Exposure of rodents to phthalates is associated with developmental and reproductive anomalies, and there is concern that these compounds may be causing adverse effects on human reproductive health. Testosterone (T), secreted almost exclusively by Leydig cells in the testis, is the primary steroid hormone that maintains male fertility. Leydig cell T biosynthesis is regulated by the pituitary gonadotropin LH. Herein, experiments were conducted to investigate the ability of di(2-ethylhexyl)phthalate (DEHP) to affect Leydig cell androgen biosynthesis. Pregnant dams were gavaged with 100 mg(-1) kg(-1) day(-1) DEHP from Gestation Days 12 to 21. Serum T and LH levels were significantly reduced in male offspring, compared to control, at 21 and 35 days of age. However, these inhibitory effects were no longer apparent at 90 days. In a second set of experiments, prepubertal rats, from 21 or 35 days of age, were gavaged with 0, 1, 10, 100, or 200 mg(-1) kg(-1) day(-1) DEHP for 14 days. This exposure paradigm affected Leydig cell steroidogenesis. For example, exposure of rats to 200 mg(-1) kg(-1) day(-1) DEHP caused a 77% decrease in the activity of the steroidogenic enzyme 17beta-hydroxysteroid dehydrogenase, and reduced Leydig cell T production to 50% of control. Paradoxically, extending the period of DEHP exposure to 28 days (Postnatal Days 21-48) resulted in significant increases in Leydig cell T production capacity and in serum LH levels. The no-observed-effect-level and lowest-observed-effect-level were determined to be 1 mg(-1) kg(-1) day(-1) and 10 mg(-1) kg(-1) day(-1), respectively. In contrast to observations in prepubertal rats, exposure of young adult rats by gavage to 0, 1, 10, 100, or 200 mg(-1) kg(-1) day(-1) DEHP for 28 days (Postnatal Days 62-89) induced no detectable changes in androgen biosynthesis. In conclusion, data from this study show that DEHP effects on Leydig cell steroidogenesis are influenced by the stage of development at exposure and may occur through modulation of T-biosynthetic enzyme activity and serum LH levels.

Aberrant secreted proteins can be destroyed by ER-associated protein degradation (ERAD), and a prominent, medically relevant ERAD substrate is the cystic fibrosis transmembrane conductance regulator (CFTR). To better define the chaperone requirements during CFTR maturation, the protein was expressed in yeast. Because Hsp70 function impacts CFTR biogenesis in yeast and mammals, we first sought ER-associated Hsp40 cochaperones involved in CFTR maturation. Ydj1p and Hlj1p enhanced Hsp70 ATP hydrolysis but CFTR degradation was slowed only in yeast mutated for both YDJ1 and HLJ1, suggesting functional redundancy. In contrast, CFTR degradation was accelerated in an Hsp90 mutant strain, suggesting that Hsp90 preserves CFTR in a folded state, and consistent with this hypothesis, Hsp90 maintained the solubility of an aggregation-prone domain (NBD1) in CFTR. Soluble ERAD substrate degradation was unaffected in the Hsp90 or the Ydj1p/Hlj1p mutants, and surprisingly CFTR degradation was unaffected in yeast mutated for Hsp90 cochaperones. These results indicate that Hsp90, but not the Hsp90 complex, maintains CFTR structural integrity, whereas Ydj1p/Hlj1p catalyze CFTR degradation. INTRODUCTIONMutated proteins that are unstable or fold slowly can accumulate in the ER, aggregate, and/or induce apoptosis (Thomas et al., 1995;Kaufman, 1999). However, eukaryotic cells have evolved the ability to identify and degrade these aberrant proteins by a pathway termed ER-associated protein degradation (ERAD). After their identification ERAD substrates are "retro-translocated" (or "dislocated") to the cytosol, ubiquitinated, and targeted to the 26S proteasome for degradation (Ellgaard et al., 1999;Romisch, 1999;Tsai et al., 2002;Kostova and Wolf, 2003;McCracken and Brodsky, 2003). How ERAD substrates are selected is not completely clear, but a family of proteins, known as molecular chaperones, are involved in this process. Several molecular chaperones bind short, linear arrays of amino acids enriched for hydrophobic residues that normally represent buried regions in folded proteins. As a result chaperones can retain mis-folded proteins in solution and facilitate their refolding and/or retro-translocation.Previous work indicated unique chaperone requirements for the degradation of soluble and integral membrane proteins in yeast (for review see Fewell et al., 2001). For example, the ER lumenal Hsp70 molecular chaperone, BiP (Kar2p in yeast) is required for the efficient degradation of CPY* (Plemper et al., 1997) and pro-␣-factor (Brodsky et al., 1999), which are soluble substrates, but is dispensable for the degradation of some mis-folded yeast membrane proteins (Plemper et al., 1998;Zhang et al., 2001;Taxis et al., 2003). In contrast, cytoplasmic Hsp70 is required for the proteolysis of several ER membrane proteins but is dispensable for CPY* and pro-␣-factor degradation (Hill and Cooper, 2000;Zhang et al., 2001). Because the ATPase activity of Hsp70s-and thus their ability to trap polypeptide substrates-is enhanced by interaction with spe...

