Negative regulation of multifunctional Ca2+/calmodulin-dependent protein kinases: physiological and pharmacological significance of protein phosphatases.

Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) play pivotal roles in intracellular Ca2+ signaling pathways. There is growing evidence that CaMKs are involved in the pathogenic mechanisms underlying various human diseases. In this review, we begin by briefly summarizing our knowledge of the involvement of CaMKs in the pathogenesis of various diseases suggested to be caused by the dysfunction/dysregulation or aberrant expression of CaMKs. It is widely known that the activities of CaMKs are strictly regulated by protein phosphorylation/dephosphorylation of specific phosphorylation sites. Since phosphorylation status is balanced by protein kinases and protein phosphatases, the mechanism of dephosphorylation/deactivation of CaMKs, corresponding to their 'switching off', is extremely important, as is the mechanism of phosphorylation/activation corresponding to their 'switching on'. Therefore, we focus on the regulation of multifunctional CaMKs by protein phosphatases. We summarize the current understanding of negative regulation of CaMKs by protein phosphatases. We also discuss the biochemical properties and physiological significance of a protein phosphatase that we designated as Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP), and those of its homologue CaMKP-N. Pharmacological applications of CaMKP inhibitors are also discussed. These compounds may be useful not only for exploring the physiological functions of CaMKP/CaMKP-N, but also as novel chemotherapies for various diseases.

Synthesis and application of caged peptides and proteins.

Caged compounds have covalently attached groups that are rapidly cleaved upon exposure to UV light. Attachment of photolabile groups makes the molecule inert until photolysis releases it in its bioactive form. When caged compounds are applied to the experimental system in advance, the concentration jump of biologically active substances can be brought about immediately in a limited area upon irradiation with pulsed and focused UV light. Therefore, caged compounds of low molecular weight, which are commercially available, have been used effectively to study the mechanisms of temporal biological phenomena, such as muscle contraction, intracellular signaling, and neurotransmission. Because many proteins and peptides play important roles in these phenomena, their caged derivatives should serve as powerful tools to clarify complex biological systems. To prepare caged proteins and peptides, several groups have improved upon a chemical modification method, as well as developed two new methods: (1) nonsense codon suppression and (2) solid-phase peptide synthesis. In this review, we summarize recent advances made in the design, preparation, and application of caged peptides and proteins.

Sulfhydryl modification of V449C in the glutamate transporter EAAT1 abolishes substrate transport but not the substrate-gated anion conductance.

Excitatory amino acid transporters (EAATs) buffer and remove synaptically released L-glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. Here, we demonstrate that modification of a cysteine substituted within a C-terminal domain of EAAT1 abolishes transport in both the forward and reverse directions without affecting activation of the anion conductance. EC(50)s for L-glutamate and sodium are significantly lower after modification, consistent with kinetic models of the transport cycle that link anion channel gating to an early step in substrate translocation. Also, decreasing the pH from 7.5 to 6.5 decreases the EC(50) for L-glutamate to activate the anion conductance, without affecting the EC(50) for the entire transport cycle. These findings demonstrate for the first time a structural separation of transport and the uncoupled anion flux. Moreover, they shed light on some controversial aspects of the EAAT transport cycle, including the kinetics of proton binding and anion conductance activation.

Effects of threo-beta-hydroxyaspartate derivatives on excitatory amino acid transporters (EAAT4 and EAAT5).

D,L-threo-beta-Benzyloxyaspartate (D,L-TBOA), an analog of threo-beta-hydroxyaspartate (THA) possessing a bulky substituent, is a potent non-transportable blocker for the excitatory amino acid transporters, EAAT1, 2 and 3, while L-threo-beta-methoxyaspartate (L-TMOA) is a blocker for EAAT2, but a substrate for EAAT1 and EAAT3. To characterize the actions of these THA analogs and the function of EAAT4 and EAAT5, we performed electrophysiological analyses in EAAT4 or EAAT5 expressed on Xenopus oocytes. In EAAT4-expressing oocytes, D,L-TBOA acted as a non-transportable blocker, while L-TMOA like D,L-THA was a competitive substrate. In contrast, D,L-THA, D,L-TBOA and L-TMOA all strongly attenuated the glutamate-induced currents generated by EAAT5. Among them, L-TMOA showed the most potent inhibitory action. Moreover, D,L-THA, D,L-TBOA and L-TMOA themselves elicited outward currents at negative potentials and remained inward at positive potentials suggesting that D,L-TBOA and L-TMOA, as well as D,L-THA, not only act as non-transportable blockers, but also block the EAAT5 leak currents. These results indicate that EAATs 4 and 5 show different sensitivities to THA analogs although they share properties of a glutamate-gated chloride channel.

