Myc-binding protein orthologue interacts with AKAP240 in the central pair apparatus of the Chlamydomonas flagella

Abstract

BackgroundFlagella and cilia are fine thread-like organelles protruding from cells that harbour them. The typical ‘9 + 2’ cilia confer motility on these cells. Although the mechanistic details of motility remain elusive, the dynein-driven motility is regulated by various kinases and phosphatases. A-kinase anchoring proteins (AKAPs) are scaffolds that bind to a variety of such proteins. Usually, they are known to possess a dedicated domain that in vitro interacts with the regulatory subunits (RI and RII) present in the cAMP-dependent protein kinase (PKA) holoenzyme. These subunits conventionally harbour contiguous stretches of a.a. residues that reveal the presence of the Dimerization Docking (D/D) domain, Catalytic interface domain and cAMP-Binding domain. The Chlamydomonas reinhardtii flagella harbour two AKAPs; viz., the radial spoke AKAP97 or RSP3 and the central pair AKAP240. Both these were identified on the basis of their RII-binding property. Interestingly, AKAP97 binds in vivo to two RII-like proteins (RSP7 and RSP11) that contain only the D/D domain.ResultsWe found a Chlamydomonas Flagellar Associated Protein (FAP174) orthologous to MYCBP-1, a protein that binds to organellar AKAPs and Myc onco-protein. An in silico analysis shows that the N-terminus of FAP174 is similar to those RII domain-containing proteins that have binding affinities to AKAPs. Binding of FAP174 was tested with the AKAP97/RSP3 using in vitro pull down assays; however, this binding was rather poor with AKAP97/RSP3. Antibodies were generated against FAP174 and the cellular localization was studied using Western blotting and immunoflourescence in wild type and various flagella mutants. We show that FAP174 localises to the central pair of the axoneme. Using overlay assays we show that FAP174 binds AKAP240 previously identified in the C2 portion of the central pair apparatus.ConclusionIt appears that the flagella of Chlamydomonas reinhardtii contain proteins that bind to AKAPs and except for the D/D domain, lack the conventional a.a. stretches of PKA regulatory subunits (RSP7 and RSP11). We add FAP174 to this growing list.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-016-0103-y) contains supplementary material, which is available to authorized users.

Structural features of FAP174, a MYCBP-1 orthologue from Chlamydomonas reinhardtii, revealed by computational and experimental analyses

“…Furthermore, a multiple sequence alignment of FAP174 with other AKAP-interacting proteins such as RII and DPY-30 revealed two conserved VLV stretches at positions 21-23 and 25-27. 12 Building upon these previous results, here, we investigate the role of C46 and the two conserved VLV stretches on the secondary structure and oligomerization properties of FAP174. We did so by applying conventional gel electrophoresis aer subjecting wild-type (WT) FAP174 and FAP174 variant proteins (FAP174VLV21-23AAA, FAP174VLV25-27AAA and FAP174C46A) with an increasing concentration of a reducing agent such as DTT, biophysical techniques such as MALDI-TOF, CD, FT-IR, and micro-Raman spectroscopy, as well as ab initio protein structure prediction and molecular dynamics (MD) simulations.…”

“…Additionally, FAP174 was found to be localized to the nucleus and basal body. 12 We then set out to search for a D/D domain on FAP174; this domain allows AKAP-binding proteins to interact with the amphipathic helix of all AKAPs as dimers. 13 We hypothesized that the only cysteine at position 46 of the FAP174 sequence could contribute to forming a dimer via a disulde bridge.…”

Radial Spokes—A Snapshot of the Motility Regulation, Assembly, and Evolution of Cilia and Flagella

“…But they share little semblance to each other, RSP7, or RSP11 except the RIIa domain (e.g., Newell et al 2008). The divergence of DD domain proteins may allow airway and testis to build slightly different RSs and perhaps CPs that also contains an RIIa-binding protein (Gaillard et al 2001;Rao et al 2016). Some of them bind to "AKAPs" in the fibrous sheath of sperm flagella (e.g., Fiedler et al 2012).…”

MYC-Binding Protein-1 is an R2D2 Protein

“…Our group found independently that FAP174 is a conserved structural part in a novel molecular complex present in the C2 microtubule of CP apparatus. We surmise that FAP174 is definitely not a physiological interactor of RSP3 [29]. Its presence in the CP, basal body or TZ, nucleus and cytoplasm could indicate that it is trafficking between the various locations of the Chlamydomonas vegetative cell after synthesis.…”

“…When the secondary structure was predicted, the Cterminus helix exhibited a strong propensity to form a coiled-coil motif, the latter being known for protein-protein interaction. Therefore, it was speculated that FAP174 harbors two molecular modules, one for binding to the AH of an AKAP and the other for partnering with proteins [29]. One of these modules was tested for interaction with a flagellar protein containing AH, viz.…”