Protein kinase Cdelta-dependent and -independent signaling in genotoxic response to treatment of desferroxamine, a hypoxia-mimetic agent

Protein kinase C (PKC) plays a critical role in diseases such as cancer, stroke, and cardiac ischemia and participates in a variety of signal transduction pathways including apoptosis, cell proliferation, and tumor suppression. Here, we demonstrate that PKC is proteolytically cleaved and translocated to the nucleus in a time-dependent manner on treatment of desferroxamine (DFO), a hypoxia-mimetic agent. Specific knockdown of the endogenous PKC by RNAi (sh-PKC) or expression of the kinase-dead (Lys376Arg) mutant of PKC (PKCKD) conferred modulation on the cellular adaptive responses to DFO treatment. Notably, the time-dependent accumulation of DFO-induced phosphorylation of Ser-139-H2AX (-H2AX), a hallmark for DNA damage, was altered by sh-PKC, and sh-PKC completely abrogated the activation of caspase-3 in DFO-treated cells. Expression of Lys376Arg-mutated PKC-enhanced green fluorescent protein (EGFP) appears to abrogate DFO/hypoxia-induced activation of endogenous PKC and caspase-3, suggesting that PKCKD-EGFP serves a dominant-negative function. Additionally, DFO treatment also led to the activation of Chk1, p53, and Akt, where DFO-induced activation of p53, Chk1, and Akt occurred in both PKC-dependent and -independent manners. In summary, these findings suggest that the activation of a PKC-mediated signaling network is one of the critical contributing factors involved in fine-tuning of the DNA damage response to DFO treatment. DNA damage; caspase-3; Akt     

Modulation of vascular smooth muscle cell migration by calcium/ calmodulin-dependent protein kinase II-delta 2

Previous studies demonstrated a requirement for multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in PDGF-stimulated vascular smooth muscle (VSM) cell migration. In the present study, molecular approaches were used specifically to assess the role of the predominant CaMKII isoform (₂ or _C) on VSM cell migration. Kinase-negative (K43A) and constitutively active (T287D) mutant forms of CaMKII₂ were expressed using recombinant adenoviruses. CaMKII activities were evaluated in vitro by using a peptide substrate and in intact cells by assessing the phosphorylation of overexpressed phospholamban on Thr¹⁷, a CaMKII-selective phosphorylation site. Expression of kinase-negative CaMKII₂ inhibited substrate phosphorylation both in vitro and in the intact cell, indicating a dominant-negative function with respect to exogenous substrate. However, overexpression of the kinase-negative mutant failed to inhibit endogenous CaMKII₂ autophosphorylation on Thr²⁸⁷ after activation of cells with ionomycin, and in fact, these subunits served as a substrate for the endogenous kinase. Constitutively active CaMKII₂ phosphorylated substrate in vitro without added Ca²⁺/calmodulin and in the intact cell without added Ca²⁺-dependent stimuli, but it inhibited autophosphorylation of endogenous CaMKII₂ on Thr²⁸⁷. Basal and PDGF-stimulated cell migration was significantly enhanced in cells expressing kinase-negative CaMKII₂, an effect opposite that of KN-93, a chemical inhibitor of CaMKII activation. Expression of the constitutively active CaMKII₂ mutant inhibited PDGF-stimulated cell migration. These studies point to a role for the CaMKII₂ isoform in regulating VSM cell migration. An inclusive interpretation of results using both pharmacological and molecular approaches raises the hypothesis that CaMKII₂ autophosphorylation may play an important role in PDGF-stimulated VSM cell migration. calcium/calmodulin-dependent protein kinase II; cell migration; adenovirus; autophosphorylation; chemotaxis; platelet-derived growth factor     

