Deactylase Inhibitors Disrupt Cellular Complexes Containing Protein Phosphatases and Deacetylases

Abstract

Affinity isolation of protein serine/threonine phosphatases on the immobilized phosphatase inhibitor microcystin-LR identified histone deacetylase 1(HDAC1), HDAC6, and HDAC10 as novel components of cellular phosphatase complexes. Other HDACs, specifically HDAC2, -3, -4, and -5, were excluded from such complexes. In vitro biochemical studies showed that recombinant HDAC6, but not HDAC4, bound directly to the protein phosphatase (PP)1 catalytic subunit. No association was observed between HDAC6 and PP2A, another major protein phosphatase. PP1 binding was mapped to the second catalytic domain and adjacent C-terminal sequences in HDAC6, and treatment of cells with trichostatin A (TSA) disrupted endogenous HDAC6·PP1 complexes. Consistent with the inhibition of tubulin deactylase activity of HDAC6, TSA enhanced cellular tubulin acetylation, and acetylated tubulin was present in the PP1 complexes from TSA-treated cells. Trapoxin B, a weak HDAC6 inhibitor, and calyculin A, a cell-permeable phosphatase inhibitor, had no effect on the stability of the HDAC6·PP1 complexes or on tubulin acetylation. Mutations that inactivated HDAC6 prevented its incorporation into cellular PP1 complexes and suggested that when bound together both were TSA disrupted the cellular complexes into the in cellular protein and and suggested that in protein to the of to and Affinity isolation of protein serine/threonine phosphatases on the immobilized phosphatase inhibitor microcystin-LR identified histone deacetylase 1(HDAC1), HDAC6, and HDAC10 as novel components of cellular phosphatase complexes. Other HDACs, specifically HDAC2, -3, -4, and -5, were excluded from such complexes. In vitro biochemical studies showed that recombinant HDAC6, but not HDAC4, bound directly to the protein phosphatase (PP)1 catalytic subunit. No association was observed between HDAC6 and PP2A, another major protein phosphatase. PP1 binding was mapped to the second catalytic domain and adjacent C-terminal sequences in HDAC6, and treatment of cells with trichostatin A (TSA) disrupted endogenous HDAC6·PP1 complexes. Consistent with the inhibition of tubulin deactylase activity of HDAC6, TSA enhanced cellular tubulin acetylation, and acetylated tubulin was present in the PP1 complexes from TSA-treated cells. Trapoxin B, a weak HDAC6 inhibitor, and calyculin A, a cell-permeable phosphatase inhibitor, had no effect on the stability of the HDAC6·PP1 complexes or on tubulin acetylation. Mutations that inactivated HDAC6 prevented its incorporation into cellular PP1 complexes and suggested that when bound together both were TSA disrupted the cellular complexes into the in cellular protein and and suggested that in protein to the of to and protein the protein of and that protein of histone and that both protein in and cellular that the and of and cellular of protein and and of histone on in in to or the of histone to that the adjacent the of histone in and of histone novel that complexes and cells the and of histone or of complexes the of into complexes to the and of complexes and to the association of the and protein phosphatase protein histone trichostatin protein histone trichostatin in histone in and cells that protein with studies that the association of the protein with a histone and the of the with the of to studies identified a that and the HDACs, and was or to the of the was not studies that deacetylase activity both and on of and in the and of as as in both and present studies were cellular complexes in and the between and phosphatases that the of cellular studies that but not complexes protein serine/threonine biochemical studies on the HDAC6 and both to studies that HDAC6 directly bound PP1 and showed that of HDAC6 or disrupted the cellular HDAC6·PP1 of HDAC6 tubulin in and acetylated tubulin was with the PP1 complexes. suggested that the HDAC6·PP1 the that the deacetylase inhibitor, trichostatin A, disrupted a novel protein and in cells. the histone TSA and deacetylase to and suggested that the of complexes and the in protein and together the activity of was from trichostatin A and from from from microcystin-LR and calyculin A from from cells and from from and from PP1 catalytic was from as and in were in and tubulin from from and from catalytic was HDAC4, -5, and as as HDAC6 were of and HDAC10 were as was HDAC6 with and and the C-terminal of HDAC6, specifically and were with a C-terminal into the were into