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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study we analyzed the covalent binding to proteins of 17 beta-estradiol (E2), retinoic acid (RA), and progesterone in MCF-7 and MCF-7/AdrR cells. MCF-7 cells have receptors for E2 and progesterone. MCF-7/AdrR cells do not have these receptors. After a 1-day incubation period with either [3H]E2, [3H]progesterone, or [3H]RA the levels of covalently bound radioactivity was between 1.4- to 2-fold greater in MCF-7 cells than in MCF-7/AdrR cells. We analyzed the labeled proteins with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. About 40 proteins were labeled by E2 in MCF-7 cells and about 10 of these proteins were the only proteins labeled by E2 in MCF-7/AdrR cells. We saw that the same 8 proteins were labeled by RA in both cell lines. Progesterone labeled 2 proteins with M(r) values of 37,000 and 20,000 in MCF-7 cells. These 2 proteins had mobilities that were the same as proteins that were labeled by either E2 or RA in both MCF-7 and MCF-7/AdrR cells. Besides these 2 proteins, we saw proteins of M(r) 51,000 (p51) and 55,000 that were covalently labeled by E2 in MCF-7 cells and by RA in both MCF-7 and MCF-7/AdrR cells. The p51 had the same mobility on 2D-PAGE as an 8-azido-[32P]cAMP-labeled protein. This protein is probably RII alpha, the type II cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase. These results suggest that the estrogen receptor, while not obligatory, might still modulate the covalent linkage of E2 to protein. In addition, our results raise the possibility that some effects of some ligands of the thyroid/steroid hormone receptor family may involve the covalent linking of these hormones to proteins, including RII alpha.
J Steroid Biochem Mol Biol 1992 Nov
PMID:The covalent labeling of proteins by 17 beta-estradiol, retinoic acid, and progesterone in the human breast cancer cell lines MCF-7 and MCF-7/AdrR. 132 24

Exposure of mouse colliculi neurons to selective 5-hydroxytryptamine (5-HT)4 agonists was accompanied by a rapid desensitization of the receptor-stimulated adenylyl cyclase response. Half-maximal desensitization occurred after 2 min. Only exposure of neurons to selective 5-HT4 agonists led to a potent desensitization of the 5-HT4-mediated response. Neurons exposed to other agents, like isoproterenol, vasoactive intestinal peptide, or forskolin, that increase cAMP levels did not undergo any desensitization of 5-HT4 receptors. Activation of protein kinase A with either 8-bromo-cAMP or dibutyryl-cAMP or application of inhibitors of protein kinase A-dependent phosphorylation did not change the rate of 5-HT4-induced desensitization. No shift to lower potency of 5-HT4 agonists in the concentration-response curve was observed. These results suggest that 5-HT4 receptor agonists induced homologous but not cAMP-mediated heterologous desensitization. A good correlation was found between the affinities of nine 5-HT4 agonists and their abilities to desensitize the adenylyl cyclase response. This may indicate that homologous desensitization is a function of the mean occupancy time of the receptors by agonists. When permeabilized neurons were loaded with heparin, an inhibitor of the beta-adrenergic receptor kinase (beta ARK), 5-HT4 receptor desensitization was reduced by 30-40%. Interestingly, Zn2+, an other inhibitor of beta ARK, totally prevented 5-HT4-induced desensitization. Pretreatment of neurons with concanavalin A, reported to inhibit sequestration of beta-adrenergic receptors from the cell surface, reduced the desensitization process by 70%. These data suggest that both sequestration and phosphorylation by beta ARK, or another specific agonist-dependent receptor kinase, are involved in homologous desensitization of 5-HT4 receptors coupled to adenylyl cyclase.
Mol Pharmacol 1992 Nov
PMID:Characterization of homologous 5-hydroxytryptamine4 receptor desensitization in colliculi neurons. 133 63

