Internalization of G Protein-Coupled Receptors in Single Olfactory Receptor Neurons

Abstract : Desensitization of many G protein-coupled receptors after ligand binding generally involves phosphorylation of the receptors and internalization of the ligandbound, phosphorylated receptors by a clathrin-mediated endocytic pathway. Olfactory receptor neurons from the channel catfish ( Ictalurus punctatus ) express the G protein-coupled odorant receptors and metabotropic glutamate receptors. To determine whether a clathrin-dependent receptor internalization pathway exists in olfactory receptor neurons, western blotting and immunocytochemistry were used to identify and localize clathrin and dynamin in isolated olfactory neurons. Clathrin and dynamin immunoreactivity was found in the cell bodies, dendrites, and dendritic knobs of the neurons. Using the activity-dependent fluorescent dye FM1-43 to monitor receptor internalization, we show that single olfactory neurons stimulated with the odorant amino acid l -glumate internalized the dye. Odorant-stimulated neurons showed a consistent pattern of internalized FM1-43 fluorescence localized in the cell bodies and dendritic knobs. Odorant-stimulated internalization was unaffected by the caveolae activator okadaic acid and was significantly decreased by a metabotropic glutamate receptor antagonist, suggesting that a functional, clathrindependent, receptor-mediated internalization pathway exists in olfactory receptor neurons.     

Phosphorylation of Serine-880 in GluR2 by Protein Kinase C Prevents Its C Terminus from Binding with Glutamate Receptor-Interacting Protein

Phosphorylation of the glutamate receptor is an important mechanism of synaptic plasticity. Here, we show that the C terminus of GluR2 of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor is phosphorylated by protein kinase C and that serine-880 is the major phosphorylation site. This phosphorylation also occurs in human embryonic kidney (HEK) cells by addition of 12-O-tetradecanoylphorbol 13-acetate. Our immunoprecipitation experiment revealed that the phosphorylation of serine-880 in GluR2 drastically reduced the affinity for glutamate receptor-interacting protein (GRIP), a synaptic PDZ domain-containing protein, in vitro and in HEK cells. This result suggests that modulation of serine-880 phosphorylation in GluR2 controls the clustering of AMPA receptors at excitatory synapses and consequently contributes to synaptic plasticity.     

The glutamate agonist homocysteine sulfinic acid stimulates glucose uptake through the calcium-dependent AMPK-p38 MAPK-protein kinase C zeta pathway in skeletal muscle cells

Homocysteine sulfinic acid (HCSA) is a homologue of the amino acid cysteine and a selective metabotropic glutamate receptor (mGluR) agonist. However, the metabolic role of HCSA is poorly understood. In this study, we showed that HCSA and glutamate stimulated glucose uptake in C2C12 mouse myoblast cells and increased AMP-activated protein kinase (AMPK) phosphorylation. RT-PCR and Western blot analysis revealed that C2C12 expresses mGluR5. HCSA transiently increased the intracellular calcium concentration. Although α-methyl-4-carboxyphenylglycine, a metabotropic glutamate receptor antagonist, blocked the action of HCSA in intracellular calcium response and AMPK phosphorylation, 6-cyano-7-nitroquinoxaline-2,3-dione, an AMPA antagonist, did not exhibit such effects. Knockdown of mGluR5 with siRNA blocked HCSA-induced AMPK phosphorylation. Pretreatment of cells with STO-609, a calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, blocked HCSA-induced AMPK phosphorylation, and knockdown of CaMKK blocked HCSA-induced AMPK phosphorylation. In addition, HCSA activated p38 mitogen-activated protein kinase (MAPK). Expression of dominant-negative AMPK suppressed HCSA-mediated phosphorylation of p38 MAPK, and inhibition of AMPK and p38 MAPK blocked HCSA-induced glucose uptake. Phosphorylation of protein kinase C ζ (PKCζ) was also increased by HCSA. Pharmacologic inhibition or knockdown of p38 MAPK blocked HCSA-induced PKCζ phosphorylation, and knockdown of PKCζ suppressed the HCSA-induced increase of cell surface GLUT4. The stimulatory effect of HCSA on cell surface GLUT4 was impaired in FITC-conjugated PKCζ siRNA-transfected cells. Together, the above results suggest that HCSA may have a beneficial role in glucose metabolism in skeletal muscle cells via stimulation of AMPK.     