the and in was to and were of in cells were in and in of were in of and of in cells were to of cells to were with or calyculin cells were with in were in and PP1 complexes were of the on were in of and to and with HDAC6·PP1 complexes were were with of and the were with with and were on PP1 or was with of or and were and the phosphatase activity binding to recombinant HDAC6 was recombinant to bound PP1 was with and phosphatase studies with recombinant to that a from a studies suggested that and in complexes that PP1 and complexes were and in cells and and HDAC4, -5, and as in cells with showed that were the cellular phosphatase were to on with a that bound of major serine/threonine phosphatase from with showed that HDAC6, and HDAC10 were present in the phosphatase complexes. HDAC2, HDAC4, and were excluded from phosphatase complexes or were components of phosphatase complexes that not with the phosphatase catalytic the PP1 the association of PP1 with HDACs, of HDAC6 and with showed PP1 bound HDAC6 of in a to suggested that PP1 binding to HDAC6 PP1 in of recombinant and that were to to and with B, PP1 was with of or complexes were and were phosphatase activity and was with and as and of serine/threonine phosphatase activity in cells and with of HDAC6 PP1 and with of recombinant or complexes were and phosphatase activity was the Consistent with the PP1 activity in a of of PP1 PP1 not in the of not of the was the that not or activity to both PP1 and not In that of of and that the PP1 from bound to in the not not with of HDAC6 with PP2A, bound not to the not that HDAC6 bound and HDAC6·PP1 in the of C-terminal of HDAC6 the domain in HDAC6, with C-terminal in cells and incorporation into endogenous PP1 complexes. with of HDAC6 with showed that PP1 from with that and HDAC6 of bound HDAC6 and to PP1 suggested that the between and in HDAC6 was PP1 the C-terminal domain of of HDAC6 with C-terminal catalytic and and the domain as B, phosphatase complexes were bound were to and with and with showed of HDAC6 from and and the bound to with showed that bound PP1 binding was to the domain to and the between and studies to the PP1 binding to not HDAC6 but and were into cellular PP1 domain in HDAC6 the second catalytic the of activity in PP1 endogenous HDAC6·PP1 complexes from cells of cells with the inhibitor TSA HDAC6 and the of a of the with TSA treatment had no effect on the of PP1 bound to no HDAC6 was in complexes. to TSA disrupted the HDAC6·PP1 that not the of cellular In a weak HDAC6 inhibitor had no effect on HDAC6 binding to endogenous PP1 or tubulin in cells. suggested that the HDAC6 to the cellular HDAC6·PP1 Consistent with the of the HDAC6·PP1 to the cell-permeable PP1 inhibitor calyculin A had no effect on the of a cellular HDAC6·PP1 inhibition PP1 cells were with trichostatin A or calyculin A that TSA was to to with PP1 complexes were on and with and were with to with tubulin HDAC6 were B, cells were with or HDAC6 were with or calyculin A as in and PP1 complexes on were with and were in cells as in and phosphatase complexes on and were with and cells were with or a PP1 was to phosphatase complexes from cells with or TSA in A and were with and and a of in cells with endogenous HDAC6, bound PP1 and was of cells with TSA or of TSA disrupted the In was not into cellular PP1 complexes. suggested that both HDAC6 and PP1 were in the cellular HDAC6·PP1 were with HDAC10 not the effect of TSA on phosphatase complexes and in cells were and into phosphatase complexes that bound TSA treatment prevented the incorporation of into the complexes. suggested that the inhibitor, disrupted the effect of TSA on PP1 binding to PP1 to protein of on showed that was into cellular PP1 complexes. TSA had no effect on the of a cellular were with the PP1 PP1 to the not that TSA and disrupted cellular of in the PP1 in cells the of the HDAC6·PP1 with A of the cellular tubulin was present in the endogenous HDAC6·PP1 from cells not with showed a of acetylated tubulin in the HDAC6·PP1 from cells HDAC6 inhibition TSA had no effect on the of tubulin bound to not but the acetylated tubulin present in PP1 complexes suggested that HDAC6·PP1 bound tubulin and in a of the HDAC6·PP1 TSA enhanced the of tubulin that bound to of the HDAC6·PP1 PP1 to acetylated of from cells with or TSA and to on or with that TSA enhanced tubulin in cells. with showed that bound of with the the of acetylated tubulin in phosphatase from cells with that protein and to the cell-permeable phosphatase inhibitor, enhanced both