Substrate specificity of Ca(2+)/calmodulin-dependent protein kinase phosphatase: kinetic studies using synthetic phosphopeptides as model substrates.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases. In order to elucidate the mechanism of substrate recognition by CaMKPase, we chemically synthesized a variety of phosphopeptide analogs and carried out kinetic analysis using them as CaMKPase substrates. This is the first report using systematically synthesized phosphopeptides as substrates for kinetic studies on substrate specificities of protein Ser/Thr phosphatases. CaMKPase was shown to be a protein Ser/Thr phosphatase having a strong preference for a phospho-Thr residue. A Pro residue adjacent to the dephosphorylation site on the C-terminal side and acidic clusters around the dephosphorylation site had detrimental effects on dephosphorylation by CaMKPase. Deletion analysis of a model substrate peptide revealed that the minimal length of the substrate peptide was only 2 to 3 amino acid residues including the dephosphorylation site. The residues on the C-terminal side of the dephosphorylation site were not essential for dephosphorylation, whereas the residue adjacent to the dephosphorylation site on the N-terminal side was essential. Ala-scanning analysis suggested that CaMKPase did not recognize a specific motif around the dephosphorylation site. Myosin light chain phosphorylated by protein kinase C and Erk2 phosphorylated by MEK1 were poor substrates for CaMKPase, while a synthetic phosphopeptide corresponding to the sequence around the phosphorylation site of the former was not dephosphorylated by CaMKPase but that of the latter was fairly good substrate. These data suggest that substrate specificity of CaMKPase is determined by higher-order structure of the substrate protein rather than by the primary structure around its dephosphorylation site. Use of phosphopeptide substrates also revealed that poly-L-lysine, an activator for CaMKPase, activated the enzyme mainly through increase in the V(max) values.

Syntheses of optically pure beta-hydroxyaspartate derivatives as glutamate transporter blockers.

DL-threo-beta-benzyloxyaspartate (DL-TBOA) is a non-transportable blocker of the glutamate transporters that serves as an indispensable tool for the investigation of the physiological roles of the transporters. To examine the precise interaction between a blocker and the transporters, we synthesized the optically pure isomers (L- and D-TBOA) and its erythro-isomers. L-TBOA is the most potent blocker for the human excitatory amino acid transporters (EAAT1-3), while D-TBOA revealed a difference in the pharmacophores between EAAT1 and EAAT3. We also synthesized the substituent variants (methyl or naphthylmethyl derivatives) of L-TBOA. The results obtained here suggest that bulky substituents are crucial for non-transportable blockers.

Synthesis of caged peptides using caged lysine: application to the synthesis of caged AIP, a highly specific inhibitor of calmodulin-dependent protein kinase II.

N(alpha)-Fmoc-N(epsilon)-(2-nitrobenzyloxycarbonyl)-lysine has been prepared and used in the solid-phase synthesis of caged peptides. The synthesized caged AIP (cagedKcagedKALRRQEAVDAL) showed characteristics required for caged peptides including a significantly reduced inhibitory activity to calmodulin-dependent protein kinase II and instantaneous recovery of the activity with photo-irradiation.

Characterization of Bacillus strains of marine origin.

A total of twenty aerobic endospore-forming bacilli, isolated from marine invertebrates and sea water of different areas of the Pacific Ocean, were taxonomically characterized. Most of the bacilli (11 strains) of marine origin belonged to the species Bacillus subtilis, according to their phenotypic characteristics, antibiotic susceptibility profiles, and fatty acids patterns. A group of four alkaliphilic strains formed a separate cluster that was tentatively classified as B. horti. One isolate, KMM 1717, associated with a sponge from the Coral Sea was identified as B. pumilus. Two strains, Bacillus KMM 1916 and KMM 1918, showed antibiotic sensitivity profiles similar to B. licheniformis, but they had a distinct fatty acid composition and peculiar phenotypic traits. The taxonomic affiliation of KMM 1810 and KMM 1763 remained unclear since their fatty acid composition and antibiotic sensitivity patterns were not resembled with none of these obtained for Bacillus strains.

Enhancement of antimicrobial activity of neuropeptide Y by N-terminal truncation.