Phosphorylation of AMPA receptors

The ionotropic α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor is densely distributed in the mammalian brain and is primarily involved in mediating fast excitatory synaptic transmission. Recent studies in both heterologous expression systems and cultured neurons have shown that the AMPA receptor can be phosphorylated on their subunits (GluR1, GluR2, and GluR4). All phosphorylation sites reside at serine, threonine, or tyrosine on the intracellular C-terminal domain. Several key protein kinases, such as protein kinase A, protein kinase C, Ca²⁺/calmodulin-dependent protein kinase II, and tyrosine kinases (Trks; receptor or nonreceptor family Trks) are involved in the site-specific regulation of the AMPA receptor phosphorylation. Other glutamate receptors (N-methyl-d-aspartate receptors and metabotropic glutamate receptors) also regulate AMPA receptors through a protein phosphorylation mechanism. Emerging evidence shows that as a rapid and short-term mechanism, the dynamic protein phosphorylation directly modulates the electrophysiological, morphological (externalization and internalization trafficking and clustering), and biochemical (synthesis and subunit composition) properties of the AMPA receptor, as well as protein-protein interactions between the AMPA receptor subunits and various intracellular interacting proteins. These modulations underlie the major molecular mechanisms that ultimately affect many forms of synaptic plasticity.     

Activation of Ca2+/calmodulin-dependent protein kinase II and protein kinase C by glutamate in cultured rat hippocampal neurons.

In cultured rat hippocampal neurons, glutamate elevated the Ca(2+)-independent activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) through autophosphorylation when the neurons were incubated in Mg(2+)-free buffer, and this response was blocked by specific antagonists of the N-methyl-D-aspartate (NMDA) receptor. In addition, glutamate stimulated the transient translocation of protein kinase C (PKC) from the cytosol to the membrane fraction. This effect was not blocked by NMDA receptor antagonists but was partially blocked by DL-2-amino-3-phosphonopropionate. Quisqualate or trans-1-amoinocyclopentane-trans1,3-dicarboxylate produced a similar effect on the translocation of PKC. In the experiments with 32P-labeled cells, the phosphorylation of microtuble-associated protein 2 and synapsin I, as well as autophosphorylation of CaM kinase II, were found to be stimulated by exposure to glutamate. These results suggest that glutamate can activate CaM kinase II through the ionotropic NMDA receptor, which in turn increases the phosphorylation of microtuble-associated protein 2 and synapsin I. PKC was activated through the metabotropic glutamate receptor in the hippocampal neurons.     

Endothelin Induces a Calcium-Dependent Phosphorylation of PEA-15 in Intact Astrocytes: Identification of Ser104 and Ser116 Phosphorylated, Respectively, by Protein Kinase C and Calcium/Calmodulin Kinase II In Vitro

Abstract: PEA-15 (phosphoprotein enriched in astrocytes, M_r = 15,000) is an acidic serine-phosphorylated protein highly expressed in the CNS, where it can play a protective role against cytokine-induced apoptosis. PEA-15 is a major substrate for protein kinase C. Endothelins, which are known to exert pleiotropic effects on astrocytes, were used to analyze further the processes involved in PEA-15 phosphorylation. Endothelin-1 or endothelin-3 (0.1 µ M ) induced a robust phosphorylation of PEA-15 that was abolished by the removal of extracellular calcium, but only diminished by inhibitors of protein kinase C. Microsequencing of phosphopeptides generated by digestion of PEA-15 following endothelin-1 treatment identified two phosphorylated residues: Ser¹⁰⁴, previously recognized as the protein kinase C site, and a novel phosphoserine, Ser¹¹⁶, located in a consensus motif for either protein kinase casein kinase II or calcium/calmodulin-dependent protein kinase II (CaMKII). Partly purified PEA-15 was a substrate in vitro for CaMKII, but not for casein kinase II. Two-dimensional phosphopeptide mapping demonstrated that the site phosphorylated in vitro by CaMKII was also phosphorylated in intact astrocytes in response to endothelin. CaMKII phosphorylated selectively Ser¹¹⁶ and had no effect on Ser¹⁰⁴, but in vitro phosphorylation by CaMKII appeared to facilitate further phosphorylation by protein kinase C. Treatment of intact astrocytes with okadaic acid enhanced the phosphorylation of the CaMKII site. These results demonstrate that PEA-15 is phosphorylated in astrocytes by CaMKII (or a related kinase) and by protein kinase C in response to endothelin.     

PKC phosphorylation of a conserved serine residue in the C-terminus of group III metabotropic glutamate receptors inhibits calmodulin binding

Group III metabotropic glutamate receptors (mGluRs) serve as presynaptic receptors that mediate feedback inhibition of glutamate release via a Ca(2+)/calmodulin (CaM)-dependent mechanism. In vitro phosphorylation of mGluR7A by protein kinase C (PKC) prevents its interaction with Ca(2+)/CaM. In addition, activation of PKC leads to an inhibition of mGluR signaling. Here, we demonstrate that disrupting CaM binding to mGluR7A by PKC in vitro is due to phosphorylation of a highly conserved serine residue, S862. We propose charge neutralization of the CaM binding consensus sequence resulting from phosphorylation to constitute a general mechanism for the regulation of presynaptic mGluR signaling.     

D(1) dopamine receptor stimulation increases GluR1 phosphorylation in postnatal nucleus accumbens cultures

Postsynaptic interactions between dopamine and glutamate receptors in the nucleus accumbens are critical for acute responses to drugs of abuse and for neuroadaptations resulting from their chronic administration. We tested the hypothesis that D(1) dopamine receptor stimulation increases phosphorylation of the AMPA receptor subunit GluR1 at the protein kinase A phosphorylation site (Ser845). Nucleus accumbens cell cultures were prepared from postnatal day 1 rats. After 14 days in culture, GluR1 phosphorylation was measured by western blotting using phosphorylation site-specific antibodies. The D(1) receptor agonist SKF 81297 increased Ser845 phosphorylation in a concentration- dependent manner, with marked increases occurring within 5 min. This was prevented by the D(1) receptor antagonist SCH 23390 and the protein kinase A inhibitor H89, and reproduced by forskolin. The D(2) receptor agonist quinpirole attenuated the response to D(1) receptor stimulation. Neither D(1) nor D(2) receptor agonists altered GluR1 phosphorylation at Ser831, the site phosphorylated by protein kinase C and calcium/calmodulin-dependent protein kinase II. In other systems, phosphorylation of GluR1 at Ser845 is associated with enhancement of AMPA receptor currents. Thus, the present results suggest that AMPA receptor transmission in the nucleus accumbens may be augmented by concurrent D(1) receptor stimulation.     

Metabotropic Glutamate Receptors Increase Amyloid Precursor Protein Processing in Astrocytes: Inhibition by Cyclic AMP

Abstract: Neurotransmitter receptors that increase phosphatidylinositol hydrolysis generate second messengers that activate protein kinase C. Here, we used metabotropic glutamate receptor agonists to increase both phosphatidylinositol hydrolysis and secretion of the soluble extracellular fragment of amyloid precursor protein (APPs) from cortical astrocyte cultures. The increase in APPs secretion was mimicked by direct activation of protein kinase C with phorbol ester and was suppressed by the metabotropic glutamate receptor antagonist l -(+)-2-amino-3-phosphonopropionic acid or by the protein kinase C inhibitor GF109203X. Ionotropic glutamate agonists did not increase APPs secretion. Forskolin or dibutyryl cyclic AMP inhibited the increase in APPs secretion caused by metabotropic glutamate receptor agonists or by phorbol ester treatment but did not affect basal APPs levels. Therefore, glutamatergic agonists that increase protein kinase C activation or decrease cyclic AMP formation may enhance the conversion of full-length APP to nonamyloidogenic APPs in Alzheimer's disease.     

