Direct interaction of Gas11 with microtubules: Implications for the dynein regulatory complex

Abstract

We previously described the Trypanin family of cytoskeleton-associated proteins that have been implicated in dynein regulation [Hill et al., J Biol Chem2000; 275(50):39369-39378; Hutchings et al., J Cell Biol2002;156(5):867-877; Rupp and Porter, J Cell Biol2003;162(1):47-57]. Trypanin from T. brucei is part of an evolutionarily conserved dynein regulatory system that is required for regulation of flagellar beat. In C. reinhardtii, the trypanin homologue (PF2) is part of an axonemal 'dynein regulatory complex' (DRC) that functions as a reversible inhibitor of axonemal dynein [Piperno et al., J Cell Biol1992;118(6):1455-1463; Gardner et al., J Cell Biol1994;127(5):1311-1325]. The DRC consists of an estimated seven polypeptides that are tightly associated with axonemal microtubules. Association with the axoneme is critical for DRC function, but the mechanism by which it attaches to the microtubule lattice is completely unknown. We demonstrate that Gas11, the mammalian trypanin/PF2 homologue, associates with microtubules in vitro and in vivo. Deletion analyses identified a novel microtubule-binding domain (GMAD) and a distinct region (IMAD) that attenuates Gas11-microtubule interactions. Using single-particle binding assays, we demonstrate that Gas11 directly binds microtubules and that the IMAD attenuates the interaction between GMAD and the microtubule. IMAD is able to function in either a cis- or trans-orientation with GMAD. The discovery that Gas11 provides a direct linkage to microtubules provides new mechanistic insight into the structural features of the dynein-regulatory complex.

Growth Arrest Specific 8 (Gas8) and G Protein-coupled Receptor Kinase 2 (GRK2) Cooperate in the Control of Smoothened Signaling

“…To avoid interference with the proteinprotein interaction, the immunoprecipitation buffer contained non-ionic detergent that mainly solubilizes the cytoplasmic detergent-soluble fraction of Gas8 (20). This fraction may be enriched by the transient transfection of Flag-Gas8 and probably by microtubule de-polymerization in the absence of microtubule-stabilizer (17). An aliquot of the extract (2%) was used as the input, while the remaining extract was incubated with Flag antibody beads (Sigma) that had been pre-blocked with 0.5% bovine serum albumin for 1 h. Extracts and beads were allowed to mix at 4°C overnight.…”

“…S1). The observation that only a small fraction of Gas8 FL is distributed to the cilia basal body could reflect a weak and transient binding to microtubules (17). Interestingly, Gas8 CT and the truncated Gas8 IMAD-GMAD constructs are not associated with microtubules, but accumulate in the cytoplasm or in aggregates, respectively.…”

“…This conserved family of microtubule-associated proteins encodes for the Dynein Regulatory Complex (DRC) subunit 4 (15,16). Several different domains within Gas8 have been identified with different affinities for microtubules, including a microtu-bule association domain (GMAD) between residues 115-258 and the N-terminal inhibitor of microtubule association domain (IMAD) between residues 1-108 (17). In this study, an engineered protein consisting of the GMAD and C terminus domains of Gas8 (Gas8 GMAD-CT ) displayed the highest affinity to microtubules.…”

“…Gas8 GMAD-CT Localization to the Cilia Basal Body Is Associated with Smo Ciliary Localization-To assess which domain of Gas8 is important for its effect on Smo and to tests whether altering the ability of Gas8 to interact with microtubules results in differential effect on Smo signaling we generated four different Flag-or GFP-tagged Gas8 fusion proteins consisting of the different mouse (m)Gas8 domains initially described in Bekker et al (17) and illustrated in Fig. 2A: mGas8 1-478 (fulllength, Gas8 FL ), mGas8 100 -478 (Gas8 GMAD-CT ), mGas8 (Gas8 IMAD-GMAD ), and mGas8 386 -478 (Gas8 CT ).…”

“…2A: mGas8 1-478 (fulllength, Gas8 FL ), mGas8 100 -478 (Gas8 GMAD-CT ), mGas8 (Gas8 IMAD-GMAD ), and mGas8 386 -478 (Gas8 CT ). The last construct consists of the 93 amino acids that bind Smo C terminus and is shorter than the C-terminal construct designed by Bekker et al (17). We followed the subcellular localization of GFP tagged-mGas8 fusion proteins in C3H10T1/2 cells, which we have previously used to monitor Smo-dependent signaling (11).…”