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Quantitation of class IA PI3Ks in mice reveals p110-free-p85s and isoform-selective subunit associations and recruitment to receptors

التفاصيل البيبلوغرافية
العنوان: Quantitation of class IA PI3Ks in mice reveals p110-free-p85s and isoform-selective subunit associations and recruitment to receptors
المؤلفون: Julian Downward, Phillip T. Hawkins, Olga Perisic, Sabine Suire, Len R. Stephens, Suhasini Kulkarni, David Oxley, T. N. Durrant, N. Tsolakos, Roger L. Williams, Tamara Chessa
المصدر: Proceedings of the National Academy of Sciences of the United States of America
بيانات النشر: National Academy of Sciences, 2018.
سنة النشر: 2018
مصطلحات موضوعية: 0301 basic medicine, Gene isoform, Protein subunit, GTPase, P110α, Biochemistry, PI3K, Receptor, Platelet-Derived Growth Factor beta, 03 medical and health sciences, Mice, In vivo, Avi-tag, Animals, Protein Isoforms, Receptors, Platelet-Derived Growth Factor, Phosphorylation, Receptor, Multidisciplinary, biology, Chemistry, Biological Sciences, Fibroblasts, Cell biology, Class Ia Phosphatidylinositol 3-Kinase, Mice, Inbred C57BL, 030104 developmental biology, Biotinylation, biology.protein, Dimerization, Platelet-derived growth factor receptor, isoform-selective, Protein Binding, Signal Transduction
الوصف: Significance The class IA PI3Ks, comprised of regulatory (p85α/p85β/p55γ) and catalytic (p110α/β/δ) subunits, which make the signaling lipid PIP3, constitute a key node in signaling. Many factors underlie their numerous cellular roles and large investments in the creation of PI3K-inhibitors. The existence of heterodimeric-isoforms (at least nine) with distinct distributions and properties and the often-debated existence of “p110-free-regulatory-subunits” as modulators provide the system with flexibility, redundancy and isoform-selective functions. Despite the scale of this endeavour, many of the system’s “rules of engagement” are unknown. Here we demonstrate preferential subunit associations, clarify the existence of “p110-free-regulatory-subunits”, show that they have properties that could allow them to modulate pathway activity, and reveal mechanisms that allow selective activation of PI3Kα and β by receptors.
Class IA PI3Ks have many roles in health and disease. The rules that govern intersubunit and receptor associations, however, remain unclear. We engineered mouse lines in which individual endogenous class IA PI3K subunits were C-terminally tagged with 17aa that could be biotinylated in vivo. Using these tools we quantified PI3K subunits in streptavidin or PDGFR pull-downs and cell lysates. This revealed that p85α and β bound equivalently to p110α or p110β but p85α bound preferentially to p110δ. p85s were found in molar-excess over p110s in a number of contexts including MEFs (p85β, 20%) and liver (p85α, 30%). In serum-starved MEFs, p110-free-p85s were preferentially, compared with heterodimeric p85s, bound to PDGFRs, consistent with in vitro assays that demonstrated they bound PDGFR-based tyrosine-phosphorylated peptides with higher affinity and co-operativity; suggesting they may act to tune a PI3K activation threshold. p110α-heterodimers were recruited 5–6× more efficiently than p110β-heterodimers to activated PDGFRs in MEFs or to PDGFR-based tyrosine-phosphorylated peptides in MEF-lysates. This suggests that PI3Kα has a higher affinity for relevant tyrosine-phosphorylated motifs than PI3Kβ. Nevertheless, PI3Kβ contributes substantially to acute PDGF-stimulation of PIP3 and PKB in MEFs because it is synergistically, and possibly sequentially, activated by receptor-recruitment and small GTPases (Rac/CDC42) via its RBD, whereas parallel activation of PI3Kα is independent of its RBD. These results begin to provide molecular clarity to the rules of engagement between class IA PI3K subunits in vivo and past work describing “excess p85,” p85α as a tumor suppressor, and differential receptor activation of PI3Kα and PI3Kβ.
اللغة: English
تدمد: 1091-6490
0027-8424
URL الوصول: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::399bd9c5b6dbbc1db2ad95379450f681
http://europepmc.org/articles/PMC6275495
Rights: OPEN
رقم الانضمام: edsair.doi.dedup.....399bd9c5b6dbbc1db2ad95379450f681
قاعدة البيانات: OpenAIRE
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