Premium
Dynamic equilibrium of eEF‐2K and CaM as a regulatory logic circuit: investigations in MCF10A cells
Author(s) -
Dalby Kevin N.,
Giles David H.,
Crittenden Chris M.,
Kaoud Tamer S.,
Ghose Ranajeet,
Brodbelt Jennifer
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.526.14
Subject(s) - mtorc1 , microbiology and biotechnology , ampk , protein kinase a , intracellular , phosphorylation , kinase , ionomycin , mapk/erk pathway , chemistry , biology , protein kinase b
Eukaryotic elongation factor 2 kinase (eEF‐2K) is a Ca 2+ /Calmodulin (CaM)‐dependent kinase that phosphorylates eEF‐2 and inactivates it, thereby blocking translation. Studies have correlated eEF‐2K expression with poorer outcomes in cancer, and it was recently shown to contribute to the Warburg effect. eEF‐2K is activated by the PKA and AMPK pathways, and inhibited by the ERK, mTORC1, and p38∂ pathways, among others. How eEF‐2K integrates these signals into a coherent output is not understood. We recently showed that phosphorylation of eEF‐2K on Ser500 by PKA, like Ca 2+ , drives the association of eEF‐2K and CaM to activate the kinase. We hypothesized that other regulatory signals converge on the equilibrium of eEF‐2K and CaM, either driving it toward the bound (fully active) or unbound (fully inactive) state. Here, we establish that at Ca 2+ levels basal to MCF10A cells, removal of either ERK or mTORC1 activity is sufficient to fully activate eEF‐2K; increasing intracellular Ca 2+ with ionomycin is sufficient to overcome these inhibitory signals. Once mTORC1 activity is already removed, additionally increasing intracellular Ca 2+ produces no increase in eEF‐2K activity. Thus, inhibitory signals maintain eEF‐2K in the unbound (inactive) state, but sufficiently high Ca 2+ can overcome this repression. Blocking glycolysis with 2‐deoxyglucose (2‐DG), which induces full eEF‐2K activation, results in the shutdown of ERK and mTORC1, increases intracellular Ca 2+ , and activates AMPK. Although AMPK phosphorylates eEF‐2K directly at Ser398, this has no effect on its association with CaM and is not required for activation by 2‐DG in cells. Similarly, directly activating AMPK (~7‐fold) with A769662 only weakly activates eEF‐2K (<2‐fold), and has no effect on ERK, mTORC1, or Ca 2+ . We show that, to a first approximation, the activation status of eEF‐2K is described by a simple Boolean “logic circuit”, where inhibitory inputs act to maintain the circuit in the OFF (unbound) state, and activating ones switch the circuit to the ON (CaM‐bound) state. AMPK appears to act on the circuit's inputs rather than forming a critical component itself. Further, we discuss how this “logic circuit” can be generalized to other cell types and ultimately reveal the mechanisms underpinning the regulation of eEF‐2K in cancer. Support or Funding Information R01 GM123252, F‐1390 Welch Foundation. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .