Determining the binding partners of PKA in the axoneme of Chlamydomonas reinhardtii flagella

Abstract

The structural axoneme found in both cilia and flagella is critical for proper embryonic development, brain development, functional eyesight, mucosal clearing of the lungs, and fertility. Though much of the axoneme structure has been determined, the regulation of these structures must be understood to develop therapies for blindness, Kartagener's syndrome, ciliary disease, and infertility. Protein Kinase A (PKA) has been shown to regulate motility of the axoneme in mammals, as well as the green alga, Chlamydomonas reinhardtii. Mammalian PKA localizes to specific sites in the cell via A Kinase Anchoring Proteins (AKAPs). In this study, the putative AKAP binding domain of PKA was cloned from Chlamydomonas into a pET22b+ expression vector and fused to a HIS tag. Protein was successfully expressed and purified. The purified AKAP binding domain was used in blot overlays and column affinity experiments to detect if Chlamydomonas PKA binds other axonemal proteins including RSP3, a known AKAP. Potential protein binding was detected; however, the binding was inconsistent and was not able to be confirmed as a true interaction. This PKA clone will be useful in future studies that may determine which proteins PKA binds in the axoneme, and therefore, how it functions to regulate motility.