Differences in the Cyclic AMP-Dependent Phosphorylation of Plasma Membrane Proteins of Differentiated and Undifferentiated L6 Myogenic Cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L₆ myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L₆ myoblasts was stimulated more than three fold by 5 × 10⁻⁵ M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L₆ cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L₆ cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f₂b. Therefore, the data show that differentiation in L₆ cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.     

Tyrosine Hydroxylase in "Leaky" Adrenal Medullary Cells: Evidence for In Situ Phosphorylation by Separate Ca2+ and Cyclic AMP-Dependent Systems

Abstract: The systems responsible for phosphorylating tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, were investigated in situ in adrenal medullary cells made permeable to solutes of up to 1,000 dalton by exposure to brief intense electric fields. Two different phosphorylation systems were found. One is dependent on Ca²⁺, the other on cyclic AMP. The Ca²⁺-dependent system is half-maximally activated by 1-2 μ M Ca²⁺ and 0.5 m M ATP, and follows a time course similar to that of secretion of catecholamines. Trifluoperazine (0.1 m M ) does not inhibit significantly Ca²⁺-dependent phosphorylation of tyrosine hydroxylase in situ. The cyclic AMP-dependent system is half-maximally activated by addition of 0.5 μ M cyclic AMP and about 0.3 m M ATP. Ca²⁺-dependent and cyclic AMP-dependent phosphorylations of tyrosine hydroxylase have roughly the same time course and are additive under conditions where one system is already saturated. Peptide maps of immunoprecipitated tyrosine hydroxylase, after in situ phosphorylation of the enzyme either in the presence of 10⁻⁸ M Ca²⁺ plus 2 × 10⁻⁵ M cyclic AMP or of 10⁻⁵ M Ca²⁺, show a marked difference indicating that the enzyme contains several phosphorylation sites. At least one of these sites is phosphorylated only by the Ca²⁺-dependent system, whereas the other site(s) are phosphorylated by both the Ca²⁺- and cyclic AMP-dependent systems. The effect of in situ phosphorylation of tyrosine hydroxylase on its enzymatic activity was also investigated.     

Light-Induced CREB Phosphorylation and Gene Expression in Rat Retinal Cells

Abstract: The signal pathway for light-induced expression of c- fos and the neuropeptide somatostatin (SS) in rat retinal cells was investigated. Flashing light induced c- fos and SS mRNA in the inner nuclear layer and the ganglion cell layer. As both c- fos and SS genes have a cyclic AMP response element (CRE) in their promoters, CRE-binding protein (CREB) phosphorylation in retinal cells was examined with a phospho-CREB-specific antibody. Both flashing light and administration of the L-type Ca²⁺ channel activator Bay K 8644 induced phosphorylation of CREB in the nuclei of the amacrine cells and the ganglion cells where c- fos /SS mRNAs were expressed. These cells could be double-stained with anti-calmodulin kinase II (anti-CaM kinase II) monoclonal antibody and phospho-CREB-specific polyclonal antiserum after Bay K 8644 administration, indicating the colocalization of phosphorylated CREB at Ser¹³³ and CaM kinase II in the neural retina.     

Ca2+ and Phospholipid-Dependent Protein Kinase Activity and Phosphorylation of Endogenous Proteins in Bovine Adrenal Medulla

