Yasuhito Naito

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.

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.

MARCKS regulates lamellipodia formation induced by IGF-I via association with PIP2 and β-actin at membrane microdomains†

Myristoylated alanine‐rich C kinase substrate (MARCKS) is considered to participate in formation of F‐actin‐based lamellipodia, which represents the first stage of neurite formation. However, the mechanism of how MARCKS is involved in lamellipodia formation is not precisely unknown. Using SH‐SY5Y cells, we demonstrated here that MARCKS was translocated from cytosol to detergent‐resistant membrane microdomains, known as lipid rafts, within 30 min after insulin‐like growth factor‐I (IGF‐I) stimulation, which was accompanied by MARCKS dephosphorylation, β‐actin accumulation in lipid rafts, and lamellipodia formation. The protein kinase C inhibitor, Ro‐31‐8220, and Rho‐kinase inhibitors, HA1077 and Y27632, themselves decreased basal phosphorylation levels of MARCKS and coincidently elicited translocation of MARCKS to lipid rafts. On the other hand, the phosphoinositide 3‐kinase inhibitor, LY294002, abolished IGF‐I‐induced dephosphorylation, translocation of MARCKS to lipid rafts, and lamellipodia formation. Treatment of cells with neomycin, a PIP2‐masking reagent, attenuated the translocation of MARCKS to lipid rafts and the lamellipodia formation induced by IGF‐I, although dephosphorylation of MARCKS was not affected. Immunocytochemical and immunoprecipitation analysis indicated that IGF‐I stimulation induced the translocation of MARCKS to lipid rafts in the edge of lamellipodia and formation of the complex with PIP2. Moreover, we demonstrated that knockdown of endogenous MARCKS resulted in significant attenuation of IGF‐I‐induced β‐actin accumulation in the lipid rafts and lamellipodia formation. These results suggest a novel role for MARCKS in lamellipodia formation induced by IGF‐I via the translocation of MARCKS, association with PIP2, and accumulation of β‐actin in the membrane microdomains. J. Cell. Physiol. 220: 748–755, 2009.

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.

Inactivation of Ca2+/calmodulin-dependent protein kinase I by S-glutathionylation of the active-site cysteine residue

We show that Ca2+/calmodulin(CaM)‐dependent protein kinase I (CaMKI) is directly inhibited by its S‐glutathionylation at the Cys179. In vitro studies demonstrated that treatment of CaMKI with diamide and glutathione results in inactivation of the enzyme, with a concomitant S‐glutathionylation of CaMKI at Cys179 detected by mass spectrometry. Mutagenesis studies confirmed that S‐glutathionylation of Cys179 is both necessary and sufficient for the inhibition of CaMKI by diamide and glutathione. In transfected cells expressing CaMKI, treatment with diamide caused a reversible decrease in CaMKI activity. Cells expressing mutant CaMKI (179CV) proved resistant in this regard. Thus, our results indicate that the reversible regulation of CaMKI via its modification at Cys179 is an important mechanism in processing calcium signal transduction in cells.

Protein phosphorylation involved in the gene expression of the hydrogen sulphide producing enzyme cystathionine γ-lyase in the pancreatic β-cell.

Cystathionine γ-lyase (CSE) is one of the major enzymes for the production of hydrogen sulphide (H(2)S), a multifunctional gasotransmitter in the pancreatic β-cell. We examined the mechanisms by which glucose induces CSE expression in mouse pancreatic islets and the insulin-secreting cell line MIN6. CSE expression was increased by anti-diabetic sulphonylureas, and decreased by the ATP-sensitive K(+)-channel opener diazoxide and the voltage-dependent Ca(2+) channel blocker nitrendipine. Application of the synthetic inhibitors of protein kinases revealed the involvement of Ca(2+)/calmodulin-dependent protein kinase (CaMK) II and extracellular signal-regulated protein kinase (ERK) in glucose- and thapsigargin-induced CSE expression. The CaMK IIδ knockdown also suppressed CSE expression. Knockdown of the transcription factors Sp1 and Elk1, both of which can be phosphorylated by ERK, blunted CSE expression. By a reporter assay, we found Sp1 may directly and Elk1 may indirectly regulate CSE expression. These findings suggest Ca(2+)-dependent CSE expression may be mediated via protein phosphorylation of Sp1 and Elk1 in pancreatic β-cells.

Calcium/calmodulin-dependent protein kinases as potential targets of nitric oxide.