These studies were undertaken to evaluate the role of protein kinase C (PKC) in the regulation by arginine vasopressin (AVP) of adrenocorticotropin (ACTH) secretion from the ovine anterior pituitary. AVP caused the rapid translocation of PKC from the cytosol to the cell membrane in ovine anterior pituitary cells that was maximal at 5 min. This phenomenon, which is a known concomitant of C-kinase activation, was produced to a greater extent by phorbol 12-myristate 13-acetate (PMA) but not by corticotropin-releasing factor (CRF). To determine whether AVP activated corticotrope PKC, we assessed the ability of three different PKC inhibitors (H-7, sphingosine, and retinal) to modify basal, AVP-, PMA-, and CRF-stimulated ACTH release. In addition to inhibiting the in vitro activity of purified PKC, each compound also caused in vitro inhibition of the protein kinase A (PKA) catalytic subunit, indicating that none could be considered to be a specific inhibitor of PKC and the PKA catalytic subunit. As determined by the mean IC50 values required for the in vitro inhibition of PKC and the PKA catalytic subunit, sphingosine was judged to be the most selective and H-7 the least selective PKC inhibitor. A 4 h exposure to each inhibitor caused a dose-dependent increase in basal ACTH release and attenuation of both AVP- and PMA-stimulated ACTH release. H-7 and retinal, in concentrations that caused a 20-50% inhibition of PKA, also attenuated CRF-stimulated ACTH release; however, this effect was not observed with sphingosine in concentrations that caused only a 10-20% inhibition of PKA. We conclude that: (1) AVP causes the direct activation of PKC in the ovine anterior pituitary and that C kinase activation is important in mediating the effect of AVP on ACTH release; (2) the finding that inhibition of PKC elevates ACTH suggests that basal ACTH secretion is also partly regulated by PKC; (3) since CRF does not cause PKC translocation in ovine anterior pituitary cells, it is unlikely that PKC plays a physiological role in the action of CRF on the corticotrope; (4) the finding that H-7 and retinal attenuate CRF-stimulated ACTH secretion suggests that CRF activates PKA in corticotropes.
Mol Cell Endocrinol 1992 Sep
PMID:Evidence that the stimulation by arginine vasopressin of the release of adrenocorticotropin from the ovine anterior pituitary involves the activation of protein kinase C. 133 7

GCN2 is a protein kinase in Saccharomyces cerevisiae that is required for increased expression of the transcriptional activator GCN4 in amino acid-starved cells. GCN2 stimulates GCN4 synthesis at the translational level by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2). We identified a truncated form of the GLC7 gene, encoding the catalytic subunit of a type 1 protein phosphatase, by its ability to restore derepression of GCN4 expression in a strain containing the partially defective gcn2-507 allele. Genetic analysis suggests that the truncated GLC7 allele has a dominant negative phenotype, reducing the level of native type 1 protein phosphatase activity in the cell. The truncated form of GLC7 does not suppress the regulatory defect associated with a gcn2 deletion or a mutation in the phosphorylation site of eIF-2 alpha (Ser-51). In addition, the presence of multiple copies of wild-type GLC7 impairs the derepression of GCN4 that occurs in response to amino acid starvation or dominant-activating mutations in GCN2. These findings suggest that the phosphatase activity of GLC7 acts in opposition to the kinase activity of GCN2 in modulating the level of eIF-2 alpha phosphorylation and the translational efficiency of GCN4 mRNA. This conclusion is supported by biochemical studies showing that the truncated GLC7 allele increases the level of eIF-2 alpha phosphorylation in the gcn2-507 mutant to a level approaching that seen in wild-type cells under starvation conditions. The truncated GLC7 allele also leads to reduced glycogen accumulation, indicating that this protein phosphatase is involved in regulating diverse metabolic pathways in yeast cells.
Mol Cell Biol 1992 Dec
PMID:Truncated protein phosphatase GLC7 restores translational activation of GCN4 expression in yeast mutants defective for the eIF-2 alpha kinase GCN2. 133 44