Immunoblotting PKC-delta: a cautionary note from the bench

Antibodies that specifically recognize signaling proteins (or individual phosphorylation events at specific residues in proteins of interest) have become important tools in the study of signaling pathways. However, the recognition properties of many commercially available antibodies have not been fully characterized. In the course of studies exploring PKC- phosphorylation mechanisms in cardiomyocytes, we have demonstrated that a BD Transduction Laboratories anti-PKC- MAb (generally viewed as an anti-PKC- protein antibody) recognizes PKC- in resting, but not in PMA-treated, cardiomyocytes. The observations that PKC- immunoreactivity is preserved when cultures are treated with PMA in the presence of a the PKC inhibitor GF-109203X and that PKC- immunoreactivity is restored by in vitro acid phosphatase treatment indicate that the epitope recognized by the BD Transduction Laboratories anti-PKC- MAb is masked by phosphorylation. The BD Transduction Laboratories MAb is poorly suited for studies that compare PKC- expression in resting and agonist-activated samples (or in studies of the relationship between PKC- phosphorylation and PKC- downregulation) because it artifactually displays PKC- phosphorylation as a decline in total PKC- protein. Other studies have shown that two anti-PKC--pY³¹¹ antibodies (manufactured by Cell Signaling Technology, Beverly, MA, and BioSource International, Camarillo, CA, respectively) specifically recognize stimulus-induced changes in PKC--Y³¹¹ phosphorylation on the endogenous PKC- enzyme, but the Cell Signaling Technology anti-PKC--pY³¹¹ antibody provides a better measure of Y³¹¹ phosphorylation in overexpressed PKC-. Collectively, these studies have identified features of anti-PKC- antibodies that affect the interpretation of immunoblot analysis experiments. These findings related to PKC- may be symptomatic of a more pervasive feature of immunoblot analysis studies of phosphoproteins in general. protein phosphorylation; signal transduction pathways; cardiomyocytes     

Activation of PLC-delta1 by Gi/o-coupled receptor agonists

The mechanism of phospholipase (PLC)- activation by G protein-coupled receptor agonists was examined in rabbit gastric smooth muscle. Ca²⁺ stimulated an eightfold increase in PLC-1 activity in permeabilized muscle cells. Treatment of dispersed or cultured muscle cells with three G_i/o-coupled receptor agonists (somatostatin, -opioid agonist [D-Pen²,D-Pen⁵]enkephalin, and A₁ agonist cyclopentyl adenosine) caused delayed increase in phosphoinositide (PI) hydrolysis (8- to 10-fold) that was strongly inhibited by overexpression of dominant-negative PLC-1(E341R/D343R; 65–76%) or constitutively active RhoA(G14V). The response coincided with capacitative Ca²⁺ influx and was not observed in the absence of extracellular Ca²⁺, but was partly inhibited by nifedipine (16–30%) and strongly inhibited by SKF-96365, a blocker of store-operated Ca²⁺ channels. Treatment of the cells with a G_q/13-coupled receptor agonist, CCK-8, caused only transient, PLC-1-mediated PI hydrolysis. Unlike G_i/o-coupled receptor agonists, CCK-8 activated RhoA and stimulated RhoA:PLC-1 association. Inhibition of RhoA activity with C3 exoenzyme or by overexpression of dominant-negative RhoA(T19N) or G₁₃ minigene unmasked a delayed increase in PI hydrolysis that was strongly inhibited by coexpression of PLC-1(E341R/D343R) or by SKF-96365. Agonist-independent capacitative Ca²⁺ influx induced by thapsigargin stimulated PI hydrolysis (8-fold), which was partly inhibited by nifedipine (25%) and strongly inhibited by SKF-96365 (75%) and in cells expressing PLC-1(E341R/D343R). Agonist-independent Ca²⁺ release or Ca²⁺ influx via voltage-gated Ca²⁺ channels stimulated only moderate PI hydrolysis (2- to 3-fold), which was abolished by PLC-1 antibody or nifedipine. We conclude that PLC-1 is activated by G_i/o-coupled receptor agonists that do not activate RhoA. The activation is preferentially mediated by Ca²⁺ influx via store-operated Ca²⁺ channels. phospholipase C; G protein     

Involvement of NH2 terminus of PKC-delta in binding to F-actin during activation of Calu-3 airway epithelial NKCC1

