Cdk5 and GSK3β inhibit fast endophilin-mediated endocytosis

Abstract

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Fast Endophilin-mediated endocytosis, FEME, is not constitutively active but triggered upon receptor activation. High levels of growth factors induce spontaneous FEME, which can be suppressed upon serum starvation. This suggested a role for protein kinases in this growth factor receptor-mediated regulation. Using chemical and genetic inhibition, we find that Cdk5 and GSK3β are negative regulators of FEME. They antagonize the binding of Endophilin to Dynamin-1 and to CRMP4, a Plexin A1 adaptor. This control is required for proper axon elongation, branching and growth cone formation in hippocampal neurons. The kinases also block the recruitment of Dynein onto FEME carriers by Bin1. As GSK3β binds to Endophilin, it imposes a local regulation of FEME. Thus, Cdk5 and GSK3β are key regulators of FEME, licensing cells for rapid uptake by the pathway only when their activity is low.

Epilepsy-related CDKL5 deficiency slows synaptic vesicle endocytosis in central nerve terminals

“…Initially, we confirmed the absence of CDKL5 in lysates of KO neurons ( Figure 2A ). We then analysed a range of presynaptic molecules including proteins important for SV recycling, such as clathrin heavy chain (CHC), dynamin 1 (Dyn1), endophilin A1, and syndapin 1; integral SV proteins, such as Syp1, VGLUT1, and the v-type proton ATPase subunit B (ATP6V1B2); and phosphoproteins that have been implicated in the regulation of SV endocytosis, such as the protein kinases glycogen synthase kinase 3 (GSK3) and Akt (Clayton et al, 2010; Ferreira et al, 2021; Smillie and Cousin, 2012). These latter enzymes were of particular interest, since the PI3K/GSK3/Akt pathway has been one of the most perturbed signalling cascades in CDKL5 deficiency model systems (Amendola et al, 2014; Jiang et al, 2019; Wang et al, 2012).…”

Multivalent Interactions between Molecular Components Involved in Fast Endophilin Mediated Endocytosis Drive Protein Phase Separation

“…In addition to endophilin, various other BAR-domain proteins participate in FEME. Two additional N-BAR family proteins, amphiphysin 1 (Amph1) and BIN1, have been found in endophilin rich spots both at the leading edge of the plasma membrane and in the majority of FEME carriers formed upon receptor engagement in BSC-1 cells 49, 50 . Amph1 was also reported to interact with the SH3 domain of endophilin in vitro via PRMs within its large disordered linker region, and this interaction has been implicated in CME of synaptic vesicles.…”

“…The observed amphiphilic, surfactant-like properties suggest a potential role of Amph1 as a size regulator of endocytic protein assemblies in FEME. Amph1 is mostly expressed in the brain whereas BIN1 is more ubiquitously expressed and plays an important role in FEME by recruiting Dynein 50 . We have already shown that the isoform 9 of BIN1, which has a short linker, similar to endophilin, also undergoes LLPS in the presence of PEG.…”

Cholera Toxin as a Probe for Membrane Biology

“…Endophilin plays several important roles in this pathway, including capture of transmembrane receptor cargo, generation of membrane curvature, and scission of tubular endocytic carriers in cooperation with dynamin and actin via a friction driven process [ 136 , 137 , 140 ]. Microtubules and dynein play important roles in the FEME pathway as well, contributing to membrane tubulation and scission [ 136 , 141 , 142 ]. Interestingly, CTxB is capable of activating FEME: in response to CTxB binding, endoA2 is recruited to the plasma membrane, resulting in uptake of CTxB into endoA2-positive carriers.…”

“…Internalization of CTxB is reduced upon knock down of EndoA2, further implicating FEME as a mechanism that controls toxin uptake [ 136 , 143 ]. Additional machinery that regulates the FEME pathway is continuing to emerge [ 142 , 143 ].…”