Crosstalk between Akt/GSK3β signaling and dynamin-1 regulates clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) regulates signaling from the plasma membrane. Analysis of clathrin-coated pit (CCP) dynamics led us to propose the existence of a rate-limiting, regulatory step(s) that monitor the fidelity of early stages in CCP maturation. Here we show that nascent endocytic vesicles formed in mutant cells displaying rapid, dysregulated CME are defective in early endosomal trafficking, maturation and acidification, confirming the importance of this "checkpoint." Dysregulated CME also alters EGF receptor signaling and leads to constitutive activation of the protein kinase Akt. Dynamin-1, which was thought to be neuron specific, is activated by the Akt/GSK3β signaling cascade in non-neuronal cells to trigger rapid, dysregulated CME. Acute activation of dynamin-1 in RPE cells by inhibition of GSK3β accelerates CME, alters CCP dynamics and, unexpectedly, increases the rate of CCP initiation. CRISPR-Cas9n-mediated knockout and reconstitution studies establish that dynamin-1 is activated by Akt/GSK3β signaling in H1299 non-small lung cancer cells. These findings provide direct evidence for an isoform-specific role for dynamin in regulating CME and reveal a feed-forward pathway that could link signaling from cell surface receptors to the regulation of CME. Synopsis Dynamin-1, previously thought to be neuron specific, is activated by an Akt/GSK3β signaling cascade in non-neuronal cells. Dynamin-1 activation alters the rate and regulation of clathrin-mediated endocytosis, providing a feed-forward pathway between endocytosis and signaling. An endocytic checkpoint monitors the fidelity of clathrin-coated vesicle (CCV) formation. Dysregulated clathrin-mediated endocytosis (CME) alters cell signaling and proliferation. Dynamin-1 and dynamin-2 differentially regulate early stages of CME. Endogenous dynamin-1 is activated by an Akt/GSK3β signaling cascade, illustrating a first non-neuronal role of dynamin-1. Activation of dynamin-1 by Akt and GSK3β in non-neuronal cells alters the rate and regulation of CME, providing a feed-forward pathway between signaling and endocytosis.