by Chong-Shan Shi, Joseph Tuscano, and John H. Kehrl Adaptor proteins CRK and CRKL associate with the serine/threonine protein kinase GCKR promoting GCKR and SAPK activation by Chong-Shan Shi, Joseph Tuscano, and John H. Kehrl Blood Volume 95(3):776-782 February 1, 2000 ©2000 by American Society of Hematology

Crk-I and Crk-II coimmunoprecipitate with GCKR Crk-I and Crk-II coimmunoprecipitate with GCKR. HEK 293T cells were transfected with constructs that directed the expression of HA-GCKR and Crk-I or Crk-II.Lysates prepared from the transfected cells were immunoprecipitated with HA, FLAG (control antibody),... Crk-I and Crk-II coimmunoprecipitate with GCKR. HEK 293T cells were transfected with constructs that directed the expression of HA-GCKR and Crk-I or Crk-II.Lysates prepared from the transfected cells were immunoprecipitated with HA, FLAG (control antibody), or a Crk antibody as indicated. The immunoprecipitates were fractionated on SDS-PAGE, transferred, and blotted with the HA antibody (top) or the Crk antibody (bottom). Locations of HA-GCKR, Crk-II, and Crk-I are indicated with arrows as the immunoglobulin light chains. These experiments were performed 3 times with similar results. Chong-Shan Shi et al. Blood 2000;95:776-782 ©2000 by American Society of Hematology

Coimmunoprecipitation of CrkL with GCKR depends on the region in GCKR between amino acids 599 and 699.HEK 293T cells were transfected with constructs that directed the expression of CrkL and GCKR or truncated versions of GCKR. Cell lysates and myc (control ... Coimmunoprecipitation of CrkL with GCKR depends on the region in GCKR between amino acids 599 and 699.HEK 293T cells were transfected with constructs that directed the expression of CrkL and GCKR or truncated versions of GCKR. Cell lysates and myc (control antibody) and HA antibody immunoprecipitates were analyzed by immunoblotting. The blot was sequentially reacted with the HA antibody (top) and the CrkL antibody (bottom). CrkL is indicated by an arrow. The truncated GCKR proteins can be seen in the cell lysate and in the HA immunoprecipitation lanes (arrows). Several nonspecific proteins are also present (top), the most prominent just below 95 kd in the cell lysate lanes. Chong-Shan Shi et al. Blood 2000;95:776-782 ©2000 by American Society of Hematology

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. 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-GCKR386–846 (lanes 1-3); CrkL (lanes 1-6); and HA-GCKR399–846 (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-GCKRT 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 HAGCKR386–846, GST (lanes 2, 4) or GST-CRKL1–109 (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-GCKR386–846expression was similar in the GST- and the GST-CRKL1-109-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. Chong-Shan Shi et al. Blood 2000;95:776-782 ©2000 by American Society of Hematology

CrkL enhances the association of Bcr-Abl and GCKR CrkL enhances the association of Bcr-Abl and GCKR.HEK 293T cells were transfected with constructs that directed the expression of Bcr-Abl and HA-GCKR in the presence or absence of CrkL (lanes 1, 2 versus lanes 3, 4). CrkL enhances the association of Bcr-Abl and GCKR.HEK 293T cells were transfected with constructs that directed the expression of Bcr-Abl and HA-GCKR in the presence or absence of CrkL (lanes 1, 2 versus lanes 3, 4). Bcr-Abl (lanes 2, 4) or control (lanes 1, 3) immunoprecipitates were analyzed by immunoblotting for HA-GCKR. Cell lysates from the same transfection were analyzed for Bcr-Abl and HA-GCKR by immunoblotting with a Bcr-Abl or an HA antibody. This experiment was preformed twice with similar results. Chong-Shan Shi et al. Blood 2000;95:776-782 ©2000 by American Society of Hematology

Crk proteins activate GCKR kinase activity Crk proteins activate GCKR kinase activity.(A) Coexpression of Crk proteins enhances GCKR kinase activity. Crk proteins activate GCKR kinase activity.(A) Coexpression of Crk proteins enhances GCKR kinase activity. HEK 293T was transfected with a control or with constructs that direct the expression of Crk-I, Crk-II, or CrkL and HA-GCKR. HA-immunoprecipitates were subjected to an in vitro kinase assay using myelin basic protein (MBP) as a substrate (autoradiograph, labeled as kinase assay). The SDS-PAGE fractionated in vitro kinase assay was analyzed by immunoblotting for phosphotyrosine (pY blot). In addition, cell lysates prepared from the same transfections were analyzed by immunoblotting for HA-GCKR (HA blot), CrkL (CrkL blot), and Crk-I and Crk-II (Crk blot). (B) Coexpression of Crk proteins triggers GCKR phosphorylation. HA-GCKR immunoprecipitates from cells transfected as in A were subjected to an in vitro kinase assay in the absence of an exogenous substrate (autoradiograph, labeled as kinase assay). Cell lysate analyzed for HA-GCKR expression by immunoblotting. Each of these experiments was performed at least twice. Chong-Shan Shi et al. Blood 2000;95:776-782 ©2000 by American Society of Hematology

