Tyrosine 420 of Rlk provides specificity for Fyn transphosphorylation. (A) Wild type Rlk, Rlk-GFPY420F or Rlk-GFPY91F were expressed alone or with wild type Fyn; equal amounts of cell lysates were resolved by SDS-PAGE and subject to immunoblotting with antiphosphotyrosine (4G10, upper panel), or anti-Rlk (α-Rlk, lower panel). (B) In vitro kinase activity of Rlk-GFPY420F. In vitro autokinase activity (middle panel) and in vitro kinase activity on enolase (bottom panel) of anti-Rlk immune complexes; immunoprecipitates of cell lysates expressing the indicated wild type or mutant Rlk with or without WT Fyn were recovered, washed, subject to in vitro kinase assays with acid-denatured enolase as described, resolved by SDS-PAGE and visualized by autoradiography. Levels of Rlk expression are shown in the top panel. Rlk mutants include WT: wild type Rlk-GFP, KI: kinase inactive Rlk-GFP (K299R), and Rlk-GFPY420F. (C) In vitro kinase activity of Rlk-GFP and Rlk-GFPY91F (bottom panel). Levels of Rlk expression are shown in the top panel. (D) Wild type Rlk or Rlk-GFPY6F were expressed alone or with wild type Fyn and immunoblotted with anti-phosphotyrosine (upper panel) or anti-Rlk, (lower panel).