Protein kinases have emerged as an important class of therapeutic targets, as they are known to be involved in pathological pathways linked to numerous human disorders. Major efforts to discover kinase inhibitors in both academia and pharmaceutical companies have centered on the development of robust assays and cost-effective approaches to isolate them. Drug discovery procedures often start with hit identification for lead development, by screening a library of chemicals using an appropriate assay in a high-throughput manner. Considering limitations unique to each assay technique and screening capability, intelligent integration of various assay schemes and level of throughput, in addition to the choice of chemical libraries, is the key to success of this initial step. Here, we describe the purification of the protein kinase, eEF-2K, and the utilization of three biochemical assays in the course of identifying small molecules that block its enzymatic reaction.
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eEF-2K Deficiency Boosts the Virus-Specific Effector CD8 T Cell Responses During Viral Infection.
Viruses
December 2024
Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77807, USA.
In this study, we revealed a critical role of eukaryotic elongation factor-2 kinase (eEF-2K), a negative regulator of protein synthesis, in regulating T cells during vaccinia virus (VACV) infection. We found that eEF-2K-deficient (eEF-2K⁻/⁻) mice exhibited a significantly higher proportion of VACV-specific effector CD8 T cells without compromising the development of VACV-specific memory CD8 T cells. RNA sequencing demonstrated that eEF-2K⁻/⁻ VACV-specific effector CD8 T cells had enhanced functionality, which improves their capacity to combat viral infection during the effector phase.
View Article and Find Full Text PDFRevealing eEF-2 kinase: recent structural insights into function.
Trends Biochem Sci
February 2024
Department of Chemistry and Biochemistry, The City College of New York, New York, NY 10031, USA; The Graduate Center of The City University of New York (CUNY), New York, NY 10016, USA. Electronic address:
The α-kinase eukaryotic elongation factor 2 kinase (eEF-2K) regulates translational elongation by phosphorylating its ribosome-associated substrate, the GTPase eEF-2. eEF-2K is activated by calmodulin (CaM) through a distinctive mechanism unlike that in other CaM-dependent kinases (CAMK). We describe recent structural insights into this unique activation process and examine the effects of specific regulatory signals on this mechanism.
View Article and Find Full Text PDFStructure of the complex between calmodulin and a functional construct of eukaryotic elongation factor 2 kinase bound to an ATP-competitive inhibitor.
J Biol Chem
June 2023
Department of Chemistry and Biochemistry, The City College of New York, New York, New York, USA; PhD Program in Biochemistry, The Graduate Center of CUNY, New York, New York, USA; PhD Program in Chemistry, The Graduate Center of CUNY, New York, New York, USA; PhD Program in Physics, The Graduate Center of CUNY, New York, New York, USA. Electronic address:
The calmodulin-activated α-kinase, eukaryotic elongation factor 2 kinase (eEF-2K), serves as a master regulator of translational elongation by specifically phosphorylating and reducing the ribosome affinity of the guanosine triphosphatase, eukaryotic elongation factor 2 (eEF-2). Given its critical role in a fundamental cellular process, dysregulation of eEF-2K has been implicated in several human diseases, including those of the cardiovascular system, chronic neuropathies, and many cancers, making it a critical pharmacological target. In the absence of high-resolution structural information, high-throughput screening efforts have yielded small-molecule candidates that show promise as eEF-2K antagonists.
View Article and Find Full Text PDFADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin.
Proc Natl Acad Sci U S A
April 2023
Department of Chemistry and Biochemistry, The City College of New York, New York, NY 10031.
Protein translation, one of the most energy-consumptive processes in a eukaryotic cell, requires robust regulation, especially under energy-deprived conditions. A critical component of this regulation is the suppression of translational elongation through reduced ribosome association of the GTPase eukaryotic elongation factor 2 (eEF-2) resulting from its specific phosphorylation by the calmodulin (CaM)-activated α-kinase eEF-2 kinase (eEF-2K). It has been suggested that the eEF-2K response to reduced cellular energy levels is indirect and mediated by the universal energy sensor AMP-activated protein kinase (AMPK) through direct stimulatory phosphorylation and/or downregulation of the eEF-2K-inhibitory nutrient-sensing mTOR pathway.
View Article and Find Full Text PDFStructural basis for the calmodulin-mediated activation of eukaryotic elongation factor 2 kinase.
Sci Adv
July 2022
Department of Chemistry and Biochemistry, The City College of New York, New York, NY 10031, USA.
Translation is a tightly regulated process that ensures optimal protein quality and enables adaptation to energy/nutrient availability. The α-kinase eukaryotic elongation factor 2 kinase (eEF-2K), a key regulator of translation, specifically phosphorylates the guanosine triphosphatase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis. eEF-2K activation requires calmodulin binding and autophosphorylation at the primary stimulatory site, T348.
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