SUMMARY ADP-ribosylation of proteins is emerging as an important regulatory mechanism. Depending on the family member, ADP-ribosyltransferases conjugate either a single ADP-ribose to a target, or generate ADP-ribose chains. Here we characterize Parp9, a mono-ADP-ribosyltransferase reported to be enzymatically inactive. Parp9 undergoes heterodimerization with Dtx3L, a histone E3 ligase involved in DNA damage repair. We show that the Dtx3L/Parp9 heterodimer mediates NAD+- dependent mono-ADP-ribosylation of ubiquitin, exclusively in the context of ubiquitin processing by E1 and E2 enzymes. Dtx3L/Parp9 ADP-ribosylates the carboxyl group of Ub Gly76. Because Gly76 is normally used for Ub conjugation to substrates, ADP-ribosylation of the Ub carboxyl terminus precludes ubiquitylation. Parp9 ADP-ribosylation activity therefore restrains the E3 function of Dtx3L. Mutation of the NAD+ binding site in Parp9 increases the DNA repair activity of the heterodimer. Moreover, poly-ADP-ribose binding to the Parp9 macrodomains increases E3 activity. Dtx3L heterodimerization with Parp9 enables NAD+ and poly- ADP-ribose regulation of E3 activity.

Activation of signal transduction kinase cascades is known to alter androgen receptor (AR) activity, but the molecular mechanisms are still poorly defined. Here we show that stress kinase signaling regulates Ser 650 phosphorylation and AR nuclear export. In LNCaP prostate cancer cells, activation of either MAPK kinase (MKK) 4:c-Jun N-terminal kinase (JNK) or MKK6:p38 signaling pathways increased Ser 650 phosphorylation, whereas pharmacologic inhibition of JNK or p38 signaling led to a reduction of AR Ser 650 phosphorylation. Both p38alpha and JNK1 phosphorylated Ser 650 in vitro. Small interfering RNA-mediated knockdown of either MKK4 or MKK6 increased endogenous prostate-specific antigen (PSA) transcript levels, and this increase was blocked by either bicalutamide or AR small interfering RNA. Stress kinase inhibition of PSA transcription is, therefore, dependent on the AR. Similar experiments involving either activation or inhibition of MAPK/ERK kinase:ERK signaling had little effect on Ser 650 phosphorylation or PSA mRNA levels. Ser 650 is proximal to the DNA binding domain that contains a nuclear export signal. Mutation of Ser 650 to alanine reduced nuclear export of the AR, whereas mutation of Ser 650 to the phosphomimetic amino acid aspartate restored AR nuclear export. Pharmacologic inhibition of stress kinase signaling reduced wild-type AR nuclear export equivalent to the S650A mutant without affecting nuclear export of the S650D mutant. Our data suggest that stress kinase signaling and nuclear export regulate AR transcriptional activity.

BackgroundClassical nuclear localization signal (NLS) dependent nuclear import is carried out by a heterodimer of importin α and importin β. NLS cargo is recognized by importin α, which is bound by importin β. Importin β mediates translocation of the complex through the central channel of the nuclear pore, and upon reaching the nucleus, RanGTP binding to importin β triggers disassembly of the complex. To date, six importin α family members, encoded by separate genes, have been described in humans.ResultsWe sequenced and characterized a seventh member of the importin α family of transport factors, karyopherin α 7 (KPNA7), which is most closely related to KPNA2. The domain of KPNA7 that binds Importin β (IBB) is divergent, and shows stronger binding to importin β than the IBB domains from of other importin α family members. With regard to NLS recognition, KPNA7 binds to the retinoblastoma (RB) NLS to a similar degree as KPNA2, but it fails to bind the SV40-NLS and the human nucleoplasmin (NPM) NLS. KPNA7 shows a predominantly nuclear distribution under steady state conditions, which contrasts with KPNA2 which is primarily cytoplasmic.ConclusionKPNA7 is a novel importin α family member in humans that belongs to the importin α2 subfamily. KPNA7 shows different subcellular localization and NLS binding characteristics compared to other members of the importin α family. These properties suggest that KPNA7 could be specialized for interactions with select NLS-containing proteins, potentially impacting developmental regulation.