The activity of neuropeptide Y (NPY) against Candida albicans, which was revealed to be fungicidal, was enhanced significantly by the truncation of amino acid residues at the N terminus. The most active peptides (MICs, approximately 1 microM) were about 10-fold more potent than the intact NPY (MIC, approximately 10 microM). The enhancement was weakened by the replacement of the N terminus by negatively charged residues and/or acylation of the alpha-amino group. These results suggest that only the alpha-helical region of NPY is necessary for the antimicrobial activity and that the net charge of the peptide is important for the activity.

A potent nonpeptide neuropeptide Y Y1 receptor antagonist, a benzodiazepine derivative.

We describe here a nonpeptide neuropeptide Y Y1 receptor antagonist, 2,4-dioxo-1,5-bis(2-oxo-2-orthotolyl-ethyl)-3-[3-[3-([3-[3-(3-p iperidin-1-ylmethyl-phenoxy)-propylcarbamoyl]-propyl]-car bamoyloxymethyl)-phenyl]-ureido]-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diaz epine (Compound 1), which was previously synthesized as a linked type of dual cholecystokinin (CCK)-B and histamine H2 receptor antagonist. Compound 1 competitively inhibited [125I]peptide YY (PYY) binding to Y1 receptors in human neuroblastoma SK-N-MC cells with Ki of 6.4 +/- 1.0 nM, while it had no effect on [125I]PYY binding to Y2 or Y5 receptors even at 1 microM. Functionally, Compound 1 inhibited the Y1 receptor-mediated increase in cytosolic free Ca2+ concentration and Y1 receptor-mediated attenuation of cAMP accumulation in a dose-dependent manner with IC50 values of 95 +/- 5 and 320 +/- 10 nM in SK-N-MC cells, respectively. Neither its CCK-B receptor antagonistic moiety of Compound 1 (Compound 2) nor its histamine H2 receptor antagonistic moiety of Compound 1 (Compound 3) had any effect on [125I]PYY binding, suggesting that the entire structure of Compound 1 is essential for Y1 receptor blocking activity. It showed no significant activity (IC50 > 1 microM) in 30 receptor binding assays and 5 enzyme assays, with the exception of CCK-B and histamine H2 receptors. We conclude that Compound 1 is a useful molecule not only for studying the physiological role of neuropeptide Y but also for exploring more specific Y1 receptor antagonists.

Critical amino acid residues of AIP, a highly specific inhibitory peptide of calmodulin-dependent protein kinase II.

The importance of the individual amino acid residues of AIP (KKALRRQEAVDAL), a highly specific inhibitor of calmodulin-dependent protein kinase II (CaMKII), was studied. Replacement of Arg6, Gln7, or Ala9 by other amino acid residues produced a marked increase in the IC50 value. Leu4 and Val10 were also sensitive to replacement, but some hydrophobic amino acids could substitute for these residues. Although replacement of Ala3, Glu8, Ala12, and Leu13 by other residues produced no significant increase in the IC50, the substitution of Lys for Ala3 decreased the IC50. An AIP analog (KKKLRRQEAFDAY), in which Ala3 and Val10 were replaced with Lys and Phe, respectively, showed an IC50 value as low as 4 nM, suggesting that it is a useful tool for studying the physiological roles of CaMKII.

DL-threo-beta-benzyloxyaspartate, a potent blocker of excitatory amino acid transporters.

DL-threo-beta-Benzyloxyaspartate (DL-TBOA), a novel derivative of DL-threo-beta-hydroxyaspartate, was synthesized and examined as an inhibitor of sodium-dependent glutamate/aspartate (excitatory amino acid) transporters. DL-TBOA inhibited the uptake of [14C]glutamate in COS-1 cells expressing the human excitatory amino acid transporter-1 (EAAT1) (Ki = 42 microM) with almost the same potency as DL-threo-beta-hydroxyaspartate (Ki = 58 microM). With regard to the human excitatory amino acid transporter-2 (EAAT2), the inhibitory effect of DL-TBOA (Ki = 5.7 microM) was much more potent than that of dihydrokainate (Ki = 79 microM), which is well known as a selective blocker of this subtype. Electrophysiologically, DL-TBOA induced no detectable inward currents in Xenopus laevis oocytes expressing human EAAT1 or EAAT2. However, it significantly reduced the glutamate-induced currents, indicating the prevention of transport. The dose-response curve of glutamate was shifted by adding DL-TBOA without a significant change in the maximum current. The Kb values for human EAAT1 and EAAT2 expressed in X. laevis oocytes were 9.0 microM and 116 nM, respectively. These results demonstrated that DL-TBOA is, so far, the most potent competitive blocker of glutamate transporters. DL-TBOA did not show any significant effects on either the ionotropic or metabotropic glutamate receptors. Moreover, DL-TBOA is chemically much more stable than its benzoyl analog, a previously reported blocker of excitatory amino acid transporters; therefore, DL-TBOA should be a useful tool for investigating the physiological roles of transporters.

