The thrombin receptor mediates functional activity‐dependent neuromuscular synapse reduction via protein kinase C activation in vitro

Activity‐dependent selective reduction of synaptic efficacy is expressed in an in vitro system involving mouse spinal cord and muscle cells. Thrombin or electrical stimulation of the innervating axons induces a decrease in neuromuscular synapse strength, and a specific thrombin inhibitor, hirudin, blocks the electrically evoked down‐regulation of synapse effectiveness. We further demonstrate that a thrombin receptor‐activating peptide (TRAP), SFLLRNPNDKYEPF, produces a decrement of synapse strength. Both TRAP and electrically evoked synapse decrement are prevented by the specific protein kinase C blocker calphostin C, and the TRAP‐evoked synapse decrement is unaffected by a specific protein kinase A blocker, H‐89. Thus, we propose that muscle activity, thrombin release, and thrombin receptor and PKC activation are initial steps in the process of the activity‐dependent synapse reduction expressed in our system. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 369–381, 1999     

Spacial isolation of protein kinase C activation in thrombin stimulated human platelets

Thrombin stimulation of human platelets is associated with turnover of inositol phospholipids, mobilization of intracellular Ca2+ stores, and activation of protein kinase C. However, within 5 minutes, the thrombin receptor desensitizes, but can be re-coupled to its effectors by stimulation of alpha 2-adrenergic receptors (Crouch and Lapetina, J. Biol. Chem. 263, 3363-3371, 1988). This effect of epinephrine was found to be inhibited by preincubation of platelets with phorbol ester, suggesting that protein kinase C was inhibitory. However, since thrombin also activated protein kinase C and epinephrine was active following thrombin stimulation of platelets, this implied that thrombin activation of protein kinase C may have been spacially isolated near the thrombin receptor and could not inactivate alpha 2-receptor activity. In the present paper, we have tested this possibility, and we present evidence which strongly favours the possibility that protein kinase C activation by receptors induces its local translocation to the cell membrane.     

C1qTNF-related protein-6 mediates fatty acid oxidation via the activation of the AMP-activated protein kinase

C1qTNF-related proteins (CTRPs) are involved in diverse processes including metabolism, inflammation host defense, apoptosis, cell differentiation, autoimmunity, hibernation, and organogenesis. However, the physiological role of CTRP6 remains poorly understood. Here we demonstrate that the globular domain of CTRP6 mediates the phosphorylation and activation of the 5′-AMP-activated protein kinase (AMPK) in skeletal muscle cells. In parallel with the activation of AMPK, CTRP6 induces the phosphorylation of acetyl coenzyme A carboxylase (ACC) and fatty acid oxidation in myocytes. Thus, CTRP6 plays a role in fatty acid metabolism via the AMPK-ACC pathway.     

Thrombin rapidly induces protein kinase D phosphorylation,and protein kinase C delta mediates the activation

Thrombin plays a critical role in hemostasis, thrombosis, and inflammation. However, the responsible intracellular signaling pathways triggered by thrombin are still not well defined. We report here that thrombin rapidly and transiently induces activation of protein kinase D (PKD) in aortic smooth muscle cells. Our data demonstrate that protein kinase C (PKC) inhibitors completely block thrombin-induced PKD activation, suggesting that thrombin induces PKD activation via a PKC-dependent pathway. Furthermore, our results show that thrombin rapidly induces PKC delta phosphorylation and that the PKC delta-specific inhibitor rottlerin blocks thrombin-induced PKD activation, suggesting that PKC delta mediates the thrombin-induced PKD activation. Using dominant negative approaches, we demonstrated that expression of a dominant negative PKC delta inhibits the phosphorylation and activation of PKD induced by thrombin, whereas neither PKC epsilon nor PKC zeta affects thrombin-induced PKD activation. In addition, our results of co-immunoprecipitation assays showed that PKD forms a complex with PKC delta in smooth muscle cells. Taken together, the findings of the present study demonstrate that thrombin induces activation of PKD and reveal a novel role of PKC delta in mediating thrombin-induced PKD activation in vascular smooth muscle cells.     

In vitro linoleic acid activation of protein kinase C

The importance of membrane fluidity in the activation of protein kinase C (PKC) was examined using the membrane fluidizer, linoleic acid, in a well-defined model membrane system. Biochemical and biophysical properties of the system were monitored. Linoleic acid activated PKC to a level of 50% of that observed for diacylglycerol. In contrast, linoleic acid did not directly interact with the phorbol ester binding site as did diacylglycerol. This was determined by the lack of involvement of the ionizable group of the fatty acid with activity and the enhancement of phorbol ester binding by linoleic acid and its ester analogs. The membrane fluidity of this model membrane system in the presence of linoleic acid was increased as determined by fluorescence polarization. This increased the availability of phospholipids, thus, explaining the linoleic acid-induced enhancement of phorbol ester binding. The PKC conformation as determined from intrinsic tryptophan fluorescence spectra was different for lipid mixtures containing linoleic acid or diacylglycerol correlating with the difference in biochemical activation properties. This study provides evidence that membrane fluidization is not the predominant function of the lipid activator in PKC activation, but may play a role in obtaining the preferred membrane state for maximal activation.     

Phosphorylation reactions in activity-dependent synapse modification at the neuromuscular junction during development

We have studied developmental activity-dependent synapse diminution in both an in vitro tissue culture chamber system and at the intact rodent neuromuscular junction (nmj). In both types of preparations, pre- and postsynaptic alterations in synapse structure and function are produced by manipulations of thrombin (Thr) and protein kinase C (PKC) activity. An opposing postsynaptic effect of PKC and protein kinase A (PKA) action on the acetycholine receptor (AChR) can be shown in vitro with PKA stabilizing and PKC destabilizing the nmj synapses. In vivo studies of normal junctional maturation show that changes in axonal inputs and postsynaptic receptor cluster morphology occur, to a substantial degree, independently of one another. Presynaptic actions of PKA are involved in the activity dependent synapse modulation that can be demonstrated in vitro. Late in the elimination process, (>12 days in vivo) the process becomes independent of PKC, implying that diverse, redundant mechanisms are involved in this important developmental process.     

RACK1 mediates activation of JNK by protein kinase C [corrected

Activation of the Jun-N-terminal kinase (JNK) signaling cascade by phorbol esters (TPA) or protein kinase C (PKC) is well documented, although the underlying mechanism is not known. Here, we demonstrate that the receptor for activated C kinase 1 (RACK1) serves as an adaptor for PKC-mediated JNK activation. Phosphorylation of JNK by PKC occurs on Ser129 and requires the presence of RACK1. Ser129 phosphorylation augments JNK phosphorylation by MKK4 and/or MKK7 and is required for JNK activation by TPA, TNFalpha, UV irradiation, and PKC, but not by anisomycin or MEKK1. Inhibition of RACK1 expression by siRNA attenuates JNK activation, sensitizes melanoma cells to UV-induced apoptosis, and reduces their tumorigenicity in nude mice. In finding the role of RACK1 in activation of JNK by PKC, our study also highlights the nature of crosstalk between these two signal-transduction pathways.     

A pheromone-binding protein mediates the bombykol-induced activation of a pheromone receptor in vitro

The enormous capacity of the male silkmoth Bombyx mori in recognizing and discriminating bombykol and bombykal is based on distinct sensory neurons in the antennal sensilla hairs. The hydrophobic pheromonal compounds are supposed to be ferried by soluble pheromone-binding proteins (PBPs) through the sensillum lymph toward the receptors in the dendritic membrane. We have generated stable cell lines expressing the candidate pheromone receptors of B. mori, BmOR-1 or BmOR-3, and assessed their responses to hydrophobic pheromone compounds dissolved by means of dimethyl sulfoxide. BmOR-1-expressing cells were activated by bombykol but also responded to bombykal, whereas cells expressing BmOR-3 responded to bombykal only. In experiments employing the B. mori PBP, no organic solvent was necessary to mediate an activation of BmOR-1 by bombykol, indicating that the PBP solubilizes the hydrophobic compound. Furthermore, the employed PBP selectively mediated a response to bombykol but not to bombykal, supporting a ligand specificity of PBPs. This study provides evidence that both distinct pheromone receptors and PBPs play an important role in insect pheromone recognition.     

The stereospecific activation of protein kinase C

Protein kinase C is synergistically activated by the presence of calcium, certain phospholipids and a diacylglycerol. The physiological activation of the enzyme appears to be determined by the availability of the diacylglycerol which is itself a product of (poly) phosphoinositol turnover. It is shown here that the diacylglycerol activation effect is stereospecific, with only the 1,2-sn-diglycerides being active. This demonstrates for the first time a stereospecific effector role for a membrane-bound lipid. Furthermore, this work strengthens the link forged between the highly potent and specific tumor promoters (such as the phorbol esters) and the diglycerides as activators of protein kinase C.     