Direct binding of nonmuscle F-actin and the C2-like domain of PKC- (C2-like domain) is involved in hormone-mediated activation of epithelial Na-K-2Cl cotransporter isoform 1 (NKCC1) in a Calu-3 airway epithelial cell line. The goal of this study was to determine the site of actin binding on the 123-amino acid C2-like domain. Truncations of the C2-like domain were made by restriction digestion and confirmed by nucleotide sequencing. His₆-tagged peptides were expressed in bacteria, purified, and analyzed with a Coomassie blue stain for predicted size and either a 6xHis protein tag stain or an INDIA His₆ probe for expression of the His₆ tag. Truncated peptides were tested for competitive inhibition of binding of activated, recombinant PKC- with nonmuscle F-actin. Peptides from the NH₂-terminal region, but not the COOH-terminal region, of the C2-like domain blocked binding of activated PKC- to F-actin. The C2-like domain and three NH₂-terminal truncated peptides of 17, 83, or 108 amino acids blocked binding, with IC₅₀ values ranging from 1.2 to 2.2 nmol (6–11 µM). NH₂-terminal C2-like peptides also prevented methoxamine-stimulated NKCC1 activation and pulled down endogenous actin from Calu-3 cells. The proximal NH₂ terminus of the C2-like domain encodes a 1-sheet region. The amino acid sequence of the actin-binding domain is distinct from actin-binding domains in other PKC isotypes and actin-binding proteins. Our results indicate that F-actin likely binds to the 1-sheet region of the C2-like domain in airway epithelial cells. truncation; protein kinase C-; C2-like domain; slot blot assay; inhibitory constant; bumetanide; Na-K-2Cl cotransporter     

PKC-delta and CaMKII-delta 2 mediate ATP-dependent activation of ERK1/2 in vascular smooth muscle

ATP, a purinergic receptor agonist, has been shown to be involved in vascular smooth muscle (VSM) cell DNA synthesis and cell proliferation during embryonic and postnatal development, after injury, and in atherosclerosis. One mechanism that ATP utilizes to regulate cellular function is through activation of ERK1/2. In the present study, we provide evidence that ATP-dependent activation of ERK1/2 in VSM cells utilizes specific isoforms of the multifunctional serine/threonine kinases, PKC, and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) as intermediates. Selective inhibition of PKC- activity with rottlerin, or adenoviral overexpression of kinase-negative PKC-, attenuated the ATP- and phorbol 12,13-dibutyrate (PDBu)-stimulated ERK1/2 activation. Inhibition of PKC- activity with Gö-6976, or adenoviral overexpression of kinase-negative PKC-, was ineffective. Alternatively, treatment with KN-93, a selective inhibitor of CaMKII activation, or adenoviral overexpression of kinase-negative CaMKII-₂, inhibited ATP-dependent activation of ERK1/2 but had no effect on PDBu- or PDGF-stimulated ERK1/2. In addition, adenoviral overexpression of dominant-negative ras (Ad.HA-Ras^N17) partially inhibited the ATP- and PDBu-induced activation of ERK1/2 and blocked ionomycin- and EGF-stimulated ERK1/2, and inhibition of tyrosine kinases with AG-1478, an EGFR inhibitor, or the src family kinase inhibitor PP2 attenuated ATP-stimulated ERK1/2 activation. Taken together, these data indicate that PKC- and CaMKII-₂ coordinately mediate ATP-dependent transactivation of EGF receptor, resulting in increased ERK1/2 activity in VSM cells. protein kinase C-; calcium/calmodulin-dependent protein kinase II- ₂; extracellular signal-regulated kinase 1/2; epidermal growth factor receptor transactivation; adenovirus     

Calcium/calmodulin-dependent protein kinase II-delta isoform regulation of vascular smooth muscle cell proliferation

There is accumulating evidence that Ca²⁺-dependent signaling pathways regulate proliferation and migration of vascular smooth muscle (VSM) cells, contributing to the intimal accumulation of VSM that is a hallmark of many vascular diseases. In this study we investigated the role of the multifunctional serine/threonine kinase, calmodulin (CaM)-dependent protein kinase II (CaMKII), as a mediator of Ca²⁺ signals regulating VSM cell proliferation. Differentiated VSM cells acutely isolated from rat aortic media express primarily CaMKII gene products, whereas passaged primary cultures of de-differentiated VSM cells express primarily CaMKII₂, a splice variant of the gene. Experiments examining the time course of CaMKII isoform modulation revealed the process was rapid in onset following initial dispersion and primary culture of aortic VSM with a significant increase in CaMKII₂ protein and a significant decrease in CaMKII protein within 30 h, coinciding with the onset of DNA synthesis and cell proliferation. Attenuating the initial upregulation of CaMKII₂ in primary cultured cells using small-interfering RNA (siRNA) resulted in decreased serum-stimulated DNA synthesis and cell proliferation in primary culture. In passaged VSM cells, suppression of CaMKII₂ activity by overexpression of a kinase-negative mutant, or suppression of endogenous CaMKII content using multiple siRNAs, significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle analysis following either inhibitory approach indicated decreased proportion of cells in G1, an increase in proportion of cells in G2/M, and an increase in polyploidy, corresponding with accumulation of multinucleated cells. These results indicate that CaMKII₂ is specifically induced during modulation of VSM cells to the synthetic phenotypic and is a positive regulator of serum-stimulated proliferation. calmodulin kinase II; phenotype modulation     

