Polyploidization of 2nH1 (ES) cells by K-252a and staurosporine

Mouse 2nH1 (ES) cells were examined for polyploidization using K-252a and staurosporine. Though 2nH1 cells were polyploidized by both K-252a and staurosporine, tetraploid cells, 4nH1K cells, were obtained only from cell populations exposed to K-252a. The probability of successful establishment of tetraploid cells was 2/9, suggesting that the highly polyploidized-tetraploid transition might occur infrequently. Cell cycle parameters of 4nH1K cells were almost the same as those of 2nH1 cells, suggesting that the rate of DNA synthesis was about twice that of the diploid cells. The cell volume of 4nH1K cells was about twice of that of diploid cells, indicating that 4nH1K cells contained about twice as much total intracellular material as 2nH1 cells. The morphology of the 4nH1K cells was flagstone-like, thus differing from that of the spindle-shaped 2nH1 cells, suggesting that morphological transformation occurred during the diploid-tetraploid transition. 4nH1K cells exhibited alkaline phosphatase activity and formed teratocarcinomas, implying that they were pluripotent. These characteristics of 4nH1K cells were similar to those of tetraploid 4nH1 cells that have been established through polyploidization by demecolcine, suggesting that 4nH1K and 4nH1 cells are similar irrespective of the different mechanisms of polyploidization.     

Establishment of a tetraploid Meth-A cell line through polyploidization by demecolcine but not by staurosporine, K-252A and paclitaxel

Polyploid cells are made by DNA reduplication without cell division, however, it is not easy to establish polyploid mammalian cell lines. It is worth studying the difference in cell character between hyperploid and parent cell lines. Meth-A cells were polyploidized by demecolcine, K-252a, staurosporine and paclitaxel. The cell-cycle responses of highly polyploid Meth-A cells after the removal of the drugs were examined by flow cytometry (FCM). Meth-A cells were highly polyploidized by these drugs. The polyploid Meth-A cells gradually decreased in ploidy after the drug release. A tetraploid Meth-A cell line was established only from the demecolcine-induced polyploid Meth-A cells. The duration of G1, S and G2/M phases of the tetraploid cell line were mostly the same as those of the parent diploid cells, except that the G2/M phase was 1.5 h longer. The chromosome number of tetraploid Meth-A cell line was about twice of the diploidy. A tetraploid Meth-A cell line was established.     

Different responses of polyploidized V79 cells after removal of two drugs, demecolcine and K-252a

To examine whether or not cells polyploidized by different mechanisms behave in a different manner after drug removal, V79 Chinese hamster cells were assessed by flow cytometry (FCM) after their polyploidization by demecolcine and K-252a, inhibitors of spindle-fiber formation and protein kinase, respectively. Cell cycle analysis of DNA histograms of V79 cells before and after the drug release was performed. With both drugs, the ploidy of V79 cells increased just after the drug removal and was maintained for a week. A difference was evident 10 days after the release. Tetraploid cells were the main population from 10 to 18 days after the release of K-252a, but not demecolcine. Cell cycle parameters were almost the same in pseudo diploid and tetraploid V79 cells, except for the tetraploid S phase which was 2h longer.     

Specific inhibition of NGF receptor tyrosine kinase activity by K-252a.

An involvement of protein tyrosine kinase in the transduction of the signals initiated by nerve growth factor (NGF) was investigated. A tyrosine kinase inhibitor, herbimycin, inhibited neurite outgrowth of rat pheochromocytoma PC12 cells induced by NGF but not that by dibutyryl-cAMP. Herbimycin and genistein blocked NGF-dependent activation of ras p21 whose essential function in neuronal differentiation has been reported. These observations suggested that tyrosine kinase activity is involved in the signaling pathways. K-252a, by contrast, inhibited NGF-induced but not EGF-dependent activation of ras p21. Tyrosine kinase activity of gp140trk, a constituent of NGF receptor, is activated by NGF for much a longer period compared to the activation of EGF receptor autokinase activity by EGF. We further demonstrated that autophosphorylation of gp140trk is selectively inhibited by K-252a.     

