Fluid flow-induced activation of subcellular AMPK and its interaction with FAK and Src

https://doi.org/10.1016/j.abb.2019.108208Get rights and content

Highlights

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    AMP-activated protein kinase (AMPK) responded to fluid flow in a subcellular location-dependent manner.

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    FAK/Src inhibition decreased the basal AMPK level and selectively blocked flow-induced AMPK activation.

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    Myosin II-dependent signaling pathway may be involved in shear stress-induced AMPK activation.

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    AMPK decreased the basal level of FAK and Src, but did not significantly affect flow-induced FAK/Src activation.

Abstract

AMP-activated protein kinase (AMPK) is a metabolic energy sensor that plays a critical role in cancer cell survival and growth. While the physical microenvironment is believed to influence tumor growth and progression, its role in AMPK regulation remains largely unknown. In the present study, we evaluated AMPK response to mechanical forces and its interaction with other mechano-responsive signaling proteins, FAK and Src. Using genetically encoded biosensors that can detect AMPK activities at different subcellular locations (cytosol, plasma membrane, nucleus, mitochondria, and Golgi apparatus), we observed that AMPK responds to shear stress in a subcellular location-dependent manner in breast cancer cells (MDA-MB-231). While normal epithelial cells (MCF-10A) also similarly responded to shear stress, they are less sensitive to shear stress compared to MDA-MB-231 cells. Inhibition of FAK and Src significantly decreased the basal activity level of AMPK at all five subcellular locations in MDA-MB-231 cells and selectively blocked shear stress-induced AMPK activation. Moreover, testing with cytoskeletal drugs revealed that myosin II might be the critical mediator of shear stress-induced AMPK activation in MDA-MB-231 cells. These findings suggest that breast cancer cells and normal epithelial cells may have different mechanosensitivity in AMPK signaling and that FAK and Src as well as the myosin II-dependent signaling pathway are involved in subcellular AMPK mechanotransduction in breast cancer cells.

Introduction

The physical tumor microenvironment affects tumor growth and progression [[1], [2], [3]]. As the cancer grows, accumulation and reorganization of extracellular matrix and abnormal growth of cancer cells result in a buildup of pressure and increased interstitial fluid flow (IFF) within the tumor tissue [4]. This IFF in turn generates shear stress to influence cancer cell invasion and proliferation possibly via mechanotransduction signaling pathways [[5], [6], [7]]. FAK and Src are such mechanotransduction signaling proteins whose activities are increased in cancer cells, thereby enhancing tumor growth and metastasis [8]. For example, activation of Src specifically enhances the ability of breast cancer cells to grown in bone marrow microenvironment and bone metastasis in breast cancer requires Src-dependent survival signals [9]. FAK promotes tumor progression and metastasis by controlling cell migration, invasion, survival, and cancer stem cell self-renewal [10]. While these cellular energy-consuming processes involve Src or FAK, it is unclear whether cellular energy homeostasis is related to these signaling proteins.

AMPK regulates cellular energy homeostasis by sensing the ratios of AMP/ATP and ADP/ATP [11]. In addition to its major role in cellular energy sensing, AMPK has been shown to have multiple roles in cell functions including cell growth, protein synthesis, autophagy, and gene transcription [12]. It is increasingly recognized that AMPK's multiple, and sometimes paradoxical, roles in cell physiology as well as pathological diseases such as cancer may be due to its compartmentalized subcellular activation because compartmentalization is considered to be one of the properties that molecules utilize to conduct multi-tasking [13,14]. However, it is not clear whether distinct subcellular pools of AMPK respond to specific extracellular stimuli. While AMPK is known to be activated by metabolic stress, recent reports have demonstrated that it is also activated by mechanical forces, suggesting its involvement in mechanotransduction [[15], [16], [17]].

In this study, we used genetically encoded biosensors that are specific to subcellular locations to visualize compartmentalized AMPK signaling activities [13]. Fluid flow-induced shear stress was applied to two types of the cells, breast cancer cells and normal epithelial cells, to study subcellular AMPK responses to mechanical forces. Finally, the role of Src and FAK as well as the cytoskeletal components in the shear stress-induced AMPK signaling activities were explored to examine the involvement of mechanotransduction in AMPK activities.

Section snippets

Biosensors and plasmids

FRET-based biosensors for monitoring subcellular compartment-specific AMPK activities were kindly provided by Dr. Jin Zhang (University of California San Diego) and Dr. Takanari Inoue (Johns Hopkins University). Briefly, the AMPK biosensors are composed of CFP, the FHA1 domain, the AMPK substrate motif, and YFP. The phosphorylation of the AMPK substrate promotes its intramolecular binding to the FHA1 domain, which results in the close association of the donor (CFP) and acceptor (YFP) and

Subcellular AMPK response to shear stress in MDA-MB-231 cells

Several studies have recently reported that AMPK responds to mechanical forces [15,16]. However, little is known about its subcellular response at the single-cell level. Thus, we sought to visualize AMPK activities at different subcellular organelles in response to fluid flow-induced shear stress. We transfected MDA-MB-231 cells with one of the five FRET-based AMPK biosensors: Cyto-AMPK, PM-AMPK, Nuc-AMPK, Mito-AMPK, and Golgi-AMPK targeting cytosol, plasma membrane, nucleus, mitochondria, and

Discussion

In this study, we used the subcellular compartment-specific AMPK biosensors to explore mechanotransduction of subcellular AMPK and its interaction with FAK and Src. The aim of this study is to visualize subcellular compartment-specific AMPK activity with high spatial and temporal precision using FRET-based biosensors. It has been demonstrated that the FRET-based biosensors allow for monitoring, in real time, phosphorylation of proteins in living cells, without the need to lyse cells for

Conclusions

Our results show that while cytosolic AMPK, the majority AMPK pool, is activated by shear stress in MDA-MB-231 and MCF-10A cells, these two cells have different subcellular AMPK activation patterns, suggesting the different mechanoresponsive metabolic signaling between breast cancer cells and normal epithelial cells. Importantly, FAK and Src as well as the myosin II-dependent signaling pathway appear to be involved in subcellular AMPK mechanotransduction. In the future, it will be of interest

Funding

This work was supported in part by the funds from a breast cancer advocacy group, 100 Voices of Hope, and Biomechanics and Biomaterials Research Center – Integrated Nanosystems Development Institute at the Indiana University Purdue University Indianapolis.

Declaration of competing interest

None.

Acknowledgements

We thank J. Zhang (University of California San Diego, USA) and T. Inoue (Johns Hopkins University, USA) for the gift of the AMPK biosensors, and Y. Wang (University of California San Diego, USA) for the gift of the FAK and Src biosensors.

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