Biochem. J. (2005) 390
(293–302) (Printed in Great Britain)
Aurora-A site specificity: a study with synthetic peptide substrates
Stefano FERRARI*1, Oriano MARIN†‡, Mario A. PAGANO†, Flavio MEGGIO†, Daniel HESS§, Mahmoud EL-SHEMERLY*, Agnieszka KRYSTYNIAK* and Lorenzo A. PINNA†‡1
*Institute of Molecular Cancer Research, University of Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland, †Department of Biological Chemistry, University of Padova, Viale G. Colombo 3, I-35121 Padova, Italy, ‡Venetian Institute for Molecular Medicine, Via Orus 2, I-35129 Padova, Italy, and §Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland
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.
Key words: Aurora-A, consensus sequence, peptide substrate, phosphorylation, protein kinase, site specificity.
Abbreviations used: AurA, Aurora-A; CDC2, cell division cycle 2 kinase; CK1, casein kinase 1; DTT, dithiothreitol; HEK-293T cells, human embryonic kidney 293T cells; kd-AurA, kinase-dead AurA; PKA, protein kinase A; wt-AurA, wild-type AurA.
1Correspondence may be addressed to either of the authors (email sferrari@imcr.unizh.ch and lorenzo.pinna@unipd.it).
Received 25 February 2005/28 April 2005; accepted 4 May 2005
Published as BJ Immediate Publication 9 August 2005, doi:10.1042/BJ20050343
The Biochemical Society, London ©2005
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