Staurosporine, K-252a, and K-252b Stabilize Calcium Homeostasis and Promote Survival of CNS Neurons in the Absence of Glucose

Abstract: Staurosporine, K-252a, and the 9-carboxylic related compound K-2525 are low-molecular-weight alkaloids from microbial origin that at high concentrations are kinase inhibitors and can antagonize the effects of neuronal growth factors. Paradoxically, we have found that very low concentrations of these agents (10 f M -10 n M ) prolong the survival of hippocampal, septal, and cortical neurons deprived of glucose. These agents did not prevent the depletion of ATP caused by glucose deprivation. The large elevation of intracellular calcium levels that normally mediates glucose deprivation-induced damage was attenuated by Staurosporine, K-252a, and K-252b. Western blot analysis using antiphosphotyrosine antibody showed that Staurosporine and the K-252 compounds (10–100 p M ) stimulated tyrosine phosphorylation of several different proteins. The tyrosine kinase inhibitor genistein significantly reduced the protective effect of Staurosporine and the K-252 compounds, indicating that tyrosine phosphorylation was required for neuroprotection by these compounds. Taken together, the data demonstrate that low concentrations of Staurosporine and the K-252 compounds can stabilize calcium homeostasis, possibly by a mechanism involving activation of receptor tyrosine kinase transduction pathways.     

A novel endocytic recycling signal distinguishes biological responses of Trk neurotrophin receptors

Endocytic trafficking of signaling receptors to alternate intracellular pathways has been shown to lead to diverse biological consequences. In this study, we report that two neurotrophin receptors (tropomyosin-related kinase TrkA and TrkB) traverse divergent endocytic pathways after binding to their respective ligands (nerve growth factor and brain-derived neurotrophic factor). We provide evidence that TrkA receptors in neurosecretory cells and neurons predominantly recycle back to the cell surface in a ligand-dependent manner. We have identified a specific sequence in the TrkA juxtamembrane region, which is distinct from that in TrkB receptors, and is both necessary and sufficient for rapid recycling of internalized receptors. Conversely, TrkB receptors are predominantly sorted to the degradative pathway. Transplantation of the TrkA recycling sequence into TrkB receptors reroutes the TrkB receptor to the recycling pathway. Finally, we link these divergent trafficking pathways to alternate biological responses. On prolonged neurotrophin treatment, TrkA receptors produce prolonged activation of phosphatidylinositol 3-kinase/Akt signaling as well as survival responses, compared with TrkB receptors. These results indicate that TrkA receptors, which predominantly recycle in signal-dependent manner, have unique biological properties dictated by its specific endocytic trafficking itinerary.     

K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity.

The rat pheochromocytoma PC12 cell line differentiates into a sympathetic neuronal phenotype upon treatment with either nerve growth factor (NGF) or basic fibroblast growth factor. The alkaloid-like compound K-252a has been demonstrated to be a specific inhibitor of NGF-induced biological responses in PC12 cells (Koizumi, S., Contreras, M. L., Matsuda, Y., Hama, T., Lazarovici, P., and Guroff, G. (1988) J. Neurosci. Res. 8, 715-721). NGF interacts with the protein product of the proto-oncogene trk and rapidly stimulates the tyrosine phosphorylation of both p140prototrk and a number of cellular substrates. Here we show that these phosphorylation events are directly inhibited in PC12 cells by K252a in a dose-dependent manner, indicating that the site of action of this inhibitor is at the NGF receptor level. K-252a inhibits p140prototrk activity in vitro, demonstrating that K-252a has a direct effect on the p140prototrk tyrosine kinase. Though many of the biochemical responses to NGF in PC12 cells are mimicked by basic fibroblast growth factor and epidermal growth factor, K-252a has no effect on the action of these growth factors in PC12 cells, demonstrating that the initial biological events initiated by NGF are distinctive during neuronal differentiation.     

Effects of the protein kinase inhibitors, staurosporine and K-252a, on PGI2 production by rat liver cells (the C-9 cell line)

Staurosporine and K-252a, known inhibitors of several protein kinases, stimulated PGI2 production (measured as 6-keto-PGF1 alpha) in rat liver cells (the C-9 cell line). Preincubation of the rat liver cells with staurosporine or K-252a enhanced the PGI2 production stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), platelet activating factor (PAF) and the Ca2(+)-ionophore A-23187, but not the PGI2 synthesis stimulated by exogenous arachidonic acid. These results suggest that phosphorylation of some proteins or certain amino acids on a protein can regulate arachidonic acid metabolism probably in the pathway leading to deesterification of phospholipids.     

Synthesis and biological activity of pyrazole analogues of the staurosporine aglycon K252c

A derivative of the staurosporine aglycon (K252c), in which the lactam ring was replaced by a pyrazole moiety, was synthesized. The resulting indolopyrazolocarbazole (3) inhibited Pim isoforms 1–3 whereas it did not impair the activity of two known targets of K252c, protein kinase C isoforms α and γ. Compound 3 exhibited moderate cytotoxic activity toward human leukemia and colon carcinoma cell lines (K562 and HCT116), strongly suggesting that this new scaffold deserves further investigations for treatment of malignancies associated with Pim activity.     

