Ataxia-telangiectasia mutated (ATM) is a cell cycle checkpoint kinase that upon activation by DNA damage leads to cell cycle arrest and DNA repair or apoptosis. The absence of Atm or the occurrence of loss-of-function mutations in Atm predisposes to tumorigenesis. MAPK7 has been implicated in numerous types of cancer with pro-survival and pro-growth roles in tumor cells, but its functional relation with tumor suppressors is not clear. In this study, we show that absence of MAPK7 delays death due to spontaneous tumor development in Atm-/-mice. Compared with Atm-/-thymocytes, Mapk7-/

Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that Cbl adaptor proteins directly control downregulation of the receptor for the epidermal growth factor (EGFR) by recruiting ubiquitin-activating and -conjugating enzymes. We infer a sequential process initiated by autophosphorylation of EGFR at a previously identified lysosome-targeting motif that subsequently recruits Cbl. This is followed by tyrosine phosphorylation of c-Cbl at a site flanking its RING finger, which enables receptor ubiquitination and degradation. Whereas all three members of the Cbl family can enhance ubiquitination, two oncogenic Cbl variants, whose RING fingers are defective and phosphorylation sites are missing, are unable to desensitize EGFR. Our study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways.

Myeloid antigen-presenting cells (APC) are known to cross-present exogenous antigen on major histocompatibility class I molecules to CD8+ T cells and thereby induce protective immunity against infecting microorganisms. Here we report that liver sinusoidal endothelial cells (LSEC) are organ-resident, non-myeloid APC capable of cross-presenting soluble exogenous antigen to CD8+ T cells. Though LSEC employ similar molecular mechanisms for cross-presentation as dendritic cells, the outcome of cross-presentation by LSEC is CD8+ T cell tolerance rather than immunity. As uptake of circulating antigens into LSEC occurs efficiently in vivo, it is likely that cross-presentation by LSEC contributes to CD8+ T cell tolerance observed in situations where soluble antigen is present in the circulation.

No abstract

We tested 42 tetrapeptides for their ability to bind to the rat brain p2l' protein farnesyltransferase as estimated by their ability to compete with p2lH"-in a farnesyltransfer assay. Peptides with the highest affinity had the structure Cys-Al-A2-X, where positions Al and A2 are occupied by aliphatic amino acids and position X is-occupied by a COOHterminal methionine, serine, or phenylalanine. Charged residues reduced affinity slightly at the Al position and much more drastically at the A2 and X positions. Effective inhibitors included tetrapeptides corresponding to the COOH termini of all animal cell proteins known to be farnesylated. In contrast, the tetrapeptide Cys-Ala-Ile-Leu (CARL), which corresponds to the COOH termini of several neural guanine nucleotide binding (G) protein y subunits, did not compete in the farnesyl-transfer assay. Inasmuch as several of these proteins are geranylgeranylated, the data suggest that the two isoprenes (farnesyl and geranylgeranyl) are transferred by different enzymes. A biotinylated heptapeptide corresponding to the COOH terminus of p21K'-1B was farnesylated, suggesting that at least some of the peptides serve as substrates for the transferase. The data are consistent with a model in which a hydrophobic pocket in the protein farnesyltranferase recognizes tetrapeptides through interactions with the cysteine and the last two amino acids.A farnesyl residue is attached in thioether linkage to the COOH-terminal cysteine of a variety of intracellular membrane-associated proteins. The list includes cellular p21lS proteins (1), nuclear lamin B (2), and the 'y subunit of bovine transducin (3). This modification is also found on mating factors secreted by fungi (4,5). In each case the farnesylated cysteine is initially the fourth residue from the COOH terminus (for review, see ref. 6). Farnesylation is followed by proteolytic removal of the three terminal residues and carboxylmethylation of the cysteine. These reactions render the COOH termini hydrophobic, presumably facilitating the initial attachment of the proteins to cell membranes. In some instances the hydrophobicity is increased by palmitoylation of nearby cysteines. Inspection of the sequences of the known farnesylated proteins has defined a weak consensus sequence for farnesylation that consists of Cys-A1-A2-X, where positions Al and A2 are occupied by aliphatic amino acids and position X is occupied by an undefined amino acid (7, 8).The likely donor of farnesyl residues is farnesyl pyrophosphate (FPP), an intermediate in the synthesis of sterols and polyisoprenes in eukaryotic cells (9). Recently, we have isolated from rat brain an enzyme that transfers a farnesyl group from [3H]FPP to p2lHa-as protein (10,11). The purified enzyme preparation contains two proteins, each with an apparent molecular mass ofabout 50 kDa, that appear to form a heterodimer (10, 11). The enzyme recognizes sequences as short as four amino acids provided that cysteine is at the fourth position from the COOH terminus. Recognition was demonst...

No abstract

The tumor suppressor gene 101 (tsg101) regulates vesicular trafficking processes in yeast and mammals. We report a novel protein, Tal (Tsg101-associated ligase), whose RING finger is necessary for multiple monoubiquitylation of Tsg101. Bivalent binding of Tsg101 to a tandem tetrapeptide motif (PTAP) and to a central region of Tal is essential for Tal-mediated ubiquitylation of Tsg101. By studying endocytosis of the epidermal growth factor receptor and egress of the human immunodeficiency virus, we conclude that Tal regulates a Tsg101-associated complex responsible for the sorting of cargo into cytoplasm-containing vesicles that bud at the multivesicular body and at the plasma membrane.[Keywords: Endocytosis; growth factor; HIV; ubiquitin; signal transduction] Supplemental material is available at http://www.genesdev.org.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3␣. Mouse UBR1p (E3␣) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans Ϸ120 kilobases of genomic DNA and contains Ϸ50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway.