Olli A. Jänne

PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases

ABSTRACT PIAS (protein inhibitor of activated STAT) proteins interact with and modulate the activities of various transcription factors. In this work, we demonstrate that PIAS proteins xα, xβ, 1, and 3 interact with the small ubiquitin-related modifier SUMO-1 and its E2 conjugase, Ubc9, and that PIAS proteins themselves are covalently modified by SUMO-1 (sumoylated). PIAS proteins also tether other sumoylated proteins in a noncovalent fashion. Furthermore, recombinant PIASxα enhances Ubc9-mediated sumoylation of the androgen receptor and c-Jun in vitro. Importantly, PIAS proteins differ in their abilities to promote sumoylation in intact cells. The ability to stimulate protein sumoylation and the interaction with sumoylated proteins are dependent on the conserved PIAS RING finger-like domain. These functions are linked to the activity of PIASxα on androgen receptor-dependent transcription. Collectively, our results imply that PIAS proteins function as SUMO-1-tethering proteins and zinc finger-dependent E3 SUMO protein ligases, and these properties are likely to explain their ability to modulate the activities of various transcription factors.

Interaction between the Amino- and Carboxyl-terminal Regions of the Rat Androgen Receptor Modulates Transcriptional Activity and Is Influenced by Nuclear Receptor Coactivators

Identical N-terminal deletions in the wild-type rat androgen receptor (rAR) and a constitutively active rAR (ARΔ641–902) devoid of the ligand-binding domain (LBD) resulted in dissimilar consequences in transcriptional activation: deletion of residues 149–295 abolished wild-type AR activity, but did not influence that of ARΔ641–902. The activity of the N-terminal transactivation domain is thus controlled by the hormone-occupied LBD, suggesting that the N- and C-terminal regions of rAR communicate. Consistent with this idea, a strong androgen-dependent interaction between the N-terminal region and LBD was demonstrated in a mammalian two-hybrid system using GAL4 and VP16 fusion proteins. This interaction can be direct or indirect. Several nuclear receptor coactivators (CBP, F-SRC-1, SRC-1, and RIP140) that interact with other steroid receptors were tested as potential mediators of the N- and C-terminal interaction of rAR using the mammalian two-hybrid system. CBP or F-SRC-1 not only enhanced AR-mediated transactivation, but also facilitated the androgen-dependent interaction between the N- and C-terminal domains, implying that part of the coactivator-dependent transcriptional activation occurs via this mechanism. In contrast, SRC-1, a coactivator for the progesterone receptor, inhibited both AR-mediated transactivation and interaction between the N and C termini. Recruitment of coregulators may involve AR domains other than the LBD, as F-SRC-1 and CBP enhanced, but SRC-1 repressed, the transcriptional activity of ARΔ641–902. Collectively, interplay between the N-terminal region and LBD of rAR results in the formation of a transactivation complex that includes coregulators and that is mandatory for optimal activation of androgen-induced promoters.

Acetylation of Androgen Receptor Enhances Coactivator Binding and Promotes Prostate Cancer Cell Growth

ABSTRACT Modification by acetylation occurs at ε-amino lysine residues of histones and transcription factors. Unlike phosphorylation, a direct link between transcription factor acetylation and cellular growth or apoptosis has not been established. We show that the nuclear androgen receptor (AR), a DNA-binding transcriptional regulator, is acetylated in vivo. The acetylation of the AR is induced by ligand dihydrotestosterone and by histone deacetylase (HDAC) inhibitors in living cells. Direct AR acetylation augmented p300 binding in vitro. Constructs mimicking neutral polar substitution acetylation (ARK630Q, ARK630T) enhanced p300 binding and reduced N-CoR/HDAC/Smad3 corepressor binding, whereas charged residue substitution (ARK630R) reduced p300 binding and enhanced corepressor binding. The AR acetylation mimics promoted cell survival and growth of prostate cancer cells in soft agar and in nude mice and augmented transcription of a subset of growth control target gene promoters. Thus, transcription factor acetylation regulates coactivator/corepressor complex binding, altering expression of specific growth control genes to promote aberrant cellular growth in vivo.

Primary structure of human insulin-like growth factor-binding protein/placental protein 12 and tissue-specific expression of its mRNA.