nPKC{varepsilon}, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells

Airway goblet cell mucin secretion is controlled by agonist activation of P2Y₂ purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (IP₃), diacylglycerol, Ca²⁺ and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKC, nPKC, nPKC, and nPKC; of these, only nPKC translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149–L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKC, nPKC, and nPKC had the same levels of ATPS- and phorbol-1,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKC (14.6 and 23.5%, for ATPS and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKC exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATPS-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y₂-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKC knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKC KO mice relative to its WT littermates. We conclude that nPKC is the effector isoform downstream of P2Y₂-R activation in the goblet cell secretory response. The translocation of nPKC observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology. protein kinase C; mucins; goblet cells; exocytosis; airways; epithelium; lung     

Angiotensin II-induced activation of p21-activated kinase 1 requires Ca2+ and protein kinase C{delta} in vascular smooth muscle cells

ANG II promotes remodeling of vascular smooth muscle cells (VSMCs) in cardiovascular diseases. It has been shown to activate p21-activated kinase (PAK)1, a critical component of signaling pathways implicated in growth and migration. However, the detailed signaling mechanism by which ANG II induces PAK1 activation in VSMCs remains unclear. Therefore, we have examined the mechanism required for activation of PAK1 by ANG II in VSMCs. ANG II, through activation of the ANG II type 1 receptor, rapidly promotes phosphorylation of PAK1 in VSMCs via a pathway independent of transactivation of the epidermal growth factor receptor. Using selective agonists and inhibitors, we demonstrated that mobilization of intracellular Ca²⁺ and PKC activation are required for ANG II-induced PAK1 phosphorylation. Rottlerin, a PKC inhibitor, significantly blocked ANG II-induced PAK1 phosphorylation. Further support for this notion was provided through infection of VSMCs with adenovirus encoding a dominant-negative (dn)PKC, which also markedly reduced phosphorylation of PAK1 by ANG II. In this pathway, Ca²⁺ acts upstream of PKC because a Ca²⁺ ionophore rapidly induced PKC phosphorylation at Tyr311 and Ca²⁺-dependent PAK1 phosphorylation was blocked by rottlerin. In addition, dnPYK-2, dnRac, and antioxidants inhibited ANG II-induced PAK1 phosphorylation, suggesting that PYK-2, Rac, and reactive oxygen species are involved in the upstream signaling. Finally, dnPAK1 markedly inhibited ANG II-induced protein synthesis in VSMCs. These data provide a novel signaling pathway by which ANG II may contribute to vascular remodeling. vascular remodeling; signal transduction     

Involvement of G protein betagamma-subunits in diverse signaling induced by G(i/o)-coupled receptors: study using the Xenopus oocyte expression system

We studied the functions of -subunits of G_i/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl^– currents in oocytes expressing ₂-adrenoceptor and the protein kinase A-dependent Cl^– channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [D-Ala², D-Leu⁵]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing ₂-adrenoceptor-CFTR and 5-HT_1A receptor (5-HT_1AR), -opioid receptor, or GABA_B receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT_1AR. The 5-HT-induced enhancement of G_s-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin (G_t). The 5-HT-induced enhancement was further augmented by coexpression of the G-activated form of adenylate cyclase (AC) type II but not AC type III. Thus -subunits of G_i/o protein contribute to the enhancement of G_s-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT_1AR or -opioid receptor alone. They elicited Ca²⁺-activated Cl^– currents in oocytes coexpressing these receptors with the G-activated form of phospholipase C (PLC)-2 but not with PLC-1. These currents were inhibited by pretreatment with PTX and coexpression of G_t, suggesting that -subunits of G_i/o protein activate PLC-2 and then cause intracellular Ca²⁺ mobilization. Our results indicate that -subunits of G_i/o protein participate in diverse intracellular signals, enhancement of G_s-coupled receptor-mediated responses, and intracellular Ca²⁺ mobilization. G protein-coupled receptor; cystic fibrosis transmembrane conductance regulator gene; cross talk; electrophysiology     

