Dynamics of the Phosphoinositide 3-Kinase p110δ Interaction with p85α and Membranes Reveals Aspects of Regulation Distinct from p110α

Burke, John E.; Vadas, Oscar; Berndt, Alex; Finegan, Tara M.; Perišić, Olga; Williams, Roger L.

doi:10.1016/j.str.2011.06.003

Cited by 94 publications

(147 citation statements)

References 36 publications

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“…Regions in both the nSH2 and cSH2 domains of p85α showed decreases in HDX due to pY binding (Fig. S6), similar to previous results with p110δ/p85α (25). Dynamic changes in WT p110α/p85α upon binding membranes.…”

Section: Resultssupporting

confidence: 89%

“…We also characterized one p85α mutant in the iSH2 domain (N564D). Lipid kinase assays were carried out with 5% PIP2-containing vesicles (25). All cancer-linked mutations, including mutants in the ABD and ABD-RBD linker, increased basal lipid kinase activity compared with WT, with H1047R acting as the most potent activator, followed by N345K and E545K (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Deuterium incorporation was quantified on pepsin-digested peptides separated on a reversed-phase ultra performance liquid chromatography (UPLC), as described previously (25). The selected peptic peptides used for both p110α and p85α analysis are shown in Dynamic changes in WT p110α/p85α upon pY activation.…”

Section: Resultsmentioning

confidence: 99%

“…Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has become a powerful tool for understanding the dynamics of complex protein systems (22) and it is particularly useful for understanding protein-lipid interactions (23)(24)(25)(26)(27). The rate of exchange of amide hydrogens with deuterium is strongly dependent on secondary structure and solvent accessibility, and this can be used to examine dynamic structural perturbations caused by different stimuli.…”

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confidence: 99%

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Oncogenic mutations mimic and enhance dynamic events in the natural activation of phosphoinositide 3-kinase p110α ( PIK3CA )

Burke

Perišić

Masson

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The p110α catalytic subunit (PIK3CA) is one of the most frequently mutated genes in cancer. We have examined the activation of the wild-type p110α/p85α and a spectrum of oncogenic mutants using hydrogen/deuterium exchange mass spectrometry (HDX-MS). We find that for the wild-type enzyme, the natural transition from an inactive cytosolic conformation to an activated form on membranes entails four distinct events. Analysis of oncogenic mutations shows that all up-regulate the enzyme by enhancing one or more of these dynamic events. We provide the first insight into the activation mechanism by mutations in the linker between the adapter-binding domain (ABD) and the Ras-binding domain (RBD) (G106V and G118D). These mutations, which are common in endometrial cancers, enhance two of the natural activation events: movement of the ABD and ABD-RBD linker relative to the rest of the catalytic subunit and breaking the C2-iSH2 interface on binding membranes. C2 domain mutants (N345K and C420R) also mimic these events, even in the absence of membranes. A third event is breaking the nSH2-helical domain contact caused by phosphotyrosine-containing peptides binding to the enzyme, which is mimicked by a helical domain mutation (E545K). Interaction of the C lobe of the kinase domain with membranes is the fourth activation event, and is potentiated by kinase domain mutations (e.g., H1047R). All mutations increased lipid binding and basal activity, even mutants distant from the membrane surface. Our results elucidate a unifying mechanism in which diverse PIK3CA mutations stimulate lipid kinase activity by facilitating allosteric motions required for catalysis on membranes.H/D exchange | PI3 kinase | signaling T he oncogenic potential of phosphoinositide 3-kinases (PI3Ks) was first established when it was shown that an activated form of the PI3K isoform p110α (v-P3K) was encoded by an avian sarcoma virus and that this viral gene and its cellular counterpart can cause cell transformation (1). The advent of large-scale sequencing established that cancer-linked somatic mutations of p110α are among the most common mutations associated with human tumors (2-4). Tumor-associated mutations appear throughout the sequence of p110α (Fig. 1A). The majority of these are in hotspots in the helical (E545K) or kinase (H1047R) domains. The synergy of these two mutations in activating p110α suggests they act independently, and both structural and functional studies indicate they work by distinct mechanisms (5-7). The presence of both of these mutations in colorectal tumors, compared with the singly mutated form, strongly correlates with decreased survival (8). However, it is not clear how other mutations throughout the sequence activate p110α, nor is it clear whether there is any unifying principle governing all of the oncogenic mutations. It is becoming increasingly apparent that the p110α mutations do not act alone in tumor development. Mice with the most common p110α mutation (H1047R) expressed in ovaries develop hyperplasias, but not tumors, wher...

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Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Oncogenic mutations mimic and enhance dynamic events in the natural activation of phosphoinositide 3-kinase p110α ( PIK3CA )

Burke

Perišić

Masson

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

259

332

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“…To verify that truncations did not change the activity or ability to bind protein partners, we carried out lipid kinase assays as well as GST pulldowns with GST‐tagged Rab11. We have previously shown that the C‐termini of both PI4K and PI3Ks are essential for their activity 28, 29. For this reason, we made a construct containing the N‐terminal and internal deletions, but with an intact C‐terminus (referred to as xtal PI4KIIIβ +cterm).…”

Section: Resultsmentioning

confidence: 99%

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

et al. 2016

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The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X‐ray crystallography has been a powerful approach to understand protein‐protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX‐MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX‐MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX‐MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ‐Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein‐protein complexes, and is an excellent strategy to optimize constructs for high‐resolution structural approaches.

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Phosphatidylinositol‐3,4,5‐trisphosphate: Tool of choice for class I PI 3‐kinases

Salamon

Backer

2013

BioEssays

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Class I PI 3-kinases signal by producing the signaling lipid phosphatidylinositol (3,4,5) trisphosphate, which in turn acts by recruiting downstream effectors that contain specific lipid-binding domains. The Class I PI 3-kinases comprise four distinct catalytic subunits linked to one of seven different regulatory subunits. All the Class I PI 3-kinases produce the same signaling lipid, PIP3, and the different isoforms have overlapping expression patterns and are coupled to overlapping sets of upstream activators. Nonetheless, studies in cultured cells and in animals have demonstrated that the different isoforms are coupled to distinct ranges of downstream responses. This review focuses on the mechanisms by which the production a common product, PIP3, can produce isoform-specific signaling by PI 3-kinases.

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Dynamics of the Phosphoinositide 3-Kinase p110δ Interaction with p85α and Membranes Reveals Aspects of Regulation Distinct from p110α

Cited by 94 publications

References 36 publications

Oncogenic mutations mimic and enhance dynamic events in the natural activation of phosphoinositide 3-kinase p110α ( PIK3CA )

Oncogenic mutations mimic and enhance dynamic events in the natural activation of phosphoinositide 3-kinase p110α ( PIK3CA )

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

Phosphatidylinositol‐3,4,5‐trisphosphate: Tool of choice for class I PI 3‐kinases

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