The Y1 adrenocortical tumor cell mutants, Kin-7 and Kin-8, harbor point mutations in the regulatory subunit (RI) of the type 1 cAMP-dependent protein kinase (cAMPdPK) that render the enzyme resistant to activation by cAMP. These mutants also are resistant to many of the regulatory effects of ACTH and cAMP. In order to examine the causal relationships between the mutations in cAMPdPK and the resistance to ACTH and cAMP, the Kin mutants were transfected with expression vectors encoding wild type subunits of cAMPdPK in order to restore cAMP-responsive protein kinase activity. The transformants then were screened for the concomitant recovery of cellular responsiveness to ACTH and cAMP. In the mutant Kin-7, cAMP-responsive protein kinase activity was recovered after transfection with an expression vector encoding wild type mouse RI. Protein kinase activity in the mutant Kin-8 remained largely cAMP-resistant after transfection with the RI expression vector but could be rendered cAMP-responsive by transfection with an expression vector encoding the wild type catalytic subunit. The recovery of cAMP-responsive protein kinase activity was accompanied by the recovery of steroidogenic and morphological responses to ACTH and cAMP, suggesting that the cAMP-dependent signaling cascade plays an obligatory role in these actions of ACTH. The growth-regulatory effects of cAMP were not reversed with the recovery of cAMP-responsive protein kinase activity, suggesting that cAMP-resistant growth regulation results from second-site, adaptive mutations either in the original Kin mutant population or in the transformants. Studies on the conversion of 22(R)-hydroxycholesterol into steroid products in parent and mutant cells indicate that the Kin mutations reduce the steroidogenic capacity of the cell as well as inhibit the hormone- and cyclic nucleotide-dependent mobilization of substrate cholesterol.
Mol Endocrinol 1992 Oct
PMID:The causal relationship between mutations in cAMP-dependent protein kinase and the loss of adrenocorticotropin-regulated adrenocortical functions. 133 50

In mammalian brain, physiological signals carried by cyclic AMP (cAMP) seem to be targeted to effector sites via the tethering of cAMP-dependent protein kinase II beta (PKAII beta) to intracellular structures. Recently characterized A kinase anchor proteins (AKAPs) are probable mediators of the sequestration of PKAII beta because they contain a high-affinity binding site for the regulatory subunit (RII beta) of the kinase and a distinct intracellular targeting domain. To establish a cellular basis for this targeting mechanism, we have employed immunocytochemistry to 1) identify the types of neurons that are enriched in AKAPs, 2) determine the primary intracellular location of the anchor protein, and 3) demonstrate that an AKAP and RII beta are coenriched and colocalized in neurons that utilize the adenylate cyclase-cyclic AMP-dependent protein kinase (PKA) signaling pathway. Antibodies directed against rat brain AKAP 150 were used to elucidate the regional, cellular and intracellular distribution of a prototypic anchor protein in the CNS. AKAP 150 is abundant in Purkinje cells and in neurons of the olfactory bulb, basal ganglia, cerebral cortex, and other forebrain regions. In contrast, little AKAP 150 is detected in neurons of the thalamus, hypothalamus, midbrain, and hindbrain. A high proportion of total AKAP 150 is concentrated in primary branches of dendrites, where it is associated with microtubules. We also discovered that the patterns of accumulation and localization of RII beta (and PKAII beta) in brain are similar to those of AKAP 150. The results suggest that bifunctional AKAP 150 tethers PKAII beta to the dendritic cytoskeleton, thereby creating a discrete target site for the reception and propagation of signals carried by cAMP.
Mol Biol Cell 1992 Nov
PMID:cAMP signaling in neurons: patterns of neuronal expression and intracellular localization for a novel protein, AKAP 150, that anchors the regulatory subunit of cAMP-dependent protein kinase II beta. 133 41

The binding of cyclin A to p34cdc2 and p32cdk2 and the protein kinase activity of the complexes has been measured by cell-free translation of the corresponding mRNA in extracts of frog eggs, followed by immunoprecipitation. A variety of mutant cyclin A molecules have been constructed and tested in this assay. Small deletions and point mutations of highly conserved residues in the 100-residue "cyclin box" abolish binding and activation of both p34cdc2 and p32cdk2. By contrast, large deletions at the N-terminus have no effect on kinase binding and activation, until they remove residues beyond 161, where the first conserved amino acids are found in all known examples of cyclin A. At the C-terminus, removal of 14 or more amino acids abolishes activity. We also demonstrate that deletion of, or point mutations, in the cyclin A homologue of the 10-residue "destruction box," previously described in cyclin B (Glotzer et al., 1991) abolish cyclin proteolysis at the transition from M-phase to interphase.
Mol Biol Cell 1992 Nov
PMID:Identification of the domains in cyclin A required for binding to, and activation of, p34cdc2 and p32cdk2 protein kinase subunits. 133 43

