Temporal Regulation and Functional Impact of ERK Activity Near the Plasma Membrane

The ERK1/2 pathway plays critical roles in eukaryotic biology by transducing extracellular signals into cell‐fate decisions. One conundrum is in understanding how disparate signals induce specific responses through a common, ERK‐dependent kinase cascade. One major mechanism to control ERK‐signaling to induce proper cellular responses is through precise regulation over the temporal dynamics of ERK activity. For example, in the pheochromocytoma PC‐12 cell line, ERK exhibits transient activity in response to Epidermal Growth Factor (EGF), ultimately resulting in proliferation. On the other hand, PC‐12 cells respond to Nerve growth factor (NGF) with sustained ERK phosphorylation and eventual differentiation into neuronal‐like cells. An additional mechanism of control over ERK is through spatial regulation of activity; however, it has been difficult to accurately examine differences in activity among specific subcellular locations. To address this issue, we have expanded the toolbox of FRET‐based ERK biosensors by creating a series of improved biosensors that are specifically targeted to various subcellular regions to measure spatiotemporal changes in ERK enzymatic activity in real‐time in living cells. Using these sensors, we have tested the hypothesis that a single extracellular signal can induce different temporal dynamics between subcellular compartments in PC‐12 cells. We report that while EGF induces transient ERK activity in the cytosol and nucleus, EGF simultaneously induces sustained ERK activity near the plasma membrane of PC‐12 cells. Furthermore, we report that PKA and cAMP play an integral role in the regulation of ERK at the plasma membrane. Specifically, we observe that increasing basal cAMP prior to EGF induces a more transient activity at the plasma membrane in a PKA‐dependent manner. However, our results also indicate that PKA activity post‐EGF is necessary for sustained ERK activity near the plasma membrane, revealing the importance of understanding the temporal dynamics of pathway crosstalk. Furthermore, we report that selective inhibition of ERK activity near the plasma membrane drastically alters PC‐12 cell morphology and adhesion. These data further our fundamental understanding of how the spatiotemporal dynamics and native biochemistry of ERK in living cells modulates cell behavior. Support or Funding Information IRACDA fellowship through NIGMS/NIH award K12GM068524, R01 DK073368, R01 MH111516, R35 CA7622 from NIH This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .