Fig. 2. Phosphorylation of fusion proteins that contain different regions of HDACm by CK2 activity extracted from oocytes. (A) Diagram showing the relative positions of potential CK2 phosphorylation sites (P) within the complete HDACm protein (+) and various deletions and mutations. The enzymatic core site is hatched and the C-terminal hydrophilic region is black. All six sites are contained in the GST-R deletion, the five sites present in the hydrophilic region are deleted in GST-R/H and the single N-terminal site is deleted from GST-V. Deletion of 10 kDa from the C-terminus removes all five of the marked sites in the hydrophylic region, whereas deletion of 5 kDa removes only the most C-terminal site. In the GST-V mutant 3m, the three sites most towards the center are mutated (Ser to Ala) and in the mutant 4m the most C-terminal site is also mutated (Thr to Ala). (B) Phosphorylation of GST-fusion proteins expressed in a K12 strain of E. coli. The stained gel (left) shows that partial hydrolysis of GST-V generates fragments that have lost a molecular mass of 5 kDa, 10 kDa and 15 kDa from the C-terminus. Abundant proteins from an added nuclear extract (GV), N1/N2 and nucleoplasmin (Npl) are also seen as stained bands. Phospholabeling with ³²PATP by a GV extract (middle) shows that labeling is restricted to the hydrophobic region but is excluded from the C-terminal 10 kDa. Phospholabeling of GST-V by CK2 isolated from GVs (right) removes labeling due to N1/N2 and nucleoplasmin and is almost completely sensitive to 10 µg/ml heparin. The known CK2 substrate Xenopus FRGY2 (Y2) is shown as a positive control. (C) Phosphorylation of GST-V and 3m and 4m mutants expressed in a BL21 strain of E. coli by oocyte nuclear (GV) and cytoplasmic extracts (D). Whereas GV extract labels neither 3m nor 4m (C), a small, but significant, labeling of 3m is detected with cytoplasmic extract in the presence of 10 nM okadaic acid (OA, D). The stained gels (top of each panel) indicates equal loading of GST-fusion proteins (GST-FP).