Protein kinase C and phosphatidylserine bind to Mr 110,000/115,000 polypeptides enriched in cytoskeletal and postsynaptic density preparations

The selective binding of protein kinase C to nitrocellulose-immobilized polypeptides from rat brain and human erythrocytes was investigated. Bound enzyme was detected immunochemically with a monospecific protein kinase C antibody, or by using radiolabeled enzyme. Two polypeptides from erythrocyte membranes with Mr values of 110,000 and 115,000 bound protein kinase C in the presence of phosphatidylserine (PS) and were highly enriched in the cytoskeletal fraction. A prominent protein kinase C-binding polypeptide at Mr about 115,000 was also evident in brain cytoplasm, postsynaptic densities, and nuclei. Overlays of electrophoretic blots with 14C-phospholipids revealed that the protein kinase C-binding polypeptides also bound PS but not other phospholipids. The binding of both protein kinase C and PS was markedly inhibited after phosphorylation of the Mr 110,000/115,000 polypeptides with the kinase itself. The relevance of the results to the binding of protein kinase C to membranes and to phospholipid-cytoskeletal interactions is discussed.     

Specific postsynaptic density proteins bind tubulin and calmodulin-dependent protein kinase type II

Cytoskeletal interactions which contribute to the assembly of the postsynaptic density (PSD) were investigated. PSDs bound 125I-tubulin specifically with an apparent Km of 2 X 10(-7) M and a Bmax of about 1 nmol/mg of protein. 125I-Tubulin blots revealed that a group of polypeptides between Mr 135,000 and 147,000 (P-140) was a major tubulin-binding PSD component. The P-140 polypeptides were highly enriched in the PSD fraction of purified synaptosomes and could not be detected in crude brain cytoplasm preparations. These polypeptides were subject to phosphorylation by endogenous calmodulin-dependent protein kinase type II, with a concomitant reduction in 125I-tubulin binding. The tubulin-binding polypeptides could also associate with the radiolabeled alpha- and beta-subunits of the calmodulin-dependent protein kinase. These observations are consistent with a role for the P-140 polypeptides in organizing the molecular structure of the PSD. The data also suggest that this structure may be modified by Ca2+-sensitive phosphorylation, thus permitting neuronal activity to modulate the cytoskeletal interactions of the PSD.     

Characterization of a soluble Mr-30,000 catalytic fragment of the neuronal calmodulin-dependent protein kinase II

Chymotryptic digestion of postsynaptic densities releases a soluble, catalytically active fragment of the alpha (Mr 50,000) subunit of the neuronal cytoskeletal calmodulin-dependent protein kinase II. The purified soluble form of the kinase likewise yields the fragment. Denaturation of the enzyme results in more extensive proteolytic degradation. 125I-Iodopeptide maps of the isolated catalytic portions of both forms of the enzyme are similar and are contained within the map of the isolated alpha subunit. Catalytic fragments of both forms of the enzyme comigrate on two-dimensional SDS-PAGE/isoelectric focusing with pI 6.7-7.2. The fragment phosphorylates microtubule-associated protein (MAP-2) but is not activated by Ca+2/calmodulin nor is it inhibited by trifluoperazine. Km values for MAP-2 and ATP are indistinguishable from those of the holoenzyme, while the Vmax is similar to that of the holoenzyme activated with Ca+2/calmodulin. Overlays of Western blots of fragment with 125I-calmodulin shows a loss of calmodulin binding. Both the number of phosphorylation sites and the ability to autophosphorylate are markedly reduced in the catalytic fragment. Evaluation of the hydrodynamic parameters of the purified fragment yielded Mr value of 25,600 with a frictional ratio (f/f0) of 1.12; the Mr value determined by SDS-PAGE was 30,000. Thus, the catalytic fragment appears to represent an activated form of the kinase with a monomeric, globular structure unlike the native enzyme which exhibits oligomerization and cytoskeletal association. These results are consistent with a tertiary structure for the calmodulin-dependent protein kinase that contains distinct domains responsible for catalytic activity, regulation by calmodulin, cytoskeletal association and the multimeric organization of enzyme subunits.     

Involvement of fodrin-binding proteins in the structure of the neuronal postsynaptic density and regulation by phosphorylation

Novel polypeptides with Mr values about 140,000 bind fodrin and spectrin and are enriched in the postsynaptic density (PSD) compared to other tissues or subcellular fractions. 125I-fodrin binding to these polypeptides is competed for by unlabeled spectrin. These polypeptides are distinct from ankyrin and its proteolytic fragments and from band 4.1 which also bind fodrin. Phosphorylation of PSDs by the endogenous calmodulin-dependent protein kinase markedly reduces 125I-fodrin binding to the transblotted preparation. Such an event may play a regulatory role in governing protein-protein interactions among elements of the PSD.     

Scaffold proteins (MAGUK, Shank and Homer) in postsynaptic density in the central nervous system

The postsynaptic density (PSD) is a dynamic multi-protein complex attached to the postsynaptic membrane composed of several hundred proteins such as receptors and channels, scaffolding and adaptor proteins, cell-adhesion proteins, cytoskeletal proteins, G-proteins and their modulators and signaling molecules including kinases and phosphtases. This review focuses on the prominent PSD scaffolds proteins such as members of the MAGUK (membrane-associated guanylyl kinase), Shank (SH3 domain and ankyrin repeat-containing protein) and Homer families. These molecules interact simultaneously with different kinds of receptors and modulate their function by linking the receptors to downstream signaling events. For example PSD 95, a main member of MAGUK family, interacts directly with carboxyl termini of NMDA receptor subunits and clusters them to the postsynaptic membrane. In addition, PSD 95 is involved in binding and organizing proteins connected with NMDAR signaling. Based on the modular character and ability to form multiproteins interactions, MAGUK, Shank and Homer are perfectly suited to act as a major scaffold in postsynaptic density.     

A Dynamic Corral Model of Receptor Trafficking at a Synapse

The postsynaptic density (PSD) is a cytoskeletal specialization within the postsynaptic membrane of a neuron that helps to concentrate and organize neurotransmitter receptors at a chemical synapse. The total number of receptors within the PSD, which is a major factor in determining the physiological strength or weight of a synapse, fluctuates due to the surface diffusion of receptors into and out of the PSD, and the interactions of receptors with scaffolding proteins and cytoskeletal elements within the PSD. In this article, we present a stochastic model of protein receptor trafficking at the PSD that takes into account these various processes. The PSD is treated as a stochastically gated corral, which contributes a source of extrinsic or environmental noise that supplements the intrinsic noise arising from small receptor numbers. Using a combination of stochastic analysis and Monte Carlo simulations, we determine the time-dependent variation in the mean and variance of synaptic receptor numbers for a variety of initial conditions that simulate fluorescence recovery after photobleaching experiments, and indicate how such data might be used to infer certain properties of the PSD.     

Identification of the Major Postsynaptic Density Protein as Homologous with the Major Calmodulin-Binding Subunit of a Calmodulin-Dependent Protein Kinase

The major postsynaptic density protein (mPSDp), comprising greater than 50% of postsynaptic density (PSD) protein, is an endogenous substrate for calmodulin-dependent phosphorylation as well as a calmodulin-binding protein in PSD preparations. The results in this investigation indicate that mPSDp is highly homologous with the major calmodulin-binding subunit (p) of tubulin-associated calmodulin-dependent kinase (TACK), and that PSD fractions also contain a protein homologous with the sigma-subunit of TACK. Homologies between mPSDp and a 63,000 dalton PSD protein and the rho- and sigma-subunits of TACK were established by the following criteria: (1) identical apparent molecular weights; (2) identical calmodulin-binding properties; (3) manifestation of Ca2+-calmodulin-stimulated autophosphorylation; (4) identical isoelectric points; (5) identical calmodulin binding and autophosphorylation patterns on two-dimensional gels; (6) homologous two-dimensional tryptic peptide maps; and (7) similar phosphoamino acid-specific phosphorylation of tubulin. The results suggest that mPSDp is a calmodulin-binding protein involved in modulating protein kinase activity in the postsynaptic density and that a tubulin kinase system homologous with TACK exists in a membrane-bound form in the PSD.     