Syntheses of potent Leu-enkephalin analogs possessing beta-hydroxy-alpha,alpha-disubstituted-alpha-amino acid and their characterization to opioid receptors.

Novel Leu-enkephalin (Leu-Enk) (1) analogs possessing various types of alpha-substituted serine instead of its glycine residue in the position 2 were synthesized via an efficient O,N-migration method. The binding characteristics of the synthetic analogs using Chinese hamster ovary (CHO) cells expressed cloned rat mu-, delta-, and kappa-receptors revealed that [(1R,2S)-Ahh2]Enk (7) was the most potent agonist of delta-opioid receptors among all the synthetic analogs tested, and was 10 times more potent than the native Leu-Enk.

Solid-phase synthesis of caged peptides using tyrosine modified with a photocleavable protecting group: application to the synthesis of caged neuropeptide Y.

A simple method for the synthesis of caged peptides using a novel derivative of tyrosine, N-Fmoc-O-(2-nitrobenzyl)-tyrosine, is described. The derivative of tyrosine can be incorporated at any position in an amino acid sequence by solid-phase peptide synthesis under the condition for Fmoc chemistry, and caged peptides that contain nitrobenzyl group on the side chain of tyrosine residue can be obtained. The nitrobenzyl group can be photocleaved by UV irradiation and the half life of the intermediate during photolysis is approximately 7 microseconds. The method was successfully applied to the synthesis of caged neuropeptide Y (NPY). The binding affinity of the caged NPY for the Y1 receptor was one or two orders of magnitude lower than that of intact NPY, but it increased to the value for intact NPY upon irradiation by UV light.

Expression of two different cholecystokinin receptors in Xenopus oocytes injected with mRNA from rabbit pancreas and rat hippocampus.

Electrophysiological responses to cholecystokinin (CCK) were studied in Xenopus oocytes injected with mRNA from rabbit pancreas or rat hippocampus. CCK-octapeptide(26-33) (sulfated form) (CCK-8) elicited inward currents in both groups. In oocytes injected with pancreatic mRNA, CCK-8-induced currents were composed of two components, fast and slow. However, in oocytes injected with hippocampal mRNA, fast currents disappeared. The potency ranking of the agonists and the antagonist indicated that the receptors expressed by pancreatic and hippocampal mRNA were CCKA- and CCKB-subtypes, respectively. Extracellular application of EGTA had little effect on the CCKB-mediated response, but attenuated the CCKA-mediated one. Intracellular injection of EGTA abolished the CCKB-mediated response, whereas small smooth currents remained in oocytes expressing the CCKA-receptor. The reversal potentials of the CCKA- and CCKB-receptor-mediated responses were consistent with that for CI- currents. However, the reversal potential of the small smooth currents in EGTA-loaded oocytes expressing the CCKA-receptors was close to that for a non-selective cation channel. These results suggest that CCK-8 activates at least two different channels, a Ca(2+)-dependent Cl- channel and a non-selective cation channel in oocytes expressing the CCKA-receptor, while the CCKB-receptor elicits only a Ca(2+)-dependent Cl- channel.

Neuropeptide YY1 receptors-mediated increase in intracellular Ca2+ concentration via phospholipase C-dependent pathway in porcine aortic smooth muscle cells.

In porcine aortic smooth muscle cells, neuropeptide Y (NPY) stimulates mobilization of CA(2+) from intracellular store sites via Y1 receptors. However, it has been debated whether or not Ca(2+) mobilization by Y1 receptors depends on the generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] following activation of phospholipase C. To examine this question, we studied the effect of U73122, an inhibitor of phospholipase C-mediated inositol phosphate accumulation on the NPY-induced rise in cytosolic free Ca(2+) concentration ([Ca(2+)]i) in comparison with that on angiotensin II (AII)-induced [Ca(2+)]i increase, which is dependent on Ins(1,4,5)P3 generation. Digital-imaging microscopy study using the Ca(2+)-sensitive dye fura-2 revealed that application of AII induced a rapid but transient [Ca(2+)]i increase in a single cell, arising from intracellular calcium stores. Application of NPY to the same cell induced a [Ca(2+)]i rise with a pattern similar to that induced by AII. AII increased the formation of Ins(1,4,5)P3 by about 3.0 fold, while the NPY-induced [Ca(2+) formation was very small. U73122 completely inhibited not only Ins(1,4,5)P3 synthesis, but also Ca(2+) mobilization induced by either agonist. The effect of U73122 on the NPY-induced Ca(2+)i increase was about 10-fold more potent that on the AII-induced one. U73343, an inactive analog of U733343, had no influence on any of the AII- and NPY-mediated effects. Herbimycin A completely inhibited the platelet-derived growth factor-induced [Ca(2+]i increase but had no effect on the NPY-induced [Ca(2+)]i increase, indicating that phospholipase C-gamma is not involved in the NPY effect. These results suggest that NPY-induced Ca2+ mobilization from intracellular stores in porcine smooth muscle cells is secondary to the very small generation of Ins(1,4,5)P3 following stimulation of phospholipase C-beta, which may account for the hypersensitivity of the NPY effect to U73122.

