Stimulation of protein kinase C recruits covert calcium channels in Aplysia bag cell neurons

The modulation of voltage-activated calcium currents by protein kinases provides excitable cells with a mechanism for regulating their electrical behaviour. At the single channel level, modulation of calcium current has, to date, been characterized only in cardiac muscle, where beta-adrenergic agonists, acting through cyclic AMP-dependent protein kinase, enhance the calcium current by increasing channel availability and opening. We now report that enhancement of calcium current in the peptidergic bag cell neurons of Aplysia by protein kinase C occurs through a different mechanism, the recruitment of a previously covert class of calcium channel. Under control conditions, bag cell neurons contain only one class of voltage-activated calcium channel with a conductance of approximately 12 pS. After exposure to agents that activate protein kinase C, these neurons also express a second class of calcium channel with a different unitary conductance (approximately 24 pS) that is never seen in untreated cells.     

Inactivation of the sarcoplasmic reticulum calcium channel by protein kinase.

The ryanodine receptor protein of skeletal muscle sarcoplasmic reticulum (SR) membranes is a calcium ion channel which allows movement of calcium from the SR lumen into the cytoplasm during muscle activation. Gating of this channel is modulated by a number of physiologically important substances including calcium. Interestingly, calcium has both activating and inactivating effects which are concentration- and tissue-specific. In skeletal muscle, calcium-dependent inactivation of calcium release occurs at concentrations reached physiologically, suggesting that calcium may modulate the release process by a negative feedback mechanism. To determine the cellular mechanism responsible for calcium-dependent inactivation, we have investigated the ability of protein phosphorylation to affect single channel gating behaviour using the patch clamp technique. Here we demonstrate that the ryanodine receptor protein/calcium release channel of skeletal muscle SR is inactivated under conditions permissive for protein phosphorylation. This inactivation is reversed by the application of phosphatase and prevented by a peptide inhibitor specific for calcium/calmodulin-dependent protein kinase II. The results provide evidence for an endogenous protein kinase which is closely associated with the ryanodine receptor protein and regulates channel gating.     

Structure of mammalian AMPK and its regulation by ADP

The heterotrimeric AMP-activated protein kinase (AMPK) has a key role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels it activates energy-producing pathways and inhibits energy-consuming processes. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer. AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain: AMP binding to the γ-regulatory domain promotes phosphorylation by the upstream kinase, protects the enzyme against dephosphorylation, as well as causing allosteric activation. Here we show that ADP binding to just one of the two exchangeable AXP (AMP/ADP/ATP) binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active mammalian AMPK displays significantly tighter binding to ADP than to Mg-ATP, explaining how the enzyme is regulated under physiological conditions where the concentration of Mg-ATP is higher than that of ADP and much higher than that of AMP. We have determined the crystal structure of an active AMPK complex. The structure shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide-binding site that mediates protection against dephosphorylation. From our biochemical and structural data we develop a model for how the energy status of a cell regulates AMPK activity.     

An essential role for postsynaptic calmodulin and protein kinase activity in long-term potentiation

The phenomenon of long-term potentiation (LTP), a long lasting increase in the strength of synaptic transmission which is due to brief, repetitive activation of excitatory afferent fibres, is one of the most striking examples of synaptic plasticity in the mammalian brain. In the CA1 region of the hippocampus, the induction of LTP requires activation of NMDA (N-methyl-D-aspartate) receptors by synaptically released glutamate with concomitant postsynaptic membrane depolarization. This relieves the voltage-dependent magnesium block of the NMDA-receptor ion channel, allowing calcium to flow into the dendritic spine. Although calcium has been shown to be a necessary trigger for LTP (refs 11, 12), little is known about the immediate biochemical processes that are activated by calcium and are responsible for LTP. The most attractive candidates have been calcium/calmodulin-dependent protein kinase II (CaM-KII) (refs 13-16), protein kinase C (refs 17-19), and the calcium-dependent protease, calpain. Extracellular application of protein kinase inhibitors to the hippocampal slice preparation blocks the induction of LTP (refs 21-23) but it is unclear whether this is due to a pre- and/or postsynaptic action. We have found that intracellular injection into CA1 pyramidal cells of the protein kinase inhibitor H-7, or of the calmodulin antagonist calmidazolium, blocks LTP. Furthermore, LTP is blocked by the injection of synthetic peptides that are potent calmodulin antagonists and inhibit CaM-KII auto- and substrate phosphorylation. These findings demonstrate that in the postsynaptic cell both activation of calmodulin and kinase activity are required for the generation of LTP, and focus further attention on the potential role of CaM-KII in LTP.     

