Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3.
(25/1402)
Signal transducers and activators of transcription (STATs) are rapidly phosphorylated on tyrosine residues in response to cytokine and growth factor stimulation of cell surface receptors. STATs hereafter are translocated to the nucleus where they act as transcription factors. Recent reports suggest that serine phosphorylation of STATs also is involved in the regulation of STAT-mediated gene transcription. Here, we studied the role of serine/threonine phosphatases in STAT3 signaling in human antigen-specific CD4(+) T cell lines and cutaneous T cell lymphoma lines, expressing a constitutively activated STAT3. We show that an inhibitor of protein phosphatases (PPs) PP1/PP2A, calyculin A, induces (i) phosphorylation of STAT3 on serine and threonine residues, (ii) inhibition of STAT3 tyrosine phosphorylation and DNA binding activity, and (iii) relocation of STAT3 from the nucleus to the cytoplasm. Similar results were obtained with other PP2A inhibitors (okadaic acid, endothall thioanhydride) but not with inhibitors of PP1 (tautomycin) or PP2B (cyclosporine A). Pretreatment with the broad serine/threonine kinase inhibitor staurosporine partly blocked the calyculin A-induced STAT3 phosphorylation, whereas inhibitors of serine/threonine kinases, such as mitogen-activated protein kinase-1 extracellular-regulated kinase-kinase, mitogen-activated protein p38 kinase, and phosphatidylinositol 3-kinase, did not. In conclusion, we provide evidence that PP2A plays a crucial role in the regulation of STAT3 phosphorylation and subcellular distribution in T cells. Moreover, our findings suggest that the level of STAT3 phosphorylation is balanced between a staurosporine-sensitive kinase(s) and PP2A. (+info)
Identification of two nuclear androgen receptors in kelp bass (Paralabrax clathratus) and their binding affinities for xenobiotics: comparison with Atlantic croaker (Micropogonias undulatus) androgen receptors.
(26/1402)
Two distinct nuclear androgen receptors (ARs) were identified in brain and ovarian tissues of kelp bass, Paralabrax clathratus, termed kbAR1 and kbAR2, which correspond to the two nuclear ARs we have previously characterized in Atlantic croaker, Micropogonias undulatus, termed acAR1 and acAR2. Scatchard analysis of nuclear fractions of whole brain tissue demonstrated that kbAR1 had a single class of high-affinity binding sites for testosterone (T; K(d) of 1. 8 nM and B(max) of 1.0 pmol/g tissue), whereas cytosolic fractions of kbAR2 ovarian tissue had a single class of high-affinity binding sites for dihydrotestosterone (DHT; K(d) of 0.1 nM and B(max) of 0.5 pmol/g tissue). Competition studies showed that both kbAR1 and kbAR2 were specific for androgens. However, kbAR1 bound only T with high affinity, whereas kbAR2 bound DHT, mibolerone, 17alpha-methyl-testosterone, T, and 11-ketotestosterone with high affinity. In addition, we examined the binding affinities of dichlorodiphenyltrichloroethane and its derivatives, several hydroxylated polychlorinated biphenyl (PCB) congeners, PCB mixtures, and the fungicide vinclozolin and its two metabolites M1 and M2 for the two ARs in Atlantic croaker ovarian, testicular, and brain tissues and in kelp bass ovarian and brain tissues. Only 4, 4'-PCB-3-OH and 2',5'-PCB-3-OH demonstrated greater than 50% displacement of [(3)H]testosterone from either acAR1 or kbAR1. In contrast, with the exception of vinclozolin, all of the xenobiotics examined demonstrated binding to acAR2 in testicular and ovarian tissues. The binding affinities were highest in the testicular tissue with M2, 2,2'5'-PCB-4-OH, and o,p'-DDD all binding with EC(50)s less than 10 microM. The binding affinities of xenobiotics to kbAR2 in ovarian tissue were similar to their binding affinities for ovarian acAR2. The finding that AR1 and AR2 possess different binding affinities for natural androgens and synthetic steroids, as well as for xenobiotics, suggests that the activities of androgens and of certain xenobiotics will depend upon the type of AR present within the target tissue. (+info)
Cardiovascular effects of clonidine-like drugs in pithed rabbits.
