Identification of phosphorylated sites in the mouse glucocorticoid receptor.

113

Abstract. Synthetic peptides corresponding to cDNAdeduced amino acid sequences unique to the human and mouse retinoic acid receptor yl (hRAR-yl and mRAR-yl, respectively) were used to generate anti-RAR-yl antibodies. Four mAbs were selected, which were directed against peptides found in region Al (Ably(Al)), region F (Ab2y(mF) and Ab4y(hF)) and region D2 (Ab5y(D2)) . These antibodies specifically immunoprecipitated and recognized by Western blotting RAR-yl proteins in COS-1 cells transfected with expression vectors containing the RAR-yl cDNAs. They all reacted with both human and mouse RARyl proteins, except Ab4y(hF) that was specific for hRAR yl. Rabbit polyclonal antibodies, directed against a peptide from the mRAR-yl F region were also obtained (RPy(mF)) and found to be specific for mouse

Section: Discussionmentioning

confidence: 64%

Retinoic acid receptor gamma: specific immunodetection and phosphorylation.

Rochette‐Egly

Lutz

Saunders

et al. 1991

113

“…Examples of both serine/threonine (Rihs and Peters, 1989;Rihs et al, 1991) and tyrosine (Fu, 1992;Shuai et al, 1993) phosphorylation effects on nuclear import have been observed. It appears unlikely that direct phosphorylation of exporting substrates is responsible for accelerated nuclear export in our assays since GR is not tyrosine phosphorylated in vivo (Bodwell et al, 1991) and under our in vitro assay conditions (data not shown). Furthermore, since sodium molybdate treatment also accelerated the energydependent nuclear export of a GR mutant with serine to alanine substitutions at its seven predominant phosphorylation sites (data not shown), it appears unlikely that indirect activation by sodium molybdate of downstream protein serine/threonine kinases acting on GR is responsible for this effect.…”

Section: Discussionmentioning

confidence: 85%

Subnuclear Trafficking of Glucocorticoid Receptors In Vitro: Chromatin Recycling and Nuclear Export

Yang

Liu

DeFranco

1997

We have used digitonin-permeabilized cells to examine in vitro nuclear export of glucocorticoid receptors (GRs). In situ biochemical extractions in this system revealed a distinct subnuclear compartment, which collects GRs that have been released from chromatin and serves as a nuclear export staging area. Unliganded nuclear GRs within this compartment are not restricted in their subnuclear trafficking as they have the capacity to recycle to chromatin upon rebinding hormone. Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality. In addition, chromatin-released receptors export from nuclei of permeabilized cells in an ATP- and cytosol-independent process that is stimulated by sodium molybdate, other group VI-A transition metal oxyanions, and some tyrosine phosphatase inhibitors. The stimulation of in vitro nuclear export by these compounds is not unique to GR, but is restricted to other proteins such as the 70- and 90-kD heat shock proteins, hsp70 and hsp90, respectively, and heterogeneous nuclear RNP (hnRNP) A1. Under analogous conditions, the 56-kD heat shock protein, hsp56, and hnRNP C do not export from nuclei of permeabilized cells. If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited. Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

“…HA-␣ -adducin (70 g of protein) was phosphorylated with GST-CAT (120 ng of protein) in 2 ml of kinase buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT) containing 100 M [ ␥ -32 P]ATP for 1 h at 30 Њ C, and the reaction product was digested with Achromobacter protease-1 (AP-1) at 37 Њ C for 20 h. The obtained peptides were applied onto a C18 reverse-phase column (4.6 ϫ 250 mm; Shiseido, Japan) and eluted with a linear gradient of 0-48% acetonitrile for 100 min at a flow rate of 1.0 ml/min by HPLC (System Gold, Beckman). The radioactive peaks were separated and phosphoamino acid sequencing was carried out with a peptide sequencer (PPSQ-10; Shimadzu, Japan) as described (Bodwell et al, 1991). The fractions obtained from each Edoman degradation cycle were measured for 32 P in a Beckman liquid scintillation counter.…”

Section: Determination Of Phosphorylation Sites Of ␣ -Adducin By Rho-kinasementioning

confidence: 99%

Phosphorylation of Adducin by Rho-Kinase Plays a Crucial Role in Cell Motility

Fukata

Oshiro

Kinoshita

et al. 1999

272

230

Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates α-adducin and thereby enhances the F-actin–binding activity of α-adducin in vitro. Here we identified the sites of phosphorylation of α-adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized α-adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated α-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated α-adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or α-adducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migra- tion in NRK49F cells. α-AdducinT445D,T480D (substi- tution of Thr445 and Thr480 by Asp), but not α-adducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates α-adducin downstream of Rho in vivo, and that the phosphorylation of adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.