A monoclonal antibody against brain calmodulin-dependent protein kinase type II detects putative conformational changes induced by Ca2+-calmodulin

A mouse monoclonal IgG1 antibody has been generated against the soluble form of the calmodulin-dependent protein kinase type II. This antibody recognizes both the soluble and cytoskeletal forms of the enzyme, requiring Ca2+ (EC50 = 20 microM) for the interaction. Other divalent cations such as Zn2+, Mn2+, Cd2+, Co2+, and Ni2+ will substitute for Ca2+, while Mg2+ and Ba2+ will not. The antibody reacts with both the alpha- and beta-subunits on Western blots in a similar Ca2+-dependent fashion but with a lower sensitivity. The affinity of the antibody for the kinase is 0.13 nM determined by displacement of 125I Bolton-Hunter-labeled kinase with unlabeled enzyme. A variety of other proteins including tubulin do not compete for antibody binding. The Mr 30,000 catalytic fragment obtained by proteolysis of either the soluble or the cytoskeletal form of the kinase fails to react with the antibody. Calmodulin and antibody reciprocally potentiate each other's interaction with the enzyme. This is illustrated both by direct binding studies and by a decrease of the Kmapp for calmodulin and an increase in the Vmax for the autophosphorylation reaction of the enzyme. The antibody thus appears to recognize and stabilize a conformation of the kinase which favors calmodulin binding although it does not itself activate the kinase in the absence of calmodulin. Since the Mr 30,000 catalytic fragment of the kinase is not immunoreactive, either the antibody combining site of the kinase must be present in the noncatalytic portion of the protein along with the calmodulin binding site or proteolysis interferes with the putative Ca2+-dependent conformational change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Capping of the N‐terminus of PSD‐95 by calmodulin triggers its postsynaptic release

Postsynaptic density protein‐95 (PSD‐95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD‐95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD‐95 is released from postsynaptic membranes in response to Ca²⁺ influx via NMDA receptors. Here, we show that Ca²⁺/calmodulin (CaM) binds at the N‐terminus of PSD‐95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD‐95 formed at its N‐terminus (residues 1–16). This N‐terminal capping of PSD‐95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD‐95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD‐95. The PSD‐95 mutant Y12E strongly impairs binding to CaM and Ca²⁺‐induced release of PSD‐95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD‐95 serves to block palmitoylation of PSD‐95, which in turn promotes Ca²⁺‐induced dissociation of PSD‐95 from the postsynaptic membrane.     

Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins. Characterization of AKAP 79.