Protein kinase C activation induces conductance changes in Hermissenda photoreceptors like those seen in associative learning

Phosphorylation of ion channels has been suggested as one molecular mechanism responsible for learning-produced long-term changes in neuronal excitability. Persistent training-produced changes in two distinct K+ currents (IA (ref. 2), IK-Ca (refs 3,4)) and a voltage-dependent calcium current (ICa; refs 3,4) have previously been shown to occur in type B photoreceptors of Hermissenda, as a result of associative learning. But the identity of the phosphorylation pathway(s) responsible for these changes has not as yet been determined. Injections of cyclic AMP-dependent protein kinase reduce a K+ current (IK) in B cells which is different from those changed by training, but fails to reduce IA and IK-Ca. Phosphorylase b kinase (an exogenous calcium/calmodulin-dependent kinase) reduces IA, but whether IK-Ca and ICa are changed in the manner of associative training is not yet known. Another protein kinase present in high concentrations in both mammalian brain and molluscan nervous systems is protein kinase C, which is both calcium- and phospholipid-sensitive. We now present evidence that activation of protein kinase C by the tumour promoter phorbol ester (PDB) and intracellular injection of the enzyme induce conductance changes similar to those caused by associative training in Hermissenda B cells (that is a reduction of IA and IK-Ca, and enhancement of ICa). These results represent the first direct demonstration that protein kinase C affects membrane K+ ion conductance mechanisms.     

Enhancement of calcium current in Aplysia neurones by phorbol ester and protein kinase C

One of the molecular mechanisms capable of regulating the physiological properties of neurones is the phosphorylation of ion channels and other cellular components by cyclic AMP-dependent protein kinase. Another protein kinase present in high concentrations in the mammalian brain is protein kinase C (a calcium/phosphatidylserine/diacylglycerol-dependent protein kinase), but there is no direct evidence, as yet, for the involvement of this enzyme in the control of neuronal excitability. We now present evidence that activation of endogenous protein kinase C by the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl- phorbol-13-acetate), or intracellular injection of the purified enzyme, enhances the voltage-sensitive calcium current in bag cell neurones of the mollusc Aplysia.     

The Ras-MAPK pathway is important for olfaction in Caenorhabditis elegans

The Ras-MAPK (mitogen-activated protein kinase) signal transduction pathway is well known to control cellular proliferation and differentiation in response to extracellular signals, but its other functions are less understood. In Caenorhabditis elegans this pathway regulates several developmental events, such as vulval induction and progression of meiosis, but its function in the nervous system is unknown. Here we report that the Ras-MAPK pathway is involved in olfaction in this organism. Mutational inactivation and hyperactivation of this pathway impairs efficiency of chemotaxis to a set of odorants. Experiments in which let-60 ras was expressed using a heat-shock promoter and a cell-specific promoter show that a normal activity of LET-60 Ras is required in mature olfactory neurons. Application of the odorant isoamylalcohol to wild-type animals leads to the activation of MAP kinase in olfactory neurons within 10 seconds. This induction is dependent on the function of the nucleotide-gated channel TAX-2/TAX-4 and the voltage-activated calcium channel subunit UNC-2. These results suggest a dynamic regulatory role for the Ras-MAPK pathway in perception and transmission of sensory signals in olfactory neurons.     

Bidirectional control of cytosolic free calcium by thyrotropin-releasing hormone in pituitary cells