Association of protein kinase C activation with IL 2 receptor expression

Tac antigen (as a measure of the IL 2 receptor) acquisition and regulation by IL 2, an antigen-receptor agonist (anti-T3), phorbol esters, and phytohemagglutinin (PHA) were studied. Phorbol esters stimulated de novo acquisition of Tac antigen, which was associated with the subcellular redistribution of protein kinase C (PK-C) from cytosol to particulate membranes of human T lymphocytes. PHA and anti-T3 (alpha-T3) antibody also stimulated a transient redistribution and activation of PK-C that reached a maximum within 20 min after stimulation. Both phorbol esters and alpha-T3 could increase Tac expression and stimulate PK-C translocation on 5 and 12 day activated T cells that were at the G0/G1 stage of the cell cycle due to IL 2 deprivation. Tac antigen-specific mRNA was seen in the nucleus within 2 hr after stimulation. In contrast, IL 2 alone could only increase Tac expression and stimulate PK-C translocation on day 5 but not day 12 activated T cells. IL 2 synergizes with alpha-T3 and phorbol ester for the regulation of Tac expression. Although IL 2 increased expression of Tac, the majority if not all of these receptors possessed low affinity for IL 2. These data suggest that the activation of PK-C is a common transmembrane signal shared by IL 2 and antigen stimulation. The results also imply that PK-C activation is necessary for the regulation of Tac antigen expression.     

A catalytically inactive form of protein kinase C-associated kinase/receptor interacting protein 4, a protein kinase C beta-associated kinase that mediates NF-kappa B activation,interferes with early B cell development

Protein kinase C-associated kinase (PKK)/receptor interacting protein 4 (RIP4) is a protein kinase C (PKC) beta-associated kinase that links PKC to NF-kappaB activation. The kinase domain of PKK is similar to that of RIP, RIP2, and RIP3. We show in this study that PKK is expressed early during lymphocyte development and can be detected in common lymphoid progenitor cells. Targeting of a catalytically inactive version of PKK to lymphoid cells resulted in a marked impairment in pro-B cell generation in the bone marrow. Although peripheral B cell numbers were markedly reduced, differentiation into follicular and marginal zone B cells was not defective in these mice. B-1a and B-1b B cells could not be detected in these mice, but this might be a reflection of the overall defect in B cell production observed in these animals. In keeping with a possible link to PKCbeta, peripheral B cells in these mice exhibit a defect in anti-IgM-mediated proliferation. These studies suggest that PKK may be required early in B cell development and for BCR-mediated B cell proliferation.     

Alpha-latrotoxin modulates the secretory machinery via receptor-mediated activation of protein kinase C

The hypothesis whether alpha-latrotoxin (LTX) could directly regulate the secretory machinery was tested in pancreatic beta cells using combined techniques of membrane capacitance (Cm) measurement and Ca2+ uncaging. Employing ramp increase in [Ca2+]i to stimulate exocytosis, we found that LTX lowers the Ca2+ threshold required for exocytosis without affecting the size of the readily releasable pool (RRP). The burst component of exocytosis in response to step-like [Ca2+]i increase generated by flash photolysis of caged Ca2+ was also speeded up by LTX treatment. LTX increased the maximum rate of exocytosis compared with control responses with similar postflash [Ca2+]i and shifted the Ca2+ dependence of the exocytotic machinery toward lower Ca2+ concentrations. LTXN4C, a LTX mutant which cannot form membrane pores or penetrate through the plasma membrane but has similar affinity for the receptors as the wild-type LTX, mimicked the effect of LTX. Moreover, the effects of both LTX and LTXN4C) were independent of intracellular or extracellular Ca2+ but required extracellular Mg2+. Our data propose that LTX, by binding to the membrane receptors, sensitizes the fusion machinery to Ca2+ and, hence, may permit release at low [Ca2+]i level. This sensitization is mediated by activation of protein kinase C.     

Opioid receptor contributes to ischemic preconditioning through protein kinase C activation in rabbits

Recent studies have reported that protection from ischemic preconditioning (PC) is blocked by the opioid receptor antagonist naloxone (NAL). We tested whether an opioid agonist could mimic PC in the rabbit heart, whether that protection involved protein kinase C (PKC) activation, and whether opioid receptors act in concert with other PKC-coupled receptors. Rabbit hearts were subjected to 30min coronary occlusions and were reperfused for either 3 (in situ) or 2 (in vitro) h. Infarct size was determined by staining with triphenyltetrazolium chloride. In untreated in situ hearts 38.5 ± 1.6% of the risk zone infarcted. PC with 5 min ischemia/10 min reperfusion significantly limited infarction to 12.7 ± 2.9% (p < 0.01). NAL infusion did not modify infarction (39.6 ± 1.6%) in non-PC hearts, but blocked the effect of one cycle of PC (34.4 ± 3.6% infarction). NAL, however, could not block cardioprotection when PC was amplified with 3 cycles of ischemia/reperfusion (9.9 ± 1.4% infarction, p < 0.01 vs. control). Morphine could also mimic ischemic preconditioning, but only at a dose much higher than would be used clinically (3 mg/kg). In isolated hearts pretreatment with morphine (0.3 M) significantly limited infarction to 9.3 ± 1.2% (p < 0.01 vs. 32.0 ± 3.1% in controls). This cardioprotective effect of morphine could be blocked by either the PKC inhibitor chelerythrine (30.4 ± 2.6% infarction) or NAL (34.0 ± 2.6% infarction). Neither chelerythrine nor NAL by itself modified infarction in non-PC hearts. NAL could not block protection from one cycle of PC in isolated hearts indicating that an intact innervation may be required for endogenous opioid production. Thus, opioid receptors, like other PKC-coupled receptors, participate in the triggering PC in the rabbit heart.     

Phenobarbital decreases hepatocyte EGF receptor expression independent of protein kinase C activation

The liver tumor promoter, phenobarbital, directly applied to cultured, adult rat hepatocytes at concentrations of greater than 1 mM, decreases cellular surface binding of EGF. This effect of phenobarbital resembles that of 4 beta-phorbol-12 alpha-myristate-13 beta-acetate (TPA) in that both decrease EGF receptor number, but do not affect receptor affinity. The effects of the two tumor promoters differ however, in that only TPA reduces high affinity EGF binding by A431 cells. They also differ in that TPA, but not phenobarbital, causes redistribution of protein kinase C from a soluble to a membranous hepatocyte subcellular fraction. These data indicate that decreased EGF binding is a common hepatocyte response to the tumor promoters, TPA and phenobarbital, but that this response can be mediated by either a TPA-activated, protein kinase C-dependent pathway or by a phenobarbital-sensitive, protein kinase C-independent pathway.     

Learning-induced activation of protein kinase C

PKC activation has been shown to mimic the biophysical consequences of classical conditioning in both rabbit hippocampus and Hermissenda type B cells. Furthermore, conditioning in rabbits results in the 24 h translocation of PKC from cytosol to membrane, which is probably responsible for mediating the biophysical consequences of conditioning. A model has been presented that suggests that long-term translocation of PKC occurs via the synergistic activation of a DG dependent pathway that activates PKC and a calcium dependent pathway that activates CaM kinase. Translocation of PKC to the plasma membrane, by altering ion channel properties, could subserve memory lasting for days, whereas translocation to the nuclear membrane could induce cellular change, by genomic regulation, lasting beyond days. We are, therefore, suggesting that protein kinase C may play a critical role in the formation of short, intermediate, and long-term associative memory.     

Ligands which effect human protein C activation by thrombin

This report documents attempts to mimic the rate enhancement effect of thrombomodulin on human alpha-thrombin-catalyzed activation of human protein C in the absence of exogenous calcium. Specifically the following tryptamine analogs at 1 mM concentration were shown to enhance the protein C activation rate relative to a control with no added effector at pH 8.3 (50 mM Tris-HCl, 0.1 M NaCl, 37 degrees C): serotonin, 1.2; tryptamine, 2.9; 5-fluorotryptamine, 4.4; 6-fluorotryptamine, 7.2. At much higher levels, e.g. 10 mM, all of the above effectors, as well as indole, showed a moderate inhibition of human protein C activation. ATP, a platelet release product, showed a sigmoidal inhibition pattern similar to that found previously for thrombin amidase, clotting, and esterase activity (Conery, B.G., and Berliner, L.J. (1983) Biochemistry 22, 369-375). Overall, the enhancement factors for human alpha-thrombin activation of protein C with the tryptamine analogs described above were remarkable when considering the effect of a simple ligand versus the natural activator, thrombomodulin.     

Rapid protein kinase D translocation in response to G protein-coupled receptor activation. Dependence on protein kinase C.

Protein kinase D (PKD)/protein kinase C (PKC) mu is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesin-induced GPCR activation.