Elsevier

Cellular Signalling

Volume 29, January 2017, Pages 78-83
Cellular Signalling

Discovery of new substrates of the elongation factor-2 kinase suggests a broader role in the cellular nutrient response

https://doi.org/10.1016/j.cellsig.2016.10.006Get rights and content

Highlights

  • eEF2K has multiple substrates beyond eEF2, including AMPK and alpha4.

  • Atypical kinases can be made analog sensitive for discovering new substrates.

  • eEF2K has an unusual substrate motif that requires threonine.

Abstract

Elongation Factor-2 Kinase (eEF2K) in an unusual mammalian enzyme that has one known substrate, elongation factor-2. It belongs to a class of kinases, called alpha kinases, that has little sequence identity to the > 500 conventional protein kinases, but performs the same reaction and has similar catalytic residues. The phosphorylation of eEF2 blocks translation elongation, which is thought to be critical to regulating cellular energy usage. Here we report a system for discovering new substrates of alpha kinases and identify the first new substrates of eEF2K including AMPK and alpha4, and determine a sequence motif for the kinase that shows a requirement for threonine residues as the target of phosphorylation. These new substrates suggest that eEF2K has a more diverse role in regulating cellular energy usage that involves multiple pathways and regulatory feedback.

Introduction

Regulation of cellular energy homeostasis is an essential feature of mammalian cells and involves nearly every major biological pathway. While several central energy-regulating pathways have been well characterized, others have remained poorly understood, including the elongation factor-2 kinase (eEF2K). Regulation of translation elongation is an important place to manage cellular energy needs, because translation is one of the most energy intensive processes in the cell, and elongation is the step in translation that requires the most energy, yet little is known about regulation at this level [1], [2], [3]. The major known mechanism to regulate translation elongation is through the phosphorylation of elongation factor-2 (eEF2), the only known substrate of eEF2K [4], [5]. The phosphorylation of eEF2 is thought to regulate energy usage by decreasing its affinity for the ribosome and blocking translation elongation [5], [6]. There is increasing appreciation for the importance of eEF2K in maintaining cellular energy levels, in particular for survival of cancer cells, for example in early stages of tumor development, suggesting a broader role in signaling pathways. eEF2K is tightly regulated through phosphorylation by several major signaling pathways including AMPK, mTOR, ERK, and PKA [7], [8], [9], [10], [11], [12]. Since no other substrates have been identified for eEF2K besides autophosphorylation, it has been thought that eEF2K represents a terminal effector of energy homeostasis. As eEF2K is an alpha kinase, standard kinase tools have not been applicable to its investigation [13], [14]. Recent evidence has shown a conserved role for eEF2K in the cellular response to nutrient deprivation and a role in tumor survival [15], [16]. In certain malignancies, including neuroblastoma and medulloblastoma, eEF2K expression correlates with poor patient survival [15], [17]. Despite the fundamental importance in responding to nutrient levels, a clear understanding of how the activity of this kinase helps the tumor survive remains elusive. Here, we report a system for finding substrates of alpha kinases like eEF2K and the discovery of several novel substrates of eEF2K, including alpha4, the essential regulatory subunit of protein phosphatase 2A (PP2A) involved in glutamine homeostasis [18], [19]; and AMPK, the key regulator of cellular energy homeostasis [20], [21]. With the identification and confirmation of new substrates of eEF2K we provide a sequence motif, which will guide discovery of further eEF2K substrates. Together, these substrates suggest that it is not only through eEF2 that eEF2K regulates energy usage, but rather multiple substrates involved in feedback of energy metabolism.

Section snippets

Plasmids and reagents

Plasmids containing the cDNA for eEF2K as well as the substrates were obtained from DNASU. AMPK kinase dead plasmid was a kind gift from David Carling. MBP was obtained from Millipore.

Protein purification

Full-length eEF2K was cloned from a plasmid containing eEF2K cDNA (DNASU) into a modified pET 47b vector (EMD) with an N-terminal Sumo fusion tag instead of the HRV3C cleavage site. The plasmid was transformed into BL21 Rosetta 2 Cells (EMD). An overnight culture was used to inoculate (1:500) 1 l of LB medium

Development of an analog-sensitive eEF2K

We speculated that eEF2K could have additional substrates along with eEF2, and the phosphorylation of these substrates with eEF2 would help explain the kinase's importance and mechanism. Therefore, we employed an analog-sensitive (AS)/covalent capture approach to identify additional substrates. This approach relies on engineering a mutant version of the kinase that can accept a bulky analog of ATP that cannot be efficiently used by any other kinase [27], [28]. This ATP analog can then be used

Discussion

The analog sensitive technique is a powerful approach for identifying novel substrates of protein kinases. Here we have extended it to an unusual family of kinases that bears no sequence identity to the large family of conventional eukaryotic protein kinases. We have noticed that the substrate capture and release approach possesses can lead to false negative identifications due to the following circumstances: 1) low protein abundance, 2) the presence of a cysteine in a tryptic peptide

Acknowledgements

This work was supported by the Howard Hughes Medical Institute and NIH, U19AI109622 and R01AI099245 to K.M.S. Mass spectrometry was provided by the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF (A.L. Burlingame, Director), supported by the Biomedical Technology Research Centers program of the NIH National Institute of General Medical Sciences, NIH NIGMS 8P41GM103481 (the Thermo Scientifi LTQ-Orbitrap Velos is specifically supported by P41GM103481 and Howard Hughes Medical

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    Present Address: Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

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