ucn-1028-c and staurosporine-aglycone

Cyclic AMP-dependent proteolysis of GATA-6(Delta50) was characterized using inhibitors for intracellular signaling pathways. Among these kinase inhibitors, only H-89 and K252a inhibited the proteolysis induced by dbcAMP, a membrane permeable cAMP analogue, others such as PD98059, SB203580, calphostine C, PP1, and KN-93 did not do so. These results suggest that A-kinase, but not C-kinase, MEK, P38 MAP-kinases or Src kinase, could participate in the observed phenomenon. We further demonstrated that an inhibitor for ubiquitin isopeptidase (Delta12-PGJ2) inhibited the degradation of GATA-6(Delta50) in the presence of dbcAMP, suggesting that the cAMP-dependent proteolysis could be mediated through the ubiquitin-proteasome pathway, although proteasome activity did not change significantly during dbcAMP treatment. The full-length GATA-6 was also responsive to the induced degradation. Furthermore, mutation of a potential phosphorylation site (Ser-290-->Ala) for A- and C-kinases, and deletion of the PEST sequence of GATA-6 did not abolish the degradation. All these results suggest that cellular factor(s) may play a crucial role in mediating the activation of the cAMP-dependent process.

Topics: Animals; Base Sequence; Benzylamines; Binding Sites; Bucladesine; Carbazoles; Cell Nucleus; CHO Cells; Cricetinae; Cyclic AMP; DNA-Binding Proteins; Enzyme Inhibitors; Flavonoids; GATA6 Transcription Factor; Humans; Imidazoles; Indole Alkaloids; Isoquinolines; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Molecular Sequence Data; Mutation; Naphthalenes; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Plasmids; Prostaglandin D2; Proteasome Endopeptidase Complex; Protein Binding; Pyridines; src-Family Kinases; Sulfonamides; Transcription Factors

Several lines of evidence suggest that phosphorylation events play an important role in transducing neurite outgrowth signals. Here we tested if such phosphorylation events altered filopodial dynamics on neuronal growth cones and thereby might affect pathfinding decisions. The general protein kinase inhibitor K252a caused an increase in the overall length of filopodia, thereby increasing the action radius of a growth cone. Application of specific kinase inhibitors demonstrated that myosin light chain kinase, Ca/calmodulin-dependent kinase II, and protein kinase A were likely not involved in this filopodial response. Inhibition of protein kinase C (PKC) with calphostin C or cerebroside, however, induced filopodial elongation similar to that seen with K252a. Activation of PKC with the phorbol ester PMA produced the opposite effect, namely filopodial shortening. Consistent with this finding, the protein phosphatase activator C(2)-ceramide resulted in a significant increase in filopodial length, whereas application of the protein phosphatase inhibitor okadaic acid caused the opposite effect, filopodial shortening. Lastly, the tyrosine kinase inhibitor genistein also caused filopodial elongation, and this effect could be negated by the tyrosine phosphatase inhibitor sodium ortho-vanadate. Using the calcium indicator fura-2, we further showed that these drugs did not cause a measurable change in the free intracellular calcium concentration ([Ca(2+)](i)) in growth cones. Taken together, these results suggest that the action radius of a growth cone and its resulting pathfinding abilities could be rapidly altered by contact with extracellular cues, leading to changes in the activity of protein kinases and phosphatases.

Topics: Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Carrier Proteins; Cells, Cultured; Cerebrosides; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Genistein; Growth Cones; Indole Alkaloids; Intracellular Signaling Peptides and Proteins; Models, Biological; Myosin-Light-Chain Kinase; Naphthalenes; Neurons; Okadaic Acid; Phosphorylation; Protein Kinase C; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Pseudopodia; Signal Transduction; Snails; Sphingosine; Tetradecanoylphorbol Acetate; Time Factors; Vanadates