K-252 Compounds: Modulators of Neurotrophin Signal Transduction

K-252 compounds, which share a common polyaromatic aglycon structure, are rather general and potent inhibitors of various protein kinases, including protein kinase C and tyrosine-specific protein kinases, and possibly act by interfering at or near the ATP binding site. However, chemical modifications in their sugar moiety can result in high specificity of the inhibitory action and, furthermore, can induce other stimulatory and inhibitory effects on nerve cells. These compounds are of particular interest because, in intact cells, they inhibit the actions of NGF and other neurotrophins without diminishing comparable actions of other growth factors. This effect seems to reflect a direct inhibitory action on trk neurotrophin receptor proteins. At concentrations lower than those necessary to inhibit neurotrophin actions, K-252a and K-252b have been shown to potentiate the stimulatory effects of NT-3 on different neurons in culture and on PC12 cells. The structural requirements for this effect seem to be different from those for the inhibition of neurotrophin actions. These findings raise the possibility of development of compounds of high selectivity, able to inhibit or potentiate the transduction mechanisms of individual neurotrophins, and identify K-252a and K-252b as lead compounds for the development of such selective molecules. Specific inhibitors and stimulators of neurotrophins would be valuable tools to investigate biological functions of the neurotrophins in vitro and in vivo. Furthermore, it is possible that, in the future, highly selective drugs with agonistic or antagonistic actions on neurotrophin mechanisms could become therapeutically useful in the treatment of neurological disease and injury.     

An immunoglobulin-like domain determines the specificity of neurotrophin receptors.

The neurotrophins influence survival and maintenance of vertebrate neurons in the embryonic, early post-natal and post-developmental stages of the nervous system. Binding of neurotrophins to receptors encoded by the gene family trk initiates signal transduction into the cell. trkA interacts preferably with nerve growth factor (NGF), trkB with brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) and trkC with neurotrophin-3 (NT-3). By constructing 17 different chimeras and domain deletions of the human trk receptors and analyzing their binding affinities to the neurotrophins we have shown that an immunoglobulin-like domain located adjacent to the transmembrane domain is the structural element that determines the interaction of neurotrophins with their receptors. Chimeras of trkC where this domain was exchanged for the homologous sequences from trkB or trkA gained high affinity binding to BDNF or NGF respectively, while deletion of this domain in trkC or trkA abolished binding to NT-3 or NGF respectively. This domain alone retained affinities to neurotrophins similar to the full-length receptors and when expressed on NIH 3T3 cells in fusion with the kinase domain showed neurotrophin-dependent activation.     

Nonphosphorylatable substrate analogs selectively block autophosphorylation and activation of the insulin receptor, epidermal growth factor receptor, and pp60v-src kinases

The receptors for insulin and epidermal growth factor undergo tyrosine autophosphorylation in response to ligand stimulation, while pp60v-src is an unregulated tyrosine kinase. In this report we show that each of the kinases phosphorylates an exogenous peptide that corresponds to the insulin proreceptor sequence 1142-1153. When the kinases were pre-phosphorylated, saturable Michaelis-Menten kinetics were observed. However, when the kinases had not been pre-phosphorylated biphasic kinetics were observed; at progressively higher substrate concentrations (greater than Km) less substrate phosphorylation was seen. Furthermore, when the kinases had not been pre-phosphorylated kinase autophosphorylation was inhibited at high substrate concentrations. On this basis we postulated that the substrate inhibition of substrate phosphorylation resulted directly from substrate inhibition of kinase autophosphorylation. To test this we designed additional peptides to function specifically as inhibitors of the kinases. Each of the 3 tyrosine residues within the substrate sequence were replaced either by 4-methoxyphenylalanine or phenylalanine, residues structurally similar to tyrosine but unable to accept phosphoryl transfer. Both analogs inhibited insulin and epidermal growth factor receptor autophosphorylation, whereas only the Phe-substituted analog inhibited pp60v-src phosphorylation. These data suggest that autophosphorylation of tyrosine residues near the kinase active site is a generalized mechanism for tyrosine kinase activation and that activation can be selectively blocked by substrates and nonphosphorylatable analogs.     

Effect of the protein kinase inhibitors, staurosporine and K-252a, on PGI2 production by rat liver cells (the C-9 cell line)

Staurosporine and K-252a, known inhibitors of several protein kinases, stimulated PGI₂ production (measured as 6-keto-PGF_1α in rat liver cells (the C-9 cell line). Preincubation of the rat liver cells with staurosporine or K-252a enhanced the PGI₂ production stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), platelet activating factor (PAF) and the Ca²⁺-ionophore a-23187, but not the PGI₂ synthesis stimulated by exogeneous arachidonic acid. These results suggest that phosphorylation of some proteins or certain amino acids on a protein can regulate arachidonic acid metabolism probably in the pathway leading to deesterification of phospholipids.     