Abstract: Soluble and membrane fractions of bovine adrenal medulla contain several substrates for the Ca²⁺/ phospholipid-dependent and cyclic AMP-dependent protein kinases. The phosphorylation of soluble proteins (36 and 17.7 kilodaltons) and a membrane protein (22.5 kilo-daltons) showed an absolute requirement for the presence of both Ca²⁺ and phosphatidylserine; other substrates showed less stringent phosphorylation requirements and many of these proteins were specific for each of the protein kinases. The Ca²⁺/phospholipid-dependent phosphorylation was rapid, with effects seen as early as at 30 s of incubation. Measurement of enzyme activities with histone HI as an exogenous substrate demonstrated that the Ca²⁺/phospholipid-dependent protein kinase was equally distributed between the soluble and membrane fractions whereas the cyclic AMP-dependent enzyme was predominantly membrane-bound in adrenal medulla and chromaffin cells. The activity of the soluble Ca²⁺/phos-pholipid-dependent protein kinase of adrenal medulla was found to be about 50% of the enzyme level present in rat brain, a tissue previously shown to contain a very high enzyme activity. These results suggest a prominent role for the Ca²⁺/phospholipid-dependent protein kinase in chromaffin cell function.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Overexpression of Ca2+/Calmodulin-Dependent Protein Kinase II Inhibits Neurite Outgrowth of PC12 Cells

Abstract: To investigate the physiological role of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) in neuronal differentiation, we transfected the cDNA of the α subunit of mouse CaM kinase II (CaM kinase IIα) into PC12 cells and established clonal cell lines that constitutively express the transfected CaM kinase IIα gene. The expression of CaM kinase IIα was confirmed by northern blot and immunoblot analyses. Northern blot analysis showed that the γ and δ subunits of CaM kinase II are mainly expressed in PC12 cells. Treatment of the cells with ionomycin activated CaM kinase IIα through autophosphorylation and generation of the Ca²⁺/calmodulin-independent form. It is interesting that the neurite outgrowth induced by dibutyryl cyclic AMP was inhibited in these cell lines in accordance with the activities of overexpressed CaM kinase IIα. The activity of cyclic AMP-dependent protein kinase showed similar levels among these cell lines. These results suggest that CaM kinase II is involved in the modulation of the neurite outgrowth induced by activation of the cyclic AMP system.     

Tyrosine Hydroxylase Phosphorylation in Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells: The Effect of Protein Kinase and Phosphatase Inhibitors on Ser19 and Ser40 Phosphorylation

Abstract: The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [γ-³²P]ATP, in the presence or absence of 10 µ M Ca²⁺, 1 µ M cyclic AMP, 1 µ M phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca²⁺ increased the phosphorylation of Ser¹⁹ and Ser⁴⁰. Cyclic AMP, and phorbol dibutyrate in the presence of Ca²⁺, increased the phosphorylation of only Ser⁴⁰. Ser³¹ and Ser⁸ were not phosphorylated. The Ca²⁺-stimulated phosphorylation of Ser¹⁹ was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273–302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca²⁺-stimulated phosphorylation of Ser⁴⁰ was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5–22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19–31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser¹⁹ and Ser⁴⁰ were dephosphorylated by PP2A.     

Interactions between cyclic AMP- and phorbol ester-dependent phosphorylation systems in S49 mouse lymphoma cells

High-resolution two-dimensional gel electrophoresis of proteins labeled with either 32Pi or [35S]methionine was used to study interactions between cyclic AMP and tetradecanoyl phorbol acetate (TPA) at the level of intracellular protein phosphorylation. Cultured S49 mouse lymphoma cells were used as a model system, and mutant sublines lacking either the catalytic subunit of cyclic AMP-dependent protein kinase or the guanyl nucleotide-binding "Ns" factor of adenylate cyclase provided tools to probe mechanisms underlying the interactions observed. Three sets of phosphoproteins responded differently to TPA treatment of wild-type and mutant cells: Phosphorylations shown previously to be responsive to activation of intracellular cyclic AMP-dependent protein kinase were stimulated by TPA in wild-type cells but not in mutant cells, a subset of phosphorylations stimulated strongly by TPA in mutant cells was inhibited in wild-type cells, and two novel phosphoprotein species appeared in response to TPA only in wild-type cells. The latter two classes of TPA-mediated responses specific to wild-type cells could be evoked in adenylate cyclase-deficient cells by treating concomitantly with TPA and either forskolin or an analog of cyclic AMP. Three conclusions are drawn from our results: 1) TPA stimulates adenylate cyclase in wild-type cells causing increased phosphorylation of endogenous substrates by cyclic AMP-dependent protein kinase, 2) activated cyclic AMP-dependent protein kinase inhibits phosphorylation (or enhances dephosphorylation) of a specific subset of TPA-dependent phosphoproteins, and 3) cyclic AMP-dependent events facilitate TPA-dependent phosphorylation of some substrate proteins.     