Postsynaptic densities (PSD) are a network of proteins located on the internal surface of excitatory synapses just inside the postsynaptic membrane. Enzymes associated with the PSD are optimally positioned to respond to signals transduced across the postsynaptic membrane resulting from excitatory synaptic transmission or neurotransmitter release. We present evidence suggesting that type II cAMP-dependent protein kinase (PKA) is anchored to the PSD through interaction of its regulatory subunit (RII) with an A-Kinase Anchor Protein (AKAPs). A cDNA for the human RII-anchoring protein, AKAP 79, was isolated by screening an expression library with radiolabeled RII. This cDNA (2621 base pairs) encodes a protein of 427 amino acids with 76% identity to bovine brain AKAP 75 and 93% identity to a carboxyl-terminal RII-binding fragment of murine brain AKAP 150. A bacterially expressed 92-amino acid fragment, AKAP 79 (335-427) was able to bind RII alpha. Disruption of secondary structure by site-directed mutagenesis at selected residues within a putative acidic amphipathic helix located between residues 392 and 408 prevented RII binding. Immunological studies demonstrate that AKAP 79 is predominantly expressed in the cerebral cortex and is a component of fractions enriched for postsynaptic densities. AKAP antisera strongly cross-react with a 150-kDa protein in murine PSD believed to be AKAP 150. Co-localization of the type II PKA in purified PSD fractions was confirmed immunologically by detection of RII and enzymologically by measuring cAMP-stimulated phosphorylation of the heptapeptide substrate Kemptide. Approximately 30% of the PSD kinase activity was specifically inhibited by PKI 5-24 peptide, a highly specific inhibitor of PKA. We propose that AKAP 79 and AKAP 150 function to anchor the type II PKA to the PSD, presumably for a role in the regulation of postsynaptic events.     

Cytoskeletal changes in platelets induced by thrombin and phorbol myristate acetate (PMA).

Changes in shape, and aggregation that accompanies platelet activation, are dependent on the assembly and reorganization of the cytoskeleton. To assess the changes in cytoskeleton induced by thrombin and PMA, suspensions of aspirin-treated,32P-prelabeled, washed pig platelets in Hepes buffer containing ADP scavengers were activated with thrombin, and with PMA, an activator of protein kinase C. The cytoskeletal fraction was prepared by adding Triton extraction buffer. The Triton-insoluble (cytoskeletal) fraction isolated by centrifugation was analysed by SDS-PAGE and autoradiography. Incorporation of actin into the Triton-insoluble fraction was used to quantify the formation of F-actin. Thrombin-stimulated platelet cytoskeletal composition was different from PMA-stimulated cytoskeletal composition. Thrombin-stimulated platelets contained not only the three major proteins: actin (43 kDa), myosin (200 kDa) and an actin-binding protein (250 kDa), but three additional proteins of Mr56 kDa, 80 kDa and 85 kDa in the cytoskeleton, which were induced in by thrombin dose-response relationship. In contrast, PMA-stimulated platelets only induced actin assembly, and the 56 kDa, 80 kDa and 85 kDa proteins were not found in the cytoskeletal fraction. Exposure of platelets to thrombin or PMA induced phosphorylation of pleckstrin parallel to actin assembly. Staurosporine, an inhibitor of protein kinase C, inhibited actin assembly and platelet aggregation induced by thrombin or PMA, but did not inhibit the incorporation of 56 kDa, 80 kDa and 85 kDa into the cytoskeletal fraction induced by thrombin. These three extra proteins seem to be unrelated to the induction of protein kinase C. We conclude that actin polymerization and platelet aggregation were induced by a mechanism dependent on protein kinase C, and suggest that thrombin-activated platelets aggregation could involve additional cytoskeletal components (56 kDa, 80 kDa, 85 kDa) of the cytoskeleton, which made stronger actin polymerization and platelet aggregation more.     

Characterization of a Nerve Growth Factor-Stimulated Protein Kinase in PC12 Cells Which Phosphorylates Microtubule-Associated Protein 2 and pp250

Treatment of PC12 cells with nerve growth factor (NGF) resulted in the rapid, but transient, activation of a protein kinase which specifically phosphorylated an endogenous 250-kDa cytoskeletal protein (pp250). We report that the microtubule-associated protein, MAP2, is an alternative substrate for the NGF-activated kinase. NGF treatment maximally activated the kinase within 5 min; however, the activity declined with longer exposure to NGF. The enzyme was localized predominantly in microsomal and soluble fractions and phosphorylated MAP2 on serine and threonine residues. The soluble enzyme was fractionated by DEAE chromatography and gel filtration and had an apparent Mr of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pI of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in Vmax for MAP2. The Km with respect to the MAP2 substrate was approximately 50 nM and was not altered by NGF treatment. A novel feature of the NGF-stimulated enzyme was its sharp dependence on Mn2+ concentration. The active enzyme is likely to be phosphorylated, because inclusion of phosphatase inhibitors was required for recovery of optimal activity and the activity was lost on treatment of the enzyme with alkaline phosphatase. Histones, tubulin, casein, bovine serum albumin, and the ribosomal subunit protein S-6 were not phosphorylated by this enzyme. The NGF-stimulated kinase was distinct from A kinase, C kinase, or other NGF-stimulated kinases. The rapid and transient activation of the protein kinase upon NGF treatment suggests that the enzyme may play a role in signal transduction in PC12 cells.     

Protease-activated protein kinase C in rat liver

In regenerating rat liver, an elevated protein kinase activity was detected which phosphorylated ribosomal protein S6 and histones. The properties of this enzyme were closely similar with those of protease-activated protein kinase C with Mr 45,000. During the study of the mechanism of proteolytic activation, type III protein kinase C (encoding alpha-sequence) was shown to be subjected to limited proteolysis by trypsin-like protease and converted to protein kinase M in ionic strength- and pH-dependent manner. This reaction was stimulated in the presence of Ca2+ and phospholipid under slightly higher ionic strength condition than physiological level (greater than 140 mM NaCl) and alkaline pH (7.5-8.0). These results suggest that activation of Na+/H+ exchanger in plasma membrane may trigger this type of proteolytic activation of protein kinase C. In addition to protein kinase M, another type of protease-activated kinase with Mr 80,000 was detected when limited proteolysis of protein kinase C was performed on inactive form of this enzyme (in the absence of either Ca2+ or phospholipid or both activators) under lower ionic strength condition. The molecular mass of this active enzyme was slightly smaller (approximately 200) than that of native protein kinase C. However, it is not clear at this time whether this small fragment was released from amino-terminal or carboxy-terminal domain to make protein kinase C partially active in the absence of Ca2+ and phospholipid. Although it has been proposed that proteolytic degradation of protein kinase C is involved in down regulation of this enzyme, the physiological significance of these two types of protease-activated forms of protein kinases in liver has remained obscure.     

Cyclic AMP-stimulated protein kinases at brain synaptic junctions.

We have examined endogenous cyclic AMP-stimulated phosphorylation of subcellular fractions of rat brain enriched in synaptic plasma membranes (SPM), purified synaptic junctions (SJ), and postsynaptic densities (PSD). The analyses of these fractions are essential to provide direct evidence for cyclic AMP-dependent endogenous phosphorylation at discrete synaptic junctional loci. Protein kinase activity was measured in subcellular fractions using both endogenous and exogenous (histones) proteins as substrates. The SJ fraction possessed the highest kinase activity toward endogenous protein substrates, 5-fold greater than SPM and approximately 120-fold greater than PSD fractions. Although the kinase activity as measured with histones as substrates was only slightly higher in SJ than SPM fractions, there was a marked preference of kinase activity toward endogenous compared to exogenous substrates in SJ fractions but in SPM fractions. Although overall phosphorylation in SJ fractions was increased only 36% by 5 micron cyclic AMP, there were discrete proteins of Mr = 85,000, 82,000, 78,000, and 55,000 which incorporated 2- to 3-fold more radioactive phosphate in the presence of cyclic AMP. Most, if not all, of the cyclic AMP-independent kinase activity is probably catalyzed by catalytic subunit derived from cyclic AMP-dependent kinase, since the phosphorylation of both exogenous and endogenous proteins was greatly decreased in the presence of a heat-stable inhibitor protein prepared from the soluble fraction of rat brain. The specific retention of SJ protein kinase(s) activity during purification and their resistance to detergent solubilization was achieved by chemical treatments which produce interprotein cross-linking via disulfide bridges. Two SJ polypeptides of Mr = 55,000 and 49,000 were photoaffinity-labeled with [32P]8-N3-cyclic AMP and probably represent the regulatory subunits of the type I and II cyclic AMP-dependent protein kinases. The protein of Mr = 55,000 was phosphorylated in a cyclic AMP-stimulated manner suggesting autophosphorylation as previously observed in other systems.     

Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin.

NMDA receptors are linked to intracellular cytoskeletal and signaling molecules via the PSD-95 protein complex. We report a novel family of postsynaptic density (PSD) proteins, termed Shank, that binds via its PDZ domain to the C terminus of PSD-95-associated protein GKAP. A ternary complex of Shank/GKAP/PSD-95 assembles in heterologous cells and can be coimmunoprecipitated from rat brain. Synaptic localization of Shank in neurons is inhibited by a GKAP splice variant that lacks the Shank-binding C terminus. In addition to its PDZ domain, Shank contains a proline-rich region that binds to cortactin and a SAM domain that mediates multimerization. Shank may function as a scaffold protein in the PSD, potentially cross-linking NMDA receptor/PSD-95 complexes and coupling them to regulators of the actin cytoskeleton.     

Isolation and major components of core structure of postsynaptic density

The core structure of postsynaptic density (PSD-core) was prepared from rat cerebral synaptosomes by application of the isolation procedure of synaptic junctions (SJ) after trypsinization, which dissociated pre- and post-synaptic structures. The PSD-core was considered to consist mainly of cytoplasmic part of postsynaptic structure, and lack the proteins localized on the external surface of the synaptic plasma membrane, such as receptors for neurotransmitters, Con A-binding proteins and connecting molecule(s) between pre- and post-synaptic structures. The PSD-core proteins which increased greatly in their contents compared with those of SJ prepared from synaptosomes (Syn-SJ) were 120 k Mr Con A-binding protein (Con A-BP) and 30 k Mr protein. Electron microscopic histochemistry suggested that 120 k Con A-BP localized widely in the main structure of the PSD-core. Protein of 30 k Mr was not extracted from PSD-core with 6 M urea, whereas actin, major PSD protein, and tubulin were easily extractable. The 30 k Mr protein was the most resistant one to trypsinization in the SJ fraction. The results suggest that the 30 k Mr protein plays an important role in stabilization and integrity of the postsynaptic density.     

ProSAP/Shank proteins - a family of higher order organizing molecules of the postsynaptic density with an emerging role in human neurological disease

The postsynaptic density (PSD) is a specialized electron-dense structure underneath the postsynaptic plasmamembrane of excitatory synapses. It is thought to anchor and cluster glutamate receptors exactly opposite to the presynaptic neurotransmitter release site. Various efforts to study the molecular structure of the PSD identified several new proteins including membrane receptors, cell adhesion molecules, components of signalling cascades, cytoskeletal elements and adaptor proteins with scaffolding functions to interconnect these PSD components. The characterization of a novel adaptor protein family, the ProSAPs or Shanks, sheds new light on the basic structural organization of the PSD. ProSAPs/Shanks are multidomain proteins that interact directly or indirectly with receptors of the postsynaptic membrane including NMDA-type and metabotropic glutamate receptors, and the actin-based cytoskeleton. These interactions suggest that ProSAP/Shanks may be important scaffolding molecules of the PSD with a crucial role in the assembly of the PSD during synaptogenesis, in synaptic plasticity and in the regulation of dendritic spine morphology. Moreover the analysis of a patient with 22q13.3 distal deletion syndrome revealed a balanced translocation with a breakpoint in the human ProSAP2/Shank3 gene. This ProSAP2/Shank3 haploinsufficiency may cause a syndrome that is characterized by severe expressive language delay, mild mental retardation and minor facial dysmorphisms.     

Properties of neurofilament protein kinase.

