The three-dimensional structure of the oligomerization domain (residues 319 to 360) of the tumor suppressor p53 has been solved by multidimensional heteronuclear magnetic resonance (NMR) spectroscopy. The domain forms a 20-kilodalton symmetric tetramer with a topology made up from a dimer of dimers. The two primary dimers each comprise two antiparallel helices linked by an antiparallel beta sheet. One beta strand and one helix are contributed from each monomer. The interface between the two dimers forming the tetramer is mediated solely by helix-helix contacts. The overall result is a symmetric, four-helix bundle with adjacent helices oriented antiparallel to each other and with the two antiparallel beta sheets located on opposing faces of the molecule. The tetramer is stabilized not only by hydrophobic interactions within the protein core but also by a number of electrostatic interactions. The implications of the structure of the tetramer for the biological function of p53 are discussed.

Fe65 is a protein mainly expressed in several districts

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Incubating human cells in diethylmaleate (DEM) depletes the intracellular pool of reduced glutathione (GSH) and increases the concentration of oxidative free radicals. We found that DEM-induced oxidative stress reduced the ability of p53 to bind its consensus recognition sequence and to activate transcription of a p53-specific reporter gene. Nevertheless, DEM treatment induced expression of WAF1/CIP1 but not GADD45 mRNA. The fact that N-acetylcysteine, a precursor of GSH that blocks oxidative stress, prevented WAF1/CIP1 induction by DEM suggests that WAF1/CIP1 induction probably was a consequence of the ability of DEM to reduce intracellular GSH levels. DEM induced WAF1/CIP1 expression in Saos-2 and T98G cells, both of which lack functional p53 protein. DEM treatment did not produce an increase in membrane-associated protein kinase C, but ERK2, a mitogen-activated protein kinase, was phosphorylated in a manner consistent with ERK2 activation. DEM treatment also produced a dose-dependent delay in cell cycle progression, which at low concentrations (0.25 mM) consisted of a G2/M arrest and at higher concentrations (1 mM) also involved G1 and S phase delays. Our results indicate that oxidative stress induces WAF1/CIP1 expression and arrests cell cycle progression through a mechanism that is independent of p53. This mechanism may provide for cell cycle checkpoint control under conditions that inactivate p53.

Recent structural studies of the minimal core DNA-binding domain of p53 (p53DBD)

The ␤-amyloid precursor protein (APP) 1 is an integral membrane protein from which the ␤-amyloid peptide is generated. The ␤-amyloid peptide forms the extracellular insoluble aggregates characteristic of Alzheimer's disease. The function of APP and the regulation of the proteolytic events generating the ␤-amyloid peptide are still unknown. APP was expected to be involved in signal transduction processes, because of its transmembrane topology. Three main isoforms of APP exist, generated by alternative splicing (APP 770 , APP 751 , and APP 695 ) and all possessing the same intracellular domain (reviewed in Ref.1). Although little is known about the putative extracellular ligand(s) for APP, several results describe the interaction of its intracellular domain with other proteins. These include the interaction with the heterotrimeric G protein Go (2), a 59-kDa ubiquitously expressed protein named APP-BP1 (3), the X11 protein (4), the neuron-abundant Fe65 protein, and an Fe65-like protein (4 -6). It was shown that intact APP binds to oligomeric Go protein and that the intracellular region of APP spanning residues 657-676 activates Go (2, 7). Furthermore, the interaction of APP with a monoclonal antibody directed against its extracellular domain mimics a ligand-receptor binding that triggers Go activation (7). APP-BP1 interacts both in vitro and in vivo with the carboxyl-terminal region of APP, which represents its intracellular domain. This protein is homologous to the product of the Arabidopsis auxin resistance gene AXR1 and to a Caenorabditis elegans protein of unknown function (3).The Fe65 gene is mainly expressed in the neurons of specific regions of the mammalian nervous system (8, 9) and encodes a protein containing two different types of protein-protein interaction domains: the WW domain (reviewed in Ref. 10) and the phosphotyrosine interaction/phosphotyrosine binding (PID/ PTB) domain (reviewed in Ref. 11). The latter was found in the oncoprotein Shc (12, 13), in its relatives , in other apparently unrelated proteins, such as Numb, X11, and Dab (15), and in insulin receptor substrate 1 (IRS-1) and 17). The PID/PTB domains interact with phosphotyrosine residues located in the intracellular domains of growth factor receptors, such as EGF-R, trkA, and plateletderived growth factor receptor in the case of Shc (13) and insulin receptor and interleukin 4 receptor in the case of IRS-1 (16). In contrast, the Fe65 region containing the two PID/PTB domains was demonstrated to interact with the intracellular domain of APP (5).All the PID/PTB domains present in the Shc family, IRS-1, and Fe65 interact with intracellular regions of membrane proteins containing the consensus motif ⌽XNPXY (where ⌽ is hydrophobic and X is any amino acid). However, Fe65 possesses at least two unique characteristics: (i) although all the known members of the PID/PTB family contain only one PID/ PTB element (13), Fe65 is an exception, because its sequence interacting with APP shows two consecutive PID/PTB domains; and (ii) although the Tyr prese...

The proteolytic processing of amyloid precursor protein (APP) through the formation of membrane-bound C-terminal fragments (CTFs) and of soluble ␤-amyloid peptides likely influences the development of Alzheimer's disease (AD). We show that in human brain a subset of CTFs are tyrosine-phosphorylated and form stable complexes with the adaptor protein ShcA. Grb2 is also part of these complexes, which are present in higher amounts in AD than in control brains. ShcA immunoreactivity is also greatly enhanced in patients with AD and occurs at reactive astrocytes surrounding cerebral vessels and amyloid plaques. A higher amount of phospho-ERK1,2, likely as result of the ShcA activation, is present in AD brains. In vitro experiments show that the ShcACTFs interaction is strictly confined to glial cells when treated with thrombin, which is a well known ShcA and ERK1,2 activator and a regulator of APP cleavage. In untreated cells ShcA does not interact with either APP or CTFs, although they are normally generated. Altogether these data suggest that CTFs are implicated in cell signaling via Shc transduction machinery, likely influencing MAPK activity and glial reaction in AD patients.The cytoplasmic region of the amyloid precursor protein contains an NPXY motif, which is present in the cytodomains of several tyrosine kinase receptors and in non-receptor tyrosine kinase (1, 2). In tyrosine kinase receptors the tyrosine residue of this motif is phosphorylated upon tyrosine kinase activation, and the NPXpY motif (where pY is phosphotyrosine) functions as a docking site for the phosphotyrosine-binding domain present in several adaptor proteins interacting with tyrosine kinase receptors and non-receptor tyrosine kinase, such as the proteins belonging to the Shc family (3, 4). In APP 1 and in APPrelated proteins APLP1 and APLP2 the NPTY motif interacts with several adaptor proteins, such as Fe65 (5), X11 (6), mDab1 (7), and JIP-1 (8), but this interaction has been demonstrated to be independent of tyrosine phosphorylation (9, 10). Recent data (11) show that in human brain CTFs can be tyrosine-phosphorylated and that in vitro the APP cytodomain is tyrosine-phosphorylated by the non-receptor tyrosine kinase Abl, which phosphorylates a tyrosine residue upstream (Tyr-682), the NPTY motif (11,12). This phosphorylation generates a motif pYXXP that is recognized by the SH2 domain of Abl itself and that might be a docking site for SH2-containing adaptors such as Shc and Grb2 proteins (11). Here we describe that in human brain tyrosine-phosphorylated CTFs represent docking sites for Shc and Grb2 proteins and generate stable complexes with these adaptors that are up-regulated in AD cases. ShcA formation is strictly confined to activated astroglial cells only, and its levels are highly enhanced in AD brains in comparison to control subjects. In AD brains it is also up-regulated in the expression of Erk1,2 kinase, likely as a consequence of ShcA activation. In vitro experiments show that thrombin triggers the ShcA-CTFs interaction and Erk ph...

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