The low‐molecular‐mass insulin‐like growth factor‐binding protein (IGF‐BP) and placental protein 12 (PP12) are identical proteins that are present in human serum, amniotic fluid, secretory endometrium and decidua. IGF‐BP/PP12 is believed to act as an autocrine or paracrine regulator of cell growth. A cDNA clone encompassing the entire protein coding region of this protein was isolated from a human decidual cDNA library. The authenticity of the cDNA was verified by in vitro transcription/translation experiments and by the identity of the 10 N‐terminal amino acids deduced for the mature peptide with those obtained by direct protein sequencing. The amino acid sequence indicates that pre‐IGF‐BP/PP12 consists of 259 amino acid residues. The putative signal peptide is 25 residues long, and the mature protein thus contains 234 amino acids and has a molecular mass of 25 293 Da. The sequence is very cysteine‐rich at the N‐terminus after which there are regions of clustered Pro, Glu, Ser and Thr residues (so‐called PEST regions), which exist in proteins with short half‐lives. The amino acid sequence also includes an Arg‐Gly‐Asp tripeptide that may function as a cell recognition signal. The IGF‐BP/PP12 gene encodes a single 1.6 kb mRNA species that is expressed in decidua, secretory endometrium, liver and a human hepatoma cell line (HepG2). Southern blot analysis suggests that there is a single IGF‐BP/PP12 gene in the human genome.

Identification of a Novel RING Finger Protein as a Coregulator in Steroid Receptor-Mediated Gene Transcription

ABSTRACT Using the DNA-binding domain of androgen receptor (AR) as a bait in a yeast two-hybrid screening, we have identified a small nuclear RING finger protein, termed SNURF, that interacts with AR in a hormone-dependent fashion in both yeast and mammalian cells. Physical interaction between AR and SNURF was demonstrated by coimmunoprecipitation from cell extracts and by protein-protein affinity chromatography. Rat SNURF is a highly hydrophilic protein consisting of 194 amino acid residues and comprising a consensus C3HC4 zinc finger (RING) structure in the C-terminal region and a bipartite nuclear localization signal near the N terminus. Immunohistochemical experiments indicated that SNURF is a nuclear protein. SNURF mRNA is expressed in a variety of human and rat tissues. Overexpression of SNURF in cultured mammalian cells enhanced not only androgen, glucocorticoid, and progesterone receptor-dependent transactivation but also basal transcription from steroid-regulated promoters. Mutation of two of the potential Zn2+coordinating cysteines to serines in the RING finger completely abolished the ability of SNURF to enhance basal transcription, whereas its ability to activate steroid receptor-dependent transcription was maintained, suggesting that there are separate domains in SNURF that mediate interactions with different regulatory factors. SNURF is capable of interacting in vitro with the TATA-binding protein, and the RING finger domain is needed for this interaction. Collectively, we have identified and characterized a ubiquitously expressed RING finger protein, SNURF, that may function as a bridging factor and regulate steroid receptor-dependent transcription by a mechanism different from those of previously identified coactivator or integrator proteins.

Ubc9 Interacts with the Androgen Receptor and Activates Receptor-dependent Transcription

Ubc9, a homologue of the class E2 ubiquitin-conjugating enzymes, has recently been shown to catalyze conjugation of a small ubiquitin-like molecule-1 (SUMO-1) to a variety of target proteins. SUMO-1 modifications have been implicated in the targeting of proteins to the nuclear envelope and certain intranuclear structures and in converting proteins resistant to ubiquitin-mediated degradation. In the present work, we find that Ubc9 interacts with the androgen receptor (AR), a member of the steroid receptor family of ligand-activated transcription factors. In transiently transfected COS-1 cells, AR-dependent but not basal transcription is enhanced by the coexpression of Ubc9. The N-terminal half of the AR hinge region containing the C-terminal part of the bipartite nuclear localization signal is essential for the interaction with Ubc9. Deletion of this part of the nuclear localization signal, which does not completely prevent the transfer of AR to the nucleus, abolishes the AR-Ubc9 interaction and attenuates the transcriptional response to cotransfected Ubc9. The C93S substitution of Ubc9, which prevents SUMO-1 conjugation by abrogating the formation of a thiolester bond between SUMO-1 and Ubc9, does not influence the capability of Ubc9 to stimulate AR-dependent transactivation, implying that Ubc9 is able to act as an AR coregulator in a fashion independent of its ability to catalyze SUMO-1 conjugation.

Androgen Receptor Acetylation Governs trans Activation and MEKK1-Induced Apoptosis without Affecting In Vitro Sumoylation and trans-Repression Function

ABSTRACT The androgen receptor (AR) is a nuclear hormone receptor superfamily member that conveys both trans repression and ligand-dependent trans-activation function. Activation of the AR by dihydrotestosterone (DHT) regulates diverse physiological functions including secondary sexual differentiation in the male and the induction of apoptosis by the JNK kinase, MEKK1. The AR is posttranslationally modified on lysine residues by acetylation and sumoylation. The histone acetylases p300 and P/CAF directly acetylate the AR in vitro at a conserved KLKK motif. To determine the functional properties governed by AR acetylation, point mutations of the KLKK motif that abrogated acetylation were engineered and examined in vitro and in vivo. The AR acetylation site point mutants showed wild-type trans repression of NF-κB, AP-1, and Sp1 activity; wild-type sumoylation in vitro; wild-type ligand binding; and ligand-induced conformational changes. However, acetylation-deficient AR mutants were selectively defective in DHT-induced trans activation of androgen-responsive reporter genes and coactivation by SRC1, Ubc9, TIP60, and p300. The AR acetylation site mutant showed 10-fold increased binding of the N-CoR corepressor compared with the AR wild type in the presence of ligand. Furthermore, histone deacetylase 1 (HDAC1) bound the AR both in vivo and in cultured cells and HDAC1 binding to the AR was disengaged in a DHT-dependent manner. MEKK1 induced AR-dependent apoptosis in prostate cancer cells. The AR acetylation mutant was defective in MEKK1-induced apoptosis, suggesting that the conserved AR acetylation site contributes to a pathway governing prostate cancer cellular survival. As AR lysine residue mutations that abrogate acetylation correlate with enhanced binding of the N-CoR repressor in cultured cells, the conserved AR motif may directly or indirectly regulate ligand-dependent corepressor disengagement and, thereby, ligand-dependent trans activation.

Increased Expression of Androgen Receptor Sensitizes Prostate Cancer Cells to Low Levels of Androgens

Androgen receptor (AR) is known to be overexpressed in castration-resistant prostate cancer. To interrogate the functional significance of the AR level, we established two LNCaP cell sublines expressing in a stable fashion two to four times (LNCaP-ARmo) and four to six times (LNCaP-ARhi) higher level of AR than the parental cell line expressing the empty vector (LNCaP-pcDNA3.1). LNCaP-ARhi cell line grew faster than the control line in low concentrations, especially in 1 nmol/L 5alpha-dihydrotestosterone (DHT). Microarray-based transcript profiling and subsequent unsupervised hierarchical clustering showed that LNCaP-ARhi cells clustered together with VCaP cells, containing endogenous AR gene amplification and overexpression, indicating the central role of AR in the overall regulation of gene expression in prostate cancer cells. Two hundred forty genes showed >2-fold changes on DHT treatment in LNCaP-ARhi at 4 h time point, whereas only 164 and 52 showed changes in LNCaP-ARmo and LNCaP-pcDNA3.1, respectively. Many androgen-regulated genes were upregulated in LNCaP-ARhi at 10-fold lower concentration of DHT than in control cells. DHT (1 nmol/L) increased expression of several cell cycle-associated genes in LNCaP-ARhi cells. ChIP-on-chip assay revealed the presence of chromatin binding sites for AR within +/-200 kb of most of these genes. The growth of LNCaP-ARhi cells was also highly sensitive to cyclin-dependent kinase inhibitor, roscovitine, at 1nmol/L DHT. In conclusion, our results show that overexpression of AR sensitizes castration-resistant prostate cancer cells to the low levels of androgens. The activity of AR signaling pathway is regulated by the levels of both ligand and the receptor.

A TESTIS-SPECIFIC ANDROGEN RECEPTOR COREGULATOR THAT BELONGS TO A NOVEL FAMILY OF NUCLEAR PROTEINS

We have characterized a novel partner for androgen receptor (AR), termed ARIP3, that interacts with the DNA-binding domain/zinc finger region of AR and is predominantly expressed in the testis. Rat ARIP3 is a nuclear protein comprising 572 amino acids. It modulates AR-dependent but not basal transcription, suggesting that ARIP3 acts as an AR transcriptional coregulator. Except for the C-terminal AR-interacting domain, ARIP3 contains distinct regions that are also present in two recently described proteins, a protein inhibitor of activated Stat3 and an RNA helicase II-interacting protein (Gu/RH-II binding protein). Conserved structural features of these proteins indicate the existence of a gene family involved in the regulation of various transcription factors. Collectively, ARIP3 belongs to a novel nuclear protein family and is perhaps the first tissue-specific coregulator of androgen receptor.

Small ubiquitin-related modifier-1 (SUMO-1) modification of the glucocorticoid receptor.

Small ubiquitin-related modifier-1 (SUMO-1) is covalently attached to many cellular targets to regulate protein-protein and protein-DNA interactions, as well as localization and stability of the target protein. The SUMO-1-conjugating E2 enzyme Ubc9 is known to interact with the glucocorticoid receptor (GR), a ligand-dependent transcription factor. In the present study, we show that GR is post-translationally modified by SUMO-1 (sumoylated) in a ligand-enhanced fashion. We identify experimentally three consensus SUMO attachment sites, two in the N-terminal transactivation region and one in the ligand-binding domain of GR. The two N-terminal sites are the major acceptor sites for SUMO-1 attachment. Mutation of these sites enhances transcriptional activity of GR on minimal promoters, but has no clear effect on the more complex mouse mammary tumour virus promoter. Thus SUMO-1 modification of GR influences receptor function in a promoter context-dependent fashion.