Effects Of K252a Research Articles

Effects of K252a and K252b on CIH induced Changes in mEPSCs from PVN‐projecting MnPO

Chronic intermittent hypoxia (CIH) has been shown to alter the response of neurons in Median Preoptic nucleus (MnPO) to activation of cardiovascular inputs. Our previous results indicate that CIH enhances spontaneous presynaptic neurotransmitter release while also increasing postsynaptic responsiveness in MnPO. In other systems, BNDF/TrkB signaling has been shown to facilitate excitatory neurotransmission, so we tested its role in the increased MnPO excitation triggered by CIH. Data were collected from PVN‐projecting MnPO neurons which play an important role in CIH‐induced hypertension. Adult 250‐350g male rats were injected bilaterally in the PVN with 200 nL retrograde adeno‐associated virus (pAAV‐CAG‐tdTomato). Three weeks after the injections, the rats were exposed to a 7‐day intermittent hypoxia protocol (6 min cycles; 3 minutes of 21% oxygen, 3 min of 10% oxygen maintenance at 10% oxygen for 8 h during the light phase). Normoxic controls were exposed to room air. At the end of the protocol, the rats were anesthetized with isoflurane (2‐3%) and horizontal brain slices of MnPO were prepared. Miniature Excitatory Postsynaptic Currents (mEPSCs) were recorded using whole cell voltage clamp configuration from PVN‐projecting MnPO neurons. The mEPSCs were recorded with the holding potential of ‐60 mV in the presence of a GABAa receptor antagonist (SR 95531 hydrobromide, 25 µM), tetrodotoxin (1µM), and a glycine receptor antagonist (strychnine, 3 µM) in aCSF. Data were analyzed off‐line using Synaptosoft software. Differences in mEPSCs properties, such as peak amplitude, frequency, decay time, area, and cell capacitance were analyzed by ANOVA and Tukey multiple comparison tests. The results showed that CIH significantly increased mEPSCs frequency (Hz: CIH 1.32 ± 0.09 vs Norm 0.83 ± 0.09, p=5E‐04), decay time (ms: CIH 8.01 ± 0.28 vs Norm 6.64 ± 0.36, p=0.004), area (pA.ms: CIH 63.85± 3.65 vs Norm 50.27 ± 3.6, p=0.01), and cell capacitance (pF: CIH 31.55 ± 1.8 vs Norm 25.34 ± 1.95, p=0.02). The contribution of BDNF/TrkB signaling to these CIH effects were tested using K252a and K252b, two structurally related kinase receptor inhibitors. MnPO slices were pre‐incubated with either K252a or K252b (200nM) for 30 min before recording. Neither K252a nor K252b influenced mEPSCs of MnPO neurons from normoxic slices. Both K252a and K252b significantly affected CIH‐induced changes mEPSCs but at different magnitudes. The K252a significantly reduced the effects of CIH on frequency (Hz: K252a 0.46 ± 0.05, p=1E‐08), decay time (ms: K252a 6.07 ± 0.34, p=1E‐04), area (pA.ms: K252a 43.26 ± 3.55, p=5E‐04), and cell capacitance (pF: K252a 19.59 ± 1.61, p=1E‐04;). In addition, K252a also significantly decreased mEPSCs peak amplitude in CIH neurons (pA: CIH 14.87 ± 0.41 vs K252a 13.27 ± 0.39, p=0.03). While K252b pretreatment also affected the same parameters, the changes produced by K252a were significantly greater. These studies suggest a role for BDNF/TrkB signaling in CIH induced changes in MnPO but additional experiments will be required to validate the specificity of our results.

TrkB-Mediated Neuroprotective and Antihypoxic Properties of Brain-Derived Neurotrophic Factor.

The neuroprotective and antihypoxic effects of brain-derived neurotrophic factor (BDNF) on dissociated hippocampal cultures in a hypoxia model were investigated. These experiments demonstrate that 10 minutes of normobaric hypoxia increased the number of dead cells in primary culture, whereas a preventive application of BDNF increased the number of viable cells. Spontaneous bioelectrical and calcium activity in neural networks was analyzed using multielectrode arrays and functional intravital calcium imaging. The results indicate that BDNF affects the functional parameters of neuronal networks in dissociated hippocampal cultures over the 7-day posthypoxic period. In addition, the effects of k252a, an antagonist of tropomyosin-related kinase B (TrkB), on functional bioelectrical activity during and after acute hypoxia were investigated. It was shown that the protective effects of BDNF are associated with binding to the TrkB receptor. Finally, intravital fluorescent mRNA probes were used to study the role of NF-κB1 in the protective effects of BDNF. Our experiments revealed that BDNF application stimulates NF-κB1 mRNA synthesis in primary dissociated hippocampal cells under normal conditions but not in hypoxic state.

Abstract 1047: The multikinase inhibitor K252a suppresses hepatocellular carcinoma tumor growth in a mouse xenograft model

Abstract There are several ongoing clinical trials of molecular-targeted therapy for hepatocellular carcinoma (HCC), but no clearly effective therapy has yet emerged. Under such conditions, we have been focusing on potentially powerful anticancer effects of the novel multikinase inhibitor K252a, since it targets unique kinases, including PKC, CaMKII, c-Met, TrkB, MLCK, and phosphorylase kinase. Indeed, the former five targets are expressed in HCC tissues and involved in cellular proliferation, invasion, and metastasis (Yoshiji et al., Cancer Res 1999, etc.). The broad spectrum of the targets may block escape pathways in HCC cells, that are resistant to conventional anticancer drugs and even sorafenib. The AIM of this study was to assess whether K252a suppressed cellular proliferation and altered malignant phenotypes such as epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Materials and Methods: The human HCC cell lines HAK-1A, HAK-1B (Yano et al., Hepatology 1993), KYN-2 (Yano et al., Pathol Int 1988), and Huh7 were used in this study. Effects of K252a on the proliferation and the cell cycle were assessed by a commercialized kit (Promega) and flow cytometry, respectively. Protein expression and localization was analyzed by Western blot and immunocytochemistry, respectively. Cellular mRNA level was evaluated by real-time PCR using TaqMan probes. HAK-1B-based xenograft model in nude mice was used to evaluate in-vivo efficacy of K252a. Results: K252a suppressed proliferation and clearly caused G2 cell-cycle arrest in all the cell lines studied. Additionally, the agent induced both polygonal transformation in cell shape and increase in cell size in several HCC cell lines having endogenous mesenchymal features. In Western blot analysis and immunocytochemistry, the expression levels of E2A, an EMT-regulating transcriptional factor, were universally decreased in the cells treated with K252a, in concert with increased expression levels of E-cadherin in some cell lines. In real-time PCR analyses, the decrease in E2A mRNA levels and the increase in E-cadherin mRNA levels were ubiquitously found. Of note, xenografted tumor tissues obtained from mice treated with K252a exhibited significant increase in E-cadherin protein expression, in comparison with those from control mice. Conclusions: Our findings suggest that K252a has robust antitumor effects on HCC through, at least in part, reversing EMT and inducing G2 cell-cycle arrest. This kind of multikinase inhibitor may help us establish the proof-of-concept for closing escape routes in drug-resistant HCC cells. Citation Format: Mitsuhiko Abe, Hironori Koga, Takafumi Yoshida, Hiroshi Masuda, Masahiro Sakata, Yu Ikezono, Toru Nakamura, Eitaro Taniguchi, Takumi Kawaguchi, Hirohisa Yano, Takuji Torimura, Michio Sata. The multikinase inhibitor K252a suppresses hepatocellular carcinoma tumor growth in a mouse xenograft model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1047. doi:10.1158/1538-7445.AM2014-1047

Expression of neurotrophins and their receptors in adult sarcomas.

10562 Background: Neurotrophins (NT) influence proliferation, survival, death, and differentiation by binding Tropomyosin-related kinase (Trk) receptors and p75NTR(CD271). While the three Trk-receptors TrkA, TrkB, and TrkC display specific binding to their ligands, i.e. TrkA to NGF (nerve growth factor), TrkB to BDNF (brain-derived neurotrophic factor), NT4/5 (neurotrophin 4/5) and TrkC to NT3 (neurotrophin 3), CD271 shows low affinity to all neurotrophins. Notably, CD271 has been suggested as marker for the prospective isolation of bone marrow mesenchymal stromal cells (MSC), and rhabdomyosarcomas have previously been found to express neurotrophins and NT receptors. Methods: RNA was isolated from sarcoma cell lines and freshly obtained sarcoma (src) cells from patients after surgical resection. RNA was reverse transcribed and cDNA products underwent PCR analysis with specific primers. Furthermore, cell lines from the same primary tissues were established and treated with doxorubicin. Effects of K252a (Trk receptor inhibitor), Pep5 (CD271-Inhibitor), and neurotrophins on sarcoma cell proliferation and survival were analyzed. Results: An array of freshly obtained clinical src samples (n=10 each subtype) was screened for expression of NT3, NT4/5, BDNF, NGF as well as TrkA, TrkB, TrkC, and CD271. Interestingly, src subtype-specific expression patterns were observed. In the established cell lines, K252a increased doxorubicin-induced tumor cell death, while CD271-Inhibitor Pep5 enhanced proliferation in doxorubicin treated cells, especially under serum free conditions and when combined with BDNF and NGF. Conclusions: NT and NT receptors are expressed on all adult src samples screened in this study. Chemotherapy resistant liposrc as well as myxofibrosrc samples displayed the strongest expression of NT3 as well as TrkB, TrkC, and CD271. The concomitant expression of NT and their receptors suggests autocrine self-stimulation, which may propagate survival, proliferation, and treatment resistance. NT receptor tyrosine kinases may represent an interesting therapeutic target in adult src.

Divergent roles of p75NTR and Trk receptors in BDNF's effects on dendritic spine density and morphology.

Activation of TrkB receptors by brain-derived neurotrophic factor (BDNF) followed by MAPK/ERK signaling increases dendritic spine density and the proportion of mature spines in hippocampal CA1 pyramidal neurons. Considering the opposing actions of p75NTR and Trk receptors in several BDNF actions on CNS neurons, we tested whether these receptors also have divergent actions on dendritic spine density and morphology. A function-blocking anti-p75NTR antibody (REX) did not affect spine density by itself but it prevented BDNF's effect on spine density. Intriguingly, REX by itself increased the proportion of immature spines and prevented BDNF's effect on spine morphology. In contrast, the Trk receptor inhibitor k-252a increased spine density by itself, and prevented BDNF from further increasing spine density. However, most of the spines in k-252a-treated slices were of the immature type. These effects of k-252a on spine density and morphology required neuronal activity because they were prevented by TTX. These divergent BDNF actions on spine density and morphology are reminiscent of opposing functional signaling by p75NTR and Trk receptors and reveal an unexpected level of complexity in the consequences of BDNF signaling on dendritic morphology.

Sustained Activation of Phosphatidylinositol 3-Kinase/Akt/Nuclear Factor κB Signaling Mediates G Protein-coupled δ-Opioid Receptor Gene Expression

