ucn-1028-c and phorbolol-myristate-acetate

In this manuscript, we present experimental evidence that PKCs phosphorylate p27 at T198 in vitro and in vivo, resulting in p27 stabilization and cell cycle arrest in MCF-7 and HeLa cells. Our findings indicate that (1) recombinant PKCα, βII, δ, η and θ isoforms phosphorylate, in in vitro kinase assays, wild-type recombinant p27 protein expressed in E. coli and wild-type p27 protein immunoprecpitated from transfected HEK-293 cells but not the T198A mutant, (2) adoptive expressed PKCα and δ phosphorylate both transfected and endogenous p27 at T198 in HEK-293 cells, (3) T198 phosphorylation of transfected and endogenous p27 is increased by PKC activators [Phorbol 12-myristate 13-acetate (PMA)] and suppressed by PKC inhibitors (Rottlerin A, G06976, Calphostin C), (4) in parallel with increased T198 phosphorylation, PMA induces stabilization of p27 protein in HeLa cells, whereas PKC inhibitors induce a decrease in p27 stability and, finally, (5) PMA-induced p27 upregulation is necessary for growth arrest of HeLa and MCF-7 cells induced by PKC activation by PMA.   Overall, these results suggest that PKC-dependent upregulation of p27 induced by its phosphorylation at T198 represents a mechanism that mediates growth arrest promoted by PMA and provide novel insights on the ability of different PKC isoforms to play a role in controlling cell cycle progression.

Topics: Amino Acid Sequence; Amino Acid Substitution; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p27; HEK293 Cells; HeLa Cells; Humans; Naphthalenes; Phosphorylation; Protein Isoforms; Protein Kinase C; Recombinant Proteins; Tetradecanoylphorbol Acetate; Transfection; Up-Regulation

This study sought to define whether inward rectifying K(+) (K(IR)) channels were modulated by vasoactive stimuli known to depolarize and constrict intact cerebral arteries. Using pressure myography and patch-clamp electrophysiology, initial experiments revealed a Ba(2+)-sensitive K(IR) current in cerebral arterial smooth muscle cells that was active over a physiological range of membrane potentials and whose inhibition led to arterial depolarization and constriction. Real-time PCR, Western blot, and immunohistochemical analyses established the expression of both K(IR)2.1 and K(IR)2.2 in cerebral arterial smooth muscle cells. Vasoconstrictor agonists known to depolarize and constrict rat cerebral arteries, including uridine triphosphate, U46619, and 5-HT, had no discernable effect on whole cell K(IR) activity. Control experiments confirmed that vasoconstrictor agonists could inhibit the voltage-dependent delayed rectifier K(+) (K(DR)) current. In contrast to these observations, a hyposmotic challenge that activates mechanosensitive ion channels elicited a rapid and sustained inhibition of the K(IR) but not the K(DR) current. The hyposmotic-induced inhibition of K(IR) was 1) mimicked by phorbol-12-myristate-13-acetate, a PKC agonist; and 2) inhibited by calphostin C, a PKC inhibitor. These findings suggest that, by modulating PKC, mechanical stimuli can regulate K(IR) activity and consequently the electrical and mechanical state of intact cerebral arteries. We propose that the mechanoregulation of K(IR) channels plays a role in the development of myogenic tone.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Cerebral Arteries; Female; Hypotonic Solutions; In Vitro Techniques; Membrane Potentials; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Naphthalenes; Patch-Clamp Techniques; Potassium Channels, Inwardly Rectifying; Protein Kinase C; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Serotonin; Tetradecanoylphorbol Acetate; Uridine Triphosphate; Vasoconstrictor Agents

In vitro effects of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, calphostin C (PKC inhibitor) and okadaic acid [OA, a protein phosphatase (PP; PP1 and PP2A) inhibitor] on 2-hydroxyestradiol-17beta (2-OHE(2))-induced oocyte maturation were investigated in the catfish Heteropneustes fossilis. Incubations of postvitellogenic follicles with PMA or OA alone did not induce oocyte maturation. However, co-incubations with 2-OHE(2) and PMA (0.05, 0.5 and 5 microM) or 2-OHE(2) and OA (0.5, 1.0 or 2.0 microM) increased germinal vesicle breakdown (GVBD) significantly over that of 2-OHE(2). Incubation of follicles with calphostin C elicited varied effects on GVBD, low (0.005 and 0.01 microM) and high (5.0 and 10.0 microM) concentrations did not affect GVBD, but medium concentrations (0.05, 0.1, 0.5, 1.0 and 2.5 microM) stimulated it. The medium concentrations elicited a biphasic stimulatory response with peak GVBD at 0.1 microM (54%). Calphostin C (>or=2.5 microM) inhibited the 2-OHE(2)-induced GVBD in a concentration-dependent manner during the 24 h incubation. Pre- or post-treatment with calphostin C inhibited the steroid-induced GVBD only at 6 h. In co-incubation studies, both PMA and OA reversed the inhibitory effect of calphostin C: the former partially and the latter fully. The results of the present study show that PKC appears to modulate the 2-OHE(2)-induced oocyte maturation. The OA-sensitive PP may be involved in the PKC modulation of steroid-induced oocyte maturation.

Topics: Animals; Catfishes; Cell Differentiation; Enzyme Inhibitors; Estradiol; Female; Naphthalenes; Okadaic Acid; Oocytes; Phosphoprotein Phosphatases; Protein Kinase C; Protein Kinase Inhibitors; Tetradecanoylphorbol Acetate

1. Staurosporine is a broad-specificity kinase inhibitor, which has acted as lead compound for the development of some novel cytotoxic compounds for treatment of cancer. This study investigates the unexpected observation that staurosporine can also induce homotypic cellular aggregation. 2. In this study, staurosporine is shown to activate rapid homotypic aggregation of U937 cells, at concentrations below those required to induce cell death. This activity is a particular feature of staurosporine, and is not shared by a number of other kinase inhibitors. The proaggregating activity of staurosporine is inhibited by deoxyglucose, cytochalasin B and colchicine. Staurosporine-induced aggregation can be distinguished from that induced by the phorbol 12-myristate 13-acetate by faster kinetics and insensitivity to cycloheximide. Staurosporine induces translocation of conventional and novel, but not atypical isoforms of protein kinase C (PKC). Aggregation induced by staurosporine is inhibited by a number of inhibitors of PKC isoforms, and by inhibitors of protein tyrosine kinases. Staurosporine also induces rapid phosphorylation of ERK and p38, and inhibitors of both these enzymes block aggregation. 3. Staurosporine induces dysregulated activation of multiple kinase signaling pathways in U937 cells, and the combined activity of several of these pathways is essential for the induction of aggregation.

Topics: Acetophenones; Adenosine Triphosphate; Benzopyrans; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; CD18 Antigens; Cell Aggregation; Cycloheximide; Cytoskeleton; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Genistein; Humans; Imidazoles; Indoles; Integrin beta1; Isoenzymes; Maleimides; Mitogen-Activated Protein Kinases; Naphthalenes; Phosphorylation; Phosphotransferases; Protein Kinase C; Protein-Tyrosine Kinases; Pyridines; Staurosporine; Temperature; Tetradecanoylphorbol Acetate; Time Factors; Tyrosine; U937 Cells

Interleukin (IL)-1 beta increases beta(2)-adrenergic receptor (beta(2)-AR) mRNA and density by protein kinase C (PKC)-dependent mechanisms in human airway epithelial cells. The present study examined the role of several nuclear transcription factors in the PKC-activated upregulation of beta(2)-AR expression. BEAS-2B cells were exposed to the PKC activator phorbol 12-myristate 13-acetate (PMA; 0.1 microM for 2-18 h). PMA had no effect on activator protein (AP)-2 or cAMP response element binding protein DNA binding activity but markedly increased nuclear factor (NF)-kappa B and AP-1 binding as assessed by electrophoretic gel mobility shift assay. PMA also increased the activity of a beta(2)-AR promoter-luciferase reporter construct in transiently transfected cells. These effects were inhibited by the PKC inhibitors Ro-31-8220 and calphostin C. Furthermore, with increasing Ro-31-8220, beta(2)-AR promoter-reporter activity correlated closely with both NF-kappa B and AP-1 activities (r > 0.89 for both). Finally, the selective NF-kappa B inhibitor MG-132 dose dependently reduced NF-kappa B binding and beta(2)-AR promoter activity but increased AP-1 binding. We conclude that PKC-induced upregulation of beta(2)-AR expression in human airway epithelial cells appears to be mediated, at least in part, by increases in NF-kappa B activity.

Topics: Cell Line; Enzyme Inhibitors; Epithelial Cells; Genes, Reporter; Humans; Indoles; Interleukin-1; Leupeptins; Naphthalenes; NF-kappa B; Promoter Regions, Genetic; Protein Kinase C; Receptors, Adrenergic, beta-2; Respiratory Mucosa; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Up-Regulation