Thromboxane A receptor-mediated cell proliferation, survival and gene expression in oligodendrocytes

Thromboxane A(2) receptors (TP) were previously localized to discrete regions in the rat brain on myelinated fiber tracts and oligodendrocytes (OLGs). The present studies extended these findings and investigated the effects of TP signaling on cell proliferation, survival, and gene expression in OLG progenitor cells (OPCs) and OLGs. It was found that the TP agonist, U46619 stimulated the proliferation of OPCs and promoted the survival of mature OLGs. Examination of the early gene expression events involved in OPC proliferation, revealed that c-fos expression was substantially increased by U46619 stimulation. Treatment of OPCs or OLGs with U46619 caused activation of the mitogen-activated protein kinases (MAPK) ERK 1/2. In OPCs this activation was blocked by inhibition of src. However, in OLGs this phosphorylation was not only blocked by inhibition of src but also by inhibition of protein kinase C (PKC). Furthermore, U46619 was found to increase CREB phosphorylation in both OPCs and OLGs. Similar to ERK 1/2 activation, there was a divergence in the mechanism of the TP-mediated CREB response for each cell type. Specifically, U46619 activation was attenuated by src and protein kinase A (PKA) inhibition in OPCs, whereas in OLGs this effect was blocked by inhibition of src, PKA as well as by inhibition of PKC. Collectively, these results provide the first demonstration that TP-activated nuclear signaling events are involved in the proliferation of OPCs, the survival of mature OLGs, and the stimulation of gene expression.     

Differences in the cyclic AMP-dependent phosphorylation of plasma membrane proteins of differentiated and undifferentiated L6 myogenic cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L6 myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L6 myoblasts was stimulated more than three fold by 5 x 10(-5) M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L6 cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L6 cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f2b. Therefore, the data show that differentiation in L6 cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.     

3',5'-Cyclic Adenosine Monophosphate- and Ca2+-Calmodulin-Dependent Endogenous Protein Phosphorylation Activity in Membranes of the Bovine Chromaffin Secretory Vesicles: Identification of Two Phosphorylated Components as Tyrosine Hydroxylase and Protein Kinase Regulatory Subunit Type II

Abstract: Membranes of the secretory vesicles from bovine adrenal medulla were investigated for the presence of the endogenous protein phosphorylation activity. Seven phosphoprotein bands in the molecular weight range of 250,000 to 30,000 were observed by means of the sodium dodecyl sulphate electrophoresis and autoradiography. On the basis of the criteria of molecular weight, selective stimulation of the phosphorylation by cyclic AMP (as compared with cyclic GMP) and immunoprecipitation by specific antibodies, band 5 (molecular weight 60,300) was found to represent the phosphorylated form of the secretory vesicle-bound tyrosine hydroxylase. The electrophoretic mobility, the stimulatory and inhibitory effects of cyclic AMP in presence of Mg²⁺ and Zn,²⁺ respectively, and immunoreactivity toward antibodies showed band 6 to contain two forms of the regulatory subunits of the type II cyclic AMP-dependent protein kinase, distinguishable by their molecular weights (56,000 and 52,000, respectively). Phosphorylation of band 7 (molecular weight 29,800) was stimulated about 2 to 3 times by Ca²⁺ and calmodulin in the concentration range of both agents believed to occur in the secretory tissues under physiological conditions.     