Serines 890 and 896 of the NMDA receptor subunit NR1 are differentially phosphorylated by protein kinase C isoforms

The NR1 subunit of the NMDA receptor has two serines (S890 and S896) whose phosphorylation by protein kinase C (PKC) differentially modulates NMDA receptor trafficking and clustering. It is not known which PKC isoforms phosphorylate these serines. In primary cultures of cerebellar neurons, we examined which PKC isoforms are responsible for the phosphorylation S890 and S896. We used specific inhibitors of PKC isoforms and antibodies recognizing specifically phosphorylated S890 or S896. The results show that PKC alpha phosphorylates preferentially S896 and PKC gamma preferentially S890. Activation of type I metabotropic glutamate receptors (mGluRs) with DHPG (3,5-dihyidroxy-phenylglycine) activates PKC gamma but not PKC alpha or beta. We found that activation of mGluRs by DHPG increases S890 but not S896 phosphorylation, supporting a role for PKC gamma in the physiological modulation of S890 phosphorylation. It is also shown that the pool of NR1 subunits present in the membrane surface contains phosphorylated S890 but not phosphorylated S896. This supports that differential phosphorylation of S890 and S896 by different PKC isoforms modulates cellular distribution of NMDA receptors and may also contribute to the selective modulation of NMDA receptor function and intracellular localization.     

Phosphorylation of basic fibroblast growth factor by purified protein kinase C and the identification of a cryptic site of phosphorylation

We have further characterized the protein kinase C (PK-C) dependent phosphorylation of basic fibroblast growth factor (FGF). Intact recombinant basic FGF and a series of ten peptide fragments of basic FGF were phosphorylated by PK-C and the products were analyzed by SDS-PAGE and autoradiography. As expected, peptide fragments containing the known site of phosphorylation (Ser64) are substrates for phosphorylation. Surprisingly however, peptides containing the receptor binding domain of the mitogen [basic FGF(106-115)] are also phosphorylated. An examination of this sequence reveals the presence of a consensus sequence (Ser108-Ala109-Lys110) that mediates the reaction. Accordingly, all peptides that contain the core amino acids basic FGF(106-111) are substrates for phosphorylation. Peptide mapping of basic FGF confirms that Ser64 is the primary site of phosphorylation, suggesting that Ser108 is a cryptic consensus sequence. Because basic FGF is metabolized to sequence specific fragments after its binding and internalization into target cells, this cryptic site may in fact be phosphorylated in vivo.     

Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit

Purified acetylcholine receptor is rapidly and specifically phosphorylated by partially purified protein kinase C, the Ca2+/phospholipid-dependent enzyme. The receptor delta subunit is the major target for phosphorylation and is phosphorylated on serine residues to a final stoichiometry of 0.4 mol of phosphate/mol of subunit. Phosphorylation is dose-dependent with a Km value of 0.2 microM. Proteolytic digestion of the delta subunit phosphorylated by either protein kinase C or the cAMP-dependent protein kinase yielded a similar pattern of phosphorylated fragments. The amino acids phosphorylated by either kinase co-localized within a 15-kDa proteolytic fragment of the delta subunit. This fragment was visualized by immunoblotting with antibodies against a synthetic peptide corresponding to residues 354-367 of the receptor delta subunit. This sequence, which contains 3 consecutive serine residues, was recently shown to include the cAMP-dependent protein kinase phosphorylation site (Souroujon, M. C., Neumann, D., Pizzighella, S., Fridkin, M., and Fuchs, S. (1986) EMBO J. 5, 543-546). Concomitantly, the synthetic peptide 354-367 was specifically phosphorylated in a Ca2+- and phospholipid-dependent manner by protein kinase C. Furthermore, antibodies directed against this peptide inhibited phosphorylation of the intact receptor by protein kinase C. We thus conclude that both the cAMP-dependent protein kinase and protein kinase C phosphorylation sites reside in very close proximity within the 3 adjacent serine residues at positions 360, 361, and 362 of the delta subunit of the acetylcholine receptor.     