protein and in protein and of cellular complexes protein phosphatases and suggested a both in the of that and that of the and the HDAC2, PP2A, and of and histone and showed that a inhibitor, but the TSA the activity of a phosphatase In the present studies the of a cellular and showed that was disrupted the treatment of cells with the of PP1 from the and the of studies showed that not but HDAC6 and HDAC10 bound cellular HDAC2, HDAC4, and were not into phosphatase complexes that bound the of in the as such as the of and or association of with of to a PP1 with the of a a and the identified as a bound HDAC2, that the of the of such as PP2A, to the no association of with in and A suggested that a of HDAC6 into the of and to into complexes that the a HDAC6 its association with the to its into the to present studies a novel association of HDAC6 with a major cellular studies the association of HDAC6 with PP1 a not and studies showed that HDAC6 bound PP1 in the of cellular and the HDAC6·PP1 to HDAC6 catalytic domain and major components of and the of observed a of the cellular in the HDAC6·PP1 in cells. treatment with the inhibitor TSA the of the HDAC6·PP1 and enhanced the of that was with the PP1 B, a weak HDAC6 inhibitor, not the HDAC6·PP1 and to tubulin acetylation. that PP1 the of HDAC6 to In the HDAC6 in to the protein and to in to the that together both the HDAC6·PP1 to C-terminal mapped PP1 binding to the second domain and adjacent C-terminal HDAC6 activity was PP1 C-terminal that or HDAC6 activity not not the the PP1 binding to the studies suggested that PP1 bound to of HDAC6, as as of of HDAC6 the of a that in PP1 suggested a novel of PP1 binding to studies suggested that tubulin deacetylase activity of HDAC6 was to the second domain inhibition of domain the of tubulin but had a effect of histone the domain TSA to the C-terminal or second HDAC6 catalytic domain to the that PP1 the HDACs, HDAC6 HDAC10 a second domain and sequences deacetylase TSA the domain to the with a the that had on the of to PP1 to the In to the in HDAC6 and HDAC10 were observed in to PP1 suggested in association of PP1 with In inhibition TSA disrupted cellular PP1 complexes and to the of PP1 binding to and the of the complexes in cell-permeable phosphatase and calyculin A, had no effect on cellular complexes and a to in protein and and HDACs, and PP1 HDAC6 in cells and and to HDACs, the of HDAC6 its activity or its association with as or its as and HDAC6 the of the C-terminal or domain that and and the and of HDAC6 In to the of cellular but studies that the activity of in with the of complexes and in protein and and protein the protein of and that protein of histone and that both protein in and cellular that the and of and cellular of protein and and of histone on in in to or the of histone to that the adjacent the of histone in and of histone novel that complexes and cells the and of histone or of complexes the of into complexes to the and of complexes and to the association of the and protein phosphatase protein histone trichostatin protein histone trichostatin in histone in and cells that protein with studies that the association of the protein with a histone and the of the with the of to studies identified a that and the HDACs, and was or to the of the was not studies that deacetylase activity both and on of and in the and of as as in both and present studies were cellular complexes in and the between and phosphatases that the of cellular studies that but not complexes protein serine/threonine biochemical studies on the HDAC6 and both to studies that HDAC6 directly bound PP1 and showed that of HDAC6 or disrupted the cellular HDAC6·PP1 of HDAC6 tubulin in and acetylated tubulin was with the PP1 complexes. suggested that the HDAC6·PP1 the that the deacetylase inhibitor, trichostatin A, disrupted a novel protein and in cells. the histone TSA and deacetylase to and suggested that the of complexes and the in protein and together the activity of was from trichostatin A and from from from microcystin-LR and calyculin A from from cells and from from and from PP1 catalytic was from as and in were in and tubulin from from and from catalytic was HDAC4, -5, and as as HDAC6 were of and HDAC10 were as was HDAC6 with and and the C-terminal of HDAC6, specifically and were with a C-terminal into the were into the and in was to and were of in cells were in and in of were in of and of in cells were to of cells to were with or calyculin cells were with in were in and PP1 complexes were of the on were