K-252a and Staurosporine Promote Choline Acetyltransferase Activity in Rat Spinal Cord Cultures

Abstract: The protein kinase inhibitor K-252a increased choline acetyltransferase (ChAT) activity in rat embryonic spinal cord cultures in a dose-dependent manner (EC₅₀ of ∼100 n M ) with maximal stimulatory activity at 300 n M resulting in as much as a fourfold increase. A single application of K-252a completely prevented the marked decline in ChAT activity occurring over a 5-day period following culture initiation. Of 11 kinase inhibitors, only the structurally related inhibitor Staurosporine also increased ChAT activity (EC₅₀ of ∼0.5 n M ). Effective concentrations of K-252a were not cytotoxic or mitogenic and did not alter the total protein content of treated cultures. Insulin-like growth factor I, basic fibroblast growth factor, ciliary neurotrophic factor, and leukemia inhibitory factor yielded dose-dependent increases in ChAT activity in spinal cord cultures. The combination of K-252a with insulin-like growth factor-l or basic fibroblast growth factor increased ChAT activity up to eightfold over that of untreated controls, which was greater than that observed with each compound alone. K-252a combined with ciliary neurotrophic factor or leukemia inhibitory factor demonstrated no additive or synergistic effects on ChAT activity. These results suggest that there are multiple mechanisms for the regulation of ChAT activity in spinal cord cultures. The enhancement of spinal cord ChAT activity by K-252a and Staurosporine defines a new neurotrophic activity for these small organic molecules and raises the possibility that they may activate some regulatory elements in common with the ciliary neurotrophic factor and leukemia inhibitory factor family of neurotrophic proteins.     

Insulin receptors prepared with iodoacetamide show enhanced autophosphorylation and receptor kinase activity.

In this study, we found that adding iodoacetamide to the homogenization buffer used in the preparation of mouse or rat liver plasma membranes resulted in an increase of insulin receptor autophosphorylation by 4-5-fold and receptor kinase activity by about 2-fold. Similar effects were obtained with iodoacetate and p-chloromercuriphenyl sulfonate. The effect of iodoacetamide was minimal when it was added to membranes prepared without the thiol reagent. The enhancing effect of iodoacetamide on insulin receptor autophosphorylation was the result of a more than 2-fold decrease in the Km and a more than 3-fold increase in Vmax for ATP. The presence of iodoacetamide in the preparation of plasma membranes also greatly increased the solubilization of the insulin receptor from the plasma membrane by Triton X-100. We propose that iodoacetamide acts to alkylate some unknown thiols released during tissue homogenization and that in its absence these thiols formed mixed disulfides with the insulin receptor, thus adversely affecting the process of receptor activation by insulin.     

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.     

Purification and autophosphorylation of insulin receptors from rat skeletal muscle

Insulin receptors of rat skeletal muscle were purified by first extracting a plasma membrane-enriched pellet obtained from a muscle homogenate with Triton X-100, followed by WGA-Sepharose and insulin-Sepharose affinity chromatography. Routinely, 4-5 micrograms of purified receptor were obtained from 15 g of tissue. The purified receptors are composed of two major polypeptides with molecular weights of 130,000 and 95,000, respectively. The binding of [125I]insulin by the purified receptors was analyzed by a Scatchard plot. There are at least two binding components. The high-affinity component, with an apparent association constant (Ka) of 2.0 X 10(9) M-1, comprises 10% of the total insulin binding sites; while the low-affinity component, with a Ka value of 1.4 X 10(8) M-1, represents 90% of the binding sites. Assuming the insulin receptor to have a molecular weight of 300,000, the receptor binds 1.7 mol of insulin per mol at saturation. Insulin is capable of stimulating the autophosphorylation of the beta-subunit of the muscle insulin receptor (Mr 95,000) by 5-10-fold. The stoichiometry of this phosphorylation reaction was determined as 0.8 phosphate per insulin binding site after a 10 min incubation with 100 nM insulin. In a previous report, I showed that the insulin stimulation of glucose transport in diaphragms from neonatal rats was small, even although the diaphragms had normal levels of insulin receptors and glucose transporters (Wang, C. (1985). Proc. Natl. Acad. Sci. USA 82, 3621-3625). To determine whether or not receptor autophosphorylation might be related to this insensitivity to insulin, the level of receptor phosphorylation was quantitated in diaphragms from rats at different stages of development. Autophosphorylation remains unchanged from birth to 21 days of age, suggesting that the lower insulin-stimulated glucose uptake by diaphragms at early stages of postnatal development as compared to that by diaphragms of older rats, is not due to a difference in receptor kinase.