Hisayuki Yokokura

Regulation of Neuronal Nitric-oxide Synthase by Calmodulin Kinases

Phosphorylation of neuronal nitric-oxide synthase (nNOS) by Ca2+/calmodulin (CaM)-dependent protein kinases (CaM kinases) including CaM kinase Iα (CaM-K Iα), CaM kinase IIα (CaM-K IIα), and CaM kinase IV (CaM-K IV), was studied. It was found that purified recombinant nNOS was phosphorylated by CaM-K Iα, CaM-K IIα, and CaM-K IV at Ser847 in vitro. Replacement of Ser847 with Ala (S847A) prevented phosphorylation by CaM kinases. Phosphorylated recombinant wild-type nNOS at Ser847 (≈0.5 mol of phosphate incorporation into nNOS) exhibited a 30% decrease ofV max with little change of both theK m for l-arginine andK act for CaM relative to unphosphorylated enzyme. The activity of mutant S847D was decreased to a level 50–60% as much as the wild-type enzyme. The decreased NOS enzyme activity of phosphorylated nNOS at Ser847 and mutant S847D was partially due to suppression of CaM binding, but not to impairment of dimer formation which is thought to be essential for enzyme activation. Inactive nNOS lacking CaM-binding ability was generated by mutation of Lys732-Lys-Leu to Asp732-Asp-Glu (Watanabe, Y., Hu, Y., and Hidaka, H. (1997) FEBS Lett. 403, 75–78). It was phosphorylated by CaM kinases, as was the wild-type enzyme, indicating that CaM-nNOS binding was not required for the phosphorylation reaction. We developed antibody NP847, which specifically recognize nNOS in its phosphorylated state at Ser847. Using the antibody NP847, we obtained evidence that nNOS is phosphorylated at Ser847 in rat brain. Thus, our results suggest that CaM kinase-induced phosphorylation of nNOS at Ser847 alters the activity control of this enzyme.

MOLECULAR AND CELLULAR PHARMACOLOGY OF A CALCIUM/ CALMODULIN-DEPENDENT PROTEIN KINASE II (CAM KINASE II) INHIBITOR, KN-62, AND PROPOSAL OF CAM KINASE PHOSPHORYLATION CASCADES

Publisher Summary It is known that Ca 2+ is an important intracellular second messenger. Ca 2+ binds to a variety of proteins and alters their functions. Calmodulin (CaM) is a ubiquitous Ca 2+ -binding protein and the Ca 2+ /CaM complex interacts with target proteins, resulting in their functional changes. In this respect, it is believed that the family of Ca 2+ /CaM-dependent protein kinases (CaM kinases) is important for Ca 2+ -mediated protein phosphorylation. The prototype of this family is smooth muscle myosin light-chain kinase (MLCK), which phosphorylates myosin regulatory light chain in a Ca 2+ /CaM-dependent manner resulting in the activation of actomyosin ATPase required for muscle fiber contraction. The multifunctional CaM kinases have different tissue distributions and substrate specificities, suggesting that each of the enzymes is involved in different types of Ca 2+ -mediated processes. Researchers have succeeded in synthesizing the potent, specific inhibitor of CaM kinase II, KN-62 (1-[ N,O -bis(5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl)-4-phenylpiperazine). CaM kinase II is distributed in a wide range of tissues, but the brain contains the highest amount. This chapter reviews previous studies with KN-62 and propose the involvement of CaM kinase II in various aspects of cell function. Researchers have succeeded in synthesizing W-7, a naphthalenesulfonamide derivative, which can inhibit a protein activator-stimulated cyclic nucleotide phosphodiesterase (PDE). When the naphthalene ring was replaced by an isoquinoline ring in the derivatives with a shorter alkyl chain, the isoquinolinesulfonamide derivatives were no longer CaM antagonists but potent protein kinase inhibitors. KN-62 was proposed to be a specific inhibitor of CaM kinase II because the compound could selectively inhibit the enzyme among a limited number of protein kinases such as PKA, PKC, and smooth muscle MLCK. Research findings suggest that KN-62 occupies the Ca 2+ /CaM binding domain of CaM kinase II, thereby inhibits its Ca 2+ /CaM-dependent activation.