Regulation of the L-Type Ca2+ Channel Current in Rat Pinealocytes

Abstract : In the present study, the role of phosphoprotein phosphatase in the regulation of L-type Ca²⁺ channel currents in rat pinealocytes was investigated using the whole-cell version of the patch-clamp technique. The effects of three phosphatase inhibitors, calyculin A, tautomycin, and okadaic acid, were compared. Although all three inhibitors were effective in inhibiting the L-type Ca²⁺ channel current, calyculin A was more potent than either tautomycin or okadaic acid, suggesting the involvement of phosphoprotein phosphatase-1. To determine the kinase involved in the regulation of these channels, cells were pretreated with H7 (a nonspecific kinase inhibitor), H89 (a specific inhibitor of cyclic AMP-dependent kinase), KT5823 (a specific inhibitor of cyclic GMP-dependent kinase), or calphostin C (a specific inhibitor of protein kinase C). Pretreatment with either H7 or calphostin C decreased the inhibitory effect of calyculin A on the L-type Ca²⁺ channel current. In contrast, pretreatment with H89 or KT5823 had no effect on the inhibition caused by calyculin A. Based on these observations, we conclude that basal phosphatase activity, probably phosphoprotein phosphatase-1, plays an important role in the regulation of L-type Ca²⁺ channel currents in rat pinealocytes by counteracting protein kinase C-mediated phosphorylation.     

Neuromodulator-Mediated Phosphorylation of Specific Proteins in a Neurotumor Hybrid Cell Line (NCB-20)

Mouse neuroblastoma X embryonic Chinese hamster brain explant hybrid cell line (NCB-20) forms functional synapses when intracellular cyclic AMP levels are elevated for a prolonged period of time. NCB-20 cells were labeled with [32P]orthophosphate under conditions where 2-chloroadenosine gave maximum increases of 32P incorporation into tyrosine hydroxylase in nerve growth factor dibutyryl cyclic AMP-differentiated PC12 (pheochromocytoma) cells. When NCB-20 cells were exposed to activators [5-hydroxytryptamine (5-HT), prostaglandin E1, or forskolin], resulting in activation of cyclic AMP-dependent protein kinase, increased 32P incorporation into two major proteins [130 kilodaltons (kDa) and 90 kDa] occurred. 5-HT (in the presence of phosphodiesterase inhibitor, isobutylmethylxanthine) gave a three- to fourfold increase, and forskolin a four- to sevenfold increase in 32P incorporation into the 90-kDa protein. [D-Ala2,D-Leu5]-enkephalin, which decreased cyclic AMP levels and reversed the 2-chloroadenosine-stimulated phosphorylation of tyrosine hydroxylase in differentiated PC12 cells, also reversed the stimulation of phosphorylation of the 90-kDa protein in NCB-20 cells. Pretreatment of NCB-20 cells with a calcium ionophore, A23187, gave increased phosphorylation of the 90- and 130-kDa proteins, but phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (tumor promoting agent), cell depolarization with high K+, or pretreatment with dibutyryl cyclic GMP had no effect on phosphorylation of these proteins. In contrast, phosphorylation of an 80-kDa protein was decreased by forskolin, but increased following activation of the calcium/phospholipid-dependent kinase with tumor promoting agent. Neither the 90-kDa nor the 80-kDa protein showed any immunological cross-reactivity with synapsin, a major synaptic protein known to be phosphorylated by cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase, but not calcium/phospholipid-dependent protein kinase. This suggests that in NCB-20 cells, several unique proteins can be phosphorylated by cyclic AMP-dependent protein kinase in response to hormonal elevation of cyclic AMP levels. In contrast, an 80-kDa protein is the primary substrate for calcium/phospholipid-dependent protein kinase, and its phosphorylation is inhibited by agents that elevate cyclic AMP levels and thereby activate cyclic AMP-dependent protein kinase.     

