Endogenous IQGAP1 and IQGAP3 do not functionally interact with Ras

Chase J Morgan,Zhigang Li,David B Sacks,Andrew C Hedman

doi:10.1038/s41598-019-46677-9

Abstract

The Ras family of small GTPases modulates numerous essential processes. Activating Ras mutations result in hyper-activation of selected signaling cascades, which leads to human diseases. The high frequency of Ras mutations in human malignant neoplasms has led to Ras being a desirable chemotherapeutic target. The IQGAP family of scaffold proteins binds to and regulates multiple signaling molecules, including the Rho family GTPases Rac1 and Cdc42. There are conflicting data in the published literature regarding interactions between IQGAP and Ras proteins. Initial reports showed no binding, but subsequent studies claim associations of IQGAP1 and IQGAP3 with K-Ras and H-Ras, respectively. Therefore, we set out to resolve this controversy. Here we demonstrate that neither endogenous IQGAP1 nor endogenous IQGAP3 binds to the major Ras isoforms, namely H-, K-, and N-Ras. Importantly, Ras activation by epidermal growth factor is not altered when IQGAP1 or IQGAP3 proteins are depleted from cells. These data strongly suggest that IQGAP proteins are not functional interactors of H-, K-, or N-Ras and challenge the rationale for targeting the interaction of Ras with IQGAP for the development of therapeutic agents.

Highlights

The Ras superfamily of small GTPases contains >150 proteins in humans[1]
HEK293 cells were transfected with GFP-tagged IQGAP1, IQGAP2, or IQGAP3 plasmids, lysed, and analyzed by SDS-PAGE and Western blotting
The data show that the anti-IQGAP3 antibody recognizes both endogenous and GFP-tagged IQGAP3 (Fig. 1A)

Summary

Introduction

The Ras superfamily of small GTPases contains >150 proteins in humans[1] These are grouped into 5 families based on sequence and functional differences. IQGAP1 is the most well studied member It is ubiquitously expressed, interacts with over 120 proteins within the cell, and is overexpressed in many different cancers[5,7]. Little is known about IQGAP3 expression in normal tissues, but like IQGAP1, it is increased in some cancers[9,10] Despite their name, IQGAPs do not function as GAPs11,12. IQGAP1 and IQGAP2 were shown to inhibit the intrinsic GTPase activity of Rac[1] and Cdc[42], keeping these proteins in the active state[11,12,13]. Publications[20,21,22,23,24,25,26], and even a US patent[27] for cancer immunotherapy, have cited these positive results without acknowledging the earlier negative binding data

Methods

Results

Conclusion