Further requirements for the interaction of GCKR and CrkL and demonstration of an association of the endogenous proteins.

(A) The regulatory domain of GCKR interacts with CrkL. Constructs directing the expression of HA-GCKR ^386–846 and CrkL were cotransfected into HEK 293T cells. The indicated immunoprecipitates and a cell lysate were examined for either HA or CrkL expression. Anti-myc antibody was used as a control. (B) The P2 motif in GCKR was required to detect a strong GCKR/CrkL interaction. Constructs directing the expression of HA-GCKR^386–846 (lanes 1-3); CrkL (lanes 1-6); and HA-GCKR^399–846 (lanes 4-6) were transfected into HEK 293T. The indicated immunoprecipitates or cell lysates were analyzed for HA or CrkL expression by immunoblotting. The designation HA-GCKR_T indicated both truncated forms of GCKR, whose mobilities on SDS-PAGE were indistinguishable. (C) The CrkL SH2 domain did not contribute to the interaction with the GCKR regulatory domain. Constructs directing the expression of HAGCKR^386–846, GST (lanes 2, 4) or GST-CRKL^1–109 (lanes 1, 3) were transfected into HEK 293T cells. Cell lysates extracted with glutathione-Sepharose 4B beads (Pharmacia Biotech AB) were analyzed for GST and HA-GCKR expression by immunoblotting. HA-GCKR^386–846expression was similar in the GST- and the GST-CRKL^1-109-transfected cells (lanes 3, 4). (D) GCKR and CrkL are constitutively associated in K562 cells. CrkL immunoprecipitates of cell lysates prepared from 10 × 10⁶ million K562 cells or cell lysates from 0.5 × 10⁶ cells were analyzed for GCKR expression using a GCKR specific antiserum. A preimmune antiserum (P.I.) was used as a control. Results from 2 different experiments (lanes 1, 2 and lanes 3, 4) are shown. Each of these experiments was preformed at least twice with similar results.