Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α Homodimer

LoPiccolo, Jaclyn; Kim, Seung Joong; Shi, Yi; Wu, Bin; Wu, Haiyan; Chait, Brian T.; Singer, Robert H.; Šali, Andrej; Brenowitz, Michael; Bresnick, Anne R.; Backer, Jonathan M.

doi:10.1074/jbc.m115.689604

Cited by 28 publications

(29 citation statements)

References 86 publications

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“…Our analysis using purified p85α and PTEN proteins showed that the p85α-I177N mutant retained its ability to both bind and regulate PTEN, similar to that of wild type p85α. This is in contrast to the proposed role for the I177 residue within the BH–BH dimer interface noted in the crystallized protein 29 , but was in good agreement with a recent study suggesting that in solution BH domains do not typically interact 44 . Cheung et al .…”

Section: Discussionsupporting

confidence: 89%

Patient-derived mutations within the N-terminal domains of p85α impact PTEN or Rab5 binding and regulation

Mellor

Marshall

Ruan

et al. 2018

Sci Rep

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The p85α protein regulates flux through the PI3K/PTEN signaling pathway, and also controls receptor trafficking via regulation of Rab-family GTPases. In this report, we determined the impact of several cancer patient-derived p85α mutations located within the N-terminal domains of p85α previously shown to bind PTEN and Rab5, and regulate their respective functions. One p85α mutation, L30F, significantly reduced the steady state binding to PTEN, yet enhanced the stimulation of PTEN lipid phosphatase activity. Three other p85α mutations (E137K, K288Q, E297K) also altered the regulation of PTEN catalytic activity. In contrast, many p85α mutations reduced the binding to Rab5 (L30F, I69L, I82F, I177N, E217K), and several impacted the GAP activity of p85α towards Rab5 (E137K, I177N, E217K, E297K). We determined the crystal structure of several of these p85α BH domain mutants (E137K, E217K, R262T E297K) for bovine p85α BH and found that the mutations did not alter the overall domain structure. Thus, several p85α mutations found in human cancers may deregulate PTEN and/or Rab5 regulated pathways to contribute to oncogenesis. We also engineered several experimental mutations within the p85α BH domain and identified L191 and V263 as important for both binding and regulation of Rab5 activity.

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Section: Discussionsupporting

confidence: 89%

Patient-derived mutations within the N-terminal domains of p85α impact PTEN or Rab5 binding and regulation

Mellor

Marshall

Ruan

et al. 2018

Sci Rep

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“…We have shown that ACK inhibits degradation of the p85α isoform, which would lead to an excess of regulatory subunits over catalytic subunits. p110free p85α exists (24) and can form homodimers (39). The formation of both p110-free monomeric p85 and dimeric p85 would be promoted by elevated levels of p85.…”

Section: Discussionmentioning

confidence: 99%

Assembly of novel, nuclear dimers of the PI3-Kinase regulatory subunits underpins the pro-proliferative activity of the Cdc42-activated tyrosine kinase, ACK

Clayton

Fox

Vicente-Garcia

et al. 2019

Preprint

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The tyrosine kinase ACK is an oncogene associated with poor prognosis in human cancers. ACK promotes proliferation, in part, by contributing to the activation of Akt, the major PI3-Kinase effector. We show that ACK also regulates PI3-Kinase directly, via interactions with the PI3-Kinase regulatory subunits. ACK interacts with all five regulatory subunit isoforms and directly phosphorylates p85α, p85β, p55α and p50α on Tyr607 (or equivalent). Phosphorylation of p85β at this residue promotes cell proliferation but, counterintuitively, ACK does not stimulate PI3-Kinase catalytic activity. We show that ACK stabilizes p85α levels by promoting an interaction between the p85 nSH2 domain and pTyr607, protecting p85 from ubiquitination. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions where the increased levels of the regulatory subunits, together with the nSH2-pTyr607 interaction, promote formation of dimeric p85. We postulate that these novel dimers undertake nuclear functions that contribute to Cdc42-ACK driven oncogenesis. We propose that ACK shapes PI3-Kinase signalling by dampening the PIP 3 response, whilst continuing to drive cell proliferation through Akt activation and hereto unexplored but crucial functions of nuclear dimeric p85. These new regulatory subunit dimers represent a previously undescribed mode of regulation for PI3-Kinase and potentially reveal additional avenues for therapeutic intervention.

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“…The integrative structure modeling protocol ( i.e ., stages 2, 3, and 4) was scripted using the Python Modeling Interface (PMI) package, version 4d97507, a library for modeling macromolecular complexes based on our open-source Integrative Modeling Platform (IMP) package 98 , version 2.6 (https://integrativemodeling.org). The current procedure is an updated version of previously described protocols 66,72,90,101–104 . Files containing the input data, scripts, and output results are available at https://salilab.org/npc2018.…”

Section: Methodsmentioning

confidence: 99%

“…The remainders of the Nup sequences not in rigid bodies (36.8% of residues, excluding FG repeats) were represented as flexible strings of beads. In a rigid-body, the beads have their relative distances constrained during configurational sampling, whereas in a flexible string, the beads are restrained by the sequence connectivity, excluded volume, and potentially additional restraints, such as chemical cross-links, as exemplified in our previous studies 43,66,72,101,117 .…”

Section: Methodsmentioning

confidence: 99%

“…2; Supplementary Table 1A) were used to restrain the distances spanned by the cross-linked residues, relying on a Bayesian scoring function 66 . The evaluation takes into account the ambiguity due to multiple copies of identical subunits and, for cross-links involving the same protein type, due to the lack of knowledge of whether they are intra- or inter-molecular 72,101,117 ; the ambiguous cross-link restraint considers all intra- and inter-molecular assignments in multiple copies of identical subunits, with only the least violated distance contributing to the score. The remaining 1,434 DSS and EDC cross-links (Fig.…”

Section: Methodsmentioning

confidence: 99%

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Integrative structure and functional anatomy of a nuclear pore complex

Kim

Fernández-Martı́nez

Nudelman

et al. 2018

Nature

Self Cite

478

894

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Summary Despite the central role of Nuclear Pore Complexes (NPCs) as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm, their large size and dynamic nature have impeded a full structural and functional elucidation. Here, we have determined a subnanometer precision structure for the entire 552-protein yeast NPC by satisfying diverse data including stoichiometry, a cryo-electron tomography map, and chemical cross-links. The structure reveals the NPC’s functional elements in unprecedented detail. The NPC is built of sturdy diagonal columns to which are attached connector cables, imbuing both strength and flexibility, while tying together all other elements of the NPC, including membrane-interacting regions and RNA processing platforms. Inwardly-directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized in distinct functional units. Taken together, this integrative structure allows us to rationalize the architecture, transport mechanism, and evolutionary origins of the NPC.

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Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α Homodimer

Cited by 28 publications

References 86 publications

Patient-derived mutations within the N-terminal domains of p85α impact PTEN or Rab5 binding and regulation

Patient-derived mutations within the N-terminal domains of p85α impact PTEN or Rab5 binding and regulation

Assembly of novel, nuclear dimers of the PI3-Kinase regulatory subunits underpins the pro-proliferative activity of the Cdc42-activated tyrosine kinase, ACK

Integrative structure and functional anatomy of a nuclear pore complex

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