There are at least three isozymes (C alpha, C beta, and C gamma) of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase (PKA) (Beebe, S., Oyen, O., Sandberg, M., Froysa, A., Hansson, V., and Jahnsen, T. (1990) Mol. Endocrinol. 4, 465-475). To compare the C gamma and C alpha isozymes, the respective cDNAs were expressed in permanently transformed Kin-8 PKA-deficient Y1 adrenal cells using the mouse metallothionein promoter. The recombinant C subunits were characterized as immunoreactive, zinc-inducible, cAMP-dependent kinase activities. In contrast to C alpha, histone was a better substrate than Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) for C gamma. Furthermore, C gamma histone kinase activity was not inhibited by the protein kinase inhibitor peptide (5-24 amide), which has been widely used as a PKA-specific inhibitor. The major C gamma peak (type I) eluted from DEAE-Sepharose at a higher NaCl concentration (120 mM) than the C alpha type I eluted (70 mM). C gamma and C alpha type II eluted between 220 and 240 mM NaCl. C gamma required higher concentrations of cAMP than C alpha did for dissociation from the mutant type I holoenzyme. These differences provided a basis for the separation of the mutant RI-associated isozymes on DEAE-Sepharose. Both C alpha (41-42 kDa) and C gamma (39-40 kDa) were identified by a C subunit antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. Zinc induced the PKA-mediated rounding phenotype in C gamma and C alpha clones, thereby restoring the cells to the parent Y1 adrenal cell phenotype. Collectively, these data indicate that C gamma is an active PKA C subunit but suggest that C gamma and C alpha have different protein and peptide recognition determinants.
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PMID:The C gamma subunit is a unique isozyme of the cAMP-dependent protein kinase. 133 96

We have used a monoclonal antibody (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder epithelial cells, to isolate a chloride channel protein by the use of an immunoaffinity column and FPLC. This protein (M(r) 219,000) has been reconstituted into a planar lipid bilayer, where it behaves as a chloride-selective channel (PCl/PNa = 20.2; PNa/PK = 1) whose unit conductance is 62.4 +/- 4.6 pS. Antibody added to the trans side (there is no effect from the cis side) causes channel open probability to drop to virtually zero, but has no effect on the conductance or the selectivity of single channels. To test the role of phosphorylation in the activity of the native channel, we studied the effects of the protein phosphatase inhibitor okadaic acid (OA) on intact gallbladders, and showed that channels opened by theophylline treatment and closed by antibody are reopened reversibly by OA (0.01-1.0 microM). Addition of the catalytic subunit of protein phosphatase 2A (PP-2A) to the cis side of a bilayer containing reconstituted chloride channels caused closure of the channels after a delay, and subsequent addition of ATP and the catalytic subunit of cAMP-dependent protein kinase (PKA) caused immediate reopening. These data indicate that (a) this chloride channel protein inserts in a directed way into the bilayer such that the cis side is 'intracellular', (b) the purified channel protein is phosphorylated, and (c) gating from the cellular side is controlled by the direct phosphorylation and dephosphorylation of the channel protein.
Mol Cell Biochem 1992 Sep 08
PMID:Reconstitution and regulation of an epithelial chloride channel. 133 26

In a previous study [Shaw, C., Pasqualotto, B. and Lanius, R.A., Mol. Neuropharmacol., in press] we have shown that phosphorylation and dephosphorylation actions of protein kinase and alkaline phosphatase lead to decreases or increases in the number of GABAA and AMPA receptors in adult rat neocortex. Using the same in vitro cortical slice preparation, we have now examined the role of these enzymes in regulating GABAA and AMPA receptors at different stages of postnatal development. GABAA receptors were labelled with [3H]SR95531 [Shaw, C. and Scarth, B.A., Mol. Brain Res., 11 (1991) 273-282]; AMPA receptors were labelled with [3H]CNQX [Lanius, R.A. and Shaw, C., Mol. Brain Res., 15 (1992) 256-262]. At postnatal day 14, GABAA receptors showed a decrease in binding in response to alkaline phosphatase treatment as opposed to an increase in binding observed in response to protein kinase treatment. Similar effects were observed for AMPA receptors at 20 days of age. The direction of regulation following the enzyme treatments were opposite to those observed in the adult cortex for both receptor populations. These fundamental changes in the enzymatic nature of regulation for such key inhibitory and excitatory receptor populations in cortex may signal an important role for age-dependent kinases and phosphatases in the events leading to modifications in neuronal function during postnatal development.
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PMID:Reversible kinase and phosphatase regulation of brain amino acid receptors in postnatal development. 133 48


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