An Ibotenate-Selective Metabotropic Glutamate Receptor: Mediates Protein Phosphorylation in Cultured Hippocampal Pyramidal Neurons

Abstract: Previous results showed that within 30 s after glutamate stimulation of cultured rat hippocampal pyramidal neurons there occurred an elevation of Ca²⁺ and diacylglycerol, and the phosphorylation of three acidic protein kinase C substrates, i.e., an 87-kDa protein known as myristoylated alanine-rich C kinase substrate and a 120-and a 48-kDa protein. In addition, it was suggested that a metabotropic-type glutamate receptor might be responsible for the phosphorylation observed. This work examines the ability of metabotropic and ionotropic glutamate receptor agonists to quickly activate phospholipases in 1.26 mM versus 50 nM extracellular Ca²⁺ by measuring the generation of inositol phosphates. NMDA, quisqualate, and trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid did not stimulate the generation of inositol phosphates in the presence of normal or low extracellular Ca²⁺ in pyramidal neurons. Kainate stimulated the production of inositol phosphates in the presence of 1.26 mM extracellular Ca²⁺ but not in 50 nM extracellular Ca²⁺. Other than glutamate, only ibotenate was able to stimulate the generation of inositol phosphates in both normal and low extracellular Ca²⁺. The maximal response to ibotenate was approximately equal to that of glutamate, when pyramidal neurons were stimulated in 50 nM extracellular Ca²⁺. The generation of inositol phosphates by glutamate and ibotenate could be partially blocked (50–60% reduction) by pretreatment of neurons with pertussis toxin (250 ng/ml),-suggesting that a GTP-binding protein might be involved. In addition, ibotenate stimulated the immediate phosphorylation of the same three protein kinase C substrates as glutamate. The NMDA receptor blocker MK-801 had no effect on this phosphorylation. These results suggest that the stimulation of phosphorylation in pyramidal neurons by glutamate occurs predominantly through the activation of an ibotenate-selective metabotropic glutamate receptor.     

Physiological regulation of Munc18/nSec1 phosphorylation on serine-313

Increased protein phosphorylation enhances exocytosis in most secretory cell types, including neurones. However, the molecular mechanisms by which this occurs and the specific protein targets remain unclear. Munc18-1/nSec1 is essential for exocytosis in neurones, and is known to be phosphorylated by protein kinase C (PKC) in vitro at Ser-313. This phosphorylation has been shown to decrease its affinity for syntaxin, and to alter the kinetics of exocytosis in chromaffin cells. However, there are no data on the physiological regulation of Ser-313 phosphorylation. Using phospho-Ser-313-specific antisera, we demonstrate here that Ser-313 is phosphorylated in intact and permeabilized chromaffin cells in response to histamine and Ca2+ respectively. Furthermore, Ser-313 is rapidly and transiently phosphorylated in intact synaptosomes in response to depolarization by KCl treatment or by 4-aminopyridine, and by the metabotropic glutamate receptor agonist dihydroxyphenylglycine. PKC was identified as the kinase, and PP1 and PP2B as the phosphatases responsible for regulating Ser-313 phosphorylation. As phosphorylation of nSec1 on Ser-313 affects the rate of transmitter release in chromaffin cells, the demonstration here that this phosphorylation event occurs in neurones suggests that synaptic neurotransmitter release may be similarly regulated by nSec1 phosphorylation. Furthermore, such changes in release kinetics are associated with long-term potentiation and depression, thus implicating nSec1 phosphorylation as a potential regulatory mechanism underlying presynaptic plasticity.     

