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. 2010 Aug 15;21(16):2966–2974. doi: 10.1091/mbc.E10-01-0015

Figure 2.

Figure 2.

HIPK2 phosphorylates CREB at Serine 271 but not Serine 133. (A) CREB-WT, CREB 133A, or CREB 271A mutant was cotransfected with FlagHIPK2-WT in K562 cells. Whole cell lysates were prepared after 48 h and subjected to SDS-PAGE and Western blotting with anti-CREB or anti-HIPK2 antibody. (B) Five or 10 ng of His-tagged recombinant CREB-WT, 133A, 271A, or 133A/271A double mutant (DM) was incubated with 9 ng recombinant HIPK2 (kinase domain aa. 165–564) in the presence of [γ-32P]ATP for 30 min at 30°C. Five nanograms of these recombinant CREB proteins was also incubated with recombinant PKA. Samples were loaded on SDS-PAGE, and phosphorylated bands were detected by autoradiograph. Comparable loading of the samples was verified by silver staining of the gel (bottom). (C) The same set of recombinant CREB proteins was incubated with recombinant HIPK2 or PKA in the presence of cold ATP, followed by SDS-PAGE and Western blotting with anti-phospho Serine 133 CREB (top) or CREB antibody (bottom).