It is now established that a key step in the action of calcium-mobilizing agonists is stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to 1,2-diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). The latter substance acts as a second messenger, controlling the release of calcium from intracellular stores (see ref. 3 for review). The bifurcating nature of the signalling system is exemplified by the fact that the other product of PtdIns(4,5)P2 hydrolysis, 1,2-diacylglycerol, can alter cellular function by activating protein kinase C, the cellular target for several tumour-promoting agents such as the phorbol esters. In various tissues, including GH3 pituitary tumour cells, a synergistic interaction between calcium ions and protein kinase C underlies agonist-induced changes in cell activity. The data presented here suggest that when GH3 cells are stimulated by thyrotropin-releasing hormone (TRH), an agonist inducing PtdIns(4,5)P2 hydrolysis, the two limbs of the inositol lipid signalling system interact to control free cytosolic calcium levels [( Ca2+]i). At low levels of TRH receptor occupancy, [Ca2+]i increases rapidly, then declines relatively slowly. As receptor occupancy increases, the calcium signal becomes more short-lived due to the appearance of a second, inhibitory, component. This latter component, which is enhanced when [Ca2+]i is elevated by high potassium depolarization, is mimicked by active phorbol esters and by bacterial phospholipase C. It seems likely that protein kinase C subserves a negative feedback role in agonist-induced calcium mobilization.     

Application of phorbol ester to mouse skin causes a rapid and sustained loss of protein kinase C

It is now widely accepted that tumour-promoting phorbol esters activate a Ca2+- and phospholipid-dependent protein kinase (protein kinase C) both in vitro and in intact cells, and that the kinase represents a major cellular phorbol ester-binding protein. The phorbol esters act as analogues of diacylglycerol, a natural regulator of protein kinase C, and stabilize the membrane-association of the kinase. Although other molecular targets may exist, protein kinase C activation is probably important in mediating the diverse responses of cultured cells to phorbol esters and in promoting in vivo tumours. The enzyme comprises a family of closely related proteins and has been detected in extracts from mouse epidermal cells, the likely targets for two-stage carcinogenesis in mouse skin. In this report we show that application of a single dose of TPA (12-O-tetradecanoyl phorbol-13-acetate) to mouse skin results in a rapid and complete loss of protein kinase C activity which is maintained for 3-4 days. This is associated with a loss of immunologically detectable protein kinase C and the accumulation of a smaller protein detectable by antibodies recognizing the regulatory domain of protein kinase C.     

Synaptic vesicle-associated Ca2+/calmodulin-dependent protein kinase II is a binding protein for synapsin I.

Synapsin I is a synaptic vesicle-associated phosphoprotein that is involved in the modulation of neurotransmitter release. Ca2+/calmodulin-dependent protein kinase II, which phosphorylates two sites in the carboxy-terminal region of synapsin I, causes synapsin I to dissociate from synaptic vesicles and increases neurotransmitter release. Conversely, the dephosphorylated form of synapsin I, but not the form phosphorylated by Ca2+/calmodulin-dependent protein kinase II, inhibits neurotransmitter release. The amino-terminal region of synapsin I interacts with membrane phospholipids, whereas the C-terminal region binds to a protein component of synaptic vesicles. Here we demonstrate that the binding of the C-terminal region of synapsin I involves the regulatory domain of a synaptic vesicle-associated form of Ca2+/calmodulin-dependent protein kinase II. Our results indicate that this form of the kinase functions both as a binding protein for synapsin I, and as an enzyme that phosphorylates synapsin I and promotes its dissociation from the vesicles.     

Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C

Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.     

Early steps of lymphocyte activation bypassed by synergy between calcium ionophores and phorbol ester

Although it has been proposed that the activation of T lymphocytes is mediated by an early rise in cytosolic calcium concentration, it has not been possible to mimic antigen- or mitogen-induced mouse lymphocyte activation by calcium ionophores that bypass receptor-mediated processes. There is now evidence from other systems that the rise in cytosolic calcium which follows receptor triggering is preceded by the breakdown of phosphatidylinositol bisphosphate into 1,2-diacylglycerol and inositol trisphosphate. The latter is known to cause release of calcium from intracellular stores. The cellular target for the former is now widely accepted to be protein kinase C. Therefore, ligand-induced cellular response follows a rise in cytosolic calcium concentration and protein kinase C activation. Here we confirm that the calcium ionophores A23187 and ionomycin do not activate mouse T lymphocytes. However, either one in combination with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which is structurally related to 1,2-diacylglycerol, induces in lymphoid cell populations the expression of receptors for interleukin-2 (IL-2), the secretion of IL-2 and cell proliferation as measured by 3H-thymidine uptake. The growth-promoting effect of IL-2 on an exogenous IL-2-dependent clone could not be substituted for by ionomycin either alone or with TPA. Thus, the combination of calcium ionophores and TPA bypasses the requirement for antigen- or lectin-induced signal at the onset of lymphocyte activation.     

Phosducin is a protein kinase A-regulated G-protein regulator.

Signal transduction by G-protein-coupled receptors is regulated by various mechanisms acting at the receptor level; those studied most thoroughly are from the beta-adrenergic receptor/Gs/adenylyl cyclase system. We report here a regulatory mechanism occurring at the level of the G proteins themselves. A protein with M(r) 33,000 that inhibits Gs-GTPase activity was purified from bovine brain. This protein is very similar or identical to phosducin, a protein previously thought to be specific for retina and pineal gland. Recombinant phosducin inhibited the GTPase activity of several G proteins, and also inhibited Gs-mediated adenylyl cyclase activation. Blockade of its inhibitory effects by protein kinase A suggests that phosducin may be part of a complex regulatory network controlling G-protein-mediated signalling.     

Exo1 and Exo2 proteins stimulate calcium-dependent exocytosis in permeabilized adrenal chromaffin cells.

In many cell types an increase in cytosolic calcium is the main signal for the exocytotic release of stored secretory components such as hormones and neurotransmitters. The site of action of calcium in exocytosis is not known, neither are the participating molecules. In the case of the intracellular membrane fusions that occur during transport through early stages of the secretory pathway, several cytosolic and peripheral membrane proteins are necessary. Permeabilized cells have been useful in understanding the requirements for calcium and nucleotides in regulated exocytosis and under certain conditions there is leakage of soluble protein components and run-down of the exocytotic response. This system can be used to identify the soluble proteins involved in exocytosis, one candidate in chromaffin cells being annexin II (calpactin). Here we use this assay to identify two other cytosolic protein factors that regulate exocytosis in permeabilized adrenal chromaffin cells, which we term Exo1 and Exo2. Exo1 from brain cytosol resolves on electrophoresis in SDS-polyacrylamide gels as a group of polypeptides of relative molecular mass approximately 30,000 and shares sequence homology with the 14-3-3 family of proteins. The ability of Exo1 to reactivate exocytosis is potentiated by protein kinase C activation and therefore Exo1 may influence the protein kinase C-mediated control of Ca(2+)-dependent exocytosis.     

Correction of cell-cell communication defect by introduction of a protein kinase into mutant cells

The cell-to-cell permeability of the junctions of various cultured mammalian cell types depends on the concentration of intracellular cyclic AMP [( cAMP]i). The permeability rises when the cells are supplied with exogenous cyclic AMP or when their cyclic AMP synthesis is stimulated with choleragen or hormones; it falls when [cAMP]i is lowered by application of serum or due to increase in cell density. The rise and fall in permeability take several hours to develop (the rise is protein synthesis-dependent) and they occur concurrently with the rise and fall in the number of intramembrane particles of the gap junctions, which probably embody the cell-to-cell channels. Is this permeability regulation mediated by phosphorylating protein kinase? In many eukaryotes, the cyclic AMP receptor is a protein kinase consisting of a pair of regulatory subunits and a pair of catalytic subunits. The latter dissociate from the holoenzyme as the cyclic AMP binds to the regulatory subunits and, in this dissociated form, catalyse the phosphorylation of the target. The regulatory subunit occurs in two isoenzyme forms, I and II. The catalytic subunit seems invariant; subunits from different isoenzymes can substitute for each other. We show here that a mutant cell lacking the isoenzyme I is deficient in permeable junctions, and that this junctional defect is corrected when the mutant is supplied with exogenous catalytic subunit.     

Evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein?

Calcium-dependent protease (calcium protease) is apparently involved in a variety of cellular processes. Here we have attempted to clarify the role and regulatory mechanism of calcium protease by analysing its structure. The complete primary structure of calcium protease (relative molecular mass (Mr) 80,000 (80K), 705 amino acids) was deduced from the nucleotide sequence of cloned complementary DNA. The protein contains four distinct domains, and we have observed a marked similarity between the second and fourth domains and the papain-like thiol proteases and calmodulin-like calcium-binding proteins, respectively. This finding suggests that calcium protease arose from the fusion of genes for proteins of completely different function and evolutionary origin. Further, it provides functional insight into cellular regulatory mechanisms mediated by Ca2+ through calcium-binding proteins.