Protein kinase C (PKC)-mediated phosphorylation of α-tubulin
at Ser165 or expression of phosphomimetic (S165D)-α-tubulin
stimulates microtubule (MT) polymerization (Cytoskeleton
2014, 71, 257–272). Ser165
lies near the interface between adjacent αβ-tubulin heterodimers
and helix H8, which contains Glu254, the catalytic residue in α-tubulin
that hydrolyzes the exchangeable GTP in β-tubulin (β:GTP)
and triggers MT depolymerization. It was hypothesized that S165D,
a phosphomimetic variant of α-tubulin, perturbs the alignment
of α:Glu254 with respect to β:GTP, thereby impairing its
hydrolysis. Molecular simulations were performed with cryoEM structures
of MTs (PDB ID: 3J6E) in which phosphomimetic S165D or control S165N had been substituted.
Unlike native and S165N structures, the distance between S165D and
α:Glu254 increased by 0.6 Å, while the distance between
α:Glu254 and β:GTP decreased by 0.4 Å. Rotation of
β:GTP by 4 Å occurred in the S165D variant, whereas β:GTP
in the S165N control was unchanged from the native structure. Additionally,
the S165D variant exhibited an altered pattern of H-bonding to β:GTP,
including the loss of three H-bonds. The significance of these findings
to β:GTP hydrolysis was analyzed in MCF-10A human breast cells
treated with an antibody that detects GTP-bound tubulin. Compared
with controls, GTP–tubulin signals were at higher levels in
cells that ectopically expressed S165D-α-tubulin (TUBA1C) or
had been treated with PKC activator DAG-lactone to induce phosphorylation
of Ser165 in native α-tubulin. These findings support a model
whereby conformational changes induced by Ser165 phosphorylation alter
the spatial relationship between β:GTP and α:Glu254, thereby
slowing GTP hydrolysis and promoting GTP caps.