in of and to and with HDAC6·PP1 complexes were were with of and the were with with and were on PP1 or was with of or and were and the phosphatase activity binding to recombinant HDAC6 was recombinant to bound PP1 was with and phosphatase was from trichostatin A and from from from microcystin-LR and calyculin A from from cells and from from and from PP1 catalytic was from as and in were in and tubulin from from and from catalytic was HDAC4, -5, and as as HDAC6 were of and HDAC10 were as was HDAC6 with and and the C-terminal of HDAC6, specifically and were with a C-terminal into the were into the and in was to and were of in cells were in and in of were in of and of in cells were to of cells to were with or calyculin cells were with in were in and PP1 complexes were of the on were in of and to and with HDAC6·PP1 complexes were were with of and the were with with and were on PP1 or was with of or and were and the phosphatase activity PP1 binding to recombinant HDAC6 was recombinant to bound PP1 was with and phosphatase studies with recombinant to that a from a studies suggested that and in complexes that PP1 and complexes were and in cells and and HDAC4, -5, and as in cells with showed that were the cellular phosphatase were to on with a that bound of major serine/threonine phosphatase from with showed that HDAC6, and HDAC10 were present in the phosphatase complexes. HDAC2, HDAC4, and were excluded from phosphatase complexes or were components of phosphatase complexes that not with the phosphatase catalytic the PP1 the association of PP1 with HDACs, of HDAC6 and with showed PP1 bound HDAC6 of in a to suggested that PP1 binding to HDAC6 and of serine/threonine phosphatase activity in cells and with of HDAC6 PP1 and with of recombinant or complexes were and phosphatase activity was the Consistent with the PP1 activity in a of of PP1 PP1 not in the of not of the was the that not or activity to both PP1 and not In that of of and that the PP1 from bound to in the not not with of HDAC6 with PP2A, bound not to the not that HDAC6 bound and HDAC6·PP1 in the of C-terminal of HDAC6 the domain in HDAC6, with C-terminal in cells and incorporation into endogenous PP1 complexes. with of HDAC6 with showed that PP1 from with that and HDAC6 of bound HDAC6 and to PP1 suggested that the between and in HDAC6 was PP1 the C-terminal domain of of HDAC6 with C-terminal catalytic and and the domain as B, phosphatase complexes were bound were to and with and with showed of HDAC6 from and and the bound to with showed that bound PP1 binding was to the domain to and the between and studies to the PP1 binding to not HDAC6 but and were into cellular PP1 domain in HDAC6 the second catalytic the of activity in PP1 endogenous HDAC6·PP1 complexes from cells of cells with the inhibitor TSA HDAC6 and the of a of the with TSA treatment had no effect on the of PP1 bound to no HDAC6 was in complexes. to TSA disrupted the HDAC6·PP1 that not the of cellular In a weak HDAC6 inhibitor had no effect on HDAC6 binding to endogenous PP1 or tubulin in cells. suggested that the HDAC6 to the cellular HDAC6·PP1 Consistent with the of the HDAC6·PP1 to the cell-permeable PP1 inhibitor calyculin A had no effect on the of a cellular HDAC6·PP1 inhibition PP1 cells were with trichostatin A or calyculin A that TSA was to to with PP1 complexes were on and with and were with to with tubulin HDAC6 were B, cells were with or HDAC6 were with or calyculin A as in and PP1 complexes on were with and were in cells as in and phosphatase complexes on and were with and cells were with or a PP1 was to phosphatase complexes from cells with or TSA in A and were with and and a of in cells with endogenous HDAC6, bound PP1 and was of cells with TSA or of TSA disrupted the In was not into cellular PP1 complexes. suggested that both HDAC6 and PP1 were in the cellular HDAC6·PP1 were with HDAC10 not the effect of TSA on phosphatase complexes and in cells were and into phosphatase complexes that bound TSA treatment prevented the incorporation of into the complexes. suggested that the inhibitor, disrupted the effect of TSA on PP1 binding to PP1 to protein of on showed that was into cellular PP1 complexes. TSA had no effect on the of a cellular were with the PP1 PP1 to the not that TSA and disrupted cellular of in the PP1 in cells the of the HDAC6·PP1 with A of the cellular tubulin was present in the endogenous HDAC6·PP1 from cells not with showed a of acetylated tubulin in the HDAC6·PP1 from cells HDAC6 inhibition TSA had no effect on the of tubulin bound to not but the acetylated tubulin present in PP1 complexes suggested that HDAC6·PP1 bound tubulin and in a of the HDAC6·PP1 TSA enhanced the of tubulin that bound to of the HDAC6·PP1 PP1 to acetylated of from cells with or TSA and to on or with that TSA enhanced tubulin in cells. with showed that bound of with the the of acetylated tubulin in phosphatase from cells with studies with recombinant to that a from a studies suggested that and in complexes that PP1 and complexes were and in cells and and HDAC4, -5, and as in cells with showed that were the cellular phosphatase were to on with a that bound of major serine/threonine phosphatase from with showed that HDAC6, and HDAC10 were present in the phosphatase complexes. HDAC2, HDAC4, and were excluded from phosphatase complexes or were components of phosphatase complexes that not with the phosphatase catalytic HDAC6 the PP1 the association of PP1 with HDACs, of HDAC6 and with showed PP1 bound HDAC6 of in a to suggested that PP1 binding to HDAC6 PP1 and of serine/threonine phosphatase activity in cells and with of HDAC6 PP1 and with of recombinant or complexes were and phosphatase activity was the Consistent with the PP1 activity in a of of PP1 PP1 not in the of not of the was the that not or activity to both PP1 and not In that of of and that the PP1 from bound to in the not not with of HDAC6 with PP2A, bound not to the not that HDAC6 bound and HDAC6·PP1 in the of PP1 C-terminal of HDAC6 the domain in HDAC6, with C-terminal in cells and incorporation into endogenous PP1 complexes. with of HDAC6 with showed that PP1 from with that and HDAC6 of bound HDAC6 and to PP1 suggested that the between and in HDAC6 was PP1 from and and the bound to with showed that bound PP1 binding was to the domain to and the between and studies to the PP1 binding to not HDAC6 but and were into cellular PP1 complexes. domain in HDAC6 the second catalytic the of activity in PP1 endogenous HDAC6·PP1 complexes from cells of cells with the inhibitor TSA HDAC6 and the of a of the with TSA treatment had no effect on the of PP1 bound to no HDAC6 was in complexes. to TSA disrupted the HDAC6·PP1 that not the of cellular In a weak HDAC6 inhibitor had no effect on HDAC6 binding to endogenous PP1 or tubulin in cells. suggested that the HDAC6 to the cellular HDAC6·PP1 Consistent with the of the HDAC6·PP1 to the cell-permeable PP1 inhibitor calyculin A had no effect on the of a cellular HDAC6·PP1 and a of in cells with endogenous HDAC6, bound PP1 and was of cells with TSA or of TSA disrupted the In was not into cellular PP1 complexes. suggested that both HDAC6 and PP1 were in the cellular HDAC6·PP1 were with HDAC10 not the effect of TSA on phosphatase complexes and in cells were and into phosphatase complexes that bound TSA treatment prevented the incorporation of into the complexes. suggested that the inhibitor, disrupted complexes. the effect of TSA on PP1 binding to PP1 to protein of on showed that was into cellular PP1 complexes. TSA had no effect on the of a cellular were with the PP1 PP1 to the not that TSA and disrupted cellular complexes. HDAC6 of in the PP1 in cells the of the HDAC6·PP1 with A of the cellular tubulin was present in the endogenous HDAC6·PP1 from cells not with showed a of acetylated tubulin in the HDAC6·PP1 from cells HDAC6 inhibition TSA had no effect on the of tubulin bound to not but the acetylated tubulin present in PP1 complexes suggested that HDAC6·PP1 bound tubulin and in a of the HDAC6·PP1 TSA enhanced the of tubulin that bound to of the HDAC6·PP1 PP1 to acetylated that protein and to the cell-permeable phosphatase inhibitor, enhanced both protein and in protein and of cellular complexes protein phosphatases and suggested a both in the of that and that of the and the HDAC2, PP2A, and of and histone and showed that a inhibitor, but the TSA the activity of a phosphatase In the present studies the of a cellular and showed that was disrupted the treatment of cells with the of PP1 from the and the of studies showed that not but HDAC6 and HDAC10 bound cellular HDAC2, HDAC4, and were not into phosphatase complexes that bound the of in the as such as the of and or association of with of to a PP1 with the of a a and the identified as a bound HDAC2, that the of the of such as PP2A, to the no association of with in and A suggested that a of HDAC6 into the of and to into complexes that the a HDAC6 its association with the to its into the to present studies a novel association of HDAC6 with a major cellular studies