Y2 receptors for neuropeptide Y are coupled to three intracellular signal transduction pathways in a human neuroblastoma cell line.

Neuropeptide Y (NPY) attenuated angiotensin II (AII)-or bradykinin (BK)-induced Ca2+ release from intracellular stores and inhibited forskolin-stimulated cAMP accumulation and omega-conotoxin-sensitive high K(+)-induced Ca2+ influx in the human neuroblastoma cell line SMS-KAN. All three NPY actions were mediated via Y2 receptors. Pretreatment with pertussis toxin completely abolished all of the NPY actions. Activation or down-regulation of protein kinase C had no effect on any NPY-mediated effect; herbimycin A, a tyrosine kinase inhibitor, only abolished the inhibitory effect of NPY on AII- or BK-induced Ca2+ mobilization. Herbimycin A also blocked platelet-derived growth factor-induced Ca2+ mobilization, which involves tyrosine kinase activation, and there was a good correlation in the concentration dependency between the two effects of herbimycin A, strongly suggesting that its ability to cancel the NPY effect is due to inhibition of tyrosine kinase activity. NPY attenuated AII- or BK-induced inositol 1,4,5-trisphosphate production, and herbimycin A reversed this NPY effect. These results provide the first evidence that Y2 receptors negatively couple to AII- or BK-induced phosphoinositide turnover leading to Ca2+ mobilization through pertussis toxin-sensitive GTP-binding protein(s). Inhibition of phospholipase C-beta activity by NPY seems to be mediated by activation of protein-tyrosine kinase or phosphotyrosine-containing protein(s).

Molecular cloning and characterization of the angiotensin receptor subtype in porcine aortic smooth muscle.

A cDNA encoding porcine aortic smooth muscle angiotensin II (AII) receptor has been isolated using the homology screening approach and sequenced. Specific binding of [125I]AII was found in COS-7 cells transfected with the cDNA (Kd = 0.37 nM, Bmax = 1 approximately 3 x 10(4)/cell) and was displaced with unlabeled AII-related peptides and DUP753 in the order of [Sar1,Ile8]AII = AII > des-Asp1-[Ile8]AII = DUP753 > angiotensin I = angiotensin III. EXP655 had no effect on [125I]AII binding. COS-7 cells transfected with the cDNA responded to AII by only a small increase in the concentration of intracellular free Ca2+. However, electrophysiological study of the receptor expressed in Xenopus laevis oocytes provided strong evidence that it could functionally couple to a second messenger system leading to the mobilization of intracellular stores of Ca2+. Northern blot analysis in cultured porcine aortic smooth muscle cells demonstrated that the expression of this gene varies with the culture media. These results indicate that the cDNA encodes the functional and regulated AT1 subtype of AII receptors.

Neuropeptide Y stimulates DNA synthesis in vascular smooth muscle cells.

Neuropeptide Y (NPY) stimulated DNA synthesis in porcine aortic smooth muscle cells in a concentration-dependent manner. [Leu31, Pro34]NPY, a Y1-specific agonist, was several hundred times more potent than NPY(13-36), which preferentially bound to Y2 receptors, for stimulating DNA synthesis. On the other hand, human pancreatic polypeptide had no effect. The potency of NPY and related peptides for stimulating DNA synthesis paralleled their potency for increasing the cytosolic free Ca2+ concentration in the cells. Pertussis toxin treatment completely blocked both effects of the peptides. Thus, NPY may induce Ca2+ mobilization and stimulation of DNA synthesis in vascular smooth muscle cells via Y1 receptors whose signal transduction system involves pertussis toxin-sensitive GTP-binding protein(s).