Nerve growth factor‐induced phosphorylation of amphiphysin‐1 by casein kinase 2 regulates clathrin–amphiphysin interactions

Abstract

Amphiphysins interact directly with clathrin and have a function in clathrin-mediated synaptic vesicle recycling and clathrin-mediated endocytosis. The neuronal isoform amphiphysin-1 is a serine/threonine phosphoprotein that is dephosphorylated upon stimulation of synaptic vesicle endocytosis. Rephosphorylation was stimulated by nerve growth factor. We analysed the regulation of amphiphysin-clathrin interactions by phosphorylation. The N-terminal domain of clathrin bound to unphosphorylated amphiphysin-1, but not to the phosphorylated protein. A search for possible phosphorylation sites revealed two casein kinase 2 consensus motifs in close proximity to the clathrin binding sites in amphiphysin-1 and -2. We mutagenized these residues (T350 and T387) to glutamate, mimicking a constitutive phosphorylation. The double mutant showed a strong reduction in clathrin binding. The assumption that casein kinase 2 phosphorylates amphiphysin-1 at T350 and T387 was corroborated by experiments showing that: (i) casein kinase 2 phosphorylated these residues directly in vitro, (ii) when expressed in HeLa cells, the glutamate mutant showed reduced phosphorylation, and (iii) casein kinase 2 inhibitors blocked nerve growth factor-induced phosphorylation of endogenous amphiphysin-1 in PC12 cells. These observations are consistent with the hypothesis that, upon activation by nerve growth factor, casein kinase 2 phosphorylates amphiphysin-1 and thereby regulates the endocytosis of clathrin-coated vesicles via the interaction between clathrin and amphiphysin.

Setting the F-BAR: Functions and regulation of the F-BAR protein family

“…In neurons, phosphorylation of amphiphysin occurs at multiple sites in vivo, with dephosphorylation after neural stimulation. 49 Phosphorylation of amphiphysin at separate residues inhibits its interaction with clathrin 53 or endophilin. 54 Phosphorylation of endophilin inhibits its interaction with dynamin.…”

The in Vivo Phosphorylation Sites in Multiple Isoforms of Amphiphysin I from Rat Brain Nerve Terminals

“…Three proline-directed protein kinases phosphorylate amphI, including cdk5/p35 (Ser-262, Ser-272, Ser-276, Ser-285, and Thr-310) (15,16,23), mitogen-activated protein kinase (MAPK) (Ser-285 and Ser-293) (24), and Dyrk1A/minibrain kinase (Ser-293 with minor sites including Thr-310, Ser-295, and Thr-312) (17). CK2 phosphorylates amphI in vitro on Thr-350 and Thr-387 (18). The majority of the in vitro phosphosites identified to date cluster to a small region within the PRD (amphI-PRD-(260 -312)).…”

CK2 Phosphorylation of an Acidic Ser/Thr Di-isoleucine Motif in the Na+/H+ Exchanger NHE5 Isoform Promotes Association with β-Arrestin2 and Endocytosis

“…Interestingly, CK2 has also been shown to regulate the cell surface abundance of the epithelial Na ϩ /H ϩ exchanger NHE3 isoform, although in this instance, direct phosphorylation of the transporter selectively increased its insertion into the plasma membrane without affecting its rate of internalization (79). Aside from cargo, CK2 also phosphorylates numerous ancillary proteins associated with clathrin-coated vesicles (80,81), including ␤-Arr2 (55,56). The consequences on ␤-Arr2 function, however, are controversial and not fully resolved.…”