AurA (Aurora-A) is a ubiquitous
protein kinase regulating entry into mitosis and shown to promote transformation upon overexpression. In order to gain information on the structural features determining its substrate specificity, we assayed human recombinant
AurA on a variety of phosphoacceptor
peptide substrates including a series of properly modified derivatives of the
Kemptide (ALRRASLGAA). The data presented here show that
AurA is a basophilic Ser/Thr
protein kinase recognizing the consensus R/K/N-R-X-S/T-B, where B denotes any hydrophobic residue with the exception of Pro. We show that the presence of a Pro at position n+1 fully abrogates phosphorylation of the
peptide substrate. Although the consensus for
AurA is reminiscent of that of PKA (
protein kinase A), it significantly differs from the latter for a much more stringent dependence on the hydrophobic residue at n+1 and for its tolerance of residues other than Arg at position n-3. Based on the finding that the
peptide ALKRASLGAA is not a substrate of PKA while still providing a sensitive assay of
AurA activity, we suggest that this
peptide may be used for differential screening of the two
kinases. We have further validated the
AurA consensus by generating a
peptide (APSSRRTT288LCGT) that comprises the main
AurA autophosphorylation site and by showing that
AurA phosphorylated this
peptide exclusively at one site fulfilling its consensus (Thr288). Moreover, we show that
AurA could autophosphorylate at Thr288 through an intermolecular mechanism of reaction and that, in vivo, PKA was not involved with Thr288 phosphorylation. The evidence obtained in the present study provides a rational tool for predicting
AurA sites in potential substrates of physiological significance.