HDAC6 interacts with FTase. A, exogenous HDAC6 interacts with exogenous FTase. HEK293 cells were transfected with different FLAG-tagged plasmids (empty vector, WT HDAC6, or TRAF3) and HA-FTβ plasmid. Cell lysates (500 μg) were immunoprecipitated (IP) with an anti-FLAG antibody and immunoblotted (IB) as indicated. B, exogenous HDAC6 interacts with endogenous FTase-α and FTase-β. A549 cells were transfected with different FLAG-tagged constructs (HDAC6, vector, or TRAF3), immunoprecipitated as in A, and immunoblotted for endogenous FTase subunits, as indicated. C, endogenous HDAC6 interacts with endogenous FTase. A549 cells were immunoprecipitated with anti-HDAC6 or IgG control and immunoblotted as indicated.

FTase interacts directly with microtubules via its α subunit. A, FTase binding to tubulin is mediated by the FTase-α subunit. Purified MBP-tagged FTα (MBP-FTα) or MBP alone immobilized on amylase beads and GST-tagged FTase-β (GST-FTβ) or GST alone immobilized on glutathione beads were incubated with cell lysates. Bound protein fractions were resolved by SDS-PAGE and immunoblotted (IB) for the presence of FTase subunits and tubulin. No tubulin was detected in control MBP or GST beads. L, protein ladder. B, deletion of the C terminus of tubulin abolishes the interaction between FTase and microtubules. The C terminus of α- and β-tubulin in preformed microtubules was removed by proteolysis with subtilisin (MTΔC). The in vitro interaction between FTase and intact (MT) or cleaved microtubules was evaluated as in A. C, MAPs interfere with the binding of tubulin to FTase. MBP control or MBP-FTα beads were incubated with preformed MAP-containing or MAP-free microtubule protein.

Effect of FTase-αKD on HDAC6 activity. A, Western blotting of cell lysates from parental A549, control (puro) and FTase-α (FTαKD) shRNA cell lines. Inhibition of FTase and GGTase activity is shown by the appearance of a nonfarnesylated (N) band for H-Ras or a nonprenylated (U) band for Rap1, respectively. Note the appearance of the nonfarnesylated and nonprenylated bands in untreated FTαKD cells, indicating basal level inhibition of protein farnesylation and geranylgeranylation, as opposed to their appearance in treated-only parental and control cells. B, effect of FTase-αKD on the tubulin deacetylase activity of HDAC6. Note the increase in basal levels of tubulin acetylation concomitant with decrease of FTase-α proteins in several of the FTαKD clones obtained form pooled shRNAs (clones All) and from single shRNAs (84-1 and 84-2 from shRNA TRCN34584 and 85-6 and 85-9 from shRNA TRCN34585) compared with the A549 parental or puro control cells. C, knockdown of FTase-α in cells results in slower cell growth evaluated by sulforhodamine B staining and OD measurement at 564 nm. D, increased sensitivity of A549-FTαKD cells to Taxol, FTI, and their combination. Cells were treated with different concentrations of LNF, Taxol, or their combination for 72 h. Cell cytotoxicity was then evaluated by sulforhodamine B staining, and cell survival was determined as the percentage of cells remaining after treatment. Student's t test was performed, and results are labeled as p < 0.05 (*) and p < 0.005 (**).

Microtubules are required for the HDAC6-FTase interaction. A, effects of microtubule inhibitors on the HDAC6-FTase interaction. HEK293 cells were transfected as in Fig. 1A, treated with 10 μm Taxol (PTX), vinblastine (VBL), or colchicine (COL) for 6 h at 37°C. Cell lysates were immunoprecipitated (IP) with anti-FLAG antibodies and immunoblotted as indicated. B, endogenous α-tubulin interacts with endogenous FTase and HDAC6. A549 cells were immunoprecipitated with anti-α-tubulin or IgG and immunoblotted as indicated. C, role of microtubules and soluble tubulin dimers on the HDAC6-FTase interaction in vitro. Purified His-HDAC6 was incubated with purified GST or GST-FTase immobilized on glutathione-agarose beads in the presence of preformed Taxol-stabilized, MAP-free microtubules (MT), or MAP-free nonpolymerized tubulin (Tubulin). The final concentration for each protein was 5 μm. Samples were centrifuged, and proteins bound to the GST-FTase beads (B) or unbound fractions (U) were detected by immunoblotting. D, FTase interacts directly with microtubules in vitro. MAP-free Taxol-stabilized preformed microtubules or nonpolymerized soluble tubulin were incubated with purified GST or GST-FTase immobilized on glutathione-agarose beads. Samples were centrifuged, and proteins bound to the beads or present in the unbound fraction were detected by immunoblotting.

Effects of FTIs on the HDAC6-microtubule-FTase complex and activity. A, FTIs abrogate the FTase-HDAC6 interaction and the FTase-microtubule (MT) interaction but not the HDAC6-microtubule interaction. A549 cells were treated with 10 μm FTIs or 0.5 μm TSA for 16 h at 37 °C. Cell lysates were immunoprecipitated (IP) with anti-HDAC6 or anti-α-tubulin antibodies and immunoblotted as indicated. Tubulin acetylation and HDJ-2 farnesylation were examined to show the activity of TSA and FTIs, respectively. N, nonfarnesylated HDJ-2; F, farnesylated HDJ-2. B, interactions between WT and Y361L mutant FTase with WT and H216A/H611A mutant HDAC6 and the effect of LNF on these interactions. HEK293 cells were transfected with various plasmid combinations, treated or untreated with 10 μm LNF for 16 h at 37 °C, immunoprecipitated, and immunoblotted as indicated. C, LNF inhibits the activity of wild type but not the Y361L mutant FTβ. D, effects of FTIs on the tubulin deacetylase activity of HDAC6 in vitro. Preformed MAP-enriched microtubules that are heavily acetylated (lane 1) were incubated with FLAG-HDAC6 immunoprecipitates (IP) from A549 cells transfected with FLAG-HDAC6 or incubated with affinity-purified His-HDAC6 in the presence of various compounds for 2 h at 37°C. HDAC6 activity was evaluated by the decrease of acetylated tubulin examined by SDS-PAGE and immunoblotting. FLAG-HDAC6 (H216A/H611A) mutant IP (Mut) was included as a control. FTIs were used at 10 μm and TSA at 0.5 μm.