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Fig. 4. Both the C-terminal region of HDACm and phosphorylation are required for nuclear uptake. (A) Phospholabeled GST-V (10 ng) was injected into the cytoplasm of stage V oocytes and incorporation of label into the GV was monitored from isolated nuclei and cytoplasms. Whereas most of the full-length fusion protein was imported within 8 hours, the GST-V-5 degradation product remained in the cytoplasm, again indicating that the C-terminal 5 kDa region is required for nuclear import. (B) Quantitation of the amounts and distribution of GST-V and GST-V-5 in GVs and cytoplasms up to 48 hours post-injection not only confirms the requirement for the C-terminal 5 kDa region but also indicates that it is required for stability of the protein. (C) The requirement of phosphorylation for nuclear import of GST-V is shown by comparing the amounts of fusion protein present in GVs at 24 hours post-injection with the amount of endogenous nuclear HDACm. Import is severely restricted in the presence of the inhibitors of CK2 activity, quercetin (Q, 60 nM) and 5,6-dichloro-1ß-D-ribofuranosylbenzimidazole (D, 50 µM), whereas the inactive analogue of quercetin, 3ß-D-rutinoside (R, 60 nM), has little inhibitory effect. Immunoblot using antibody directed against the C-terminal peptide of HDACm. (D) Quantitation of three repeat experiments confirm the requirement of phosphoryation for efficient nuclear import of GST-V. Density scan of each GST-V band is expressed as a fraction of the density scan of the corresponding endogenous HDACm band.

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