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. 2009 May 8;284(19):12940–12948. doi: 10.1074/jbc.M900207200

FIGURE 2.

FIGURE 2.

Ryk binds to Cdc37. A, FLAG-tagged Cdc37 and Myc-tagged Ryk were transfected into 293T cells. Binding of Cdc37 and Ryk was analyzed by immunoprecipitation (IP) followed by Western blotting (IB) using anti-Myc or anti-FLAG antibodies. FL, full-length. B, a Western blot analysis of anti-FLAG and anti-Myc immunoprecipitates from membrane and cytosolic extracts is shown. C, Cdc37 binds to the ICD region of Ryk. 293T cells expressing Cdc37-FLAG were transfected with the wild-type Ryk, Ryk ICD, or a control plasmid. An anti-Myc immunoprecipitate from whole cell lysates was analyzed by Western blotting using anti-FLAG and anti-Myc antibodies. D, Cdc37 recognizes the tyrosine kinase domain of Ryk. A schematic of the wild-type and mutant of Ryk ICD constructs shows that the Ryk tyrosine kinase domain consists of 11 conserved subdomains (top panel). Each mutant was constructed by deletion of the indicated subdomains. Wild-type and subdomain I/II-deleted mutants, but not other mutants, bind Cdc37 (bottom panel). Plasmids encoding wild-type and mutant ICD proteins or a control vector were cotransfected with FLAG-Cdc37. Cell lysates were analyzed by immunoprecipitation and Western blotting with anti-Myc or anti-FLAG antibodies. E, the fragment encoding amino acids 367–579 of the Ryk kinase domains is sufficient to bind to Cdc37 directly. HA-tagged Ryk-(367–579) was purified from bacteria. Cdc37-FLAG was purified by immunoprecipitation from 293T cells transfected with the construct encoding Cdcd37-FLAG. A Coomassie Blue-stained SDS-polyacrylamide gel containing purified proteins (top panel) confirms the purity of the protein preparations. Arrows indicate purified Ryk-(367–579) (lane 1) and Cdc37-FLAG (lane 2). Purified protein samples were incubated under the indicated conditions, and immunoprecipitation and Western blot analyses were performed using anti-HA or anti-FLAG antibody (lower panel). F, the Cdc37 C terminus is required for binding of Ryk. 293T cells expressing wild-type (WT) Ryk-Myc were transfected with plasmids encoding either wild-type Cdc37-FLAG or C-terminal (CT) deletion mutant (Cdc37ΔCT-FLAG). Anti-FLAG and anti-Myc immunoprecipitates were subjected to Western blotting with anti-Myc and anti-FLAG antibodies.