Characterization of Four Mammalian Numb Protein Isoforms

Abstract

Numb is a membrane-associated, phosphotyrosine binding (PTB) domain-containing protein that functions as an intrinsic determinant of cell fate during Drosophila development. We have identified four isoforms of mammalian Numb with predicted molecular masses of 65, 66, 71, and 72 kDa that are generated by alternative splicing of the Numb mRNA. The different isoforms result from the presence of two sequence inserts within the PTB domain and the central region of the protein.The endogenous expression pattern of these isoforms, examined using specific antisera, varied in different tissues and cell lines. In addition, differentiation of P19 cells with retinoic acid leads to the specific loss of expression of the 71-and 72-kDa Numb proteins, suggesting that the expression of certain forms of Numb protein is regulated in a cell type-specific manner.Expression of Numb proteins fused to green fluorescent protein revealed that the form of the PTB domain with the alternatively spliced insert constitutively associated with the plasma membrane in polarized MadinDarby canine kidney cells. In contrast, the isoform without the insert was cytoplasmic, suggesting that different PTB domain isoforms may regulate the subcellular localization of Numb proteins. The membrane localization may be due, in part, to differential affinity for acidic phospholipids. The distinct expression and localization patterns of the different mammalian Numb isoforms suggest that they have distinct functional properties.

The γ-secretase-generated intracellular domain of β-amyloid precursor protein binds Numb and inhibits Notch signaling

“…Intracellular modulators of Notch, such as Nbl and the four Numb isoforms (32, 34, 35) (p65, p66, p71, and p72, are henceforth referred to collectively as Numbs) are potential molecular links between AID and NICD. Like other APP-interacting proteins, Nbl and Numbs contain a phosphotyrosine binding (PTB) domain (32,(34)(35) that could bind the Y 682 ENPTY 687 motif present in the cytoplasmic tail of APP (15)(16)(17). Notch1, APP, and Numbs͞Nbl are present in the mouse brain and are coexpressed by cortical neurons (Fig.…”

“…APP, APPCT-Gal4, AID, NICD, N⌬E, CBF1-luciferase, and TP1-luciferase have been described (5,13,16,27,(29)(30)(31). Mouse Nbl and Numb (e.g., p65, p66, p71, and p72) constructs (32) were cloned into Flag-tagged pcDNA3.1 (Invitrogen), pECFP-N1, or pEYFP-N1 (CLON-TECH). Deletion mutants of Nbl and AID were generated by using PCR.…”

Phosphorylation of Numb Family Proteins

“…previous report in which four mouse Numb isoforms having molecular masses of 65, 66, 71, and 72 kDa were generated by alternative splicing of the Numb mRNA (43). CREB from rat liver was also detected only in the eluate from Thr 177 -phosphorylated CaM-KI CD-coupled resin (Fig.…”

“…We detected the phosphorylation of a 65-kDa Numb family protein in all of the tissue extracts tested, along with the weak phosphorylation of a 75-kDa Numb family protein in several tissues, indicating that the Numb family proteins are phosphorylated in vivo. However, we were unable to distinguish between the phosphorylation of Numbl and Numb isoforms (43) in the crude tissue extract, due to the similarity of the immunoreactivity of the antibody against phospho-Numb and phospho-Numbl (Fig. 6, A and B).…”

“…Therefore, we cloned Numb cDNA (1779 bp; accession number AB210108) by reverse transcription-PCR, which revealed that this cDNA encoded a p65 isoform composed of 592 amino acid residues (Fig. 3A) (43) and then produced recombinant GST-fused Numb protein. Similar to the results of the phosphorylation experiment with rat Numbl (Fig.…”