Cyclin K interacts with the activation domain of STAT3.

(A) COS, A375, or HepG2 cells were cotransfected with vectors expressing a Gal4 luciferase reporter gene and the Gal4-STAT3 expression vector, in the presence or absence of the cyclin K expression vector (lanes 1-9). Following transfection, cells were serum starved and stimulated as described for Figure 1 for 4 hours. Identical experiments were performed in parallel in HepG2 cells using a Gal4-VP16 plasmid (lanes 10-12). (B) A375 cells were transfected as described in panel A with increasing amounts of cyclins (150 ng, lanes 3-4; 250 ng, lanes 5-6; 500 ng, lanes 7-8) in the presence of the Gal4-STAT3 expression vector. (C) Representation of the carboxy-terminal his-STAT3^716–770 and full-length GST–cyclin K fusion protein used in pull-down experiments. SH2 indicates Src homology domain. Dotted lines illustrate the correspondence of fusion protein to the C terminal part of the protein below. (D) Purified GST–cyclin K fusion proteins (50-100 ng) were analyzed for binding to histidine or his-STAT3^716–770fusion proteins (50-100 ng) corresponding to the activation domain of STAT3 immobilized on nickel-agarose beads (lanes 1-3). In parallel (lanes 4-6), his-STAT3^716–770 (50-100 ng) was tested for binding to GST or full-length GST–cyclin K (50-100 ng) immobilized on sepharose beads. The same experiments were then repeated using equivalent amounts (3 pM) of purified GST–cyclin D or GST–cyclin K, with his or his-STAT3^716–770 immobilized on nickel-agarose beads (lanes 7-13). Note that the Western blot experiment in panel D, lanes 7-13, was performed with an antibody directed against GST, and that lanes 12 to 13 correspond to a 10% input.