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

Abstract

Background: Flagella 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.

Results: We 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.

Conclusion: It 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.

Keywords: A-kinase anchoring proteins (AKAPs); Central pair; Chlamydomonas reinhardtii; FAP174; Flagella; MYCBP-1.

Fig. 1

FAP174 harbours a RII-like fold (a) A BLAST of FAP174 was carried out and representative MYCBP-1-like sequences from species of plants, animals, algae and protozoans were selected and a phylogenetic tree was generated using MEGA6. Organisms and their Accession numbers used for this exercise are C. reinhardtii ACR55627, V. carterii XP_002950671.1, B. rapa NP_001288931.1, A. thaliana NP_671849.1, C. arabica ADY38785.1, C. sativus XP_004172365.1, O. sativa EEC81635.1, S. bicolor XP_002459454.1, S. lycopersicon XP_004236796.1, P. sitchensis ABR18049.1, A. mellifera XP_624300.1, S. kowaleskii XP_002733639.1, X. tropicalis NP_001017035.1, M. Domestica XP_001364995.1, P. troglodytes XP_003949426.1, M. mulatta XP_001113156.2, H. sapiens BAA09338.1, P. tetraaurelia XP_001442915.1, T. cruzi XP_805155.1, P. sojae XP_009522570.1, A. anophagefferens XP_009033259.1, E. siliculosus CBN77061.1 and T. gondii XP_002370315.1. (b) Alignment of representative sequences from those used for the Phylogenetic analysis. The sequence homology is the highest at the N-terminal region that is predicted to fold into a helix-loop-helix structure as expected of dimerization and docking domains. The C-terminus of FAP174 shows a propensity to form coiled-coils. Identical residues are shaded, arrows represent the helix-forming residues and the lines represent coiled-coil forming residues. (c) Multiple alignment of FAP174 with proteins contianing the D/D domain using the best matches from HHpred. (http://toolkit.tuebingen.mpg.de/hhpred). The sequences with the RIIa D/D domain and DPY-30 domains that showed significant match were aligned using Multiple Sequence Alignment software (Clustal Omega). Identical regions are shaded

Fig. 2

The Radial spoke and Central pair AKAPs. Schematics depicting the two AKAPs in the 9 + 2 axoneme. AKAP97 is RSP3 proposed to be a scaffold protein binding to radial spoke proteins (RSPs) with RII and DPY30 domains. The identity and partners of AKAP240 in C2 of the central pair apparatus remain elusive. This study attempts to identify these interactors. Only part of the 9 + 2 axoneme cross section is illustrated. Also note, that the entire RSP harbours 23 different proteins; however, the figure depicts molecules significant for the current study

Fig. 3

Sub-flagellar localization of FAP174. (a) FAP174 Western blot of axonemes from WT and flagellar mutants. A polypeptide of ~10 kDa was present in the axonemes of WT and the radial spokes mutant pf14; whereas, among the central pair mutants it was reduced in the pf15 and pf20, and absent in pf18 and pf19 that lack the central pair microtubules. The presence of FAP174 in pf16 axoneme that lack the C1 microtubule suggest that FAP174 associates with the C2 microtubule. (b) The Ponceau-stained tubulin bands show equal loading of axonemal proteins. (c-e) Immunofluorescent microscopy of whole cells using pre-immune serum as a control to show autofluorescence and non-specificity of the antibody (images captured at 63x magnification). (f-q) Immunofluorescent microscopy of FAP174 comparing whole cells and nucleo-flagella apparatus (NFA) from wild type and pf18. The samples were decorated with anti-acetylated tubulin or anti-FAP174 as indicated. FAP174 localizes to the flagella as well as to the base of flagella possibly the transition zone (arrows). FAP174 was not detectable in the pf18 flagella but appeared as the bright spot at the base of flagella. (r-t) Localization of FAP174 around nucleus is more prominent in cells with low autofluorescence background and one such representative image is depicted here. The scale bars in the figure indicates 10 μm

Fig. 4

Interaction of FAP174 with GST-RSP3-AH 96–180 in vitro. (a) Purified proteins used for the assay. (b)  Negative control for the assay, which depicts no interaction between Arl6 and GST-RSP3 AH(96-180). (c) Co-purification of FAP174 with GST or GST-RSP3-AH 96–180. Testing the in vitro interaction of the GST-RSP3-AH 96–180 with 6His-FAP174 protein. Note the rather weak interaction as evidenced in the last lane (arrows)

Fig. 5

The recognition of AKAP240 by recombinant FAP174 and RII in vitro. (a) The blots were probed with recombinant 6His-tagged RIIa D/D protein and revealed by HRP-conjugated anti-His antibody. The polypeptide migrated above 250 kDa in the axonemes of WT, pf14 and pf20 but were absent in mutants lacking the central pair, as expected of AKAP240. (b) 6His-FAP174 overlays also show the same band above 250 kDa. 6His-FAP174 also reacted with two other proteins. The polypeptide migrated above 250 kDa in the axonemes of WT, pf14 and pf20 but were absent in mutants lacking the central pair. (c) Control overlay blots with WT and mutant axonemes were probed with the anti-His antibody coupled to HRP without the interacting proteins