Abstract
Aurora-A kinase is necessary for centrosome maturation, for assembly and maintenance of a bipolar spindle, and for proper chromosome segregation during cell division. Aurora-A is an oncogene that is overexpressed in multiple human cancers. Regulation of kinase activity apparently depends on phosphorylation of Thr-288 in the T-loop. In addition, interactions with targeting protein for Xenopus kinesin-like protein 2 (TPX2) allosterically activate Aurora-A. The Thr-288 phosphorylation is reversed by type-1 protein phosphatase (PP1). Mutations in the yeast Aurora, Ipl1, are suppressed by overexpression of Glc8, the yeast homolog of phosphatase inhibitor-2 (I-2). In this study, we show that human I-2 directly and specifically stimulated recombinant human Aurora-A activity in vitro. The I-2 increase in kinase activity was not simply due to inhibition of PP1 because it was not mimicked by other phosphatase inhibitors. Furthermore, activation of Aurora-A was unaffected by deletion of the I-2 N-terminal PP1 binding motif but was eliminated by deletion of the I-2 C-terminal domain. Aurora-A and I-2 were recovered together from mitotic HeLa cells. Kinase activation by I-2 and TPX2 was not additive and occurred without a corresponding increase in T-loop phosphorylation. These results suggest that both I-2 and TPX2 function as allosteric activators of Aurora-A. This implies that I-2 is a bifunctional signaling protein with separate domains to inhibit PP1 and directly stimulate Aurora-A kinase.
Publication types
-
Research Support, Non-U.S. Gov't
-
Research Support, U.S. Gov't, P.H.S.
MeSH terms
-
Animals
-
Aurora Kinases
-
Cell Cycle Proteins / genetics
-
Cell Cycle Proteins / metabolism
-
Enzyme Activation
-
Enzyme Inhibitors / metabolism
-
HeLa Cells
-
Humans
-
In Vitro Techniques
-
Microtubule-Associated Proteins / genetics
-
Microtubule-Associated Proteins / metabolism
-
Mutagenesis, Site-Directed
-
Neoplasm Proteins / genetics
-
Neoplasm Proteins / metabolism
-
Nuclear Proteins / genetics
-
Nuclear Proteins / metabolism
-
Phosphoprotein Phosphatases / metabolism*
-
Phosphoproteins / genetics
-
Phosphoproteins / metabolism
-
Phosphorylation
-
Protein Kinases / chemistry
-
Protein Kinases / genetics
-
Protein Kinases / metabolism*
-
Protein Serine-Threonine Kinases
-
Protein Tyrosine Phosphatase, Non-Receptor Type 1
-
Protein Tyrosine Phosphatases / metabolism
-
Proteins / genetics
-
Proteins / metabolism*
-
Receptor-Like Protein Tyrosine Phosphatases, Class 2
-
Recombinant Proteins / chemistry
-
Recombinant Proteins / genetics
-
Recombinant Proteins / metabolism
-
Signal Transduction
-
Xenopus Proteins / genetics
-
Xenopus Proteins / metabolism
Substances
-
Cell Cycle Proteins
-
Enzyme Inhibitors
-
Microtubule-Associated Proteins
-
Neoplasm Proteins
-
Nuclear Proteins
-
Phosphoproteins
-
Proteins
-
Recombinant Proteins
-
TPX2 protein, Xenopus
-
TPX2 protein, human
-
Xenopus Proteins
-
protein phosphatase inhibitor-2
-
Protein Kinases
-
AURKA protein, Xenopus
-
Aurora Kinases
-
Protein Serine-Threonine Kinases
-
Phosphoprotein Phosphatases
-
PTPRU protein, human
-
Protein Tyrosine Phosphatase, Non-Receptor Type 1
-
Protein Tyrosine Phosphatases
-
Receptor-Like Protein Tyrosine Phosphatases, Class 2