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Molecular mechanism of activation of class IA phosphoinositide 3-kinases (PI3Ks) by membrane-localized HRas #MMPMID28515318
Siempelkamp BD; Rathinaswamy MK; Jenkins ML; Burke JE
J Biol Chem 2017[Jul]; 292 (29): 12256-66 PMID28515318show ga
Class IA PI3Ks are involved in the generation of the key lipid signaling molecule phosphatidylinositol 3,4,5-trisphosphate (PIP3), and inappropriate activation of this pathway is implicated in a multitude of human diseases, including cancer, inflammation, and primary immunodeficiencies. Class IA PI3Ks are activated downstream of the Ras superfamily of GTPases, and Ras?PI3K interaction plays a key role in promoting tumor formation and maintenance in Ras-driven tumors. Investigating the detailed molecular events in the Ras?PI3K interaction has been challenging because it occurs on a membrane surface. Here, using maleimide-functionalized lipid vesicles, we successfully generated membrane-resident HRas and evaluated its effect on PI3K signaling in lipid kinase assays and through analysis with hydrogen?deuterium exchange MS. We screened all class IA PI3K isoforms and found that HRas activates both p110? and p110? isoforms but does not activate p110?. The p110? and p110? activation by Ras was synergistic with activation by a soluble phosphopeptide derived from receptor tyrosine kinases. Hydrogen?deuterium exchange MS revealed that membrane-resident HRas, but not soluble HRas, enhances conformational changes associated with membrane binding by increasing membrane recruitment of both p110? and p110?. Together, these results afford detailed molecular insight into the Ras?PI3K signaling complex, provide a framework for screening Ras inhibitors, and shed light on the isoform specificity of Ras?PI3K interactions in a native membrane context.