(27/1402)
Administration (3 to 100 microg/kg IV) of clonidine, rilmenidine, and an imidazoline derivative, 2-(2-chlorophenylamino)imidazoline, in pithed nonstimulated rabbits caused a dose-dependent increase in mean arterial pressure without affecting heart rate. Prazosin (0.1 mg/kg IV) almost abolished the pressor responses to 2-(2-chlorophenylamino)imidazoline, partially inhibited those induced by clonidine, but failed to affect those elicited by rilmenidine. In contrast, yohimbine (1 mg/kg IV) blunted the pressor responses of the 3 drugs. In sympathetically stimulated pithed rabbits, 2-(2-chlorophenylamino)imidazoline induced only pressor effects, whereas clonidine and rilmenidine caused a transient pressure increase followed by a dose-dependent depressor effect. Yohimbine abolished the depressor effect of both drugs, which may have involved presynaptic alpha(2)-adrenoceptors. In conclusion, peripheral effects of 2-(2-chlorophenylamino)imidazoline and clonidine involved at least alpha(1)- and alpha(2)-adrenoceptor activation, whereas pressor and depressor effects of rilmenidine were mediated by alpha(2)-adrenoceptors. (+info)
Distinct roles for PP1 and PP2A in phosphorylation of the retinoblastoma protein. PP2a regulates the activities of G(1) cyclin-dependent kinases.
(28/1402)
The function of the retinoblastoma protein (pRB) in controlling the G(1) to S transition is regulated by phosphorylation and dephosphorylation on serine and threonine residues. While the roles of cyclin-dependent kinases in phosphorylating and inactivating pRB have been characterized in detail, the roles of protein phosphatases in regulating the G(1)/S transition are not as well understood. We used cell-permeable inhibitors of protein phosphatases 1 and 2A to assess the contributions of these phosphatases in regulating cyclin-dependent kinase activity and pRB phosphorylation. Treating asynchronously growing Balb/c 3T3 cells with PP2A-selective concentrations of either okadaic acid or calyculin A caused a time- and dose-dependent decrease in pRB phosphorylation. Okadaic acid and calyculin A had no effect on pRB phosphatase activity even though PP2A was completely inhibited. The decrease in pRB phosphorylation correlated with inhibitor-induced suppression of G(1) cyclin-dependent kinases including CDK2, CDK4, and CDK6. The inhibitors also caused decreases in the levels of cyclin D2 and cyclin E, and induction of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). The decrease in cyclin-dependent kinase activities were not dependent on induction of cyclin-dependent kinase inhibitors since CDK inhibition still occurred in the presence of actinomycin D or cycloheximide. In contrast, selective inhibition of protein phosphatase 1 with tautomycin inhibited pRB phosphatase activity and maintained pRB in a highly phosphorylated state. The results show that protein phosphatase 1 and protein phosphatase 2A, or 2A-like phosphatases, play distinct roles in regulating pRB function. Protein phosphatase 1 is associated with the direct dephosphorylation of pRB while protein phosphatase 2A is involved in pathways regulating G(1) cyclin-dependent kinase activity. (+info)
Mode-specific inhibition of sodium-calcium exchange during protein phosphatase blockade.
(29/1402)
The effects of the protein phosphatase inhibitors calyculin A and okadaic acid on Na(+)/Ca(2+) exchange activity were examined in transfected Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger. Incubating the cells for 5-10 min with 100 nM calyculin A reduced exchange-mediated (45)Ca(2+) uptake or Ba(2+) influx by 50-75%. Half-maximal inhibition of (45)Ca(2+) uptake was observed at 15 nM calyculin A. The nonselective protein kinase inhibitors K252a and staurosporine provided partial protection against the effects of calyculin A. Okadaic acid, another protein phosphatase inhibitor, nearly completely blocked exchange-mediated Ba(2+) influx. Chinese hamster ovary cells expressing a mutant exchanger in which 420 out of 520 amino acid residues were deleted from the central hydrophilic domain of the exchanger remained sensitive to the inhibitory effects of calyculin A and okadaic acid. Surprisingly, Na(o)(+)-dependent Ca(2+) efflux appeared to be only modestly inhibited, if at all, by calyculin A or okadaic acid. We conclude that protein hyperphosphorylation during protein phosphatase blockade selectively inhibits the Ca(2+) influx mode of Na(+)/Ca(2+) exchange, probably by an indirect mechanism that does not involve phosphorylation of the exchanger itself. (+info)
Serine/threonine protein phosphatases and regulation of K-Cl cotransport in human erythrocytes.