Neurofilament (NF) protein kinase, partially purified from NF preparations [Toru-Delbauffe & Pierre (1983) FEBS Lett. 162, 230-234], was found to be distinct from both the casein kinase present in NFs and the cyclic AMP-dependent protein kinase which is able to phosphorylate NFs. NF-kinase phosphorylated the three NF protein components. The amount of phosphate incorporated per molecule was higher for NF 200 than for NF 145 and NF 68. Other proteins present in the NF preparations were also used as NF-kinase substrates. Two of them might correspond to the myelin basic proteins with Mr values of 18,000 and 21,000. Four other substrates in the NF preparation were not identified (respective Mr values 53,000, 55,000, 65,000 and greater than 300,000). NF kinase also phosphorylated two additional brain-cell cytoskeletal elements: GFAp and vimentin. Casein, histones and phosvitin, currently used as substrates for protein kinase assays, were very poor phosphate acceptors. Half-maximal NF-kinase activity was obtained at an NF protein concentration of about 0.25 mg/ml in heated, salt-washed, NF preparations. The specific activity was about 5 pmol of 32P incorporated/min per microgram of NF kinase preparation protein. ATP was a phospho-group donor (Km 8 X 10(-5) M), but GTP was not. NF-kinase activity remained stable at 65 degrees C for more than 1 h. The enzyme was not degraded by storage at -20 degrees C for several months in a buffer containing 50% (w/v) sucrose. Maximal activity was obtained with 5 mM-Mg2+ (Mg2+ could be replaced by Co2+); Zn2+ and Cu2+ inhibited the reaction. NF-kinase was not dependent on cyclic AMP, cyclic GMP, Ca2+ or Ca2+ plus dioleoylglycerol and phosphatidylserine.     

Calcium/Calmodulin-Dependent Protein Kinase II Is Associated with NR2A/B Subunits of NMDA Receptor in Postsynaptic Densities

Abstract: NMDA receptors and Ca²⁺/calmodulin-dependent kinase II (CaMKII) have been reported to be highly concentrated in the postsynaptic density (PSD). Although the possibility that CaMKII in PSD might be associated with specific proteins has been put forward, the protein or proteins determining the targeting of the kinase in PSD have not yet been identified. Here we report that CaMKII binds to NR2A and NR2B subunits of NMDA receptors in PSD isolated from cortex and hippocampus. The association of NMDA receptor subunits and CaMKII was assessed by immunoprecipitating PSD proteins with antibodies specific for NR2A/B and CaMKII: CaMKII coprecipitated with NR2A/B and NR1 but not with other glutamate ionotropic receptor subunits, such as GluR1 and GluR2-3. A direct association between CaMKII and NR2A/B subunits was further confirmed by overlay experiments using either ³²P-autophosphorylated CaMKII or ³²P-NR2A/B and by evaluating the formation of a CaMKII-NR2A/B complex by means of the cross-linker disuccimidyl suberate. These data demonstrate an association between the NMDA receptor complex and CaMKII in the postsynaptic compartment, suggesting that this colocalization may be relevant for synaptic plasticity.     

Phosphorylation of myocardial fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and protein kinase C. Activation by phosphorylation and amino acid sequences of the phosphorylation sites

Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by cAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit Mr of 58,000) was rapidly phosphorylated by both cAMP-dependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by cAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit Mr of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by cAMP-dependent protein kinase lowered the Km value for fructose 6-phosphate from 87 to 42 microM without affecting the Vmax, whereas the phosphorylation by protein kinase C increased the Vmax value from 55 to 85 milliunits/mg without altering the Km value. The phosphorylated peptides were isolated, and their amino acid sequences were determined. The phosphorylation sites for both cAMP-dependent protein kinase and protein kinase C were located in a single peptide whose sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile-Arg-Arg-Pro. The seryl residue nearest the N terminus was the residue specifically phosphorylated by cAMP-dependent protein kinase, whereas the threonine residue nearest the C terminus was phosphorylated by protein kinase C.