Abstract 2179 The Role of an Intrinsically Disordered Loop in Regulating the Cellular Activity of Eukaryotic Elongation Factor-2 Kinase

Protein homeostasis relies on an intricate network of cellular pathways that balance the accurate synthesis of proteins with degradation and the removal of misfolded proteins, a hallmark of neurodegenerative diseases. One mechanism for maintaining the proteome's fidelity is modulating the rate at which the ribosome synthesizes proteins, which is important for ensuring proper protein folding. A key regulator of this rate is elongation factor-2 (eEF-2), an essential component of the translation elongation machinery that catalyzes the ribosome's movement along the mRNA. The function of eEF-2 is inhibited through phosphorylation by the calmodulin-dependent eukaryotic elongation factor-2 kinase (eEF-2K). Many cellular signaling pathways, such as those activated by growth factors or nutrient deprivation, phosphorylate eEF-2K to regulate its activity towards eEF-2. The mechanism by which these cellular signals control eEF-2K activity to modulate elongation rates is poorly understood. These phosphorylations are primarily concentrated on a large, intrinsically disordered region in eEF-2K, termed the regulatory loop. We hypothesized that the regulatory loop acts to negatively regulate the affinity of eEF-2K for its essential activator, calmodulin (CaM), and that phosphorylations on this loop may modulate its conformation, allowing for dynamic control of eEF-2K activation by CaM in response to cellular cues. To address this, we created a version of eEF-2K with the regulatory loop removed (ΔR eEF-2K). In cells, this construct shows higher activity towards eEF-2 and increased co-immunoprecipitated CaM. We also found a 10-fold increase in the apparent affinity of CaM for the recombinant ΔR eEF-2K compared to WT eEF-2K. This data supports a mechanism in which the regulatory loop acts as a suppressive element in eEF-2K by reducing its affinity for CaM. Next, we incorporated phosphorylation into the regulatory loop of recombinant eEF-2K at Ser359, a mTORC1-dependent inhibitory phosphorylation site. We found that the P-Ser359 resulted in a 5-fold decrease in the apparent affinity of CaM. To determine if this mechanism was relevant in cells, we immunoprecipitated eEF-2K from cells grown normally or from cells starved of nutrients and growth factors — a treatment known to activate eEF-2K and remove inhibitory phosphorylations, including Ser359. We found an approximately 18-fold increase in the apparent affinity of CaM for eEF-2K from starved cells. Taken together, our data support a mechanism where the regulatory loop and inhibitory phosphorylation of the loop reduce the affinity of eEF-2K for CaM, potentially explaining how eEF-2K activity is suppressed under normal cellular conditions. Our data also reveals that activation of the cellular enzyme via starvation is correlated with the removal of inhibitory phosphorylations and a significant increase in the affinity of eEF-2K for CaM. These findings give insight into the regulatory mechanisms that allow eEF-2K to remarkably integrate signals from upstream nutrient and energy-sensing cellular pathways to ultimately control protein synthesis rates. This work is supported by NIH award R01 GM123252.