Selective regulation by delta-PKC and PI 3-kinase in the assembly of the antiapoptotic TNFR-1 signaling complex in neutrophils

TNF is implicated in the attenuation of neutrophil constitutive apoptosis during sepsis. Antiapoptotic signaling is mediated principally through the TNF receptor-1 (TNFR-1). In adherent neutrophils, when -integrin signaling is activated, TNF phosphorylates TNFR-1 and activates prosurvival and antiapoptotic signaling. Previously, we identified the -PKC isotype and phosphatidylinositol (PI) 3-kinase as critical regulators of TNF signaling in adherent neutrophils. Both kinases associate with TNFR-1 in response to TNF and are required for TNFR-1 serine phosphorylation, NF-B activation, and inhibition of apoptosis. The purpose of this study was to examine the role of -PKC and PI 3-kinase in the assembly of TNFR-1 signaling complex that regulates NF-B activation and antiapoptotic signaling. Coimmunoprecipitation studies established that PI 3-kinase, -PKC, and TNFR-1 formed a signal complex in response to TNF. -PKC recruitment required both -PKC and PI 3-kinase activity, whereas PI 3-kinase recruitment was -PKC independent, suggesting that PI 3-kinase acts upstream of -PKC. An important regulatory step in control of antiapoptotic signaling is the assembly of the TNFR-1-TNFR-1-associated death domain protein (TRADD)-TNFR-associated factor 2 (TRAF2)-receptor interacting protein (RIP) complex that controls NF-B activation. Inhibition of either -PKC or PI 3-kinase decreased TNF-mediated recruitment of RIP and TRAF2 to TNFR-1. In contrast, TRADD recruitment was enhanced. Thus -PKC and PI 3-kinase are positive regulators of TNF-mediated association of TRAF2 and RIP with TNFR-1. Conversely, these kinases are negative regulators of TRADD association. These results suggest that -PKC and PI 3-kinase regulate TNF antiapoptotic signaling at the level of the TNFR-1 through control of assembly of a TNFR-1-TRADD-RIP-TRAF2 complex. inflammation; tumor necrosis factor receptor-1-associated death domain protein; receptor interacting protein; tumor necrosis factor receptor-associated factor 2; antiapoptotic signaling     

Protein kinase D protects against oxidative stress-induced intestinal epithelial cell injury via Rho/ROK/PKC-delta pathway activation

Protein kinase D (PKD) is a novel protein serine kinase that has recently been implicated in diverse cellular functions, including apoptosis and cell proliferation. The purpose of our present study was 1) to define the activation of PKD in intestinal epithelial cells treated with H₂O₂, an agent that induces oxidative stress, and 2) to delineate the upstream signaling mechanisms mediating the activation of PKD. We found that the activation of PKD is induced by H₂O₂ in both a dose- and time-dependent fashion. PKD phosphorylation was attenuated by rottlerin, a selective PKC- inhibitor, and by small interfering RNA (siRNA) directed against PKC-, suggesting the regulation of PKD activity by upstream PKC-. Activation of PKD was also blocked by a Rho kinase (ROK)-specific inhibitor, Y-27632, as well as by C3, a Rho protein inhibitor, demonstrating that the Rho/ROK pathway also mediates PKD activity in intestinal cells. In addition, H₂O₂-induced PKC- phosphorylation was inhibited by C3 treatment, further suggesting that PKC- is downstream of Rho/ROK. Interestingly, H₂O₂-induced intestinal cell apoptosis was enhanced by PKD siRNA. Together, these results clearly demonstrate that oxidative stress induces PKD activation in intestinal epithelial cells and that this activation is regulated by upstream PKC- and Rho/ROK pathways. Importantly, our findings suggest that PKD activation protects intestinal epithelial cells from oxidative stress-induced apoptosis. These findings have potential clinical implications for intestinal injury associated with oxidative stress (e.g., necrotizing enterocolitis in infants). Rho kinase; protein kinase C-     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A     

