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

FIGURE 3.

FIGURE 3.

Cdc37 increases protein levels and nuclear localization of the Ryk ICD, and an Hsp90 inhibitor inhibits the activity of Cdc37 on the Ryk ICD. A, expression of Cdc37-FLAG increases Ryk ICD protein levels, whereas it has no effect on Ryk ICDΔ494–579, which does not bind to Cdc37. Addition of MG132 (MG) 6 h prior to cell lysis prevents degradation of both wild-type Ryk and Ryk ICDΔ494–579. 293T cells were transfected with FLAG-Cdc37, wild-type Ryk, or Ryk ICDΔ494–579-Myc plus GFP. DMSO, dimethyl sulfoxide. B, Cdc37 enhances nuclear localization of the Ryk ICD. Subcellular fractions from 293T cells transiently expressing Ryk ICD-Myc alone or together with Cdc37-FLAG were analyzed by Western blotting using anti-Myc, anti-FLAG, anti-Hsp90, and anti-lamin A/C antibodies. Detection of lamin A/C confirmed the purity of nuclear fractionation. C, the Ryk ICD and ICDΔ319–366 are localized in the nucleus and cytoplasm, whereas Ryk ICD mutants that do not bind to Cdc37 (see Fig. 2D) remain cytoplasmic. The Ryk ICD and its mutants were fused with GFP. Nuclei were visualized using mCherry-tagged histone H2B. EGFP, enhanced green fluorescent protein. D, treatment with the Hsp90 inhibitor 17-AGG decreases the levels of the wild-type Ryk ICD but not Ryk ICDΔ494–579. Cells were transiently transfected with wild-type Ryk-Myc alone or together with Cdc37-FLAG or with Ryk ICDΔ494–579-Myc plus Cdc37-FLAG. Cells were then treated with the indicated concentration of 17-AGG for 24 h prior to lysis. ICD levels were analyzed by Western blotting of samples from whole cell lysates (top panels) and subcellular fractions (bottom panels). 17-AGG treatment had no effect on expression levels of Cdc37, Hsp90, and GFP. E, treatment with the Hsp90 inhibitor 17-AGG induces ubiquitination of the Ryk ICD. 293T cells transfected with HA-ubiquitin (Ub) and/or Ryk ICD-Myc were treated for 6 h with MG132 and different doses of 17-AGG. Immunoprecipitation (IP) was performed using anti-Myc antibody followed by Western blotting (IB) using anti-HA antibody.