Isolated single smooth muscle cells from the fundus of a guinea-pig stomach were permeabilized by use of streptolysin-O (0.5 U/ml). Most of the permeabilized cells responded to 0.6 microM Ca2+, but not to 0.2 microM Ca2+, with a resulting maximal cell shortening to approximately 71% of the resting cell length. These cells were relaxed again by washing with the Ca2+-free solution (2.5 nM free Ca2+) for 3-5 min. Addition of 10 microM acetylcholine (ACh) resulted in both a marked decrease in the concentration of Ca2+ required to trigger a threshold response and an increase in the maximal cell shortening, indicating that the cells retained the muscarinic receptor function. When the cell treated with a protein kinase C (PKC) inhibitor, K-252b (1 microM), for 3 min was exposed to 10 microM ACh in the presence of K-252b, the cell shortened within 2 min with a maximal cell shortening. When the cell shortening was induced by 10 microM ACh plus 1 microM Ca2+ in the presence of K-252b (1 microM) or more selective PKC inhibitors, such as calphostin C (1 microM) or PKC pseudosubstrate peptide (100 microM), the extension of the shortened cells, by washing with the Ca2+-free solution, was significantly inhibited. In contrast, K-252b (1 microM) did not inhibit the relaxation of Ca2+-induced shortened cells. These results suggest that the receptor-mediated activation of PKC in the process of ACh-induced cell shortening plays a role in the subsequent relaxation of the shortened cells.

Topics: Acetylcholine; Animals; Bacterial Proteins; Calcium; Carbazoles; Cell Membrane Permeability; Enzyme Inhibitors; Guinea Pigs; In Vitro Techniques; Indole Alkaloids; Muscle Relaxation; Muscle, Smooth; Naphthalenes; Protein Kinase C; Stomach; Streptolysins

Both nerve growth factor (NGF) and K-252a stimulate the uptake of calcium into PC12 cells. Stimulation by either is prevented by pretreatment of the cells with the tumor promoter phorbol 12-myristate 13-acetate (PMA), suggesting an involvement of protein kinase C in the stimulation. The effect of PMA is specific in that the calcium uptake stimulated by either the L-type channel agonist BAY K 8644 or by ATP is not altered in PMA-pretreated cells. An involvement of kinase C is also suggested by the inhibition of NGF- or K-252a-stimulated calcium uptake by the kinase C inhibitors staurosporine and calphostin C. Inhibition by the isoform-specific agents GO 6976 and thymeleatoxin implicates one of the classic calcium-sensitive isoforms of kinase C. The close similarity in the profiles of inhibition of NGF-stimulated and K-252a-stimulated calcium uptake by the various effectors suggests that NGF and K-252a act on calcium uptake through some of the same signaling elements.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adenosine Triphosphate; Animals; Calcium; Calcium Channel Agonists; Carbazoles; Carcinogens; Enzyme Inhibitors; Indole Alkaloids; Indoles; Isoenzymes; Naphthalenes; Nerve Growth Factors; PC12 Cells; Phorbol Esters; Protein Kinase C; Protein Kinase C-alpha; Rats; Receptors, Nerve Growth Factor; Signal Transduction; Staurosporine; Tetradecanoylphorbol Acetate

Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin (alpha2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RI alpha of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RI alpha protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RI alpha antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated alpha2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.

Topics: Adenoviruses, Human; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Endocytosis; Enzyme Inhibitors; Epidermal Growth Factor; Genistein; Humans; Indole Alkaloids; Indoles; Isoflavones; Isoquinolines; Lactoferrin; Low Density Lipoprotein Receptor-Related Protein-1; Maleimides; Melanoma; Naphthalenes; Phenols; Protein Kinase C; Receptors, Immunologic; Signal Transduction; Staurosporine; Sulfonamides; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Calcium/calmodulin-dependent protein kinase-II (CamK-II) is a major neuronal protein which plays a significant role in the cellular process of long-term potentiation (LTP), and vesicular release of neurotransmitters. Here, we show that KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine, a specific cell-permeable inhibitor of CamK-II substantially protected neurons from (1) acute NMDA toxicity and (2) hypoxia/hypoglycemia-induced neuronal injury in fetal rat cortical cultures. KN-62 did not directly inhibit glutamate, kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), glycine, or [piperidyl-3,4-(N)]-(N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine) (TCP) binding to rat brain membranes. Finally, KN-62 significantly reduced cellular calcium accumulation following either NMDA challenge or hypoxia/hypoglycemia insult. Our results show that CamK-II plays a key role in mediating some of the biochemical events leading to cell death following an acute excitotoxic insult.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Cell Death; Cell Hypoxia; Cell Survival; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fetus; Genistein; Imidazoles; Indole Alkaloids; Isoflavones; Isoquinolines; Kinetics; N-Methylaspartate; Naphthalenes; Neurons; Piperazines; Polycyclic Compounds; Protein Kinase C; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Spectrin

