Discovery of new substrates of the elongation factor-2 kinase suggests a broader role in the cellular nutrient response
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
References (47)
- et al.
The role of protein synthesis in cell cycling and cancer
Mol. Oncol.
(2009) - et al.
Purification and characterization of calmodulin-dependent protein kinase III from rabbit reticulocytes and rat pancreas
J. Biol. Chem.
(1993) - et al.
Stimulation of the AMP-activated protein kinase leads to activation of eukaryotic elongation factor 2 kinase and to its phosphorylation at a novel site, serine 398
J. Biol. Chem.
(2004) - et al.
Alpha-kinases: analysis of the family and comparison with conventional protein kinases
Prog. Biophys. Mol. Biol.
(2004) - et al.
The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation
Cell
(2013) - et al.
Alpha4 is an essential regulator of PP2A phosphatase activity
Mol. Cell
(2009) - et al.
The B55alpha subunit of PP2A drives a p53-dependent metabolic adaptation to glutamine deprivation
Mol. Cell
(2013) - et al.
Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli
Protein Expr. Purif.
(2003) - et al.
Chemical genetic identification of NDR1/2 kinase substrates AAK1 and Rabin8 uncovers their roles in dendrite arborization and spine development
Neuron
(2012) - et al.
The emerging power of chemical genetics
Curr. Opin. Cell Biol.
(2002)
Determinants for substrate phosphorylation by Dictyostelium myosin II heavy chain kinases A and B and eukaryotic elongation factor-2 kinase
Biochim. Biophys. Acta
Identification of a major determinant for serine-threonine kinase phosphoacceptor specificity
Mol. Cell
The E3 ubiquitin ligase- and protein phosphatase 2A (PP2A)-binding domains of the Alpha4 protein are both required for Alpha4 to inhibit PP2A degradation
J. Biol. Chem.
Human differentiation-related gene NDRG1 is a Myc downstream-regulated gene that is repressed by myc on the core promoter region
Gene
Phosphorylation of RTP, an ER stress-responsive cytoplasmic protein
Biochem. Biophys. Res. Commun.
Inhibition of AMPK catabolic action by GSK3
Mol. Cell
Ca2 +/calmodulin-dependent phosphorylation of elongation factor 2
FEBS Lett.
Regulation of peptide-chain elongation in mammalian cells
Eur. J. Biochem.
A hierarchy of ATP-consuming processes in mammalian cells
Biochem. J.
Phosphorylation of elongation factor 2 by EF-2 kinase affects rate of translation
Nature
Functional properties of phosphorylated elongation factor 2
Eur. J. Biochem.
A novel mTOR-regulated phosphorylation site in elongation factor 2 kinase modulates the activity of the kinase and its binding to calmodulin
Mol. Cell. Biol.
Mapping the functional domains of elongation factor-2 kinase†
Biochemistry
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Present Address: Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.