Expression of the delta-opioid receptor gene (dor) is tightly controlled during neuronal differentiation and developmental stages. Such distinct temporal and spatial expression of dor during development suggests a role for the delta-opioid receptor in early developmental events. However, little is known about intracellular signaling pathways that control dor expression. A well established cell line model for the study of gene expression during neuronal differentiation is the rat adrenal pheochromocytoma PC12 cell line. Here we found that the constitutively activated TrkA/phosphatidylinositol 3-kinase/Akt (protein kinase B)/NF-kappaB survival cascade mediates dor expression during nerve growth factor (NGF)-induced differentiation of PC12h cells. Biochemical experiments showed that constitutive phosphorylation of Akt and IkappaBalpha correlates with NGF-induced dor expression. Overexpression of the transcriptional activator NF-kappaB/p65 increased dor promoter activity. Overexpression of the NF-kappaB signaling super inhibitor mutant IkappaBalpha (S32A/S36A) abolished the effect of p65 and blocked NGF-induced activation of NF-kappaB signaling, resulting in a significant reduction in dor promoter activity. Treatment with SN50, an NF-kappaB-specific nuclear translocation peptide inhibitor, inhibited the translocation of NF-kappaB, resulting in a reduction of dor mRNA. The gel shift assay supported the fact that there exists an NF-kappaB-binding site on the dor promoter. RNA interference experiments using NF-kappaB/p65 small interfering RNA confirmed that NF-kappaB signaling is required for dor expression. Our findings not only provide a new mechanistic explanation for NGF-induced dor expression but also shed some light on the molecular mechanism of the temporal and spatial expression of dor and the roles of the delta-opioid receptor during neuronal differentiation.

Effect of K-252a and abscisic acid on the efflux of citrate from soybean roots.

The Al-induced release of organic acid has been suggested as an important mechanism for Al resistance in plants. In this study, the effect of K-252a and abscisic acid (ABA) on the efflux of citrate was investigated in soybean (Glycine max L.) roots. Al initiated citrate efflux from the root apices 30 min after the addition of Al. The Al-triggered efflux of citrate was sensitive to metabolic inhibitors and anion channel inhibitors. Pretreatment or treatment with K-252a, an inhibitor of protein kinase, severely inhibited the Al-induced efflux of citrate accompanying an increase in Al accumulation and intensified Al-induced root growth inhibition. Al-treatment increased the endogenous level of abscisic acid (ABA) in soybean roots in a dose- and time-dependent manner, while K-252a failed to inhibit the Al-induced increase in endogenous ABA. Exogenous application of ABA increased the activity of citrate synthase (EC 4.1.3.7) by 26.2%, and decreased Al accumulation by 32.3%, respectively. ABA-induced increases in citrate efflux and root elongation were suppressed by K-252a, while ABA could not reverse the K-252a effects. Taken together, these results suggest that ABA is probably involved in the early response, after which K-252a-sensitive protein kinases play a key step in regulating the activity of an anion channel, through which citrate is released from the apical cells of soybean roots.

Anti-proliferative effect of the kinase inhibitor K252a on human prostatic carcinoma cell lines.

Members of the K252 family of kinase inhibitors have been demonstrated to inhibit a number of neurotrophin-mediated cellular responses, and to preferentially inhibit the activity of neurotrophin receptors. In this study, we examined the effects of K252a and K252b on the growth of human prostate carcinoma cells, whose growth is, in part, mediated by a prostatic nerve growth factor(NGF)-like protein(s). K252a inhibited [3H]thymidine incorporation by three androgen-independent prostate tumor cell lines (TSU-pr1, DU-145, and PC-3), under basal growth conditions, and in response to growth stimulation by human prostatic stromal (hPS) cell proteins and serum. K252b, which does not readily penetrate cell membranes, had no significant effect on [3H]thymidine incorporation by the prostate tumor cell lines. The decrease in [3H]thymidine incorporation by the cell lines in response to K252a did not appear to be the result of K252a cytotoxicity at concentrations as high as 100 nM, as measured by the Trypan blue assay for cell viability. Treatment of cells for 25 hours with 100 nM K252a resulted in accumulation of TSU-pr1, DU-145, and PC-3 cells in G0/G1, concurrent with a substantial decrease in cells synthesizing DNA. Treatment of androgen-responsive LNCaP prostatic carcinoma cells for 25 hours with 100 nM K252a also resulted in a significant decrease in DNA synthesis. Human recombinant NGF-mediated phosphorylation of a 140-kDa Trk NGF receptor in the TSU-pr1 cell line was inhibited by treatment with 100 nM K252a. Hence, K252a inhibition of Trk phosphorylation most probably contributed, in part, to the inhibition of prostate tumor cell growth in vitro. These results suggest that the mechanism of K252a action may be useful in the design of potential therapies for prostate cancer treatment.

Epidermal growth factor induces PC12 cell differentiation in the presence of the protein kinase inhibitor K-252a.

The protein kinase inhibitors K-252a and K-252b have been shown earlier to block the actions of nerve growth factor and other neurotrophins and, at lower concentrations, to selectively potentiate neurotrophin-3 actions. In the present study we show that K-252a, but not K-252b, enhances epidermal growth factor (EGF)-and basic fibroblast growth factor (BFGF)-induced neurite outgrowth of PC12 cells at higher concentrations than required for neurotrophin inhibition. In parallel, tyrosine phosphorylation of extracellular signal-regulated kinases (Erks) elicited by EGF of bFGF was also increased in the presence of K-252a, and this signal was prolonged for 6 h. EGF- and bFGF-induced phosphorylation of phospholipase C-gamma 1 were not changed. The effect of K-252a on Erks was resistant to chronic treatment with phorbol ester, indicating that protein kinase C is not involved in this potentiation. In partial contrast to the actions of K-252a, the neurotrophin-3-potentiating effect of K-252b was accompanied by an increase in tyrosine phosphorylation of the Erks and of phospholipase C-gamma 1. Finally, although K-252a alone did not induce neurite outgrowth or tyrosine phosphorylation of Erks or phospholipase C-gamma 1, this compound alone stimulated phosphatidylinositol hydrolysis. Our findings identify activities of K-252a besides the direct interaction with neurotrophin receptors and suggest that a K-252a-sensitive protein kinase or phosphatase might be involved in signal transduction of EGF and bFGF. Our results are further compatible with the hypothesis that sustained activation of Erks may be important in PC12 differentiation.

Early responses of PC-12 cells to NGF and EGF: effect of K252a and 5'-methylthioadenosine on gene expression and membrane protein methylation.

Although epidermal growth factor (EGF) and nerve growth factor (NGF) have markedly different biological effects on PC-12 cells, many of the signaling events following ligand binding are similar. Both EGF and NGF result in the induction of the primary response gene egr-1/TIS8 and increased methylation of a variety of membrane-associated proteins as early as 5 min after EGF or NGF treatment using a methylation assay that detects methyl esters as well as methylated arginine residues. At 20 min after stimulation with these factors, the stimulation of methylation by NGF is greater than that of EGF, especially in the polypeptides of 36-42 and 20-22 kDa. To help dissect the pathways involved in these cellular responses, the protein kinase inhibitor K252a and the methyltransferase inhibitor 5'-methylthioadenosine (MTA) were used. Both K252a and MTA inhibit NGF-, but not EGF-mediated, primary response gene expression. In contrast, MTA, but not K252a, can block NGF-induced membrane associated protein methylation. These data suggest a role for differential protein methylation reactions in EGF and NGF signal transduction.

Reducing Reagent-Induced Activation of Guanylate Cyclase in the Cellular Slime Mold, Dictyostelium discoideum

Binding of folic acid (an intrinsic agonist) to the cell surface receptors evokes transmembrane signals for activation and adaptation of guanylate cyclase in Dictyostelium discoideum. The activation signal activates this enzyme and then the adaptation signal terminates the activation. As a result, these two signals cooperatively induce a transient activation of guanylate cyclase. We investigated transmembrane signal transduction for guanylate cyclase using 2,3-dimercapto-1-propanol (BAL, a thiol-reducing reagent) since BAL induces or modifies the transmembrane signal(s). We found that BAL induced prolonged or continuous activation of guanylate cyclase. Thus, the mode of the activation is drastically different (transient versus continuous) between folic acid and BAL. We also found that the BAL-induced continuous activation was not observed when the cells were stimulated with BAL + folic acid, while folic acid + BAL transiently induced more cGMP accumulation than folic acid alone. We lastly showed that K252a, a protein kinase inhibitor, enhanced both the folic acid-induced and the BAL-induced activation of guanylate cyclase. Our results suggest that BAL induces or mimics the activation signal for guanylate cyclase. The lack of termination in the BAL-induced activation suggests that BAL does not induce the adaptation signal or that the adaptation does not inhibit the BAL-induced activation. The former possibility is more likely since folic acid suppresses the BAL-induced continuous activation. The effect of K252a suggests that protein phosphorylation plays a role in suppression of guanylate cyclase.

P-258 - Effects of K252a, a protein kinase inhibitor, on the growth of cultured endothelial cells.

P-258 - Effects of K252a, a protein kinase inhibitor, on the growth of cultured endothelial cells.

Effect of K252a, a protein kinase inhibitor, on the proliferation of vascular smooth muscle cells

In the growth arrested cultures of bovine carotid smooth muscle, K252a (10 - 100 ng/ml), a protein kinase inhibitor with wide spectrum suppressed the cell proliferation induced by TPA and increase of serum. K252a was more potent in the antiproliferative activity than H7, a C-kinase-specific inhibitor, but less than staurosporine, another wide-spectrum protein kinase inhibitor. Since C-kinase plays an important role in the signal transduction leading to the cell proliferation and K252a inhibits C-kinase in vitro, the antiproliferative effect of K252a to carotid smooth muscle cells is likely to be exerted through c-kinase dependent pathway.

Induction of differentiation of HL-60 cells by protein kinase C inhibitor, K252a

To clarify the role of protein kinase C and protein kinase A in cell proliferation and differentiation, the effects of K252a and its derivatives (K252b, KT5720), which have different inhibitory activity to these protein kinases, on the proliferation and differentiation of HL-60 cells were investigated. The proliferation and DNA synthesis of the HL-60 cells were inhibited by K252a in a dose dependent manner. However, K252b and KT5720 which are more specific inhibitors of protein kinase C or protein kinase A, respectively, had no observable effect on cell proliferation. K252a (40nM) enhanced the differentiation of HL-60 cells induced by 1,25(OH) 2D 3, retinoic acid and DMSO. K252b and KT5720 did not affect 1,25(OH) 2D 3-induced differentiation. K252a significantly inhibited the differentiation induced by PMA. These results demonstrate that K252a but not its derivatives can function as an antitumor drug and enhancer of the differentiation induced by various inducers.

P-482 - Effect of K252a, a protein kinase inhibitor, on the proliferation of cultured vascular smooth muscle cells

P-482 - Effect of K252a, a protein kinase inhibitor, on the proliferation of cultured vascular smooth muscle cells

Selective Inhibition of Nerve Growth Factor‐Stimulated Protein Kinases by K‐252a and 5′‐S‐Methyladenosine in PC12 Cells

K-252a, a protein kinase inhibitor isolated from the culture broth of Nocardiopsis sp., inhibits the nerve growth factor (NGF)-stimulated phosphorylation of microtubule-associated protein 2 (MAP2) and Kemptide (synthetic Leu-Arg-Arg-Ala-Ser-Leu-Gly) by blocking the activation of two independent kinases in PC12 cells: MAP2/pp250 kinase and Kemptide kinase. The NGF-stimulated activation of these kinases is inhibited in a dose-dependent manner following treatment of the cells with K-252a. Although these kinases also are activated by epidermal growth factor (EGF) and 12-O-tetradecanoyl-phorbol 13-acetate, K-252a has no inhibitory effect when these agents are used. Half-maximal inhibition of the activation of both kinases was observed at 10-30 nM K-252a. K-252a was shown to directly inhibit the activity of MAP2/pp250 kinase and Kemptide kinase when added to the phosphorylation reaction mixture in vitro; however, half-maximal inhibition under these conditions was observed at greater than or equal to 50 nM K-252a. These data suggest that K-252a exerts its effects at a step early in the cascade of events following NGF binding. The effects of K-252a are similar to those reported for 5'-S-methyladenosine (MTA) and other methyltransferase inhibitors. Treatment of PC12 cells with MTA inhibited NGF-, but not EGF-mediated activation of MAP2/pp250-kinase (Ki greater than 500 microM). MTA, when added to the phosphorylation reaction mixture in vitro, directly inhibited kinase activity (Ki = 50 microM), suggesting that the effects of MTA may be the result of its action on protein kinases rather than methyltransferases.

The effect of K-252a, a potent microbial inhibitor of protein kinase, on activated cyclic nucleotide phosphodiesterase.

K-252a, an indole carbazol compound of microbial origin, inhibited activation of bovine brain phosphodiesterase induced by calmodulin (CaM), sodium oleate, or limited proteolysis with almost equal potency. Kinetic analysis revealed that the CaM-activated phosphodiesterase (CaM-PDE) was competitively inhibited by K-252a with respect to CaM. On the other hand, inhibition of the trypsin-activated phosphodiesterase was competitive with respect to cyclic AMP. Addition of a lower amount of phosphatidylserine or sodium oleate to the reaction medium was efficacious in attenuating the inhibition of the CaM-PDE by W-7, compound 48/80, or calmidazolium but, in contrast, had no effect on the inhibition by K-252a. Furthermore, CaM-independent systems such as [3H]nitrendipine receptor binding or Na+ + K+-ATPase were influenced less by K-252a compared with W-7, compound 48/80 and calmidazolium. In conclusion, K-252a is an inhibitor of CaM-dependent cyclic nucleotide phosphodiesterase and it appears that it inhibits the enzyme not only via CaM antagonism but possibly also by interfering with the enzyme.

K-252a, a novel microbial product, inhibits smooth muscle myosin light chain kinase.

Effects of K-252a, (8R*, 9S*, 11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta [cde]trinden-1-one, purified from the culture broth of Nocardiopsis sp., on the activity of myosin light chain kinase were investigated. 1) K-252a (1 x 10(-5) M) affected three characteristic properties of chicken gizzard myosin-B, natural actomyosin, to a similar degree: the Ca2+-dependent activity of ATPase, superprecipitation, and the phosphorylation of the myosin light chain. 2) K-252a inhibited the activities of the purified myosin light chain kinase and a Ca2+-independent form of the enzyme which was constructed by cross-linking of myosin light chain kinase and calmodulin using glutaraldehyde. The degrees of inhibition by 3 x 10(-6) M K-252a were 69 and 48% of the control activities with the purified enzyme and the cross-linked complex, respectively. Chlorpromazine (3 x 10(-4) M), a calmodulin antagonist, inhibited the native enzyme, but not the cross-linked one. These results suggested that K-252a inhibited myosin light chain kinase by direct interaction with the enzyme, whereas chlorpromazine suppressed the enzyme activation by interacting with calmodulin. 3) The inhibition by K-252a of the cross-linked kinase was affected by the concentration of ATP, a phosphate donor. The concentration causing 50% inhibition was two orders magnitude lower in the presence of 100 microM ATP than in the presence of 2 mM ATP. 4) Kinetic analyses using [gama-32P]ATP indicated that the inhibitory mode of K-252a was competitive with respect to ATP (Ki = 20 nM). These results suggest that K-252a interacts at the ATP-binding domain of myosin light chain kinase. The direct action of the compound on the enzyme would explain the multivarious inhibition of myosin ATPase, of superprecipitation, and of the contractile response of smooth muscle.

Inhibition by K-252a, a new inhibitor of protein kinase, of nerve growth factor-induced neurite outgrowth of chick embryo dorsal root ganglion cells

The effect of K-252a, a new alkaloid-like kinase inhibitor from the culture broth of Nocardiopsis sp., on the action of nerve growth factor on normal nerve cells was examined. Nerve cells were isolated from the dorsal root ganglia of chick embryo by trituration without using digesting enzymes, and were seeded on collagen- or poly- l-lysine-coated plastic dishes containing serum-free medium. K-252a at 50–100 nM reversibly inhibited the formation of neurites by dorsal root ganglion cells elicited by the treatment with nerve growth factor.

P-76 - Effect of K-252a, a new microbial metabolite, on the secretory responses in isolated rat mast cells and neutrophils

P-76 - Effect of K-252a, a new microbial metabolite, on the secretory responses in isolated rat mast cells and neutrophils