FIG. 5.

Rux inhibits CycA dependent kinase activity in cell extracts. The indicated volumes of extracts prepared from cells expressing wild-type (lanes 1 to 3 and 10 to 12) or mutant (lanes 4 to 9) forms of Rux were mixed with 2 μl of extract prepared from cells coexpressing CycA and Cdc2 (lanes 1 to 9) or CycA and Cdc2c (lanes 10 to 12) as described in Materials and Methods and tested for cyclin-CDK activity with histone H1 as an exogenous substrate. The reaction products were analyzed by SDS-PAGE and autoradiography. Wild-type and mutant Rux proteins were expressed at equivalent levels and migrated at the same relative size, as determined by Western blot analysis (data not shown). Increasing the volume of the Rux-containing extract from 0 to 3 μl resulted in an approximately fivefold inhibition of CycA-associated kinase activities as determined by PhosphorImager quantitation. When extract was prepared from untransfected or mocktransfected cells, a similar inhibition of endogenous CycA-CDK activity by Rux could be detected after prolonged exposure of autoradiographs (data not shown). Note that histone H1 phosphorylation products appear as a doublet in these experiments. The data are representative of at least three independent experiments.