TGF-beta-regulated collagen type I accumulation: role of Src-based signals

Transforming growth factor- (TGF-) stimulates myofibroblast transdifferentiation, leading to type I collagen accumulation and fibrosis. We investigated the function of Src in TGF--induced collagen I accumulation. In human mesangial cells, PTyr416 Src (activated Src) was 3.3-fold higher in TGF--treated cells than in controls. Src activation by TGF- was blocked by rottlerin and by a dominant negative mutant of protein kinase C (PKC), showing that TGF- activates Src by a PKC-based mechanism. Pharmacological inhibitors and a dominant negative Src mutant prevented the increase in collagen type I secretion in cells exposed to TGF-. Similarly, on-target Src small interference RNA (siRNA) prevented type I collagen secretion in response to TGF-, but off-target siRNA complexes had no effect. It is well established in mesangial cells that upregulation of type I collagen by TGF- requires extracellular signal-regulated kinase 1/2 (ERK1/2), and we found that activation of ERK1/2 by TGF- requires Src. In conclusion, these results suggest that stimulation of collagen type I secretion by TGF- requires a PKC-Src-ERK1/2 signaling motif. mesangial cells; fibrosis; glomerulus; transforming growth factor-     

Laminin-alpha1 globular domains 3 and 4 induce heterotrimeric G protein binding to alpha-syntrophin's PDZ domain and alter intracellular Ca2+ in muscle

-Syntrophin is a component of the dystrophin glycoprotein complex (DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with -dystroglycan, which binds to extracellular matrix laminin. Syntrophin contains two pleckstrin homology (PH) domains and one PDZ domain. Because PH domains of other proteins are known to bind the -subunits of the heterotrimeric G proteins, whether this is also a property of syntrophin was investigated. Isolated syntrophin from rabbit skeletal muscle binds bovine brain G-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, - and -dystroglycan, or dystrophin precipitate a complex with G from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of G binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of G_s and G occurs and active G_s, measured as GTP-³⁵S bound, decreases. Because intracellular Ca²⁺ is elevated in Duchenne muscular dystrophy and G_s is known to activate the dihydropyridine receptor Ca²⁺ channel, whether laminin also altered intracellular Ca²⁺ was investigated. Laminin-1 decreases active (GTP-S-bound) G_s, and the Ca²⁺ channel is inhibited by laminin-1. The laminin ₁-chain globular domains 4 and 5 region, the region bound by DGC -dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to -dystroglycan inhibits G binding by syntrophin in C₂C₁₂ myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor. Duchenne muscular dystrophy; protein G -subunit; pleckstrin homology domain     

CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to increase plasma membrane localization and 86Rb+ uptake

The carboxy terminus (CT) of the colonic H⁺-K⁺-ATPase is required for stable assembly with the -subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HK₂, we selected 84 amino acids in the CT of the -subunit of mouse colonic H⁺-K⁺-ATPase (CT-HK₂) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HK₂ with CD63, recombinant CT-HK₂ and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HK₂ protein (but not CT-HK₁) coprecipitated with CD63, confirming stable assembly of HK₂ with CD63. In HEK-293 transfected with HK₂ plus ₁-Na⁺-K⁺-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HK₂/NK₁ and ⁸⁶Rb⁺ uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HK₂-NK₁ complex in the cell membrane. protein assembly; cell surface localization     

Involvement of PKCdelta and PKD in pulmonary microvascular endothelial cell hyperpermeability

The involvement of PKC, the isoforms of which are categorized into three subtypes: conventional (, I, II, and ), novel [, , , and µ (also known as PKD),], and atypical ( and /), in the regulation of endothelial monolayer integrity is well documented. However, isoform activity varies among different cell types. Our goal was to reveal isoform-specific PKC activity in the microvascular endothelium in response to phorbol 12-myristate 13-acetate (PMA) and diacylglycerol (DAG). Isoform activity was demonstrated by cytosol-to-membrane translocation after PMA treatment and phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein after PMA and DAG treatment. Specific isoforms were inhibited by using both antisense oligonucleotides and pharmacological agents. The data showed partial cytosol-to-membrane translocation of isoforms , I, and and complete translocation of PKC and PKD in response to PMA. Furthermore, antisense treatment and pharmacological studies indicated that the novel isoform PKC and PKD are both required for PMA- and DAG-induced MARCKS phosphorylation and hyperpermeability in pulmonary microvascular endothelial cells, whereas isoforms , I, and were dispensable with regard to these same phenomena. signal transduction; permeability; myristolated alanine-rich C kinase substrate; microvasculature; pulmonary endothelium     

