Determinants for Aurora-A Activation and Aurora-B Discrimination by TPX2

Abstract

View related articles © L a n d e s B i o s c i e n c e 2 0 0 4 . N o t f o r d i s t r i b u t i o n . ABSTRACTThe mitotic kinases Aurora-A and Aurora-B have similar amino-acid sequences but are differently localised and regulated during cell division. The basis for their interactions with different and specific regulators is unclear. Surprisingly, our recent structural studies indicate that TPX2 regulates Aurora-A activity by binding at a site that is conserved almost completely on Aurora-B. Here we investigate molecular determinants of TPX2-Aurora-A recognition. Using structure-based mutagenesis, we show that a single amino-acid difference on the surface of the kinase catalytic domain is key to the precision with which TPX2 discriminates between Aurora-A and Aurora-B. The conservation at this amino-acid position suggests that this discriminatory mechanism is likely to be conserved in higher eukaryotes.

The Aurora A and Aurora B Protein Kinases: A Single Amino Acid Difference Controls Intrinsic Activity and Activation by TPX2

“…The specific activity of G205N Aurora A was 10-fold lower, approximating the specific activity of WT Aurora B towards histone H3. This finding was not unexpected, due to the close proximity and suggested importance of this surface residue for binding of Aurora A to its specific substrate TPX2, 27,31 and our data confirms that G205 is important for substrate binding. Nevertheless, it is remarkable that the large difference in intrinsic activity of the two kinases in the absence of regulatory subunits can be accounted for by a single amino acid difference.…”

“…This suggests that even weak binding of TPX2 may be sufficient for enzymatic activation, whereas tighter binding is needed to affect the structural changes in the activation loop required for protection of T288 from PP1, as reported for the TPX2 W34E mutant. 31 In particular, it is possible that the C-terminal helix may not protect T295 in the G205N Aurora A mutant partially because of the weaker binding of TPX2 in general. In any case, our biochemical dissociation of the activating effect of TPX2 from its phosphatase protection activity (Fig.…”

“…27 It was important to test whether the amino acid at this position in Xenopus Aurora B (N158) was responsible for the marked difference in basal catalytic activity between Aurora A and Aurora B, in part because the equivalent residue in Xenopus Aurora A (G 205) is a direct determinant for high affinity interaction of Aurora A with TPX2. 27,31 As shown in Figure 1C, mutation of N158 to A did not lead to activation of Aurora B; indeed the resulting mutant exhibited activity equivalent to mutant or dephosphorylated versions of the kinase. However, mutation of N 158 to G, thereby replicating the primary sequence of Aurora A in this region (Fig.…”

A single amino acid change converts Aurora-A into Aurora-B-like kinase in terms of partner specificity and cellular function

“…S2 A). Consistent with previous reports (29,30), the kinase activity of Aurora-A G198N was reduced by 10-fold compared with that of Aurora-A WT (Fig. S3).…”

“…Because the protein sequences of their catalytic domains are very similar, we are interested in understanding how distinct localization and function are determined. Although it has been reported that G198 in human Aurora-A (29) [G205 in Xenopus (30)] is a determinant for its regulation by TPX2 in vitro, the molecular mechanism and the biological effects of the G-N mutation were unknown. Here, we propose that the G-N mutation converts Aurora-A into Aurora-B-like kinase in cell (Fig.…”