Ras N-17 blocks Crk-induced GCKR activation. Ras N-17 blocks Crk-induced GCKR activation. HEK 293T cells were cotransfected constructs directing the expression of HA-GCKR in conjunction with Crk-I (lanes 2, 3), Crk-II (lanes 4, 5), CrkL (lanes 6, 7), or a control plasmid (lane 1) in the presence (lanes 3, 5, 7) or absence of Ras N-17 (lanes 1, 2, 4, 6).HA-GCKR immunoprecipitates were assayed for activity using an in vitro kinase with MBP as a substrate. The fold induction compared to HA-GCKR immunoprecipitate from the control transfection is indicated. Levels of Crk-I, Crk-II, CrkL, Ras N-17, and HA-GCKR expression as detected by immunoblotting are shown. These experiments were performed twice. Chong-Shan Shi et al. Blood 2000;95:776-782 ©2000 by American Society of Hematology

Evidence for GCKR involvement in Crk-I- and Crk-II-induced SAPK activation.HEK 293 cells were cotransfected with constructs that direct the expression of HA-SAPK (lanes 1 to 6), Crk-II (lanes 1, 2), Crk-I (lanes 3, 4), or GCKR (lane 5) in the presence (lane... Evidence for GCKR involvement in Crk-I- and Crk-II-induced SAPK activation.HEK 293 cells were cotransfected with constructs that direct the expression of HA-SAPK (lanes 1 to 6), Crk-II (lanes 1, 2), Crk-I (lanes 3, 4), or GCKR (lane 5) in the presence (lanes 1, 3) or absence (lanes 2, 4 to 6) of a construct that expressed a GCKR antisense RNA. HA-SAPK immunoprecipitates were subjected to an in vitro kinase assay using GST-jun1–79 as a substrate. The fold induction compared to HA-SAPK alone is indicated. The levels of HA-SAPK, Crk-II, and Crk-I as detected by immunoblotting are shown below. These experiments were performed twice with similar results. Chong-Shan Shi et al. Blood 2000;95:776-782 ©2000 by American Society of Hematology

Evidence that GCKR is required for CrkL-induced SAPK activation Evidence that GCKR is required for CrkL-induced SAPK activation.(A) A GCKR antisense construct inhibits CrkL-induced SAPK activation. Evidence that GCKR is required for CrkL-induced SAPK activation.(A) A GCKR antisense construct inhibits CrkL-induced SAPK activation. HEK 293 cells were cotransfected with constructs that direct the expression of HA-SAPK (lanes 1 to 5), CrkL (lanes 2 to 5), and a GCKR antisense RNA (lanes 4, 5). HA-SAPK immunoprecipitates were subjected to an in vitro kinase assay using GST-jun1–79 as a substrate. The fold induction compared to the control is shown below the in vitro kinase assay result. Levels of HA-SAPK and CrkL expression are shown as detected by immunoblotting. (B) GCKRT178A inhibits CrkL-induced SAPK activation. HEK 293 cells were cotransfected with constructs that direct the expression of HA-SAPK (lanes 1 to 5), CrkL (lanes 2 to 5), and increasing concentrations of GCKRT178A (1, 2, and 3 μg/mL, lanes 3 to 5, respectively). HA-SAPK immunoprecipitates were subjected to an in vitro kinase assay using GST-jun1–79 as a substrate. The fold induction compared to cells transfected only with the construct directing HA-SAPK expression is indicated below the autoradiograph. Levels of HA-SAPK, CrkL, and GCKRT178A as detected by immunoblotting are shown. Low levels of endogenous GCKR are detected (lanes 1, 2, bottom). These experiments were performed twice with similar results. Chong-Shan Shi et al. Blood 2000;95:776-782 ©2000 by American Society of Hematology

GCKR involvement in CrkL-induced SAPK activation in K562 cells GCKR involvement in CrkL-induced SAPK activation in K562 cells.K562 cells were transfected with contructs directing the expression of CrkL (lanes 1 to 3), antisense GCKR (lane 1), kinase inactive GCKR (lane 2), or wild-type GCKR (lane 4). GCKR involvement in CrkL-induced SAPK activation in K562 cells.K562 cells were transfected with contructs directing the expression of CrkL (lanes 1 to 3), antisense GCKR (lane 1), kinase inactive GCKR (lane 2), or wild-type GCKR (lane 4). Levels of phosphorylated SAPK were detected with an antibody specific for pSAPK. Levels of SAPK, GCKR, and CrkL in the cell lysates were detected by immunoblotting with SAPK, GCKR, and CrkL-specific antibodies. This experiment was performed twice with similar results. Chong-Shan Shi et al. Blood 2000;95:776-782 ©2000 by American Society of Hematology