Activation of large-conductance, Ca2+-activated K+ channels by cannabinoids

We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca²⁺-activated K⁺ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the -subunit of the BK channel (BK-), both - and ₁-subunits (BK-₁), or both - and ₄-subunits (BK-₄) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-₁ within a similar concentration range. Because methAEA could potentiate BK-, BK-₁, and BK-₄ with similar efficacy, the -subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK- and HEK-BK-₁ did not change intracellular Ca²⁺ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB₁ antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB₂ agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed. anandamide; channel opener     

PKC zeta participates in activation of inflammatory response induced by enteropathogenic E. coli

We showed previously that enteropathogenic Escherichia coli (EPEC) infection of intestinal epithelial cells induces inflammation by activating NF-B and upregulating IL-8 expression. We also reported that extracellular signal-regulated kinases (ERKs) participate in EPEC-induced NF-B activation but that other signaling molecules such as PKC may be involved. The aim of this study was to determine whether PKC is activated by EPEC and to investigate whether it also plays a role in EPEC-associated inflammation. EPEC infection induced the translocation of PKC from the cytosol to the membrane and its activation as determined by kinase activity assays. Inhibition of PKC by the pharmacological inhibitor rottlerin, the inhibitory myristoylated PKC pseudosubstrate (MYR-PKC-PS), or transient expression of a nonfunctional PKC significantly suppressed EPEC-induced IB phosphorylation. Although PKC can activate ERK, MYR-PKC-PS had no effect on EPEC-induced stimulation of this pathway, suggesting that they are independent events. PKC can regulate NF-B activation by interacting with and activating IB kinase (IKK). Coimmunoprecipitation studies showed that the association of PKC and IKK increased threefold 60 min after infection. Kinase activity assays using immunoprecipitated PKC-IKK complexes from infected intestinal epithelial cells and recombinant IB as a substrate showed a 2.5-fold increase in IB phosphorylation. PKC can also regulate NF-B by serine phosphorylation of the p65 subunit. Serine phosphorylation of p65 was increased after EPEC infection but could not be consistently attenuated by MYR-PKC-PS, suggesting that other signaling events may be involved in this particular arm of NF-B regulation. We speculate that EPEC infection of intestinal epithelial cells activates several signaling pathways including PKC and ERK that lead to NF-B activation, thus ensuring the proinflammatory response. inflammation; enteropathogenic Escherichia coli; nuclear factor-B; protein kinase C; IB kinase; extracellular signal-regulated kinase     

Functional analysis of Na+/K+-ATPase isoform distribution in rat ventricular myocytes

The Na⁺/K⁺-ATPase (NKA) is the main route for Na⁺ extrusion from cardiac myocytes. Different NKA -subunit isoforms are present in the heart. NKA-1 is predominant, although there is a variable amount of NKA-2 in adult ventricular myocytes of most species. It has been proposed that NKA-2 is localized mainly in T-tubules (TT), where it could regulate local Na⁺/Ca²⁺ exchange and thus cardiac myocyte Ca²⁺. However, there is controversy as to where NKA-1 vs. NKA-2 are localized in ventricular myocytes. Here, we assess the TT vs. external sarcolemma (ESL) distribution functionally using formamide-induced detubulation of rat ventricular myocytes, NKA current (I_Pump) measurements and the different ouabain sensitivity of NKA-1 (low) and NKA-2 (high) in rat heart. Ouabain-dependent I_Pump inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-2, K_1/2 = 0.38 ± 0.16 µM) that accounts for 29.5 ± 1.3% of I_Pump and a low-affinity isoform (NKA-1, K_1/2 = 141 ± 17 µM) that accounts for 70.5% of I_Pump. Detubulation decreased cell capacitance from 164 ± 6 to 120 ± 8 pF and reduced I_Pump density from 1.24 ± 0.05 to 1.02 ± 0.05 pA/pF, indicating that the functional density of NKA is significantly higher in TT vs. ESL. In detubulated myocytes, NKA-2 accounted for only 18.2 ± 1.1% of I_Pump. Thus, 63% of I_Pump generated by NKA-2 is from the TT (although TT are only 27% of the total sarcolemma), and the NKA-2/NKA-1 ratio in TT is significantly higher than in the ESL. The functional density of NKA-2 is 4.5 times higher in the T-tubules vs. ESL, whereas NKA-1 is almost uniformly distributed between the TT and ESL. T-tubules; Na⁺/K⁺ pump current; ouabain; external sarcolemma; detubulation