mice, we confirm genetically that Nef requires PACS-2 to localize to the paranuclear region and assemble the multikinase cascade. Moreover, genetic loss of PACS-2 or inhibition of class I PI3K prevents Nef-mediated MHC-I down-regulation, demonstrating that short interfering RNA knockdown of PACS-2 phenocopies the gene knock-out. This PACS-2-dependent targeting pathway is not restricted to Nef, because PACS-2 is also required for trafficking of an endocytosed cation-independent mannose 6-phosphate receptor reporter from early endosomes to the TGN. Together, these results demonstrate PACS-2 is required for Nef action and sorting of itinerant membrane cargo in the TGN/endosomal system. HIV-12 negative factor (Nef), a 27-kDa N-myristoylated protein, enhances viral replication and virion infectivity, and it is required for the onset of AIDS following HIV-1 infection (1, 2). Nef affects cells in many ways, including altering T-cell activation and maturation (3-5), subverting the apoptotic machinery, and down-regulating CD4 molecules and major histocompatibility complex class I (MHC-I) molecules encoded by the HLA-A and -B loci (2,6). But the precise mechanism of how Nef mediates these pathways has remained elusive.Nef diverts cell-surface MHC-I molecules to trans-Golgi network (TGN)-associated endosomal compartments by an endocytic pathway that is stimulated by class I phosphoinositide 3-kinase (PI3K) and dependent on ADP-ribosylation factor-6 (ARF6) (2,7,8). This MHC-I down-regulation requires the action of three motifs (1, 2) as follows: an N-proximal ␣-helical region (residues 7-26) (9) containing a critical methionine (Met 20 ) that promotes association of MHC-I with the heterotetrameric adaptor AP-1 (10, 11); an acidic cluster (EEEE 65 ) required for binding to phosphofurin acidic cluster sorting protein-1 (PACS-1) (12, 13); and an SH3-binding domain formed by a type II polyproline helix (PXXP 75 ) (9, 12) that promotes association of Nef with Src family tyrosine kinases (SFKs) (14 -17). The conservation of these three motifs in the pandemic M group HIV-1, which accounts for over 90% of all AIDS cases worldwide, suggests they control an essential pathway required for HIV-1 pathogenesis (18,19).The EEEE 65 and PXXP 75 sites act sequentially to recruit and stimulate class I PI3K by directing the assembly of an SFK-ZAP-70/Syk-PI3K cascade in primary CD4 ϩ T-cells (8). Assembly of this multikinase complex is initiated by the EEEE 65 -dependent targeting of Nef to the paranuclear region, which enables the PXXP 75 SH3 domain-binding motif to recruit a TGN-localized SFK. This Nef-activated SFK then stimulates tyrosine phosphorylation of ZAP-70/Syk, recruiting class I PI3K by an unknown mechanism to increase endocytosis of MHC-I through an ARF6-regulated pathway (7,8). MHC-I molecules internalized by this signaling pathway are then redistributed to paranuclear endosomal compartments by a process * This work was supported by National Institutes of Health National Research Service Awards DK076343 (to R. T. Y.), T32 NS007...

Summary TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis.