Cell-cycle-dependent regulation of androgen receptor function.

The androgen receptor (AR) is a critical oncogene in prostate cancer (PCa) development and progression. In this study, we demonstrate cell-cycle-dependent regulation of AR activity, localization, and phosphorylation. We show that for three AR-target genes, androgen-stimulated AR transactivation is highest during the G1 phase, decreased during S-phase, and abrogated during G2/M. This change in AR transactivation parallels changes in AR localization and phosphorylation. A combination of imaging techniques and quantitative analysis reveals nuclear AR localization during interphase and the exclusion of the majority, but not all, AR from chromatin during mitosis. Flow cytometry analyses using a phospho-S308 AR-specific antibody in asynchronous and chemically enriched G2/M PCa cells revealed ligand-independent induction of S308 phosphorylation in mitosis when CDK1 is activated. Consistent with our flow cytometry data, IP-western blotting revealed an increase in S308 phosphorylation in G2/M, and the results of an in vitro kinase assay indicated that CDK1 was able to phosphorylate the AR on S308. Pharmacological inhibition of CDK1 activity resulted in decreased S308 phosphorylation in PCa cells. Importantly, using a combination of anti-total AR and phospho-S308-specific antibodies in immunofluorescence experiments, we showed that the AR is excluded from condensed chromatin in mitotic cells when it was phosphorylated on S308. In summary, we show that the phosphorylation of the AR on S308 by CDK1 during mitosis regulates AR localization and correlates with changes in AR transcriptional activity. These findings have important implications for understanding the function of AR as an oncogene.

Androgen receptor phosphorylation: biological context and functional consequences.

The androgen receptor (AR) is a ligand-regulated transcription factor that belongs to the family of nuclear receptors. In addition to regulation by steroid, the AR is also regulated by post-translational modifications generated by signal transduction pathways. Thus, the AR functions not only as a transcription factor but also as a node that integrates multiple extracellular signals. The AR plays an important role in many diseases, including complete androgen insensitivity syndrome, spinal bulbar muscular atrophy, prostate and breast cancer, etc. In the case of prostate cancer, dependence on AR signaling has been exploited for therapeutic intervention for decades. However, the effectiveness of these therapies is limited in advanced disease due to restoration of AR signaling. Greater understanding of the molecular mechanisms involved in AR action will enable the development of improved therapeutics to treat the wide range of AR-dependent diseases. The AR is subject to regulation by a number of kinases through post-translational modifications on serine, threonine, and tyrosine residues. In this paper, we review the AR phosphorylation sites, the kinases responsible for these phosphorylations, as well as the biological context and the functional consequences of these phosphorylations. Finally, what is known about the state of AR phosphorylation in clinical samples is discussed.

Effects of the ß-agonist, isoprenaline, on the down-regulation, functional responsiveness and trafficking of ß2-adrenergic receptors with N-terminal polymorphisms.

The ß2-AR (ß2-adrenergic receptor) is an important target for respiratory and CVD (cardiovascular disease) medications. Clinical studies suggest that N-terminal polymorphisms of ß2-AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down-regulation of ß2-AR variants following ß-agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human ß2-AR (designated ß2-AR-RE, ß2-AR-GE, ß2-AR-RQ and ß2-AR-GQ) were studied using site-directed mutagenesis and recombinant expression in HEK-293 cells (human embryonic kidney cells). Ligand-binding assays demonstrated that after 24 h exposure to 1 µM isoprenaline, isoforms with Arg16 (ß2-AR-RE and ß2-AR-RQ) underwent increased down-regulation compared with isoforms with Gly16 (ß2-AR-GE and ß2-AR-GQ). Consistent with these differences in down-regulation between isoforms, prolonged isoprenaline treatment resulted in diminished cAMP response to subsequent isoprenaline challenge in ß2-AR-RE relative to ß2-AR-GE. Confocal microscopy revealed that the receptor isoforms had similar co-localization with the early endosomal marker EEA1 following isoprenaline treatment, suggesting that they had similar patterns of internalization. None of the isoforms exhibited significant co-localization with the recycling endosome marker Rab11 in response to isoprenaline treatment. Furthermore, we found that prolonged isoprenaline treatment led to a higher degree of co-localization of ß2-AR-RE with the lysosomal marker LAMP1 (lysosome-associated membrane protein 1) compared with that of ß2-AR-GE. Taken together, these results indicate that a mechanism responsible for differential responses of these receptor isoforms to the ß-agonist involves differences in the efficiency with which agonist-activated receptors are trafficked to the lysosomes for degradation, or differences in degradation in the lysosomes.

Matrix rigidity regulates cancer cell growth and cellular phenotype.

BACKGROUND: The mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness) of the microenvironment and how this response varies among cancer cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate. This plate was used to determine how changes in the rigidity of the extracellular matrix modulate the biological properties of tumor cells. The cell lines tested fall into one of two categories based on their proliferation on substrates of differing stiffness: "rigidity dependent" (those which show an increase in cell growth as extracellular rigidity is increased), and "rigidity independent" (those which grow equally on both soft and stiff substrates). Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions. More importantly, seeding the cell lines into the lungs of nude mice revealed that the ability of cells to grow on soft gels in vitro correlated with their ability to grow in a soft tissue environment in vivo. The lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug. CONCLUSIONS/SIGNIFICANCE: These observations suggest that the mechanical properties of the matrix environment play a significant role in regulating the proliferation and the morphological properties of cancer cells. Further, the multiwell format of the soft-plate assay is a useful and effective adjunct to established 3-dimensional cell culture models.

Growth and tumor suppressor NORE1A is a regulatory node between Ras signaling and microtubule nucleation.

NORE1A is a Ras-binding protein that belongs to a group of tumor suppressors known as the Ras association domain family. Their growth- and tumor-suppressive function is assumed to be dependent on association with the microtubule cytoskeleton. However, a detailed understanding of this interplay is still missing. Here, we show that NORE1A directly interacts with tubulin and is capable of nucleating microtubules. Strikingly, the ability to stimulate nucleation is regulated in a dual specific way either via phosphorylation of NORE1A within the Ras-binding domain by Aurora A kinase or via binding to activated Ras. We also demonstrate that NORE1A mediates a negative effect of activated Ras on microtubule nucleation. On the basis of our results, we propose a novel regulatory network composed of the tumor suppressor NORE1A, the mitotic kinase Aurora A, the small GTPase Ras, and the microtubule cytoskeleton.

Characterization of a panel of six beta2-adrenergic receptor antibodies by indirect immunofluorescence microscopy.

BACKGROUND: The beta2-adrenergic receptor (beta2AR) is a primary target for medications used to treat asthma. Due to the low abundance of beta2AR, very few studies have reported its localization in tissues. However, the intracellular location of beta2AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to beta-agonist medications. Thus, a method for visualizing beta2AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant beta2AR in primary cell cultures. METHODS: A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat beta2AR expressed in HEK 293 cells. Antibodies capable of recognizing rat beta2AR were identified and used to localize native beta2AR in primary cultures of rat airway smooth muscle and epithelial cells. beta2AR expression was confirmed by performing ligand binding assays using the beta-adrenergic antagonist [3H] dihydroalprenolol ([3H]DHA). RESULTS: Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human beta2AR (Ab-Bethyl) specifically recognized human but not rat beta2AR. An antibody developed against the C-terminal domain of the mouse beta2AR (Ab-sc570) specifically recognized rat but not human beta2AR. An antibody developed against 78 amino acids of the C-terminus of the human beta2AR (Ab-13989) was capable of recognizing both rat and human beta2ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat beta2AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface. CONCLUSION: Antibodies have been identified that recognize human beta2AR, rat beta2AR or both rat and human beta2AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of beta2AR in tissues.

Cog1p plays a central role in the organization of the yeast conserved oligomeric Golgi complex.

The conserved oligomeric Golgi (COG) complex is an evolutionarily conserved peripheral membrane oligomeric protein complex that is involved in intra-Golgi protein trafficking. The COG complex is composed of eight subunits that are located in two lobes; Lobe A contains COG1-4, and Lobe B is composed of COG5-8. Both in vivo and in vitro protein-protein interaction techniques were applied to characterize interactions between individual COG subunits. In vitro assays revealed binary interactions between Cog2p and Cog3p, Cog2p and Cog4p, and Cog6p and Cog8p and a strong interaction between Cog5p and Cog7p. The two-hybrid assay confirmed these findings and revealed that Cog1p interacted with subunits from both lobes of the complex. Antibodies to COG subunits were utilized to determine the protein levels and membrane association of COG subunits in yeast delta cog1-8 mutants. As a result, we created a model of the protein-protein interactions within the yeast COG complex and proposed that Cog1p is a bridging subunit between the two COG lobes. In support of this hypothesis, we have demonstrated that Cog1p is required for the stable association between two COG subcomplexes.