Biophysical and Structural Characterization of Novel RAS-Binding Domains (RBDs) of PI3Kα and PI3Kγ

JOURNAL OF MOLECULAR BIOLOGY

卷:433

Article

Martinez, Nicholas G.¹  Thieker, David F.^1,2  Carey, Leiah M.^1,2  Rasquinha, Juhi A.¹  Kistler, Samantha K.¹  Kuhlman, Brian A.^1,2  Campbell, Sharon L.^1,2 

[1] Univ North Carolina Chapel Hill, Dept Biochem & Biophys, Chapel Hill, NC 27599 USA

[2] Univ North Carolina Chapel Hill, Lineberger Comprehens Canc Ctr, Chapel Hill, NC 27599 USA

关键词: RAS;    phosphoinositide 3-kinase (PI3K);    protein/protein interactions;    cell signaling;    cancer;   

DOI  :  10.1016/j.jmb.2021.166838

来源: Elsevier

【 摘 要 】

Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate to generate a key lipid second messenger, phosphatidylinositol 3,4,5-bisphosphate. PI3K alpha and PI3K gamma require activation by RAS proteins to stimulate signaling pathways that control cellular growth, differentiation, motility and survival. Intriguingly, RAS binding to PI3K isoforms likely differ, as RAS mutations have been identified that discriminate between PI3K alpha and PI3K gamma, consistent with low sequence homology (23%) between their RAS binding domains (RBDs). As disruption of the RAS/PI3K alpha interaction reduces tumor growth in mice with RAS- and epidermal growth factor receptor driven skin and lung cancers, compounds that interfere with this key interaction may prove useful as anti-cancer agents. However, a structure of PI3K alpha bound to RAS is lacking, limiting drug discovery efforts. Expression of full-length PI3K isoforms in insect cells has resulted in low yield and variable activity, limiting biophysical and structural studies of RAS/PI3K interactions. This led us to generate the first RBDs from PI3K alpha and PI3Kc that can be expressed at high yield in bacteria and bind to RAS with similar affinity to full-length PI3K. We also solved a 2.31 angstrom X-ray crystal structure of the PI3K alpha-RBD, which aligns well to full-length PI3K alpha. Structural differences between the PI3K alpha and PI3K gamma RBDs are consistent with differences in thermal stability and may underly differential RAS recognition and RAS-mediated PI3K activation. These high expression, functional PI3K RBDs will aid in interrogating RAS interactions and could aid in identifying inhibitors of this key interaction. (C) 2021 Elsevier Ltd. All rights reserved.

【 授权许可】

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