The kinase inhibitors employed in Work Package 5 have limited affinity to their target kinases. In addition, many of these inhibitors will also inhibit kinases other than those against which they were developed. Therefore it is crucial to develop correlative strategies to validate the connection between specific mitotic kinases and certain phosphorylation events established in WP5.

In this Work Package, we will first generate inhibitor-sensitive or inhibitor-resistant mitotic kinases based on studies of the structure and catalytic mechanism of protein kinases. These mutant kinases will allow us to evaluate the specificity of a given kinase inhibitor, to inhibit specific kinases exclusively and thus to verify whether a phosphorylation event is catalyzed by a specific kinase. We will also generate modified kinases engineered to work with specific ATP analogues that are poor substrates for the endogenous kinases (Shokat strategy). These modified kinases utilizing labeled ATP-analogues provide us with a unique means to test whether these kinases directly phosphorylated specific protein substrates. Such kinases can potentially also be used to search for kinase substrates.

- To design, produce and test inhibitor-sensitive or inhibitor-resistant mitotic kinases

- To characterize ATP-analogue sensitive kinases

- To identify mitotic kinase substrates using Shokat strategy in a Xenopus egg extract system

- To validate the phosphorylation state of protein complexes using modified kinases

Andrea Musacchio (European Institute of Oncology, Milan, Italy)

Ariane Abrieu (CNRS - CRBM, Montpellier, France)

Tim Hunt (Clare Hall Laboratories, South Mimms, UK)

Jan-Michael Peters (Research Institute of Molecular Pathology, Vienna, Austria)

1) Structure analysis of kinase/inhibitor complexes

The Musacchio group at the EIO has successfully elucidated the structure of Aurora B together with its binding partner INCENP as well as the structure of Aurora B/INCENP complex in the presence of the Aurora B inhibitor Hesperadin. The structure reveals how INCENP activates and Hesperadin inhibits Aurora B activity. These findings were reported in the following paper:
Mechanism of Aurora-B activation by INCENP and inhibition by Hesperadin: Sessa F, Mapelli M, Ciferri C, Tarricone C, Areces LB, Schneider TR, Stukenberg PT and Musacchio A (2005) Mol Cell 18:379-391

2) Generation of inhibitor-resistant kinase mutants

The Musacchio group at the EIO is using the structure information they obtained as a guide to create inhibitor-resistant kinases. A series of Aurora B mutants have been generated. The catalytic activity of these mutants is being tested currently.

3) Functional identification of mitotic kinase substrates using Shokat strategy in a Xenopus egg extract system

The Abrieu group at the CNRS-CRBM and the Hunt group at Clare Hall Laboratories are using the Shokat strategy to identify substrates of Mps1 and Cdk2 in Xenopus egg extract systems. Despite the technical difficulties, a number of potential Cdk2 substrates were revealed and are subjected to further investigation.

- Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.
DeLuca JG, Gall WE, Ciferri C, Cimini D, Musacchio A & Salmon ED. Cell. 2006. 127, 969-82 (PMID: 17129782)

- In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics.
Vink M, Simonetta M, Transidico P, Ferrari K, Mapelli M, De Antoni A, Massimiliano L, Ciliberto A, Faretta M, Salmon ED & Musacchio A. Curr Biol. 2006, 16, 755-66 (PMID: 16631582)

- Determinants of conformational dimerization of Mad2 and its inhibition by p31(comet).
Mapelli M, Filipp FV, Rancati G, Massimiliano L, Nezi L, Stier G, Hagan RS, Confalonieri S, Piatti S, Sattler M & Musacchio A. EMBO J 2006, 25, 1273-8 (PMID: 16525508)

- Mechanism of Aurora-B activation by INCENP and inhibition by Hesperadin.
Sessa F, Mapelli M, Ciferri C, Tarricone C, Areces LB, Schneider TR, Stukenberg PT and Musacchio A (2005) Mol Cell 18:379-391 (PMID: 15866179)