin could not restore signal-induced NF-κB activation. Furthermore, linear chain-mediated activation of IKK2 involved homotypic interaction of the IKK2 kinase domain. Collectively, these results demonstrate that linear polyubiquitination of NEMO plays crucial roles in IKK activation and that this modification involves the HOIP NZF1 domain and recognition of NEMO-conjugated linear ubiquitin chains by NEMO on another IKK complex.     

Direct Binding and Regulation of RhoA Protein by Cyclic GMP-dependent Protein Kinase Iα

Vascular smooth muscle cell (VSMC) tone is regulated by the state of myosin light chain (MLC) phosphorylation, which is in turn regulated by the balance between MLC kinase and MLC phosphatase (MLCP) activities. RhoA activates Rho kinase, which phosphorylates the regulatory subunit of MLC phosphatase, thereby inhibiting MLC phosphatase activity and increasing contraction and vascular tone. Nitric oxide is an important mediator of VSMC relaxation and vasodilation, which acts by increasing cyclic GMP (cGMP) levels in VSMC, thereby activating cGMP-dependent protein kinase Iα (PKGIα). PKGI is known to phosphorylate Rho kinase, preventing Rho-mediated inhibition of MLC phosphatase, promoting vasorelaxation, although the molecular mechanisms that mediate this are unclear. Here we identify RhoA as a target of activated PKGIα and show further that PKGIα binds directly to RhoA, inhibiting its activation and translocation. In protein pulldown and immunoprecipitation experiments, binding of RhoA and PKGIα was demonstrated via a direct interaction between the amino terminus of RhoA (residues 1–44), containing the switch I domain of RhoA, and the amino terminus of PKGIα (residues 1–59), which includes a leucine zipper heptad repeat motif. Affinity assays using cGMP-immobilized agarose showed that only activated PKGIα binds RhoA, and a leucine zipper mutant PKGIα was unable to bind RhoA even if activated. Furthermore, a catalytically inactive mutant of PKGIα bound RhoA but did not prevent RhoA activation and translocation. Collectively, these results support that RhoA is a PKGIα target and that direct binding of activated PKGIα to RhoA is central to cGMP-mediated inhibition of the VSMC Rho kinase contractile pathway.