Identification of the interface between cGMP-dependent protein kinase Iβ and its interaction partners TFII-I and IRAG reveals a common interaction motif

Protein-protein interactions have emerged as an important mechanism providing for specificity in cellular signal transduction. Two splice variants of type I cGMP-dependent protein kinase (PKG I alpha and I beta) differ only in their N-terminal similar to 100 amino acids, which mediate binding to different target proteins. PKG I beta, but not I alpha, binds to the general transcriptional regulator TFII-I and the inositol 1,4,5-trisphosphate receptor-associated PKG substrate IRAG. Using a combination of site-directed mutagenesis and in vitro binding assays, we identified a group of acidic amino acids in the N-terminal leucine zipper dimerization domain of PKG I beta required for its binding to both TFII-I and IRAG. Small clusters of basic amino acids in possible alpha-helical regions in TFII-I and IRAG were found to mediate their interaction with PKG I beta. Mutation of two negatively charged residues in the PKG I beta leucine zipper (D26K/E31R) to positively charged residues, found in corresponding positions in PKG I alpha, completely abrogated binding to TFII-I and IRAG without disrupting PKG dimerization. Mutation of specific basic residues in TFII-I or IRAG abolished binding of the full-length proteins to PKG I beta in intact cells. Based on these results, we propose a model for specific PKG I beta interaction with target proteins.