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Expression, Purification, Crystallisation and X-ray Crystallographic Analysis of a Truncated Form of Human Src Homology 2 Containing Inositol 5-Phosphatase 2

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Abstract

The Src homology 2 containing inositol 5-phosphatase 2 (SHIP2) catalyses the dephosphorylation of the phospholipid phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3) to form PI(3,4)P2. PI(3,4,5)P3 is a key lipid second messenger, which can recruit signalling proteins to the plasma membrane and subsequently initiate numerous downstream signalling pathways responsible for the regulation of a plethora of cellular events such as proliferation, growth, apoptosis and cytoskeletal rearrangements. SHIP2 has been heavily implicated with several serious diseases such as cancer and type 2 diabetes but its regulation remains poorly understood. In order to gain insight into the mechanisms of SHIP2 regulation, a fragment of human SHIP2 containing the phosphatase domain and a region proposed to resemble a C2 domain was crystallized. Currently, no structural information is available on the putative C2-related domain or its relative position with respect to the phosphatase domain. Initial crystals were polycrystalline, but were optimized to obtain diffraction data to a resolution of 2.1 Å. Diffraction data analysis revealed a P212121 space group with unit cell parameters a = 136.04 Å, b = 175.84 Å, c = 176.89 Å. The Matthews coefficient is 2.54 Å3 Da−1 corresponding to 8 molecules in the asymmetric unit with a solvent content of 51.7 %.

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Abbreviations

DTT:

1,4-Dithiothreitol

GST:

Glutathione S-transferase

MES:

2-(N-Morpholino)ethanesulfonic acid

PI(3,4,5)P3 :

Phospholipid phosphatidylinositol 3,4,5-triphosphate

PI3 K:

Phosphoinositide 3-kinase

PH-R:

Pleckstrin homology related domain

PEG:

Polyethylene glycol

Ptase:

Phosphatase

PMSF:

Phenylmethylsulfonyl fluoride

PTEN:

Phosphatase and tensin homolog

SDS-PAGE:

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

SH2:

Src homology 2

SHIP2:

SH2 containing inositol 5-phosphatase 2

TCEP:

tris(2-Carboxyethyl)phosphine

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Acknowledgments

We thank Javier Muñoz and Fernando García Martínez of the Proteomics Unit at the National Cancer Research Centre for mass spectrometry analysis. We further thank the ESRF for providing the synchrotron-radiation facilities and the staff of beamline ID29 for their assistance in the data collection. The work was supported by the Spanish Ministry of Economy and Competitiveness Grants BFU2010-15923 (to D. L.), the Comunidad Autónoma de Madrid Grant S2010/BMD-2457 (to D. L.), and by the National Cancer Research Centre. D.L. is also a recipient of awards from the Ramón y Cajal Program (RYC-2010-06948), the Volkswagen Foundation (Az: 86 416-1) and Worldwide Cancer Research (15-1177).

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Authors and Affiliations

  1. Cell Signalling and Adhesion Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Calle Melchor Fernández Almagro 3, 28029, Madrid, Spain

    Johanne Le Coq, Luis Heredia Gallego & Daniel Lietha

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  1. Johanne Le Coq
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  2. Luis Heredia Gallego
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  3. Daniel Lietha
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Correspondence to Daniel Lietha.

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Le Coq, J., Heredia Gallego, L. & Lietha, D. Expression, Purification, Crystallisation and X-ray Crystallographic Analysis of a Truncated Form of Human Src Homology 2 Containing Inositol 5-Phosphatase 2. Protein J 35, 225–230 (2016). https://doi.org/10.1007/s10930-016-9665-y

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