Frizzled-9 Is Activated by Wnt-2 and Functions in Wnt/β-Catenin Signaling

Frizzled has been known to function as a Wnt receptor. Although there have been a number of mammalian Frizzled members identified, their binding specificities with Wnt and functions in mammalian cells have been poorly understood. Here, we demonstrate that rat Frizzled-9 (Rfz9) functions in Wnt/β-catenin signaling in 293T cells. Rfz9 overexpression induces the hyperphosphorylation and relocalization of mouse Dishevelled-1 (Dvl-1) from the cytoplasm to the cell membrane and the accumulation of cytosolic β-catenin. Transfections of Rfz9 with each of several Wnt members show that only Wnt-2 activates Rfz9 in T cell factor (TCF)-dependent transcription. Deletion mutant analysis determines that there is a difference in Rfz9 C-terminal residues required for the modifications of Dvl-1 and those required for the inductions of β-catenin stabilization and TCF transactivation. Deletion of the Wnt-binding domain does not abolish Rfz9 activity completely, although it causes the inactivation of Wnt-2-dependent TCF transcription. Rfz9 also relocalizes Axin from the cytoplasm to the plasma membrane in the presence of Dvl-1, suggesting that one of the consequences of Dvl-1 relocalization by Rfz9 is to bring Axin to the cell membrane.

The Regulatory Region of Calcium/Calmodulin-dependent Protein Kinase I Contains Closely Associated Autoinhibitory and Calmodulin-binding Domains

The mechanism for the regulation of Ca2+/calmodulin-dependent protein kinase I (CaM kinase I) was investigated using a series of COOH-terminal truncated mutants. These mutants were expressed in bacteria as fusion proteins with glutathione S-transferase and purified by affinity chromatography using glutathione Sepharose 4B. A mutant (residues 1-332) showed complete Ca2+/CaM-dependent activity. Truncation mutants (residues 1-321, 1-314, and 1-309) exhibited decreasing affinities for Ca2+/CaM and also exhibited decreasing Ca2+/CaM-dependent activities. Truncation mutants (residues 1-305 or 1-299) were unable to bind Ca2+/CaM and were inactive. In contrast, truncation mutants (residues 1-293 or 1-277) were constitutively active at a slightly higher level (2-fold) than fully active CaM kinase I. These results indicate the location of the Ca2+/CaM-binding domain on CaM kinase I (residues 294-321) and predict the existence of an autoinhibitory domain near, or overlapping, the Ca2+/CaM-binding domain. These conclusions were supported by studies which showed that a synthetic peptide (CaM kinase I(294-321)) corresponding to residues 294-321 of CaM kinase I inhibited the fully active kinase in a manner that was competitive with Ca2+/CaM and also inhibited the constitutively active mutant (residues 1-293) in a manner that was competitive with Syntide-2, a peptide substrate, (Ki = 1.2 μM) but was non-competitive with ATP. Thus, these results suggest that CaM kinase I is regulated through an intrasteric mechanism common to other members of the family of Ca2+/CaM-dependent protein kinases.

Isoform-specific activation and structural diversity of calmodulin kinase I

We earlier confirmed that there are isoforms of Ca2+/calmodulin (CaM)-dependent protein kinase I (CaM kinase I) (CaM kinase Iβ1 and Iγ) beside CaM kinase Iα by cDNA cloning (Yokokura, H., Terada, O., Naito, Y., and Hidaka, H. (1997) Biochim. Biophys. Acta 1338, 8–12). Here, we demonstrate the existence of an isoform-specific activation mechanism of CaM kinase I and alternative splicing specifically regulating CaM kinase I (CaM kinase Iβ2) in the central nervous system. To cast light on isoform structure-enzyme activity relationships, CaM kinase Iβ1, Iβ2, and Iα were expressed separately using a baculovirus/Sf9 cell expression system. The novel CaM kinase Iβ2 isoform demonstrated similar catalytic activity to those of CaM kinase Iβ1 and Iα. Interestingly, CaM kinase Iβ1 and Iβ2 both can activate CaM kinase Iα activity via phosphorylation at Thr177. Reverse transcribed-polymerase chain reaction analysis showed that CaM kinase Iβ2 is dominant in the cerebrum and cerebellum, whereas CaM kinase Iβ1 is present in peripheral tissues such as liver, heart, lung, kidney, spleen, and testis. CaM kinase Iβ2 was also detected with an anti-CaM kinase Iβ2 antibody in PC12 cells. The results indicate that alternative splicing is a means for tissue-specific expression of CaM kinase Iβ. Thus the Thr177 residue of CaM kinase Iα is phosphorylated by not only CaM kinase kinase but also CaM kinase Iβ for activation of the enzyme.

Evidence for calmodulin inter-domain compaction in solution induced by W-7 binding

Small‐angle X‐ray scattering and nuclear magnetic resonance were used to investigate the structural change of calcium‐bound calmodulin (Ca2+/CaM) in solution upon binding to its antagonist, N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7). The radius of gyration was 17.4±0.3 Å for Ca2+/CaM‐W‐7 with a molar ratio of 1:5 and 20.3±0.7 Å for Ca2+/CaM. Comparison of the radius of gyration and the pair distance distribution function of the Ca2+/CaM‐W‐7 complex with those of other complexes indicates that binding of two W‐7 molecules induces a globular shape for Ca2+/CaM, probably caused by an inter‐domain compaction. The results suggest a tendency for Ca2+/CaM to form a globular structure in solution, which is inducible by a small compound like W‐7.

Isolation and comparison of rat cDNAs encoding Ca2+/calmodulin-dependent protein kinase I isoforms.

For possible multiple isoforms of Ca2+/calmodulin-dependent protein kinase I (CaM kinase I), only one cDNA (CaM kinase I alpha) hitherto has to been cloned. By screening of embryonic (E18) rat brain cDNA libraries, we have now isolated two additional examples (CaM kinase I beta and gamma). Northern blot analysis revealed CaM kinase I alpha to predominate over I beta and gamma in rat brain. Analysis of the tissue distribution of the isoforms by reverse transcription-polymerase chain reaction (RT-PCR) protocols demonstrated CaM kinase I alpha in a variety of tissues while the expression of CaM kinase I beta and gamma was more limited to the brain. The obtained results support the idea that CaM kinase I exists as a set of isoforms.

Mechanism of antitumor action of PKC activator, gnidimacrin.

Daphnane‐type diterpene gnidimacrin isolated from the Chinese plant Stellera chamaejasme L. is an antitumor agent that activates protein kinase C (PKC). The mechanism of antitumor action of gnidimacrin and the possible involvement of PKC were examined using sensitive K562 and refractory HLE cells. Gnidimacrin did bind to K562 cells 3 times more than to HLE cells. Immunoblot analyses revealed pronounced PKCβII expression in gnidimacrin sensitive cell lines including K562 cells, while refractory HLE cells strongly expressed PKCα, but not PKCβII. In a 24‐hr exposure of K562 cells to gnidimacrin, G1 phase arrest and inhibition of cdk2 kinase activity was found at growth‐inhibitory concentration (0.0005 μg/ml). Complete inhibition of cdk2 activity and maximum G1 phase arrest were observed at 0.005 μg/ml, however, these biological effects were reduced at 0.05 μg/ml (260 times the 50% inhibitory concentration). Cellular PKC after a 24‐hr exposure was examined by immunoblot analysis and specific binding of [3H]phorbol‐12,13‐dibutyrate as a ligand of PKC. Expression and the amount of functional PKC of K562 cells were not changed at 0.002 μg/ml, but down‐regulated to less than 1/10th of the control at 0.05 μg/ml. The reduction of biological effects at 0.05 μg/ml is most likely due to PKC down‐regulation. Our results suggest that PKC (particularly βII) is one of the major determinants of the ability of cells to respond to gnidimacrin and that the antitumor action might be associated with cell‐cycle regulation through suppression of cdk2 activity. Int. J. Cancer 77:243–250, 1998.© 1998 Wiley‐Liss, Inc.

A new and potent calmodulin antagonist, HF-2035, which inhibits vascular relaxation induced by nitric oxide synthase

HF-2035, 2-[N-(2-aminoethyl)-N-(2,4,5-trichlorobenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, was synthesized and its effects on calmodulin-dependent enzymes were investigated. HF-2035 inhibited calmodulin kinase I, calmodulin kinase II and myosin light-chain kinase with IC50 values of 1.3 microM, 1.6 microM and 68 microM, respectively. HF-2035 also inhibited the activity of recombinant rat neuronal nitric oxide synthase, one of the calmodulin-dependent enzymes, with a Ki of 0.78 microM. Partially purified nitric oxide synthase of rat brain was also inhibited by HF-2035 with an IC50 of 3.2 microM. Kinetic analysis indicated that this inhibitory effect of HF-2035 was competitive with respect to calmodulin. We examined the effects of HF-2035 on constitutive nitric oxide synthase in a bioassay using vascular strips of rabbit carotid artery with and without endothelium. HF-2035 inhibited acetylcholine- and calcium ionophore, A23187 (6S-[6 alpha (2S*,3S*),8 beta (R*),9 beta, 11 alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)- ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazol ecarboxylic acid)-induced relaxation of endothelium-intact strips with an ED50 of 1.5 +/- 0.5 microM and 2.8 +/- 1 microM, respectively. This compound, however, did not inhibit N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A), an exogenous nitric oxide donor, -induced relaxation of endothelium-denuded strips. W-7 (N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide) inhibited acetylcholine-induced relaxation with an ED50 of 46 +/- 7 microM, which was 30-fold less potent than HF-2035. HF-2035 was unable to inhibit the activity of the inducible form of nitric oxide synthase in isolated thoracic aorta of rat treated with Escherichia coli lipopolysaccharide. These findings suggest that HF-2035 is a new and potent calmodulin antagonist, and may be used as a mother compound to develop more selective inhibitors of constitutive nitric oxide synthase.

Unique Inhibitory Action of the Synthetic Compound 2-[N-(2-Aminoethyl)-N-(5-isoquinolinesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (CKA-1306) against Calcium/Calmodulin-dependent Protein Kinase I

A newly synthesized compound, 2-[N-(2-aminoethyl)-N-(5-isoquinolinesulfonyl)]amino-N-(4-chlorocinnamyl )-N-methylbenzylamine (CKA-1306), was found to inhibit cyclic AMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase I (CaMK I) with IC50 values of 1.6+/-0.14 and 2.5+/-0.16 microM, respectively. In contrast, the established PKA inhibitors H-8 and H-89 inhibited CaMK I with relatively high IC50 values of >100 and 24.4+/-3.2 microM, respectively. An additional inhibitor, KN-62, against Ca2+/calmodulin-dependent protein kinase II (CaMK II) did not inhibit either PKA or CaMK I at the concentrations tested. In our library of many isoquinolinesulfonamide derivatives, only CKA-1306 inhibited CaMK I to a satisfactory degree, suggesting a unique mode of action. Indeed, the inhibition of CaMK I by CKA-1306 was competitive in every respect to Mg2+/ATP, peptide substrate (syntide-2), and Ca2+/calmodulin. This phenomenon may be understood from the context of the recently determined structure of the enzyme in its autoinhibited state. Such kinetic analysis was also extended to cases using a phosphorylated and activated enzyme at Thr177 or a constitutively active, COOH-terminal truncated mutant at Gln293. CKA-1306 still competed with Mg2+/ATP for the two enzymes, but it no longer achieved any competitive advantage over syntide-2. These results may reflect some differences in the active conformation of CaMK I. However, the compound should be constant in its recognition of an Mg2+/ATP-binding site of the enzyme. Though CKA-1306 is not specific to CaMK I, the compound will be useful in studying the enzyme further under limited conditions.