Lymphocyte protein kinase activity in cells from young and elderly men and women

Protein kinase activity of lymphocytes isolated from human subjects was assayed using histone as substrate. The activity was stimulated about twofold by cyclic AMP and total enzyme activity, determined in the presence of cyclic AMP, was inhibited by 65% by the specific heat-stable inhibitor of cyclic AMP-dependent protein kinase. Histone phosphorylation was not stimulated by cyclic GMP in the presence of the inhibitor. Cyclic AMP-dependent protein kinase could be activated in vitro by incubating intact cells with isoproterenol or with forskolin and was reflected by a significant (P less than 0.05) increase in the protein kinase activity ratio. In contrast to these well-characterized adenylate cyclase activators, incubating cells for up to 2 hr in vitro in the presence of the specific beta-blocker propranolol had no significant effect on the amount of cyclic AMP-dependent protein kinase that was in the activated state. When compared in subjects between the ages of 21 and 74 years, lymphocyte protein kinase activity was unaltered by age or gender. These results indicate that cyclic nucleotide-dependent protein kinase is of the cyclic AMP-dependent variety in the human lymphocyte. A low amount of the cyclic AMP-dependent activity (about 15%) is in the already activated state in freshly isolated cells, and this is not further reduced by incubation in vitro or by beta-blockade. In contrast to previously reported changes in the capacity to synthesize cyclic AMP, lymphocyte protein kinase is unaltered by gender or age in human subjects.     

Ca2+, calmodulin-dependent regulation of microtubule formation via phosphorylation of microtubule-associated protein 2, tau factor, and tubulin, and comparison with the cyclic AMP-dependent phosphorylation

Abstract: Isolated microtubule-associated protein 2 (MAP2), τ factor, and tubulin were phosphorylated by a purified Ca²⁺, calmodulin-dependent protein kinase (640K enzyme) from rat brain. The phosphorylation of MAP2 and τ factor separately induced the inhibition of microtubule assembly, in accordance with the degree. Tubulin phosphorylation by the 640K enzyme induced the inhibition of microtubule assembly, whereas the effect of tubulin phosphorylation by the catalytic subunit was undetectable. The effects of tubulin and MAPs phosphorylation on microtubule assembly were greater than that of either tubulin or MAPs phosphorylation. Because MAP2, τ factor, and tubulin were also phosphorylated by the catalytic subunit of type-II cyclic AMP-dependent protein kinase from rat brain, the kinetic properties and phosphorylation sites were compared. The amount of phosphate incorporated into each microtubule protein was three to five times higher by the 640K enzyme than by the catalytic subunit. The K _m values of the 640K enzyme for microtubule proteins were four to 24 times lower than those of the catalytic subunit. The peptide mapping analysis showed that the 640K enzyme and the catalytic subunit incorporated phosphate into different sites on MAP2, τ factor, and tubulin. Investigation of phosphoamino acids revealed that only the seryl residue was phosphorylated by the catalytic subunit, whereas both seryl and threonyl residues were phosphorylated by the 640K enzyme. These data suggest that the Ca²⁺, calmodulin system via phosphorylation of MAP2, τ factor, and tubulin by the 640K enzyme is more effective than the cyclic AMP system on the regulation of microtubule assembly.     

Evidence against direct involvement of cyclic AMP-dependent protein phosphorylation in the exocytosis of amylase.

To examine whether or not the activation of cyclic AMP-dependent protein kinase is coupled to the exocytosis of amylase from rat parotid cells, the effect of protein kinase inhibitors on amylase release and protein phosphorylation was studied. A membrane-permeable inhibitor of cyclic AMP-dependent protein kinase, N-[2-(methylamino)ethyl]-5-isoquinolinesulphonamide (H-8), and peptide fragments of the heat-stable protein kinase inhibitor [PKI-(5-24)-peptide and PKI-(14-24)-amide] strongly inhibited cyclic AMP-dependent protein kinase activity in the cell homogenate. However, H-8 had no inhibitory effect on amylase release from either intact or saponin-permeabilized parotid cells stimulated by isoproterenol or cyclic AMP. Moreover, PKI-(5-24)-peptide and PKI-(14-24)-amide did not inhibit cyclic AMP-evoked amylase release from saponin-permeabilized cells, whereas cyclic AMP-dependent phosphorylations of 21 and 26 kDa proteins in intact or permeabilized cells were markedly inhibited by these inhibitors. These results suggest that cyclic AMP-dependent protein phosphorylation is not directly involved in the exocytosis of amylase regulated by cyclic AMP.     

