Figure 2.

Gwl binds to Hsp90/Cdc37 and importins. (A) Hsp90 binds to the N-terminal half of Gwl kinase. Oocytes were injected with mRNA encoding Flag-tagged N- or C-Terminal Gwl (Fig. 5A) and incubated overnight. Some oocytes were then treated with progesterone and monitored for entry into meiosis I (MI). Oocyte extracts were immunoprecipitated with anti-Flag antibody beads, and the immunoprecipitates were analyzed by immunoblotting for Flag and Hsp90. (B) The GXXGXG motif is essential for binding to Hsp90/Cdc37. Oocytes were injected with mRNA encoding WT, G41S, or T748D Flag-Gwl and incubated overnight. Oocyte extracts were immunoprecipitated with anti-Flag antibody beads and immunoprecipitates were immunoblotted for Flag, Hsp90, and Cdc37. (C) mRNAs encoding WT, G41S, or T748D Flag-Gwl were injected into G2 oocytes followed by overnight incubation. Maturation was then induced in some oocytes by progesterone. Flag-Gwl immunoprecipitates were prepared from G2 and MI oocytes and analyzed for Flag (upper panel) or kinase activity against myelin basic protein (MBP, middle panel, autoradiograph). The level of MBP in the assays is shown as a loading control (lower panel). (D) Gwl binds importins. Immunoprecipitates from oocytes in G2 and MI expressing Flag-Gwl were prepared as in Figure 1, and Flag-Gwl bound proteins were analyzed by immunoblotting using antibodies against Flag, importin α, and importin β, as indicated. (E) Oocytes were injected with mRNA encoding Flag-Gwl and incubated overnight. Oocytes were then treated with progesterone and MI extracts were immunoprecipitated with preimmune or importin α serum, and followed by immunoblotting using Flag, importin β, or importin α antibodies, as indicated. (F) Oocytes were injected with mRNA encoding Flag-Gwl and incubated overnight, and analyzed by immunoblotting using antibodies against Gwl and Flag. (G) Immunoprecipitation of importin α was performed from Xenopus egg extracts, followed by immunoblotting using Gwl and importin α antibodies, as indicated.