Dynamics of Ras Complexes Observed in Living Cells

Xiangyong Li,Honglin Jin,Zhiyong Cheng

doi:10.3390/s120709411

Abstract

K-Ras works as a switch in many important intracellular signaling pathways and plays important roles in cell growth, proliferation, differentiation and carcinogenesis. For signal transduction from K-Ras to Raf1, the best-characterized effector of K-Ras, the general view is that Ras recruits Raf1 from the cytoplasm to the cell membrane. To elucidate this process, we constructed a series of fusion proteins (including Raf1 and K-Ras fused with either fluorescent proteins or fluorescent protein fragments) to compare subcellular localizations of these proteins. Bimolecular fluorescence complementation (BiFC) and a co-transfection system were used. In the BiFC system, the K-Ras/Raf1 complexes were mainly located in the cell membrane, while the Raf1 control was uniformly distributed in the cytoplasm. However, the complexes of Raf1 and K-RasC185S, a K-Ras mutant which loses membrane-localization, were also able to accumulate in the cell membrane. In contrast, an apparent cytosolic distribution pattern was observed in cells co-transfected with mcerulean-Raf1 and EGFP-K-RasC185S, suggesting that the membrane localization of K-Ras/Raf1 complexes is not entirely dependent on K-Ras, and that other factors, such as the irreversible conformation formed between K-Ras and Raf1 may play a role. This study sheds light on the interaction between K-Ras and Raf1 and provides a practical method to elucidate the mechanism underlying K-Ras and Raf1 binding to the cell membrane.

Highlights

The Ras protein family is a major component of numerous cellular signaling pathways that control cell differentiation, proliferation, survival, cell cycle entry and cytoskeletal dynamics [1].Dysregulation of these cellular functions is a hallmark of diseases including cancers
This study provides new insights into the understanding of Raf1 binding to cell membrane and offers a practical method to elucidate the functional role of the signaling proteins in K-Ras pathways
To investigate the cellular localization of K-Ras and Raf1, two sets of plasmids were constructed; (1) Vectors encoding Raf1 and K-Ras Bimolecular fluorescence complementation (BiFC) fusions, and (2) Vectors encoding Raf1 and K-Ras fused to standard fluorescent proteins (Figure 1)

Summary

Introduction

The Ras protein family is a major component of numerous cellular signaling pathways that control cell differentiation, proliferation, survival, cell cycle entry and cytoskeletal dynamics [1]. Dysregulation of these cellular functions is a hallmark of diseases including cancers. There are still some unresolved issues regarding their interactions such as where and how the activation of Raf and K-Ras occurs in cells, whether K-Ras and Raf traffic together or are part of a larger multicomponent signaling complexes, as well as whether the ultimate cellular localization of the. This study provides new insights into the understanding of Raf binding to cell membrane and offers a practical method to elucidate the functional role of the signaling proteins in K-Ras pathways

Plasmids Construction

Results and Discussion

Subcellular Localization of Co-Expressed K-Ras and Raf1