Isoform-specific patterns of phosphorylation on axonemal dynein heavy chains

Abstract

Axonemal dyneins power ciliary motility and phosphorylation of key intermediate and light chain components affects the regulation and properties of these motors in very distantly related organisms. It is also known that many axonemal dynein heavy chains are subject to this post-translational modification although this has been little studied. Here we examine axonemal dynein heavy chains from a broad range of ciliated eukaryotes and identify phosphorylated sites embedded within various kinase recognition motifs such as those for PKA, PKC and casein kinase II. Mapping these sites onto discrete heavy chain types reveals class-specific locations apparently mediated by different kinases. For example, we find that all Chlamydomonas heavy chain phosphorylation sites are in an extended loop derived from AAA5 that arches over the coiled-coil buttress which in turn interacts with the microtubule-binding stalk. In contrast, most sites in the monomeric inner arm dyneins occur very close to the N-terminus and may be involved in assembly processes. In Chlamydomonas, the two cilia (termed cis and trans) exhibit different intrinsic beat frequencies and we identify cilium-specific phosphorylation patterns on both the heavy chain and outer arm docking complex consistent with differential regulation of these motors in the two organelles. SignificanceO_LIMany axonemal dynein heavy chains are extensively phosphorylated. However, the location, sequence context and potential role of these modifications is uncertain. C_LIO_LIHere we use mass spectrometry to define phosphorylated sites in inner and outer arm dyneins from a broad range of motile ciliated eukaryotes. C_LIO_LIWe identify numerous modified sites that correspond to various kinase consensus sequences and reveal that multiple sites occur in conserved locations depending on dynein heavy chain class. We also find phosphorylation modifications that are specific to either the cis or trans cilium of Chlamydomonas which may be related to their inherent beat frequency imbalance. C_LI