The use of beta, gamma-methyleneadenosine 5'-triphosphate to determine ATP competition in a scintillation proximity kinase assay.

A novel method for characterizing the kinetics of protein kinase inhibitors is described. This method uses glycogen synthase kinase beta as the model protein kinase and looks at the shift in IC50 of inhibitors using the nonhydrolyzable ATP analog, beta, gamma-methyleneadenosine 5'-triphosphate, also known as AMP-PCP. Due to its inability to be hydrolyzed, AMP-PCP is being used to characterize known glycogen synthase kinase inhibitors by determining the shift in IC50 at concentrations above its calculated Ki of 490 microM. The assay format for the detection of inhibition is a scintillation proximity assay which is robust and reproducible at very low levels of [gamma-33P]ATP. The use of AMP-PCP coupled with the use of the scintillation proximity assay allows this characterization of inhibition without increasing [gamma-33P]ATP and without significantly diluting the overall assay signal. We have used this method in kinetic analyses to demonstrate that we can detect a significant shift in IC50 with the known ATP competitive inhibitors, staurosporine, Ro 31-8220, and olomoucine. The IC50 for glycogen synthase peptide and lithium chloride, which has been reported to be uncompetitive, remains unchanged.

A systematic analysis of the AF-2 domain of human retinoic acid receptor alpha reveals amino acids critical for transcriptional activation and conformational integrity.

We previously identified a carboxy-terminal transactivation function termed AF-2 within the last 15 amino acids of the ligand binding domain of the human retinoic acid receptor alpha (hRAR alpha). Truncation of this region abolished transcriptional activity. Here we provide a systematic analysis using alanine scanning mutagenesis of amino acids from Ser405 to Gly419 on a truncated hRAR alpha (delta419) to identify residues within this region that are responsible for transcriptional activity. Whereas mutations in positions 405, 408, 411, and 415-419 have little or no effect on the ability of modified receptors to activate a DR5 response element, mutations in positions 406, 407, 409, 410, and 412-413 modify either the potency or efficacy of all-trans retinoic acid (tRA) -induced gene transcription. Therefore, receptors with mutations in positions 409, 410, 413, and 414 have low transcriptional activity over a wide range of tRA concentrations. Receptors with mutations in positions 406, 407, and 412 exhibit a maximum transcriptional activity similar to wild-type hRAR alpha, but require higher concentrations of tRA. Replacing residues 405-419 on delta419 with the conserved AF-2 domain from the vitamin D3 receptor or the estrogen receptor results in a receptor with wild-type or low transcriptional activity, respectively. A full-length hRAR alpha mutant with an alanine substitution at position 406 (hRAR alpha M406A) binds tRA, but unlike the truncated M406A, which lacks the "F" region, it is not transcriptionally active. Protease mapping experiments detect a consistent difference in the conformation of hRAR alpha M406A compared to wild-type hRAR alpha. These data define amino acids from Ser405 to Gly419 on delta419 that are critical for transcriptional activity and point to the importance of the conformational integrity of receptor domains in maintaining ligand-induced transcriptional activation.

The AF-2 region of the retinoic acid receptor alpha mediates retinoic acid inhibition of estrogen receptor function in breast cancer cells.

The growth of estrogen receptor (ER)-positive breast cancer cells is inhibited by all-trans-retinoic acid (RA). In the present study, estrogen (E2) induction of pS2 mRNA levels was significantly reduced within 6 h following cotreatment with RA. In transient transfection experiments, RA repressed transactivation from a vitellogenin E2-responsive element by approximately 50% and wild-type RA receptor alpha (RARalpha) or RARbeta enhanced this inhibition. Transfection of truncated RARalpha mutants terminating before or at amino acid 412 markedly decreased RA inhibition of E2-induced reporter gene activity. Expression of RARs with deletions of amino acids 413 and 414 in the transactivation-2 (AF-2) domain also reduced RA inhibition, while deletions and point mutations beyond amino acid 414 behaved like the wild-type RARalpha. RA-treated MCF-7 cells transfected with an RARalpha AF-2 region mutant were twice as sensitive to growth inhibition as untransfected and vector-transfected control cells. Thus, the AF-2 domain in the C terminus of the RARalpha mediates RA inhibition of ER-induced transcription in breast cancer cells. In addition, transcriptional interference between RARs and ERs may contribute to RA inhibition of ER-positive breast cancer cell growth.

Mutagenesis of the ligand binding domain of the human retinoic acid receptor alpha identifies critical residues for 9-cis-retinoic acid binding.

We have recently identified a small region (amino acids 405-419) within the ligand binding domain of a truncated human retinoic acid receptor alpha (delta 419) that is required for binding of 9-cis-retinoic acid (RA), but not all-trans-retinoic acid (t-RA). To probe the structural determinants of this high affinity 9-cis-RA binding site, a series of delta 419 mutants were prepared whereby an individual alanine residue was substituted for each amino acid within this region. These modified receptors were expressed in mammalian COS-1 cells and assayed for their ability to bind 9-cis-RA as well as t-RA. Only two of the mutants, M406A (mutation of methionine 406 to alanine), and I410A (mutation of isoleucine 410 to alanine) exhibit no detectable binding of 9-cis-RA when analyzed using saturation binding kinetics. Substitution of methionine 406 with the amino acids leucine, isoleucine, and valine yields mutant receptors that exhibit decreased binding for 9-cis-RA as the length or hydrophobicity of the R group decreases. Further substitution of methionine 406 with the small polar amino acid, threonine, results in a loss of detectable 9-cis-RA binding. Since amino acids 405-419 on a human RAR alpha (hRAR alpha) are predicted to form a short amphipathic alpha-helix, modeling of this structure into a helical wheel indicates that these two amino acids, methionine 406 and isoleucine 410, are actually positioned proximal to each other. Data presented here suggest that high affinity 9-cis-RA binding to a hRAR alpha depends on an interaction with the two amino acids methionine 406 and isoleucine 410.

The discovery of 9-cis retinoic acid: a hormone that binds the retinoid-X receptor.

Two classes of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid-X receptors (RXRs), mediate the physiologic activity of retinoids. The RXRs can form biologically active heterodimers with the RARs and with other nuclear receptors, including the vitamin-D, thyroid hormone, and peroxisome proliferator-activated receptors. Thus, the RXRs may play a pivotal role in modulating the action of other hormones or ligands. The RXRs were originally classified as orphan receptors whose cognate ligand was unknown until recently, when 9-cis retinoic acid (9-cis RA) was discovered to bind directly and activate this family of receptors. Since 9-cis RA also binds and activates the RARs, it is interesting to speculate that this natural ligand may regulate a broad range of physiologic processes by mediating transcriptional activity through both RAR- and RXR-linked pathways.

Characterization of the ligand binding domain of human retinoid X receptor alpha expressed in Escherichia coli.

In order to study the structural details of ligand protein interactions of the human retinoid X receptor alpha (hRXR alpha), the DEF and EF domains of the receptor were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The fusion proteins were expressed at high levels and were affinity-purified by chromatography over glutathione-agarose. The DEF and EF domains were cleaved from the fusion proteins by digestion with thrombin. Retinoic acid binding was quantitated using two different methods. The apparent dissociation constant (Kd) and the stoichiometry of 9-cis-retinoic acid binding were performed by monitoring quenching of protein fluorescence. To directly compare the binding affinity of the E. coli-derived truncated hRXR alpha with full-length hRXR alpha expressed in transiently transfected COS cells, Scatchard analyses of [3H]9-cis-retinoic acid binding assays were performed. Both methods of analysis indicate that while the cleaved DEF peptide bound 9-cis-retinoic acid tightly, the cleaved EF peptide exhibited variable binding activity between preparations. By fluorimetric analysis, the Kd of the cleaved DEF peptide was estimated to be 3 +/- 0.5 nM with a stoichiometry of 1:1.1 +/- 0.1. By Scatchard analysis, the Kd values for [3H]9-cis-retinoic acid to the GST-hRXR alpha (DEF) peptide and the cleaved DEF peptide were estimated to be 1.8 nM and 5.6 nM, respectively. The estimated molecular mass from high speed sedimentation equilibrium experiments was 36 +/- 2 kDa for the apo-DEF peptide alone and 38 +/- 3 kDa for the holo-DEF peptide complexed with 9-cis-retinoic acid. This suggests that the recombinant ligand binding domain was predominantly in the monomer form. However, dimers of the cleaved DEF peptides were detected in chemical cross-linking experiments both in the presence and absence of 9-cis-retinoic acid. Since the purified E. coli-derived truncated hRXR alpha DEF peptide appears to fully retain its ligand binding activity, it should provide a useful model system for further structural analysis of ligand-protein interactions.

Distinct binding determinants for 9-cis retinoic acid are located within AF-2 of retinoic acid receptor alpha.