Differential regulation of metabotropic glutamate receptor 5-mediated phosphoinositide hydrolysis and extracellular signal-regulated kinase responses by protein kinase C in cultured astrocytes

The metabotropic glutamate receptor 5 (mGluR5) exhibits a rapid loss of receptor responsiveness to prolonged or repeated agonist exposure. This receptor desensitization has been seen in a variety of native and recombinant systems, and is thought to result from receptor-mediated, protein kinase C (PKC)-dependent phosphorylation of the receptor, uncoupling it from the G protein in a negative feedback regulation. We have investigated the rapid PKC-mediated desensitization of mGluR5 in cortical cultured astrocytes by measuring downstream signals from activation of mGluR5. These include activation of phosphoinositide (PI) hydrolysis, intracellular calcium transients, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation. We present evidence that PKC plays an important role in rapid desensitization of PI hydrolysis and calcium signaling, but not in ERK2 phosphorylation. This differential regulation of mGluR5-mediated responses suggests divergent signaling and regulatory pathways which may be important mechanisms for dynamic integration of signal cascades.     

Insulin-sensitive phosphorylation of serine 1293/1294 on the human insulin receptor by a tightly associated serine kinase

In these studies we demonstrate that insulin stimulates both tyrosine and serine phosphorylation of the insulin receptor after its partial purification on wheat germ-agarose, and after affinity purification on insulin-agarose. Analysis of the serine phosphate incorporated into partially purified or highly purified insulin receptor suggests that an insulin-sensitive serine kinase (IRSK) copurifies with the insulin receptor. Following trypsin digestion, reversed-phase high pressure liquid chromatography (HPLC) analysis of the phosphorylated, affinity-purified insulin receptor preparation reveals phosphopeptide profiles similar to those of trypsin-digested receptors immunoprecipitated from 32P-labeled fibroblasts overexpressing the human insulin receptor. The major insulin-stimulated HPLC phosphopeptide peak from insulin receptors labeled in intact cells contains a hydrophilic phosphoserine-containing peptide which rapidly elutes from a C18 column. HPLC and two-dimensional separation indicate that the same phosphopeptide is obtained when affinity-purified insulin receptors are phosphorylated by IRSK. The serine containing tryptic peptide within the cytoplasmic domain of the human insulin receptor predicted to elute most rapidly upon HPLC had the sequence SSHCQR corresponding to residues 1293-1298. A synthetic peptide containing this sequence is phosphorylated by the insulin receptor/IRSK preparation. After alkylation and trypsin digestion, the synthetic phosphopeptide comigrates with the alkylated, tryptic phosphopeptide derived from insulin receptor phosphorylated in vitro by IRSK. We propose that serine 1293 or 1294 of the human insulin receptor is a major site(s) phosphorylated on the insulin receptor in intact cells and is phosphorylated by IRSK. Furthermore, insulin added directly to affinity-purified insulin receptor/IRSK preparations stimulates the phosphorylation of synthetic peptides corresponding to this receptor phosphorylation site and another containing threonine 1336. Kemptide phosphorylation is not stimulated by insulin under these conditions. No phosphorylation of peptide substrates for Ca2+/calmodulin-dependent protein kinase, protein kinase C, casein kinase II, or cGMP-dependent protein kinase by IRSK is detected. These data indicate that IRSK exhibits specificity for the insulin receptor and may be activated by the insulin receptor tyrosine kinase in an insulin-dependent manner.     