Nitric oxide (NO) synthesis is controlled by Ca(2+)/calmodulin (CaM) binding with and kinase-dependent phosphorylation of constitutive NO synthases, which catalyze the formation of NO and L-citrulline from L-arginine. NO operates as a mediator of important cell signaling pathways, such as cGMP signaling cascade. Another mechanism by which NO exerts biological effects is mediated via post-translational modification of redox-sensitive cysteine thiols of proteins. The Ca(2+)/CaM-dependent protein kinases (CaM kinases) such as CaM kinase I, CaM kinase II, and CaM kinase IV, are a family of protein kinases which requires binding of Ca(2+)/CaM to and subsequent phosphorylation of the enzymes to initiate its activation process. We report other regulation mechanisms of CaM kinases, such as S-glutathionylation of CaM kinase I at Cys(179) and S-nitrosylation of CaM kinase II at Cys(6/30). Such unique post-translational modification of CaMKs by NO shed light on a new area of mutual regulation of NO- and CaM kinases-signals. Based on the novel direct regulation of these kinases, we propose that CaM kinases/NO signaling would be good targets for understanding how they can participate in neuronal physiology and disease.

Phosphorylation of ribosomal protein S19 at Ser59 by CaM Kinase Iα

In order to examine the possible involvements of Ca2+/calmodulin‐dependent protein kinases (CaM kinases) in the regulation of ribosomal functions, we tested the phosphorylation of rat ribosomal protein S19 (RPS19) by various CaM kinases in vitro. We found that CaM kinase Iα, but not CaM kinase Iβ1, Iβ2, II, or IV, robustly phosphorylated RPS19. From the consensus phosphorylation site sequence, Ser59, Ser90, and Thr124 were likely to be phosphorylated; therefore, we mutated each amino acid to alanine and found that the mutation of Ser59 to alanine strongly attenuated phosphorylation by CaM kinase Iα, suggesting that Ser59 was a major phosphorylation site. Furthermore, we produced a specific antibody against RPS19 phosphorylated at Ser59, and found that Ser59 was phosphorylated both in GT1‐7 cells and rat brain. Phosphorylation of RPS19 in GT1‐7 cells was inhibited by KN93, an inhibitor of CaM kinases. Immunoblot analysis after subcellular fractionation of rat brain demonstrated that phosphorylated RPS19 was present in 80S ribosomes. Phosphorylation of RPS19 by CaM kinase Iα augmented the interaction of RPS19 with the previously identified S19 binding protein. These results suggest that CaM kinase Iα regulates the functions of RPS19 through phosphorylation of Ser59.

Dynamics of Ca2+/calmodulin-dependent protein kinase II following acute myocardial ischemia—translocation and autophosphorylation

Ca(2+)/calmodulin-dependent protein kinase (CaMK) family is responsive to changes in the intracellular Ca(2+) concentration. However, their functions have not been well established in the ischemia/reperfusion heart. The effects of myocardial ischemia on CaMKII, the most strongly expressed form, were investigated using isolated rat hearts. Rat hearts were rendered globally ischemic by stopping perfusion for 15 min, and then reperfused, heart ventricles being analyzed in each phase. Western blotting detected a decrease in the cytosolic and concomitant increase in the particulate fraction of CaMKII following transient ischemia. Redistribution to the cytosol was revealed on reperfusion. Northern blot showed CaMKII gene expression decreased by ischemia. Furthermore, autoradiography and confocal immunohistochemical findings provided autophosphorylation of CaMKII in the cytosol, ischemia causing decrease, with gradual recovery on reperfusion. These results indicate a transient partial translocation of CaMKII accompanied by kinase activity, with residual myocardial CaMKII undergoing autophosphorylation during ischemia and reperfusion, demonstrating two different characteristic dynamics of CaMKII.

IOP-lowering effect of isoquinoline-5-sulfonamide compounds in ocular normotensive monkeys.

Rho-associated coiled coil-formed protein kinase (ROCK) inhibitors are under development as a new class of antiglaucoma agents. Based on the potent ROCK inhibitor H-1152, previously developed by us, we explored the possibility of related compounds as antiglaucoma agents and synthesized seven types of H-1152-inspired isoquinoline-5-sulfonamide compounds (H-0103-H-0107, H-1001, H-1005). Although all of these compounds potently inhibited ROCK (IC50=18-48 nM), only H-0104 and H-0106 exerted strong intraocular pressure (IOP)-lowering effects into the eyes of monkeys. These results suggested the possibility that there is no direct relationship between ROCK inhibition and IOP-lowering effects, indicating that the initial screening of compounds based on ROCK inhibitory activity may be an unsuitable strategy for developing antiglaucoma agents with potent IOP-lowering effects.