Stimulatory Effects of Protein Kinase C and Calmodulin Kinase II on N-Methyl-d-Aspartate Receptor/Channels in the Postsynaptic Density of Rat Brain

Abstract: To clarify the regulatory mechanism of the N -methyl- d -aspartate (NMDA) receptor/channel by several protein kinases, we examined the effects of purified type II of protein kinase C (PKC-II), endogenous Ca²⁺/calmodulin-dependent protein kinase II (CaMK-II), and purified cyclic AMP-dependent protein kinase on NMDA receptor/ channel activity in the postsynaptic density (PSD) of rat brain. Purified PKC-II and endogenous CaMK-II catalyzed the phosphorylation of 80–200-kDa proteins in the PSD and l -glutamate-(or NMDA)-induced increase of (+)-5-[³H]methyl-10, 11-dihydro-5 H -dibenzo[a, d]cyclohepten-5, 10-imine maleate ([³H]MK-801; open channel blocker for NMDA receptor/channel) binding activity was significantly enhanced. However, the pretreatment of PKC-II-and CaMK-II-catalyzed phosphorylation did not change the binding activity of l -[³H]glutamate, cis -4-[³H](phospho-nomethyl)piperidine-2-carboxylate ([³H]CGS-19755; competitive NMDA receptor antagonist), [³H]glycine, α-[³H]-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, or [³H]-kainate in the PSD. Pretreatment with PKC-II-and CaMK-II-catalyzed phosphorylation enhanced l -glutamate-induced increase of [³H]MK-801 binding additionally, although purified cyclic AMP-dependent protein kinase did not change l -glutamate-induced [³H]MK-801 binding. From these results, it is suggested that PKC-II and/or CaMK-II appears to induce the phosphorylation of the channel domain of the NMDA receptor/channel in the PSD and then cause an enhancement of Ca²⁺ influx through the channel.     

Characterization of the Phosphorylation Site of PP59, a Substrate for Cyclic AMP-Dependent Protein Kinase That Is Enriched in the Synaptic Membrane Fraction of Rat Cerebellum

Abstract: We have identified previously a synaptic membrane-associated protein, PP59, that serves as a substrate for cyclic AMP-dependent protein kinase and is enriched in rat cerebellum. We show here that PP59 can be extracted from synaptic plasma membranes with a combination of 2% Triton X-100 plus 1 M KCl. A 290-fold purification of PP59 was achieved by selective solubilization, followed by continuous-elution preparative gel electrophoresis. To determine the amino acid sequence surrounding the cyclic AMP-dependent protein kinase phosphorylation site within PP59, the partially purified ³²P-phosphorylated protein was digested with chymotrypsin, and radiolabeled peptides were purified by sequential reversed-phase HPLC in two different solvent systems. Automated Edman degradation revealed a single phosphorylation site contained within the sequence Ala-Arg-Glu-Arg-Ser-Asp-Ser(P)-Thr-Gly-Ser-Ser-Ser-Val-Tyr. No strong sequence homology to this peptide fragment with other known peptides or proteins in the SwissProt, PIR, or GenPept databases could be found. A synthetic peptide containing this unique 14-amino acid sequence was used to develop polyclonal anti-peptide antibodies that were affinity-purified and shown to recognize intact PP59 as determined by western blotting. These antibodies specifically inhibited the phosphorylation of PP59 by cyclic AMP-dependent protein kinase in an in vitro phosphorylation assay containing synaptic plasma membranes.