During tumor development, cells acquire multiple phenotypic changes upon misregulation of oncoproteins and tumor suppressor proteins. Hakai was originally identified as an E3 ubiquitin-ligase for the E-cadherin complex that regulates cell-cell contacts. Here, we present evidence that Hakai plays a crucial role in various cellular processes and tumorigenesis. Overexpression of Hakai affects not only cell-cell contacts but also proliferation in both epithelial and fibroblast cells. Furthermore, the knockdown of Hakai significantly suppresses proliferation of transformed epithelial cells. Expression of Hakai is correlated to the proliferation rate in human tissues and is highly up-regulated in human colon and gastric adenocarcinomas. Moreover, we identify PTB-associated splicing factor (PSF), an RNA-binding protein, as a novel Hakai-interacting protein. By using cDNA arrays, we have determined various specific PSF-associated mRNAs encoding proteins that are involved in several cancer-related processes. Hakai affects the ability of PSF to bind these mRNAs, and expression of PSF short hairpin RNA or a dominant-negative PSF mutant significantly suppresses proliferation of Hakai-overexpressing cells. Collectively, these results suggest that Hakai is an important regulator of cell proliferation and that Hakai may be an oncoprotein and a potential molecular target for cancer treatment.

A comparative investigation of the substrate requirements for the enzyme 5-aminoimidazole ribonucleotide (AIR) carboxylase from E. coli and G. gallus has been conducted using in vivo and in vitro studies. In Escherichia coli, two enzymes PurK and PurE are required for the transformation of AIR to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). The Gallus gallus PurCE is a bifunctional enzyme containing AIR carboxylase and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide (SAICAR) synthetase. The E. coli PurE and the C-terminal domain of the G. gallus PurCE protein maintain a significant degree of amino acid sequence identity and also share CAIR as a product of their enzymatic activities. The substrate requirements of AIR carboxylases from E. coli and G. gallus have been compared by a series of in vitro experiments. The carbamic acid, N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) is a substrate for the E. coli PurE (Mueller et al., 1994) but not for the G. gallus AIR carboxylase. In contrast, AIR and CO2 are substrates for the G. gallus AIR carboxylase. The recognition properties of the two proteins were also compared using inhibition studies with 4-nitro-5- aminoimidazole ribonucleotide (NAIR). NAIR is a tight-binding inhibitor of the G. gallus AIR carboxylase (K(i) = 0.34 nM) but only a steady-state inhibitor (K(i) = 0.5 microM) of the E. coli PurE. These data suggest significant differences in the transition states for the reactions catalyzed by these two evolutionarily related enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

The IB kinase (IKK) holocomplex, containing the kinases IKK␣, IKK␤, and the scaffold NEMO (NF-B essential modifier), mediates activation of NF-B by numerous physiological stimuli. Heat shock protein 90 (Hsp90) and the co-chaperone Cdc37 have been indicated as additional subunits, but their specific functions in signal transduction are indistinct. Using an RNA interference approach, we demonstrate that Cdc37 recruits Hsp90 to the IKK complex in a transitory manner, preferentially via IKK␣. Binding is conferred by N-terminal as well as C-terminal residues of Cdc37. Cdc37 is essential for the maturation of de novo synthesized IKKs into enzymatically competent kinases but not for assembly of an IKK holocomplex. Mature IKKs, T-loop-phosphorylated after stimulation either by receptor-mediated signaling or upon DNA damage, further require Hsp90-Cdc37 to generate an activated state. Thus, the present data denote Hsp90-Cdc37 as a transiently acting essential regulatory component of IKK signaling.NF-B maintains key functions in various biological and pathological processes, including immune and inflammatory reactions, development, proliferation, apoptosis, stress responses, and oncogenesis (1-3). Three major NF-B-activating pathways can be distinguished. In the first, canonical pathway, a broad range of extracellular stimuli, including bacterial pathogens, antigens, mitogens, and inflammatory cytokines, induce diverse intracellular cascades, which activate the IB kinase (IKK) 4 complex. IKK-mediated phosphorylation triggers IB and p105 polyubiquitination by the SCF ␤TrCP E3 ligase complex and subsequent proteasomal destruction, resulting in the release of p50-, p65-, and c-Rel-containing heterodimers (1, 4). With much slower kinetics, a second, noncanonical NF-B pathway induces C-terminal processing of NF-B2/p100 to generate p52-containing complexes (5). Finally, a "nuclear-tocytoplasmic" pathway promotes NF-B activation in response to DNA damage (6).The IKK complex has a large apparent molecular mass of 700 -900 kDa and contains the kinases IKK␣ and IKK␤ as well as the regulatory nonenzymatic scaffold protein IKK␥, also known as NEMO (NF-B essential modifier) (1, 7). Stoichiometric analyses indicated equimolar content of IKK␥ and kinase molecules (8, 9) in the IKK complex, where a tetramer of IKK␥ is thought to bind to two kinase dimers (10). The scaffold protein ELKS has been proposed as a further regulatory component of cellular IKK complexes (11). IKK activation is dependent on phosphorylation at activation loop (T-loop) serines, either by upstream IKK kinases or by autophosphorylation. The activation process involves catalytic, nondestructive Lys-63-linked polyubiquitination (Ub K63 ) of IKK␥ as well as Ub K63 binding by IKK␥ (12, 13). Moreover, conformational changes by induced protein interactions may also be a mechanism to stimulate IKK activity. A number of regulatory proteins have been suggested to interact with IKK components (reviewed in Refs. 1 and 7). Among them, the chaperones Hsp90 and Cdc37 were proposed ...

The purL gene of Escherichia coli encoding the enzyme formylglycinamidine ribonucleotide (FGAM) synthetase which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and MgATP to FGAM, glutamate, ADP, and Pi has been cloned and sequenced. The mature protein, as deduced by the structural gene sequence, contains 1628 amino acids and has a calculated Mr of 141,418. Comparison of the purL control region to other pur loci control regions reveals a common region of dyad symmetry which may be the binding site for the "putative" repressor protein. Construction of an overproducing strain permitted purification of the protein to homogeneity. N-Terminal sequence analysis and comparison of glutamine binding domain sequences (Ebbole & Zalkin, 1987) confirm the amino acid sequence deduced from the gene sequence. The purified protein exhibits glutaminase activity of 0.02% the normal turnover, and NH3 can replace glutamine as a nitrogen donor with a Km = 1 M and a turnover of 3 min-1 (2% glutamine turnover). The enzyme forms an isolable (1:1) complex with glutamine: t1/2 is 22 min at 4 degrees C. This isolated complex is not chemically competent to complete turnover when FGAR and ATP are added, demonstrating that ammonia and glutamine are not covalently bound as a thiohemiaminal available to complete the chemical conversion to FGAM. hydroxylamine trapping experiments indicate that glutamine is bound covalently to the enzyme as a thiol ester. Initial velocity and dead-end inhibition kinetic studies on FGAM synthetase are most consistent with a sequential mechanism in which glutamine binds followed by rapid equilibrium binding of MgATP and then FGAR. Incubation of [18O]FGAR with enzyme, ATP, and glutamine results in quantitative transfer of the 18O to Pi.

The apolipoprotein (apo) E receptor-2 (apoER2) is a member of the low density lipoprotein receptor gene family and an important regulator of neuronal migration. It acts as a receptor for the signaling factor Reelin and provides positional cues to neurons that migrate to their proper position in the developing brain. Besides brain formation defects, apoER2-deficient mice also exhibit male infertility. The role of the receptor in male reproduction, however, remained unclear. Here we demonstrate that apoER2 is highly expressed in the initial segment of the epididymis, where it affects the functional expression of clusterin and phospholipid hydroperoxide glutathione peroxidase (PHGPx), two proteins required for sperm maturation. Reduced PHGPx expression in apoER2 knockout mice results in the inability of the sperm to regulate the cell volume and in abnormal sperm morphology and immotility. Because insufficient expression of PHGPx is a major cause of infertility in men, these findings not only highlight an important new function for apoER2 that is unrelated to neuronal migration, but they also suggest a possible role for apoER2 in human infertility.Apolipoprotein (apo) E receptor-2 (apoER2) 1 is a member of the low density lipoprotein (LDL) receptor gene family and a prototype receptor that acts both in endocytosis and in signal transduction (1-4). In particular, apoER2 functions as a cellular receptor for Reelin, a signaling factor that regulates neuronal migration processes in the embryonic brain. Reelin binds to apoER2 and to the related very low density lipoprotein receptor on the surface of post-mitotic neurons that migrate to their proper position in the developing brain (2, 5). Binding of Reelin results in tyrosine phosphorylation of Disabled 1, an adaptor protein bound to the receptor tails, and in activation of downstream signaling pathways involving the Src family of tyrosine kinases and phosphatidylinositol 3-kinase (4, 6 -8). Defects in these signaling cascades as in apoER-2-deficient mice cause abnormal layering of neurons in the cortex, hippocampus, and cerebellum (9).Aside from post-mitotic neurons in the brain, apoer2 transcripts are also abundant in the placenta, the ovaries, and the epididymis (1, 3, 10). The function of the receptor in these tissues, however, remains unclear. In the present study, we aimed at elucidating novel roles for apoER2 in tissues other than the brain. We focused our attention on the male reproductive system because apoER2 is highly expressed in the principal cells of the epididymis (3) and because receptor-deficient mice suffer from male infertility (9). Here we have identified a crucial role for the receptor in sperm maturation, in particular in the acquisition and development of sperm motility. EXPERIMENTAL PROCEDURESMaterials-The generation of apoER2-deficient mice has been described before (9). Because of male infertility, the line was bred in-house by mating of homozygous-deficient (apoer2 Ϫ/Ϫ ) females with males heterozygous for the receptor gene defect (apoer2 ϩ/Ϫ )...