A cancer-associated aurora A mutant is mislocalized and misregulated due to loss of interaction with TPX2

Abstract

Mutations in protein kinases can drive cancer through alterations of the kinase activity or by uncoupling kinase activity from regulation. Changes to protein expression in Aurora A, a mitotic Ser/Thr kinase, are associated with the development of several human cancers, but the effects of somatic cancer-associated mutations have not been determined. In this study we show that Aurora A kinase activity is altered in different ways in three somatic cancer-associated mutations located within the catalytic domain; Aurora A(V174M) shows constitutively increased kinase activity, Aurora A(S155R) activity is decreased primarily due to misregulation, and Aurora A(S361*) activity is ablated due to loss of structural integrity. These alterations suggest vastly different mechanisms for the role of these three mutations in human cancer. We have further characterized the Aurora A(S155R) mutant protein, found that its reduced cellular activity and mislocalization are due to loss of interaction with TPX2, and deciphered the structural basis of the disruption at 2.5 A resolution. Previous studies have shown that disruption of the Aurora A/TPX2 interaction results in defective spindles that generate chromosomal abnormalities. In a panel of 40 samples from microsatellite instability-positive colon cancer patients, we found one example in which the tumor contained only Aurora A(S155R), whereas the normal tissue contained only wild-type Aurora A. We propose that the S155R mutation is an example of a somatic mutation associated with this tumor type, albeit at modest frequency, that could promote aneuploidy through the loss of regulated interactions between Aurora A and its protein partners.

FIGURE 1.

Biochemical activity and localization of Aurora A. a, shown is the location of somatic mutations within the secondary structure of the Aurora A kinase domain, residues 122–403. β-Strands are marked as arrows, α-helices are shown as zigzags, and the mutations are marked in red. The activation loop, including the DFG motif, is shown as a thicker line. b, in vitro kinase activity of Aurora A proteins expressed in human cells is shown. Immunoprecipitation is shown of wild-type Aurora A, Aurora A(S155R), Aurora A(V174M), and Aurora A(S361*) or empty vector followed by a kinase assay using MBP as a substrate. Immunoprecipitated wild-type and mutant Aurora A proteins were visualized by immunoblotting as loading control. c, shown is in vitro activity of recombinant Aurora A kinase domain proteins expressed in E. coli using MBP as a substrate in the presence of increasing concentrations of TPX2 activator. Band intensity was quantified using ImageJ software (46). Data are expressed as average intensity. Error bars represent S.D. (n = 3). d, HeLa cells expressing GFP (green) and Myc-tagged wild-type Aurora A (WT), Aurora A(S155R), Aurora A(V174M), and Aurora A(S361*) stained with α-Myc antibody (red). 4′,6-Diamidino-2-phenylindole staining (blue) indicates the DNA content (arrows show the centrosomes in mitotic cells; the bar = 20 μm).

FIGURE 2.

Aurora A interaction with TPX2 and PP1 and identification of Aurora A(S155R) mutation in DNA samples from colon cancers. a, co-precipitation assay for the interaction of TPX2 and Aurora A recombinant proteins expressed in E. coli. Glutathione S-transferase-tagged TPX2 or control glutathione S-transferase-tagged TPX2 mutant were incubated with glutathione beads and Aurora A kinase domain. After washing, proteins bound to beads were separated by SDS-PAGE and visualized using Coomassie (upper two lanes) or immunoblotting (IB) using a phosphospecific Thr(P)-288 antibody. All three input Aurora A proteins were autophosphorylated on Thr-288. WT, wild type. b, immunoprecipitation (IP) assay for the interaction of TPX2 and Aurora A from human cell extracts. Wild-type and mutant Myc-tagged Aurora A was immunoprecipitated from 293T cell extracts followed by immunoblotting using α-TPX2 antibody. Total Aurora A levels are shown in the lower panel. c, in vitro dephosphorylation assay of recombinant wild-type Aurora A and protection from dephosphorylation by TPX2 is shown. Wild-type Aurora A, Aurora A(S155R), kinase inactive Aurora A(D274N), and Aurora A(V174M) were incubated with PP1 phosphatase. Proteins were separated by SDS-PAGE and visualized with Coomassie (upper two lanes), Thr(P)-288 Western blot (third lane), and anti-PP1 Western blot (bottom lane). The asterisk indicates PP1 protein that migrates at the same level as Aurora A. d, DNA sequence analysis from a primary human tumor from the large intestine and plasma from the same patient is shown.

FIGURE 3.

S155R mutation introduces an ordered, localized alteration in the structure of Aurora A. a, shown is superposition of Aurora A(S155R) (yellow) and TPX2/wild-type (WT) Aurora A (purple/cyan). The region around the mutation is indicated by a gray box. b, 2F_o − F_c electron density map contoured at 1.0σ in the vicinity of residue 155 is shown. c, superposition of Aurora A(S155R) and TPX2/WT Aurora A in the vicinity of residue 155 shows the steric clash between Arg-155/Phe-157 and the TPX2 binding site around F19. d, structure of Aurora A(S155R) around the binding site for a small, flat, hydrophobic molecule, modeled as the adenine of ADP is shown. e, the structure of TPX2/WT Aurora A is shown in the same view as c and d.