A single discrete Rab5-binding site in phosphoinositide 3-kinase β is required for tumor cell invasion

Samantha D Heitz,David J Hamelin,Reece M Hoffmann,Nili Greenberg,Gilbert Salloum,Zahra Erami,Bassem D Khalil,Aliaksei Shymanets,Elizabeth A Steidle,Grace Q Gong,Bernd Nürnberg,John E Burke,Jack U Flanagan,Anne R Bresnick,Jonathan M Backer

doi:10.1074/jbc.ra118.006032

Abstract

Phosphoinositide 3-kinase β (PI3Kβ) is regulated by receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), and small GTPases such as Rac1 and Rab5. Our lab previously identified two residues (Gln596 and Ile597) in the helical domain of the catalytic subunit (p110β) of PI3Kβ whose mutation disrupts binding to Rab5. To better define the Rab5-p110β interface, we performed alanine-scanning mutagenesis and analyzed Rab5 binding with an in vitro pulldown assay with GST-Rab5GTP Of the 35 p110β helical domain mutants assayed, 11 disrupted binding to Rab5 without affecting Rac1 binding, basal lipid kinase activity, or Gβγ-stimulated kinase activity. These mutants defined the Rab5-binding interface within p110β as consisting of two perpendicular α-helices in the helical domain that are adjacent to the initially identified Gln596 and Ile597 residues. Analysis of the Rab5-PI3Kβ interaction by hydrogen-deuterium exchange MS identified p110β peptides that overlap with these helices; no interactions were detected between Rab5 and other regions of p110β or p85α. Similarly, the binding of Rab5 to isolated p85α could not be detected, and mutations in the Ras-binding domain (RBD) of p110β had no effect on Rab5 binding. Whereas soluble Rab5 did not affect PI3Kβ activity in vitro, the interaction of these two proteins was critical for chemotaxis, invasion, and gelatin degradation by breast cancer cells. Our results define a single, discrete Rab5-binding site in the p110β helical domain, which may be useful for generating inhibitors to better define the physiological role of Rab5-PI3Kβ coupling in vivo.

Highlights

Phosphoinositide 3-kinase ␤ (PI3K␤) is regulated by receptor tyrosine kinases (RTKs), G protein– coupled receptors (GPCRs), and small GTPases such as Rac1 and Rab5
To define the Rab5-binding interface within p110␤ (PI3K␤), we first examined whether p110␤ selectively bound to any of the three Rab5 isoforms (A, B, and C), which have been shown to have distinct cellular roles [8, 16, 17]
Using lysates from HEK293T cells expressing wild type (WT) phosphoinositide 3-kinases (PI3Ks)␤ heterodimer and an in vitro pulldown assay, we were unable to detect any difference in PI3K␤ binding to the three Rab5 isoforms

Summary

Introduction

Phosphoinositide 3-kinase ␤ (PI3K␤) is regulated by receptor tyrosine kinases (RTKs), G protein– coupled receptors (GPCRs), and small GTPases such as Rac and Rab. Our lab previously identified two residues (Gln596 and Ile597) in the helical domain of the catalytic subunit (p110␤) of PI3K␤ whose mutation disrupts binding to Rab. Of the 35 p110␤ helical domain mutants assayed, 11 disrupted binding to Rab without affecting Rac binding, basal lipid kinase activity, or G␤␥-stimulated kinase activity. These mutants defined the Rab5-binding interface within p110␤ as consisting of two perpendicular ␣-helices in the helical domain that are adjacent to the initially identified Gln596 and Ile597 residues. G.) and the Macromolecular Therapeutics Core of the Albert Einstein Cancer Center through NCI, National Institutes of Health Grant CA013330. Our results define a single, discrete Rab5-binding site in the p110␤ helical domain, which may be useful for generating inhibitors to better define the physiological role of Rab5–PI3K␤ coupling in vivo

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Conclusion