Protein Kinase CKII Interacts with and Phosphorylates the SAG Protein Containing Ring-H2 Finger Motif

Abstract

To investigate the biological function of CKII, we have identified proteins that interact with the subunits of CKII using the yeast two-hybrid system. Here we report that SAG, an antioxidant protein containing Ring-H2 finger motif, is a cellular partner associating with the β subunit of CKII. SAG does not interact with the α subunit of CKII. Analysis of SAG deletion mutants indicates that the Ring-H2 motif of SAG is necessary and sufficient for its binding to the β subunit of CKII. Recombinant SAG can be phosphorylated by CKII in vitro, providing evidence that the β subunit mediates the interaction of CKII enzyme with substrate proteins. Overlay experiment shows that SAG and the β subunit of CKII associate directly in vitro and that CKII-mediated phosphorylation of SAG does not affect the interaction between SAG and the β subunit of CKII. Northern blot analysis indicates that both SAG and the β subunit of CKII were relatively rich in human heart, liver, skeletal muscle, and pancreas, but were detected in only trace amounts in brain, placenta, and lung. Our present results suggest that CKII may play a role in the regulation of SAG function.