Activation of Mitogen-Activated Protein Kinase in Cultured Rat Hippocampal Neurons by Stimulation of Glutamate Receptors

Abstract: Mitogen-activated protein kinase (MAP kinase) was activated by stimulation of glutamate receptors in cultured rat hippocampal neurons. Ten micromolar glutamate maximally stimulated MAP kinase activity, which peaked during 10 min and decreased to the basal level within 30 min. Experiments using glutamate receptor agonists and antagonists revealed that glutamate stimulated MAP kinase through NMDA and metabotropic glutamate receptors but not through non-NMDA receptors. Glutamate and its receptor agonists had no apparent effect on MAP kinase activation in cultured cortical astrocytes. Addition of calphostin C, a protein kinase C (PKC) inhibitor, or down-regulation of PKC activity partly abolished the stimulatory effect by glutamate, but the MAP kinase activation by treatment with ionomycin, a Ca²⁺ ionophore, remained intact. Lavendustin A, a tyrosine kinase inhibitor, was without effect. In experiments with ³²P-labeled hippocampal neurons, MAP kinase activation by glutamate was associated with phosphorylation of the tyrosine residue located on MAP kinase. However, phosphorylation of Raf-1, the c- raf protooncogene product, was not stimulated by treatment with glutamate. Our observations suggest that MAP kinase activation through glutamate receptors in hippocampal neurons is mediated by both the PKC-dependent and the Ca²⁺-dependent pathways and that the activation of Raf-1 is not involved.     

Glutamate-stimulated activation of DNA synthesis via mitogen-activated protein kinase in primary astrocytes: involvement of protein kinase C and related adhesion focal tyrosine kinase

Glutamate is the major excitatory neurotransmitter in the CNS. Although its role in neurons has been studied extensively, little is known about its function in astrocytes. We studied the effects of glutamate on signaling pathways in primary astrocytes. We found that the tyrosine kinase related adhesion focal tyrosine kinase (RAFTK) is tyrosine phosphorylated in response to glutamate in a time- and dose-dependent manner. This phosphorylation was pertussis toxin (PTX) sensitive and could be attenuated by the depletion of Ca2+ from intracellular stores. RAFTK tyrosine phosphorylation was mediated primarily by class I/II metabotropic glutamate receptors and depends on protein kinase C (PKC) activation. Glutamate treatment of primary astrocytes also results in a significant increase in the activity of the mitogen-activated protein kinases [extracellular signal-related kinases 1/2 (ERK1/2)]. Like RAFTK phosphorylation, ERK1/2 activation is PTX sensitive and can be attenuated by the depletion of intracellular Ca2+ and by PKC inhibition, suggesting that RAFTK might mediate the glutamate-dependent activation of ERK1/2. Furthermore, we demonstrated that glutamate stimulation of primary astrocytes leads to a significant increase in DNA synthesis. Glutamate-stimulated DNA synthesis is PTX sensitive and can be inhibited by the MAP kinase kinase inhibitor PD98059, suggesting that in primary astrocytes, glutamate might signal via RAFTK and MAP kinase to promote DNA synthesis and cell proliferation.     

Phosphorylation of membrane-bound acetylcholine receptor by protein kinase C: characterization and subunit specificity

Acetylcholine receptor (AChR) from Torpedo electric organ in its membrane-bound or solubilized form is phosphorylated by the Ca2+/phospholipid-dependent protein kinase (PKC). The subunit specificity for PKC is different from that observed for cAMP-dependent protein kinase (PKA). Whereas PKC phosphorylates predominantly the delta subunit and the phosphorylation of the gamma subunit by this enzyme is very low, PKA phosphorylates both subunits to a similar high extent. We have extended our phosphorylation studies to a synthetic peptide from the gamma subunit, corresponding to residues 346-359, which contains a consensus PKA phosphorylation site. This synthetic peptide is phosphorylated by both PKA and PKC, suggesting that in the intact receptor both kinases may phosphorylate the gamma subunit at a similar site, as has been previously demonstrated by us for the delta subunit [Safran, A., et al. (1987) J. Biol. Chem. 262, 10506-10510]. The diverse pattern of phosphorylation of AChR by PKA and PKC may play a role in the regulation of its function.     

Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C

Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.