The mutant form of lamin A responsible for the premature aging disease Hutchinson-Gilford progeria syndrome (termed progerin) acts as a dominant negative protein that changes the structure of the nuclear lamina. How the perturbation of the nuclear lamina in progeria is transduced into cellular changes is undefined. Using patient fibroblasts and a variety of cell-based assays, we determined that progerin expression in Hutchinson-Gilford progeria syndrome inhibits the nucleocytoplasmic transport of several factors with key roles in nuclear function. We found that progerin reduces the nuclear/cytoplasmic concentration of the Ran GTPase and inhibits the nuclear localization of Ubc9, the sole E2 for SUMOylation, and of TPR, the nucleoporin that forms the basket on the nuclear side of the nuclear pore complex. Forcing the nuclear localization of Ubc9 in progerin-expressing cells rescues the Ran gradient and TPR import, indicating that these pathways are linked. Reducing nuclear SUMOylation decreases the nuclear mobility of the Ran nucleotide exchange factor RCC1 in vivo, and the addition of SUMO E1 and E2 promotes the dissociation of RCC1 and Ran from chromatin in vitro. Our data suggest that the cellular effects of progerin are transduced, at least in part, through reduced function of the Ran GTPase and SUMOylation pathways.The nuclear lamina provides an architectural framework that defines the size, shape, and physical properties of the nucleus (29). A critical function of the nuclear lamina is to provide a scaffold for chromatin attachment, and there is a growing body of evidence linking the nuclear lamina to the regulation of gene expression and chromosome positioning within interphase cells (49). The nuclear periphery, including the region proximal to the lamina, is rich in heterochromatin and provides a nuclear subcompartment that promotes transcriptional silencing (19). The mechanisms responsible for transcriptional silencing associated with the lamina appear to involve epigenetic regulation and modulation of the higherorder chromatin structure (2). Other functions of the lamina include roles in DNA replication and apoptosis (22,29). The principal components of the lamina are lamin A/C and lamin B, which are encoded by the LMNA and LMNB genes, respectively (22, 29). More than 300 mutations in LMNA have been described (http://www.umd.be/LMNA/) and have been linked to 12 diseases collectively known as laminopathies. These diseases include dilated cardiomyopathy with conduction defects (DCM-CD), familial partial lipodystrophy (FPLD), atypical Werner's syndrome, Emery-Dreifuss muscular dystrophy (EDMD), and Hutchinson-Gilford progeria syndrome (HGPS) (9, 70, 77).The nuclear lamina also provides a scaffold for organizing nuclear pore complexes (NPCs) within the nuclear membrane (1). NPCs span the nuclear lamina and both the inner and outer nuclear membranes and serve as conduits for nuclear import and export (73). Nucleoporins that comprise the NPC are organized into subcomplexes that disassemble and reassemble duri...

We describe a mechanism for protein phosphatase 2A (PP2A) targeting to the androgen receptor (AR) and provide insight into the more general issue of kinase and phosphatase interactions with AR. Simian virus 40 (SV40) small t antigen (ST) binding to N-terminal HEAT repeats in the PP2A A subunit induces structural changes transduced to C-terminal HEAT repeats. This enables the C-terminal HEAT repeats in the PP2A A subunit, including HEAT repeat 13, to discriminate between androgen-and androgen antagonist-induced AR conformations. The PP2A-AR interaction was used to show that an AR mutant in prostate cancer cells (T877A) is activated by multiple ligands without acquiring the same conformation as that induced by androgen. The correlation between androgen binding to AR and increased phosphorylation of the activation function 1 (AF-1) region implies that changes in AR conformation or chaperone composition are causal to kinase access to phosphorylation sites. However, AF-1 phosphorylation sites are kinase accessible prior to androgen binding. This suggests that androgens can enhance the phosphorylation state of AR either by negatively regulating the ability of the ligand-binding domain to bind phosphatases or by inducing an AR conformation that is resistant to phosphatase action. SV40 ST subverts this mechanism by promoting the direct transfer of PP2A onto androgen-bound AR, resulting in multisite dephosphorylation.The nuclear receptor superfamily of transcription factors directs the expression of genes whose products regulate diverse biological pathways. The domain organization of nuclear receptors is conserved and includes an N-terminal activation function 1 (AF-1) region, a central DNA binding domain (DBD), and a C-terminal ligand-binding domain (LBD). Ligand binding to the LBD initiates a series of changes in nuclear receptor structure, chaperone composition, localization, transactivation potential, and protein half-life (t 1/2 ) (10,28,36,44). Understanding how ligand binding elicits these changes is fundamental to understanding nuclear receptor regulation and activity. Defining how ligands control nuclear receptor activity should also provide insight into certain disease mechanisms and aid in the design of drugs such as selective androgen receptor modulators and selective estrogen receptor modulators (4, 27).In addition to ligand binding, nuclear receptors can be regulated by signal transduction pathways. Kinases including those controlled by growth factor-dependent pathways act directly or indirectly on a variety of nuclear receptors (37). Kinases reported to regulate androgen receptor (AR)-dependent transcription include the mitogen-activated protein kinases (p42/ 44, p38, and Jun N-terminal protein kinase), protein kinase A, and protein kinase C (8). The mitogen-activated protein kinases p38 and Jun N-terminal protein kinase also regulate the nucleocytoplasmic distribution of AR (15). Determining exactly how kinases regulate nuclear receptor transcription activity has been challenging because of cross talk bet...