ABSTRACT
Numb is a phosphotyrosine-binding (PTB) domain-containing protein implicated in the control of cell fate decisions during development. A modified two-hybrid screen in yeast was used to identify Numb PTB domain-interacting proteins important for Numb function. Here we report the identification of a novel protein, LNX, which interacts specifically with the Numb PTB domain. Two differentially expressed LNX messages encode overlapping proteins with predicted molecular masses of 80 kDa (LNX) and 70 kDa (LNX-b). LNX and LNX-b contain unique amino-terminal sequences and share four PDZ domains. The unique amino-terminal region of LNX includes a RING finger domain. The Numb PTB domain binding region of LNX was mapped to the sequence motif LDNPAY, found in both protein isoforms. Mutational analysis of LNX and peptide competition experiments showed that phosphorylation of the tyrosine residue within this motif was not required for binding to the Numb PTB domain. Finally, we also provide evidence that tyrosine phosphorylation of the LDNPAY sequence motif in LNX could generate a binding site for the phosphorylation-dependent binding of other PTB domain-containing proteins such as SHC. We speculate that LNX may be important for clustering PTB-containing proteins with functionally related transmembrane proteins in specific membrane compartments.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Phosphotyrosine , Ubiquitin-Protein Ligases , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Carrier Proteins/biosynthesis , Cell Line , Cell Membrane/metabolism , Cloning, Molecular , Embryo, Mammalian , Gene Library , Humans , Mice , Molecular Sequence Data , Molecular Weight , Mutagenesis, Site-Directed , Organ Specificity , RNA, Messenger/biosynthesis , Rats , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Transcription, Genetic , Transfection , Tumor Cells, Cultured , Zinc FingersABSTRACT
Here we report the cloning and primary characterization of TIC, a new member of the bHLH/PAS domain family of transcription factors. Northern blot analysis indicates that the 3.3-kb Tic mRNA is widely expressed. A polyclonal antibody against TIC identifies multiple protein species in most cell lines and tissues tested, suggesting that alternative splicing may result in the production of protein isoforms. Interspecies backcross and FISH mapping have been used to localize the Tic gene to mouse Chromosome (Chr) 7 F2-F3, given the locus name Arntl. FISH mapping was also used to localize the human gene to Chr 11p15.
