Figure 3.

Activation of Hck-YEEI in yeast requires the Nef PxxPxR motif and hydrophobic pocket. A) Conserved features of the Nef:SH3 interface. The SH3 domain is shown in green, while Nef is colored blue. Side chains of conserved prolines in the Nef N-terminal region that contact the SH3 hydrophobic surface are shown (Pro72; Pro75) along with Arg77 which forms a salt bridge with SH3 Asp100. The SH3 domain RT loop Ile reside (Ile96; green spheres) interacts with several conserved residues that extend from the intersection of the Nef αA and αB helices to form a hydrophobic pocket (Phe90, Trp113, Tyr120). This model was produced using the crystallographic coordinates of Lee, et al. (PDB: 1EFN) [35]. B) Upper panel, left four lanes: growth of yeast cultures expressing wild-type Nef (WT), a Nef-PA mutant in which the PxxP motif is replaced by AxxA (PA), the Nef hydrophobic pocket mutant Y120I (YI), or no Nef (Con). The cultures shown in the right four lanes also co-expressed Hck-YEEI. All cultures were spotted and scanned as per the legend to Figure 1. Lower panels: Lysates from the yeast cultures shown in the top panel were immunoblotted with anti-phosphotyrosine antibodies (pTyr) as well as Hck, and Nef antibodies.