Structural and functional study of the MAST2 kinase/PTEN phosphatase interplay, a critical event for neurosurvival. – MAST-2-PTEN

The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is widely expressed in nervous system, where it inhibits neuronal survival and impedes natural mechanisms of repair. Changes in PTEN levels in brain have been linked to neurological diseases. The phosphorylation of the C-terminal domain of PTEN (PTEN-Ctail) by cellular kinases modulates its cellular trafficking, stability and conformation and consequently its activity. The Microtubule-associated Ser/Thr kinase 2 (MAST2) is a known PTEN regulator. MAST2 belongs to the family of AGC kinases, and like PTEN, also inhibits neurogenesis and neuroprotection. MAST2 and PTEN form a complex through the interaction of the PDZ domain encoded by MAST2 with the PDZ binding site (PDZ-BS) located in the C-terminal domain of PTEN. The phosphorylation of PTEN by MAST2 is drastically increased when the complex PTEN/MAST2 is stabilized by the PDZ/PDZ-BS interaction. We have recently demonstrated that a viral protein specifically targets the PTEN/MAST2 interaction to promote the survival of the neurons it infects and that disruption of this interaction alters the intracellular trafficking of PTEN (Terrien et al., Science Signaling, 2012). Moreover, we have identified by NMR the full phosphorylation pattern of PTEN-Ctail in vitro and in cell extracts (Cordier et al., J.A.C.S., 2012). It has been proposed that a conformational mechanism of PTEN stability (open/closed model) which depends on the phosphorylation state of PTEN-Ctail controls the cellular localization of PTEN. The contribution of MAST2 in this conformational change remains elusive.
Our aim is to characterize at a structural level the molecular mechanisms by which MAST2 regulates PTEN activity. In particular, we would like to elucidate how the formation of the MAST2/PTEN complex controls the phosphorylation and the conformation of PTEN, and subsequently affects its cellular localization through the PDZ/PDZ-BS interaction. To do so, we will determine by NMR the PTEN-Ctail phosphorylation pattern and relative kinetics by MAST2 kinase in vitro, in cell extracts and even more challenging, in intact living neuronal cells. The model of open/closed conformation of PTEN that is thought to be crucial in the control of the phosphatase activity will be tested and characterized in vitro. The modular organization of large proteins and the transient nature of phosphorylation and of conformational equilibrium represent major challenges to study systems such MAST2/PTEN. Using an integrated structural biology approach, we will combine X-ray crystallography to determine isolated or rigid domain structures and NMR coupled to SAXS to apprehend the potential transient nature of modular proteins and to study segmental motions of sub-domains.We will establish the atomic resolution structure of MAST2 kinase, characterize its catalytic activity and decipher its role in cellular trafficking. In human neurons culture, we will analyze whether the catalytic domain of MAST2 is required for neuritogenesis and for the subcellular localization of PTEN.
To achieve these objectives, the proposal associates three academic laboratories with complementary expertise: the team of Dr. N. Wolff (Institut Pasteur, Paris), specialized in structural biology and protein interactions, the group of Dr. M. Blackledge (Institut de Biologie Structurale, Grenoble), expert in protein dynamics and the unit headed by Dr M. Lafon (Institut Pasteur, Paris), specialized in neuronal biology. We plan to address fundamental ambitious questions of molecular mechanism and spatial cell biology of proteins controlling neurosurvival. This project should improve our knowledge on neuronal survival by giving new insights on the function, structure, control of PTEN phosphorylation, molecular conformation and trafficking of MAST kinase family. It might allow discovering new compounds able to disrupt the MAST2/PTEN complex, opening promising avenues for new neuroprotective therapies.