Retinoids exert their physiological action by interacting with two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which regulate gene expression by forming transcriptionally active heterodimeric RAR/RXR or homodimeric RXR/RXR complexes on DNA. Retinoid receptor activity resides in several regions, including the DNA and ligand binding domains, a dimerization interface, and both a ligand-independent (AF-1) and a ligand-dependent (AF-2) transactivation function. While 9-cis retinoic acid (RA) alone is the cognate ligand for the RXRs, both 9-cis RA and all-trans RA (t-RA) compete for binding with high affinity to the RARs. This latter observation suggested to us that the two isomers may interact with a common binding site. Here we report that RAR alpha has two distinct but overlapping binding sites for 9-cis RA and t-RA. Truncation of a human RAR alpha to 419 amino acids yields a receptor that binds both t-RA and 9-cis RA with high affinity, but truncation to amino acid 404 yields a mutant receptor that binds only t-RA with high affinity. Remarkably, this region also defines a C-terminal boundary for AF-2, as addition of amino acids 405 to 419 restores receptor-mediated gene activity to a truncated human RAR alpha lacking this region. It is interesting to speculate that binding of retinoid stereoisomers to unique sites within an RAR may function with AF-2 to cause differential activation of retinoid-responsive gene pathways.

Thrombin activation of human platelets causes tyrosine phosphorylation of PLC-gamma 2.

Human platelets contain phospholipase C (PLC)-gamma 2, a distinct isoform closely related to PLC-gamma 1. Both inositol phospholipid-specific phospholipases C contain the src-related SH2 regions. Stimulation of platelets with the potent agonist, thrombin, led to a rapid and transient phosphorylation of PLC-gamma 2 on tyrosine residues. Activated platelets lysed in the absence of sodium orthovanadate had levels of tyrosine-phosphorylated PLC-gamma 2 paralleling those seen in unstimulated platelets. Previously, it had been shown that PLC-gamma 1 was phosphorylated on tyrosine residues by the agonist-occupied platelet-derived growth factor (PDGF) receptor and epidermal growth factor (EGF) receptor in cells other than platelets. In addition, more recent data have indicated that PLC-gamma 2 is also capable of being tyrosine-phosphorylated in cells of hematopoietic origin, such as B cells and natural killer (NK) cells. Here we report that PLC-gamma 2 expressed in a terminally-differentiated hematopoietic cell is also tyrosine-phosphorylated in response to an agonist.

Characterization of a Polyphosphoinositide Phospholipase C from the Plasma Membrane of Avena sativa.

A phosphoinositide-specific phospholipase C activity was identified in oat root (Avena sativa, cv Victory) plasma membranes purified by separation in an aqueous two-phase polymer system. The enzyme is highly active toward inositol phospholipids but only minimally active toward phosphatidylethanolamine and phosphatidylcholine. Activity approaches maximal levels at 200 micromolar phosphatidylinositol 4-phosphate (PIP) and is highly dependent on calcium; it is inhibited by 1 millimolar EGTA and is activated by calcium with an apparent activation constant of 2 micromolar. At 10 micromolar calcium and 200 micromolar inositol phospholipid, the enzyme is specific for phosphatidylinositol 4,5-bisphosphate (PIP(2)) and PIP, which are hydrolyzed at 10 and 4 times, respectively, the rate of phosphatidylinositol (PI) hydrolysis. The principle water soluble products of hydrolysis, as determined by high performance liquid chromatography, are inositol 1,4,5-trisphosphate from PIP(2), inositol 1,4-bisphosphate from PIP, and inositol phosphate from PI.

Separation and Characterization of Inositol Phospholipids from the Pulvini of Samanea saman.

To supplement current thin-layer chromatographic methods for separation and quantitation of plant phospholipids, an alternative method, high-performance liquid chromatography was developed. The major inositol-containing lipids from the pulvini of Samanea saman Merr. were identified as phosphatidylinositol, phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate based on comigration with authentic standards on high-performance liquid chromatography and on thin-layer chromatography. The patterns of incorporation of radioactivity into the putative phosphatidylinositol and phosphatidylinositol phosphate were consistent with these identifications when pulvini were labeled with [(3)H]glycerol, [(3)H]inositol, or [(32)P]orthophosphate. Analysis of the products of enzymic hydrolysis, of chemical deacylation, and of ;fingerprint' methanolysis of these phospholipids confirmed the identifications.