the association of HDAC6 with PP1 a not and studies showed that HDAC6 bound PP1 in the of cellular and the HDAC6·PP1 to HDAC6 catalytic domain and major components of and the of observed a of the cellular in the HDAC6·PP1 in cells. treatment with the inhibitor TSA the of the HDAC6·PP1 and enhanced the of that was with the PP1 B, a weak HDAC6 inhibitor, not the HDAC6·PP1 and to tubulin acetylation. that PP1 the of HDAC6 to In the HDAC6 in to the protein and to in to the that together both the HDAC6·PP1 to C-terminal mapped PP1 binding to the second domain and adjacent C-terminal HDAC6 activity was PP1 C-terminal that or HDAC6 activity not not the the PP1 binding to the studies suggested that PP1 bound to of HDAC6, as as of of HDAC6 the of a that in PP1 suggested a novel of PP1 binding to studies suggested that tubulin deacetylase activity of HDAC6 was to the second domain inhibition of domain the of tubulin but had a effect of histone the domain TSA to the C-terminal or second HDAC6 catalytic domain to the that PP1 the HDACs, HDAC6 HDAC10 a second domain and sequences deacetylase TSA the domain to the with a the that had on the of to PP1 to the In to the in HDAC6 and HDAC10 were observed in to PP1 suggested in association of PP1 with In inhibition TSA disrupted cellular PP1 complexes and to the of PP1 binding to and the of the complexes in cell-permeable phosphatase and calyculin A, had no effect on cellular complexes and a to in protein and and HDACs, and PP1 HDAC6 in cells and and to HDACs, the of HDAC6 its activity or its association with as or its as and HDAC6 the of the C-terminal or domain that and and the and of HDAC6 In to the of cellular but studies that the activity of in with the of complexes and in protein and and that protein and to the cell-permeable phosphatase inhibitor, enhanced both protein and in protein and of cellular complexes protein phosphatases and suggested a both in the of that and that of the and the HDAC2, PP2A, and of and histone and showed that a inhibitor, but the TSA the activity of a phosphatase In the present studies the of a cellular and showed that was disrupted the treatment of cells with the of PP1 from the and the of studies showed that not but HDAC6 and HDAC10 bound cellular HDAC2, HDAC4, and were not into phosphatase complexes that bound the of in the as such as the of and or association of with of to a PP1 with the of a a and the identified as a bound HDAC2, that the of the of such as PP2A, to the no association of with HDAC6 in and A suggested that a of HDAC6 into the of and to into complexes that the a HDAC6 its association with the to its into the to present studies a novel association of HDAC6 with a major cellular studies the association of HDAC6 with PP1 a not and studies showed that HDAC6 bound PP1 in the of cellular and the HDAC6·PP1 to HDAC6 catalytic domain and major components of and the of observed a of the cellular in the HDAC6·PP1 in cells. treatment with the inhibitor TSA the of the HDAC6·PP1 and enhanced the of that was with the PP1 B, a weak HDAC6 inhibitor, not the HDAC6·PP1 and to tubulin acetylation. that PP1 the of HDAC6 to In the HDAC6 in to the protein and to in to the that together both the HDAC6·PP1 to C-terminal mapped PP1 binding to the second domain and adjacent C-terminal HDAC6 activity was PP1 C-terminal that or HDAC6 activity not not the the PP1 binding to the studies suggested that PP1 bound to of HDAC6, as as of of HDAC6 the of a that in PP1 suggested a novel of PP1 binding to studies suggested that tubulin deacetylase activity of HDAC6 was to the second domain inhibition of domain the of tubulin but had a effect of histone the domain TSA to the C-terminal or second HDAC6 catalytic domain to the that PP1 the HDACs, HDAC6 HDAC10 a second domain and sequences deacetylase TSA the domain to the with a the that had on the of to PP1 to the In to the in HDAC6 and HDAC10 were observed in to PP1 suggested in association of PP1 with In inhibition TSA disrupted cellular PP1 complexes and to the of PP1 binding to and the of the complexes in cell-permeable phosphatase and calyculin A, had no effect on cellular complexes and a to in protein and and HDACs, and PP1 HDAC6 in cells and and to HDACs, the of HDAC6 its activity or its association with as or its as and HDAC6 the of the C-terminal or domain that and and the and of HDAC6 In to the of cellular but studies that the activity of in with the of complexes and in protein and and and and with studies on HDAC6 that were not in and a of the the tubulin