Novel GKAPs for cGMP-dependent protein kinase type II: identification and functional significance

Abstract

Many protein kinases are maintained in specific subcellular microenvironments by association to anchoring proteins. Proper functioning of the cGMP-dependent protein kinase II (cGK II) involves membrane targeting through myristoylation. To determine whether additional binding to anchoring proteins is also important for its targeting, in analogy with G-kinase anchoring proteins (GKAPs) for cGK I, and to identify potential new components of the cGMP/cGK signalling pathway we performed a yeast twohybrid screen of novel cGK II binding partners. By screening a brain cDNA library using cGK II as a bait the intermediate filament protein GFAP (glial fibrillary acidic protein) was obtained as a positive clone. The interaction was confirmed by overlayand pull-down assays and by immunoprecipitation. Interestingly, the highly homologous intermediate filament protein vimentin has previously been identified as a high affinity binding partner for cGK Iα. We determined which domains are required for the interaction and found, surprisingly, that the least conserved N-terminal domain of the cGK's (aa 1–108 of cGKII and 1–94 of cGK Iα) interacts with the most strongly conserved core domain of the intermediate filaments (aa 237–386 of GFAP). The physiological function of the interaction is under current investigation.