Functional Analysis of the Numb Phosphotyrosine-binding Domain Using Site-directed Mutagenesis

Abstract

The Numb protein is involved in cell fate determination during Drosophila neural development. Numb has a protein domain homologous to the phosphotyrosinebinding domain (PTB) in the adaptor protein Shc. In Shc, this domain interacts with specific phosphotyrosine containing motifs on receptor tyrosine kinases and other signaling molecules. Residues N-terminal to the phosphotyrosine are also crucial for phosphopeptide binding to the Shc PTB domain. Several amino acid residues in Shc have been implicated by site-directed mutagenesis to be critical for Shc binding to receptor tyrosine kinases. We have generated homologous mutations in Numb to test whether, in vivo, these changes affect Numb function during Drosophila sensory organ development. Two independent amino acid changes that interfere with Shc binding to phosphotyrosine residues do not affect Numb activity in vivo. In contrast, a mutation shown to abrogate the ability of the Shc PTB domain to bind residues upstream of the phosphotyrosine virtually eliminates Numb function. Similar results were observed in vitro by examining the binding of the Numb PTB domain to proteins from Schneider S2 cells. Our data confirm the importance of the PTB domain for Numb function but strongly suggest that the Numb PTB domain is not involved in phosphotyrosine-dependent interactions.

Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases

“…The PTB domain of Odin is most related to that found in Numb (both of these are similar to the PTB domain of Shc). It is interesting to note that the binding of Numb PTB domain is phosphotyrosine independent but is critical for its ability to alter cell fate in Drosophila (Yaich et al, 1998). Thus, Odin likely represents a cytosolic adapter protein that negatively regulates growth factor receptor signaling.…”

Regulation of centrosome movements by Numb and the Collapsin Response Mediator Protein during Drosophila sensory progenitor asymmetric division

“…The following stocks were used in this study: neur-Gal4 (Bellaïche et al, 2001a), pUAS-His2B::mRFP (Langevin et al, 2005a), UAS-GFP::cnn (Megraw et al, 2002), Ubi-YFP::Asl , UAS-D82glued (Allen et al, 1999), UAS-Myc-numb (Yaich et al, 1998) (Morris et al, 2012), crmp supIA1 (Morris et al, 2012), cnn hk21 (Bloomington #5039), mud f01205 (Ségalen et al, 2010), sas-4 S2214 (Basto et al, 2006), pins P62 (Yu et al, 2000), FRT40A numb 15 , FRT40A ada ear4 , FRT82B spdo 3R6 and FRT9-2 baz xi106 (Wodarz et al, 1999) mutant alleles have been previously described. N ts and Dl RF are a Notch and a Delta thermosensitive alleles, respectively (Gho et al, 1996;Parody and Muskavitch, 1993).…”

The glycosyltransferase Fringe promotes Delta-Notch signaling between neurons and glia, and is required for subtype-specific glial gene expression

“…Gal4 lines used: elav C155 -Gal4 , htl-Gal4 (Shishido et al, 1997), repo-Gal4 (Sepp et al, 2001), scrt -Gal4 (Boyle et al, 2006). UAS lines: UAS-fng (Kim et al, 1995), UAS-N ICD (Go et al, 1998), UAS-H (Maier et al, 1999), UAS-Numb (Yaich et al, 1998), UAS-Ser (Speicher et al, 1994), UAS-Dl , UAS-mCD8::GFP (Lee and Luo, 1999), UAS-Tom (Lai et al, 2000) and UAS-mycGFP, which has a nuclear localization signal. Mutant alleles: Df(3L)riXT1 (fng Df ), fng L73 , fng 13 and fng 80 (Correia et al, 2003;Irvine and Wieschaus, 1994), fng RF584 (Grammont and Irvine, 2001), ser +r83k , ser Rx82 (Speicher et al, 1994), ser (Fleming et al, 1990), loco rC56 (Granderath et al, 1999) here called loco-lacZ, and Dl Rev10 (Heitzler and Simpson, 1991).…”