(30/1402)
Activation of K-Cl cotransport is associated with activation of membrane-bound serine/threonine protein phosphatases (S/T-PPases). We characterize red blood cell S/T-PPases and K-Cl cotransport activity regarding protein phosphatase inhibitors and response to changes in ionic strength and cell size. Protein phosphatase type 1 (PP1) activity is highly sensitive to calyculin A (CalA) but not to okadaic acid (OA). PP2A activity is highly sensitive to CalA and OA. CalA completely inhibits K-Cl cotransport activity, whereas OA partially inhibits K-Cl cotransport. Membrane PP1 and membrane PP2A activities are elevated in cells suspended in hypotonic solutions, where K-Cl cotransport is elevated. Increases in membrane PP1 activity (62 +/- 10% per 100 meq/l) result from decreases in intracellular ionic strength and correlate with increases in K-Cl cotransport activity (54 +/- 10% per 100 meq/l). Increases in membrane PP2A activity (270 +/- 77% per 100 mosM) result from volume increases and also correlate with increases in K-Cl cotransport activity (420 +/- 47% per 100 mosM). The characteristics of membrane-associated PP1 and PP2A are consistent with a role for both phosphatases in K-Cl cotransport activation in human erythrocytes. (+info)
[Ca(2+)](i) oscillation frequency regulates agonist-stimulated NF-kappaB transcriptional activity.
(31/1402)
In nonexcitable cells, stimulation by high agonist concentrations typically produces a biphasic increase in cytosolic Ca(2+) ([Ca(2+)](i)). This response is characterized by a transient initial increase because of intracellular Ca(2+) release followed by a sustained elevation which varies in amplitude depending on the nature of the stimulus. In contrast, low-level stimulation often evokes oscillatory changes in [Ca(2+)](i). The specific information provided by repetitive [Ca(2+)](i) spikes appears to be encoded in the frequency rather than in the amplitude of [Ca(2+)](i) oscillations. The specific, membrane-permeable inositol 1,4, 5-trisphosphate (Ins-1,4,5-P(3)) receptor blocker Xestospongin C (XeC, 2-20 microM) was used to affect [Ca(2+)](i) signaling in human aortic endothelial cells (HAEC) during an established response to low-level (1 microM) histamine stimulation. XeC produced a dose-dependent decrease in the frequency of [Ca(2+)](i) oscillations during histamine stimulation without affecting oscillation amplitude. Histamine stimulated a 14-fold increase in NF-kappaB-chloramphenicol acetyltransferase reporter gene activity that was dose-dependently decreased by XeC. Thus, during low-level agonist stimulation, [Ca(2+)](i) oscillation frequency regulates nuclear transcription in HAEC. (+info)
Polycations induce calcium signaling in glomerular podocytes.
(32/1402)
BACKGROUND: The neutralization of the polyanionic surface of the podocyte by perfusion of kidneys with polycations, such as protamine sulfate, leads to a retraction of podocyte foot processes and proteinuria. This study investigates the effects of protamine sulfate or anionic, neutral, or cationic dextrans on the cytosolic calcium activity ([Ca2+]i) in podocytes. METHODS: [Ca2+]i was measured in single cultured differentiated mouse podocytes with the fluorescence dye fura-2/AM. RESULTS: Protamine sulfate caused a concentration-dependent and partially reversible increase of [Ca2+]i (EC50 approximately 1.5 micromol/liter). Pretreatment of the cells with heparin (100 U/liter) inhibited the protamine sulfate-mediated increase of [Ca2+]i. Like protamine sulfate, diethylaminoethyl dextran (DEAE-dextran) concentration dependently increased [Ca2+]i in podocytes (EC50 approximately 20 nmol/liter), whereas dextran sulfate or uncharged dextran (both 10 micromol/liter) did not influence [Ca2+]i. A reduction of the extracellular Ca2+ concentration (from 1 mmol/liter to 1 micomol/liter) partially inhibited the protamine sulfate and the DEAE-dextran-induced [Ca2+]i response. Flufenamate (100 micromol/liter) or Gd3+ (10 micromol/liter), which are known to inhibit nonselective ion channels, did not influence the [Ca2+]i increase induced by protamine sulfate. In the presence of thapsigargin (50 nmol/liter), an inhibitor of the endoplasmic reticulum Ca2+-ATPase, both protamine sulfate and DEAE-dextran increased [Ca2+]i. CONCLUSIONS: The data indicate that polycations increase podocyte [Ca2+]i. The increase of [Ca2+]i may be an early event in the pathogenesis of protamine sulfate-mediated retraction of podocyte foot processes. (+info)