Picomolar concentrations of lead stimulate brain protein kinase C

Recent growth studies in children suggest that there is no threshold for adverse effects from the universal exposure to inorganic lead. The biochemical mechanisms mediating low-level toxicity are unclear, but in several biological systems, lead alters calcium-mediated cellular processes and may mimic calcium in binding to regulatory proteins. Here we present evidence that lead stimulates diacylglycerol-activated calcium and phospholipid-dependent protein kinase, protein kinase C, partially purified from rat brain. Picomolar concentrations of lead are equivalent to micromolar calcium in kinase activation, so this regulatory enzyme is sensitive to the lead levels expected from current environmental exposure.     

Inositol trisphosphate, a novel second messenger in cellular signal transduction

There has recently been rapid progress in understanding receptors that generate intracellular signals from inositol lipids. One of these lipids, phosphatidylinositol 4,5-bisphosphate, is hydrolysed to diacylglycerol and inositol trisphosphate as part of a signal transduction mechanism for controlling a variety of cellular processes including secretion, metabolism, phototransduction and cell proliferation. Diacylglycerol operates within the plane of the membrane to activate protein kinase C, whereas inositol trisphosphate is released into the cytoplasm to function as a second messenger for mobilizing intracellular calcium.     

Induction of vanilloid receptor channel activity by protein kinase C

Capsaicin or vanilloid receptors (VRs) participate in the sensation of thermal and inflammatory pain. The cloned (VR1) and native VRs are non-selective cation channels directly activated by harmful heat, extracellular protons and vanilloid compounds. However, considerable attention has been focused on identifying other signalling pathways in VR activation; it is known that VR1 is also expressed in non-sensory tissue and may mediate inflammatory rather than acute thermal pain. Here we show that activation of protein kinase C (PKC) induces VR1 channel activity at room temperature in the absence of any other agonist. We also observed this effect in native VRs from sensory neurons, and phorbol esters induced a vanilloid-sensitive Ca2+ rise in these cells. Moreover, the pro-inflammatory peptide, bradykinin, and the putative endogenous ligand, anandamide, respectively induced and enhanced VR activity, in a PKC-dependent manner. These results suggest that PKC may link a range of stimuli to the activation of VRs.     

Inhibitors of protein kinase C

Protein kinase catalyzes the transfer of the γ-phosphoryl group from ATP to the hydroxyl groups o fprotein side chains, which plays critical roles in signal transduction pathways by transmitting extracellular signals across the plasma membrane and nuclear membrane to the destination sites in the cytoplasm and the nucleus. Protein kinase C (PKC) is a superfamily of phospholipid-dependent Ser/Thr kinase. There are at least 12 isozymes in PKC family.They are distributed in different tissues and play different roles in physiological processes. On account of their concern with a variety of pathophysiologic states, such as cancer,inflammatory conditions, autoimmune disorder, and cardiac diseases, the inhibitors, which can inhibit the activity of PKC and the interaction of cytokine with receptor, and interfere signal transduction pathway, may be candidates of therapeutic drugs. Therefore, intense efforts have been made to develop specific protein kinase inhibitors as biological tools and therapeutic agents. This article reviews the recent development of some of PKC inhibitors based on their interaction with different conserved domains and different inhibition mechanisms.     

Transduction in taste receptor cells requires cAMP-dependent protein kinase

In taste chemoreception, cyclic adenosine monophosphate (cAMP) appears to be one of the intracellular messengers coupling reception of stimulus to the generation of the response. The recent finding that sweet agents cause a GTP-dependent generation of cAMP poses the question of how this cytosolic messenger acts at the membrane of taste receptor cells. We have shown that cAMP causes a substantial depolarization in these cells. Here we show with whole-cell recordings and inside-out membrane patches that the depolarization caused by cAMP is accounted for by the action of cAMP-dependent protein kinase, which inactivates potassium channels predominantly of 44 pS conductance. Thus, intracellular signalling of the gustatory cells differs from that of olfactory and photoreceptor cells, where cyclic nucleotides control unspecific channels by binding to them rather than by inducing their phosphorylation.     

Interleukin-2 stimulates association of protein kinase C with plasma membrane

Interleukin-2 (IL-2) is a regulatory peptide important for the growth and differentiation of antigen-specific T lymphocytes and large granular lymphocytes. Interaction of IL-2 with its specific receptor results in the promotion of S-phase progression as well as, in certain circumstances, the production and release of gamma-interferon (IFN-gamma). Although the binding of IL-2 with high-affinity specific receptors has been well characterized, the intracellular mechanisms by which this ligand-receptor interaction promotes growth and differentiation are unknown. Here, we present evidence that IL-2/receptor interaction produces a rapid and transient redistribution of protein kinase C (PK-C) from the cytosol to the plasma membrane. Phorbol myristate acetate (PMA) also induces PK-C transposition in an analogous manner, except that PMA-induced PK-C transposition to the plasma membrane is apparently protracted. As phorbol esters have been shown to mimic IL-2 in the regulation of cellular proliferation as well as IFN-gamma production, the activation of PK-C by either phorbol esters or IL-2/receptor interaction seems to have a crucial role in signal transduction elicited by these extracellular messengers.     

Tyrosine kinase receptor indistinguishable from the c-met protein

Growth factor receptors with protein tyrosine kinase activity are central to the control of proliferation of both normal and malignant cells. Using anti-phosphotyrosine antibodies, we have previously identified a transmembrane glycoprotein with abnormally high protein tyrosine kinase activity in a human gastric tumour cell line (GTL-16). Electrophoresis under non-reducing conditions revealed that this kinase (relative molecular mass 145,000 (145 K)) is disulphide-linked to a 50K chain in an alpha beta-complex of 190K (p190). From its novel two-chain structure, we deduced that p190 was the prototype of a new class of tyrosine kinase receptors. We now show that p190 is indistinguishable from the protein encoded by the c-met proto-oncogene and that the alpha beta-subunit structure is conserved in other human cell lines. We also show that the high level of p190 found in the GTL-16 cell line is accompanied by amplification and overexpression of c-met. This provides the first example of a functional alteration of c-met in a human tumour cell line.     

Growth inhibition by protein kinase C late in mitogenesis

The importance of alpha-thrombin in the clotting cascade is well-known, but it is also a potent mitogen. Like many other mitogens, thrombin causes receptor-mediated activation of a phosphatidylinositol-specific phospholipase C (PLC), leading to the release of diacylglycerol and the subsequent activation of protein kinase C (refs 3-6). Protein kinase C is probably important in cell proliferation, as activation of this enzyme by phorbol esters promotes growth in many systems. Some growth factors have tyrosine kinase activity and function without activation of PLC or protein kinase C. In this report we show that alpha-thrombin retains its mitogenicity in vascular smooth muscle cells depleted of protein kinase C. Phorbol-12-myristate-13-acetate (PMA) is found to be a potent growth inhibitor when added to vascular smooth muscle cells with alpha-thrombin. Moreover, growth inhibition is maximal when protein kinase C is activated 4 hours after exposure to thrombin, long after the completion of 'early events' induced by thrombin. Thus, PMA probes an event late in the G1 phase of the cell cycle or at the G1-S transition.     

42,000-molecular weight EGF receptor has protein kinase activity

The epidermal growth factor (EGF) receptor and other growth factor receptors have been shown to possess tyrosine-specific protein kinase activity. Before the demonstration of kinase activity in growth factor receptors, tyrosine kinases of molecular weight (MW) 60,000 (60K) were found to be encoded by the src oncogene and other oncogenes related to src. Our earlier work on intracellular processing of the EGF receptor, a 170,000-MW polypeptide, provided evidence for proteolytic separation of well defined structural domains, and suggested to us the possibility of separating functional domains by limited proteolysis. The isolation of such kinase domains should facilitate comparison of the receptor/kinase with other well characterized kinases including those of oncogene origin. We report here the identification of a catalytically functional 42K kinase derived proteolytically from the isolated human EGF receptor. This fragment, comparable in size to pp60src, carries the kinase ATP-binding site, and functions catalytically even after detachment from the EGF-binding site and the major autophosphorylation region.     

Activation of protein kinase C augments evoked transmitter release

In view of the emerging role of the phosphoinositide system in cellular communication we examined its involvement in quantal-transmitter release, which is a key element in synaptic transmission. Transmitter release is normally activated by an increase in intracellular calcium, achieved either by entry of calcium ions through the presynaptic membrane or by intracellular calcium liberation. One of the targets of the phosphoinositide signalling system is the enzyme protein kinase C (PKC), which can be activated experimentally by tumour promoting phorbol esters, including 12-O-tetradecanoylphorbol-13-acetate (TPA). Such activation of PKC may be implicated in transmitter release in two ways. First, phorbol esters were found to increase secretion and enhance calcium currents; it might therefore be expected that they would increase synaptic transmitter release. But phorbol esters also inhibit the calcium current in dorsal root ganglion neurones. We report that the phorbol ester TPA augments synaptic transmission at the neuromuscular junction by increasing transmitter liberation. Activation of PKC also depends synaptic depression.     

A mutant protein kinase C that can transform fibroblasts

Expression of normal protein kinase C (PKC) isoenzymes in fibroblasts has been shown to alter growth regulation but has failed to induce complete transformation of the recipient cells. Here we report on a murine ultraviolet-induced fibrosarcoma cell line which has an unusual PKC subcellular distribution with 87% of the PKC activity associated with the membrane. We have cloned and sequenced the alpha-PKC complementary DNA from ultraviolet-induced-fibrosarcoma cells and from mouse Balb/c brain and found four point mutations in the fibrosarcoma PKC, of which three are in the highly conserved regulatory domain and one is in the conserved region of the catalytic domain. Expression of this mutant alpha-PKC gene in normal Balb/c 3T3 fibroblasts results in a fibrosarcoma-like PKC membrane localization and in cell transformation, as judged by their formation of dense foci, anchorage-independent growth and ability to induce solid tumours when inoculated into nude mice. By contast, transfectants expressing the normal alpha-PKC cDNA do not display a morphology typical of malignant transformed cells and fail to induce tumours in vivo. These findings demonstrate that point mutations in the primary structure of PKC modulate enzyme function and are responsible for inducing oncogenicity.     

Roles of protein kinase C in oocyte meiotic maturation and fertilization

Protein kinase C (PKC) is a superfamily of Ser/Thr protein kinases that is distributed widely in eukaryotes. It plays key regulatory roles at multiple steps of oocyte meiotic maturation and fertilization. During the process of meiotic maturation, the activation of PKC in cumulus cells stimulates meiotic maturation, whereas the activation of PKC in oocytes results in the inhibition of germinal vesicle breakdown. PKC activity increases following the meiotic maturation, and decreases at the transition of metaphase/anaphase in meiosis I, so as to facilitate the release of the first polar body and the entry of meiosis II. In fertilization of mammalian oocytes, PKC may act as one of the downstream targets of Ca2+ to stimulate the cortical granule exocytosis, release the oocytes from MII arrest and to induce pronucleus formation. PKC is also involved in the regulation of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Several PKC isoforms have been identified in mammalian oocytes, and there is evidence showing that classical PKCs may be the principal mediator of oocyte cortical reaction.     

Stimulus-dependent myristoylation of a major substrate for protein kinase C

Bacterial lipopolysaccharide (LPS), the major surface component of gram-negative bacteria, exerts a profound effect on the immune system by enhancing the release of proteins and arachidonic acid metabolites from macrophages (for review see ref. 1). The molecular mechanism(s) by which LPS induces these various secretory responses is unknown. We previously reported that LPS promotes the myristoylation of several macrophage proteins including one with a relative molecular mass (Mr) of 68K2. We have now found that by several criteria the 68K myristoylated protein is similar or identical to the 80/87K protein, a major specific substrate for protein kinase C (PKC) found in brain and fibroblasts (for review see refs 7,8). We have also found that the myristoylated PKC substrate is quantitatively associated with the membrane fraction. Myristoylation of the PKC substrate may target it to the membrane and constitute a transduction pathway for stimulus-response coupling.     

Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons

Inositol 1,4,5-trisphosphate (InsP3) mediates the effects of several neurotransmitters, hormones and growth factors by mobilizing Ca2+ from a vesicular, non-mitochondrial intracellular store. Many studies have indirectly suggested the endoplasmic reticulum (ER) to be the site of InsP3 action, though some have implicated the plasma membrane or a newly described smooth surfaced structure, termed the calciosome. Using antibodies directed against a purified InsP3-receptor glycoprotein, of relative molecular mass 260,000, in electron microscope immunocytochemical studies of rat cerebellar Purkinje cells, we have now localized the InsP3 receptor to ER, including portions of the rough endoplasmic reticulum, a population of smooth-membrane-bound organelles (smooth ER), a portion of subplasmalemmal cisternae and the nuclear membrane, but not to mitochondria or the cell membrane. These results suggest that in cerebellar Purkinje cells, InsP3-induced intracellular calcium release is not the property of a single organelle, but is effected by specialized portions of both rough and smooth ER, and possibly by other smooth surfaced structures. The present findings are the first immunocytochemical demonstration of an InsP3 receptor within a cell.     

Phosphorylation of the glucose transporter in vitro and in vivo by protein kinase C

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) is present in many mammalian tissues, and its important physiological protein substrates are only now beginning to be identified. A useful advance in identifying these intracellular substrates has been the recognition that the kinase is the receptor for phorbol esters, which stimulate phosphotransferase activity. Phorbol ester-induced changes in protein phosphorylation in intact cells may thus be taken, in part, as a probable indication of protein kinase C activation. The many cellular effects of phorbol esters include the stimulation of glucose uptake, although the response of glucose uptake to phorbol esters appears to be complex, apparently varying in response time and requirement for protein synthesis. Such observations prompted us to explore one possible explanation for the alteration of glucose uptake, namely, phosphorylation of the glucose transporter by protein kinase C. We report here that incubation of purified human erythrocyte glucose transporter with rat brain protein kinase C results in the phosphorylation of a protein of relative molecular mass (Mr) 50,000-60,000 which has subsequently been identified as the glucose transporter by specific immunoprecipitation with a monoclonal antibody. Immunoprecipitation of membrane proteins from 32P-labelled human erythrocytes revealed a phorbol ester-stimulated phosphorylation of the transporter. This covalent modification of the glucose transporter may thus, in part, underlie the ability of phorbol esters and certain hormones to stimulate glucose uptake.     

Tissue-specific expression of three distinct types of rabbit protein kinase C

We examined the structure of protein kinase C in an attempt to understand the molecular events connecting protein kinase C activation with the cellular response. Rabbit complementary DNA clones coding for three distinct types of protein kinase C, named alpha, beta and gamma, have been identified and sequenced. The deduced amino acid sequence for alpha, beta and gamma (673, 671 and 672 amino acids, respectively) are closely related. Kinases alpha and beta share an identical N-terminal sequence of 621 amino acid residues and their messenger RNAs arise from a single gene. The C-terminal halves of alpha, beta and gamma are protein kinase domains and are highly homologous to other protein kinases. The mRNAs for alpha, beta and gamma are expressed in various tissues with strikingly different tissue specificities. The one for gamma is found ubiquitously among various tissues, while those for alpha and beta predominate in the brain.     

Activation of protein kinase C potentiates isoprenaline-induced cyclic AMP accumulation in rat pinealocytes

The pineal gland has proven to be an excellent model for the study of adrenergic control systems. Noradrenaline, released from sympathetic nerve terminals in the pineal gland, regulates a large nocturnal increase in melatonin synthesis by stimulating the activity of arylalkylamine N-acetyltransferase (NAT, EC 2.3.1.87) 30-70-fold. An essential step in both the induction and maintenance of high NAT activity is an increase in intracellular cyclic AMP. Noradrenaline acts via beta-adrenoceptors to increase pineal cyclic AMP by activating adenylate cyclase, and the activation of pineal alpha 1-adrenoceptors potentiates beta-adrenergic stimulation not only of NAT but of both cyclic AMP and cyclic GMP. Here we describe investigations designed to test whether alpha 1-adrenergic potentiation of beta-adrenergic stimulation of pineal cyclic AMP involves protein kinase C. Our results suggest that kinase activation is involved and the data provide the first demonstration of a synergistic interaction between Ca2+-phospholipid-dependent protein kinase (protein kinase C) and neurotransmitter-dependent stimulation of cyclic AMP.     

MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin.

AGONISTS that stimulate protein kinase C (PKC) induce profound changes in cell morphology correlating with the reorganization of submembranous actin, but no direct connection between PKC and actin assembly has been identified. The myristoylated, alanine-rich C kinase substrate (MARCKS) binds calmodulin and is a predominant, specific substrate of PKC which is phosphorylated during macrophage and neutrophil activation , growth factor-dependent mitogenesis and neurosecretion; it is redistributed from plasma membrane to cytoplasm when phosphorylated and is involved in leukocyte motility. Here we report that MARCKS is a filamentous (F) actin crosslinking protein, with activity that is inhibited by PKC-mediated phosphorylation and by binding to calcium-calmodulin. MARCKS may be a regulated crossbridge between actin and the plasma membrane, and modulation of the actin crosslinking activity of the MARCKS protein by calmodulin and phosphorylation represents a potential convergence of the calcium-calmodulin and PKC signal transduction pathways in the regulation of the actin cytoskeleton.