Journal Article

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

Williams, R. L., Tsolakos, N., Perisic, O., Kulkarni, S., Stephens, L., Oxley, D., Durrant, T. N., Suire, S. M., Downward, J., Chessa, T., Hawkins, P. T.

2018

Overview

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 cooperativity; 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.

• Publisher: National Academy of Sciences
• Subject: 1-Phosphatidylinositol 3-kinase / Activation / Affinity / Animals / Biochemistry / Biological Sciences / Cdc42 protein / Cellular biology / Class Ia Phosphatidylinositol 3-Kinase - genetics / Class Ia Phosphatidylinositol 3-Kinase - metabolism / Dimerization / Fibroblasts - metabolism / Kinases / Liver / Lysates / Mice / Mice, Inbred C57BL / Molecular biology / Peptides / Phosphatidylinositol 3,4,5-triphosphate / Phosphorylation / Platelet-derived growth factor / Protein Binding / Protein Isoforms - genetics / Protein Isoforms - metabolism / Quantitation / Receptor mechanisms / Receptor, Platelet-Derived Growth Factor beta - genetics / Receptor, Platelet-Derived Growth Factor beta - metabolism / Receptors / Receptors, Platelet-Derived Growth Factor - genetics / Receptors, Platelet-Derived Growth Factor - metabolism / Recruitment / Signal Transduction / Streptavidin / Tumor suppressor genes / Tyrosine