BINDING-SPECIFICITY AND MUTATIONAL ANALYSIS OF THE PHOSPHOTYROSINE BINDING DOMAIN OF THE BRAIN-SPECIFIC ADAPTER PROTEIN SHCC

She proteins (hereafter referred to as ShcA) represent major substrate s of tyrosine phosphorylation by a wide variety of growth factors and cytokines, We have recently described a novel ShcA-like protein, ShcC, which like ShcA contains an NH2-terminal phosphotyrosine binding doma in (PTB), a central effector region (CH1) and a COOH-terminal Src homo logy 2 domain (SH2). Both the SH2 and PTB domains of ShcC bind a simil ar profile of proteins as the comparable regions of ShcA. In an effort to define the functional differences or similarities between ShcA and ShcC, we have further characterized the PTB domain of ShcC. Using a d egenerate phosphopeptide library screen, we show that the PTB domain o f ShcC preferentially binds the sequence ydrophobic-Asn/hydrophobic-As n-Pro-Ser/Thr-Tyr(P). This sequence is similar to the binding site for the ShcA PTB domain, suggesting that these two proteins may have over lapping specificities. In addition, random mutagenesis of the ShcC PTB domain has identified several amino acids important for PTB function (Gly(32), Glu(63), Ala(136), Gly(139), and Asp(140)). Mutation of thes e amino acids dramatically reduces the affinity of the ShcC PTB domain for the activated epidermal growth factor receptor in vitro.