FIGURE 5:

CREB-HΔTMC phosphorylation by CKII and GSK-3 on distinct motifs within and adjacent to the DSG region, respectively. (a) Equal amounts of purified GST, GST-wt, or GST-DSG protein bound to glutathione-agarose beads were incubated with purified GSK-3b or CKII and analyzed by SDS–PAGE and autoradiography. The amount of proteins in the reaction is shown by total protein staining (CBB), and the phosphorylated species is shown for each kinase. The GST control migrates at a lower position but has been transposed for ease of direct comparison. (b) Replicate equivalent samples of the GST-wt protein were incubated in vitro without or with a primary kinase as indicated (1st) and the beads washed to remove the kinase and exchange buffers and then incubated without or with a second kinase (2nd). The different combinations and order are indicated. Samples were then analyzed by SDS–PAGE and autoradiography. (c) Equal amounts of the GST-wt (lanes 1, 3, 5, and 7) and GST-DSG mutant (lanes 2, 4, 6, and 8), as in a, were incubated with the various combinations of primary and secondary kinases as indicated. (d) Equal amounts of purified GST, GST-3Sd, GST-DSG, or GST-3Sd+DSG protein were incubated with purified GSK-3 or CKII and subsequently analyzed by SDS–PAGE and autoradiography. The equalized amount of the proteins in the reaction is shown by total protein staining (CBB), and the phosphorylated species is shown for each kinase and mutant as indicated.