Stamen hair cells of the spiderwort plant Tradescantia virginiana exhibit unusually predictable rates of progression through mitosis, particularly from the time of nuclear envelope breakdown (NEBD) through the initiation of cytokinesis. The predictable rate of progression through prometaphase and metaphase has made these cells a useful model system for the determination of the timing of regulatory events that trigger entry into anaphase. A number of studies suggest that the elevation of one or more protein kinase activities is a necessary prerequisite for entry into anaphase. The current experiments employ two strategies to test when these elevations in protein kinase activity actually occur during metaphase. In perfusions, we added the protein kinase inhibitors K-252a, staurosporine, or calphostin C to living stamen hair cells for 10-min intervals at known times during prometaphase or metaphase and monitored the subsequent rate of progression into anaphase. Metaphase transit times were altered as a function of the time of addition of K-252a or staurosporine to the cells; metaphase transit times were extended significantly by treatments initiated in prometaphase through early metaphase and again late in metaphase. Transit times were normal after treatments initiated in mid-metaphase, approximately 15 to 21 min after NEBD. Calphostin C had no significant effect on the metaphase transit times. In parallel, cells were microinjected with known quantities of a general-purpose protein kinase substrate peptide, VRKRTLRRL, at predefined time points during prometaphase and metaphase. At a cytosolic concentration of 100 nM to 1 microM, the peptide doubled or tripled the metaphase transit times when injected into the cytosol of mitotic cells within the first 4 min after NEBD, at any point from 7.5 to 9 min after NEBD, at any point from 14 to 16 min after NEBD, at 21 min after NEBD, or at 24 min after NEBD. At the concentration used and during these brief intervals, the peptide appeared to act as a competitive inhibitor to reveal inflection points when protein kinase activation was occurring or when endogenous substrate levels approached levels of the peptide. The timing of these inflection points coincides with the changes in protein kinase activities during prometaphase and metaphase, as indicated by our perfusions of cells with the broad spectrum kinase inhibitors. Collectively, our results suggest that the cascade that culminates in anaphase is complex and involv

Topics: Alkaloids; Anaphase; Carbazoles; Cell Division; Cells, Cultured; Enzyme Activation; Indole Alkaloids; Mitosis; Naphthalenes; Oligopeptides; Plant Cells; Plants; Polycyclic Compounds; Protein Kinase C; Staurosporine

Involvement of protein kinase C in receptor-operated Ca2+ sensitization of cell shortening was investigated by use of alpha-toxin-permeabilized smooth muscle cells from the fundus of the guinea-pig. Most of the isolated cells responded to 0.6 microM Ca2+ with a maximal shortening to approximately 65% of the resting cell length. Addition of acetylcholine (ACh) at a maximal concentration (10 microM) resulted in a marked decrease in the concentration of Ca2+ required to trigger a threshold response from 0.6 microM to 0.2 microM. The augmentation of Ca2+ sensitivity by ACh was not inhibited by specific protein kinase C inhibitors, calphostin C and K-252b at a concentration of 1 microM. These findings suggest that protein kinase C is not involved in the muscarinic receptor-operated augmentation of Ca2+ sensitivity.

Topics: Acetylcholine; Animals; Calcium; Carbazoles; Gastric Fundus; Guinea Pigs; In Vitro Techniques; Indole Alkaloids; Muscle Contraction; Muscle, Smooth; Naphthalenes; Polycyclic Compounds; Protein Kinase C; Receptors, Muscarinic; Type C Phospholipases