Siah1 is the central component of a multiprotein E3 ubiquitin ligase complex that targets ␤-catenin for destruction in response to p53 activation. The E3 complex comprises, in addition to Siah1, Siah-interacting protein (SIP), the adaptor protein Skp1, and the F-box protein Ebi. Here we show that SIP engages Siah1 by means of two elements, both of which are required for mediating ␤-catenin destruction in cells. An N-terminal dimerization domain of SIP sits across the saddle-shaped upper surface of Siah1, with two extended legs packing against the sides of Siah1 by means of a consensus PXAXVXP motif that is common to a family of Siah-binding proteins. The C-terminal domain of SIP, which binds to Skp1, protrudes from the lower surface of Siah1, and we propose that this surface provides the scaffold for bringing substrate and the E2 enzyme into apposition in the functional complex.Polyubiquitination of specific proteins in cells involves the concerted action of E1, 5 E2, and E3 enzymes. First, E1 covalently binds and activates ubiquitin for subsequent transfer to one of several E2s. The latter can in turn directly transfer its bound ubiquitin to the amino groups of lysine side chains in target proteins. More often, however, E3 ligases recognize substrates and direct their interaction with E2s, resulting in the highly specific regulation of target protein polyubiquitination (1, 2). Humans carry two highly related genes, siah1 and siah2 (3), that encode the mammalian homologs of the Drosophila Sina protein, which is required for R7 photoreceptor cell differentiation within the sevenless pathway (4, 5). Sina/Siah proteins are E3 ligases, acting either as single proteins or as part of a multiprotein complex that is analogous to the Skp1-cullin-1-F-box (SCF) complex. Among the targets of Sina/Siah are NcoR (6), DCC (7), c-Myb (8), BOB-1/OBF-1 (9, 10), Peg3/Pw1 (11), Kid (12), Numb (13) (24), and ␣-ketoglutarate dehydrogenase (25). In addition, Siah interacts with adenomatous polyposis coli, a tumor suppressor involved in colon cancers (26); VAV, a nucleotide exchange factor involved in control of Rho/Rac proteins (27); BAG-1, a Hsp70/Hsc70-binding protein that modulates pathways involved in the control of cell proliferation, death, and migration (28, 29); and Dab-1, an inhibitor of Siah1 (30). However, Sina/Siah does not appear to target phyllopod, adenomatous polyposis coli, VAV, BAG-1, or Dab-1 for polyubiquitination and degradation. Thus, not all Siahbinding proteins are targets of Siah-mediated degradation.Recently, we discovered a novel pathway for ␤-catenin degradation involving a complex formed by Siah1, SIP, the adaptor protein Skp1 that is common to the SCF complex, and the F-box protein Ebi that binds ␤-catenin independent of phosphorylation (31). Siah1 expression is upregulated by p53, revealing a link between genotoxic injury and destruction of ␤-catenin, reduced Tcf/LEF activity, and cell cycle arrest (31). Siah1 is a dimeric protein that contains an N-terminal RING domain (an E2 binding domain) followed by...

The co-chaperone p23 forms a complex with the chaperone Hsp90 that mediates the folding pathway leading to the production of functional steroid receptors. Solution NMR spectroscopy has been used to characterize sites of interaction between Hsp90 and

The metabolic precursors and cerebral compartmentation of the augmented GABA pool induced by vigabatrin, an irreversible inhibitor of GABA transaminase, have been investigated by 13C NMR. Adult rats receiving rat chow ad libitum were given either drinking water only or drinking water containing 2.5 g/L vigabatrin for 7 days. Both groups of animals were infused either with [1,2‐13C2]acetate (15 µmol/min/100 g body weight), an exclusive precursor of GABA formation through the glial glutamine pathway, or with [1,2‐13C2]glucose (15 µmol/min/100 g body weight), a substrate that can produce GABA through the glial glutamine pathway or by direct metabolism in the neurons. The brains were frozen in situ, extracted with perchloric acid, and analyzed by 13C NMR. In vigabatrin‐treated animals [13C]glutamine, a common intermediate for [13C]GABA synthesis from glucose or acetate, was accumulated to similar amounts during infusions with [1,2‐13C2]glucose or [1,2‐13C2]acetate. However, [13C]GABA accumulation was sevenfold higher during [1,2‐13C2]glucose infusions or twofold higher during [1,2‐13C2]acetate infusions. These results show that the direct pathway of GABA formation by neuronal metabolism of glucose predominates over the alternative pathway through glial glutamine. Near‐equilibrium relationships of the aminotransferases of GABA and aspartate imply that the observed [13C]GABA accumulation occurs initially in the neuronal compartment.

Cell culture techniques, high-resolution in vitro 1H NMR spectroscopy, and chromatographic analyses were used to compare the properties of three types of human brain and nervous system tumours. Cell lines were immunocytochemically characterized at all stages in culture with specific antibodies. Intracellular metabolites present in cell extracts were analysed by 1H NMR spectroscopy and by high performance liquid chromatography (HPLC). The spectra from meningiomas, neuroblastomas, and glioblastomas displayed, in addition to similarities-including the presence of signals from leucine, isoleucine, valine, threonine, lactate, acetate, glutamate, choline-containing compounds and glycine-certain distinguishing metabolic features. Spectra from meningiomas featured relatively high signals from alanine. Intense signals from creatine were present in neuroblastoma spectra, while in spectra from glioblastoma they were not detectable. We found statistically significant differences by 1H NMR spectroscopy in the amounts of alanine, glutamate, creatine, phosphorylcholine and threonine among the types of tumours examined. HPLC determinations confirmed that there were also other metabolites specific to a type of tumour, such as taurine, gamma-aminobutyric acid, and serine. We suggest that these findings have potential relevance for the development of non-invasive diagnosis of tumour lineage by 1H NMR spectroscopy in vivo.