Unconventional EGF-induced ERK1/2-mediated Kv1.3 endocytosis

Abstract

The potassium channel Kv1.3 plays roles in immunity, neuronal development and sensory discrimination. Regulation of Kv1.3 by kinase signaling has been studied. In this context, EGF binds to specific receptors (EGFR) and triggers tyrosine kinase-dependent signaling, which down-regulates Kv1.3 currents. We show that Kv1.3 undergoes EGF-dependent endocytosis. This EGF-mediated mechanism is relevant because is involved in adult neural stem cell fate determination. We demonstrated that changes in Kv1.3 subcellular distribution upon EGFR activation were due to Kv1.3 clathrin-dependent endocytosis, which targets the Kv1.3 channels to the lysosomal degradative pathway. Interestingly, our results further revealed that relevant tyrosines and other interacting motifs, such as PDZ and SH3 domains, were not involved in the EGF-dependent Kv1.3 internalization. However, a new, and yet undescribed mechanism, of ERK1/2-mediated threonine phosphorylation is crucial for the EGF-mediated Kv1.3 endocytosis. Our results demonstrate that EGF triggers the down-regulation of Kv1.3 activity and its expression at the cell surface, which is important for the development and migration of adult neural progenitors.

Ubiquitination mediates Kv1.3 endocytosis as a mechanism for protein kinase C-dependent modulation

“…The “antibody-feeding assay” was essential to unequivocally decipher the PKC-dependent endocytosis of the dopamine transporter (DAT) and the EGFR-dependent internalization of Kv1.3 4 21 . HA-Kv1.3-YFP channels that simultaneously contain an extracellular HA tag and an intracellular YFP fluorophore were expressed in HEK-293 cells.…”

“…To further decipher this mechanism, stable Kv1.3 HEK cells were transfected with siRNAs against dynamin II (Dyn II) and Clathrin ( Fig. 4I–Q ), both crucial for the EGFR-mediated clathrin-dependent endocytosis of Kv1.3 4 . Under effective Clathrin and Dyn II depletion ( Fig.…”

“…Kv1.3 signaling relies on the activity, abundance and proper localization of channels at the cell surface. In this respect, Epidermal growth factor receptor (EGFR) activity regulates Kv1.3 by both tyrosine phosphorylation and ERK1/2-dependent endocytosis, with consequences for neuronal fate 4 5 . Kv1.3 is also regulated by PKC, modulating T-cell activation 6 .…”

“…The localization of Kv1.3 in rafts and caveolae is dependent on the accessibility of a caveolin-binding domain near the T1 domain and of the Kvβ subunit recognition motif at the N-terminal of the channel 12 . Evidence demonstrates that the control of Kv1.3 surface abundance takes place at multiple stages, balancing forward trafficking mechanisms to the cell membrane and the internalization of fully functional channels 4 13 . These results strongly support the idea that the prevalence of Kv1.3 channels at the membrane surface has enormous consequences for cell physiology.…”

Endocytosis: A Turnover Mechanism Controlling Ion Channel Function

“…Similarly, EGFR activation leads to Kv1.3 phosphorylation [ 51 ], but in contrast to Kv1.2 phosphorylation, Kv1.3 endocytosis is independent of this tyrosine phosphorylation. EGFR signals induce the downregulation of Kv1.3 via two complementary mechanisms: (i) tyrosine phosphorylation of the channel reduces the Kv1.3 current, and (ii) an unconventional ERK1/2 kinase-dependent mechanism triggers channel endocytosis [ 52 ]. In contrast to their effect on Kv1.3 and Kv1.2, the activation of tyrosine kinase receptors enhances, rather than suppresses, the Kv7.1 current [ 53 ].…”