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. 2008 Jul 25;13(4):632–648. doi: 10.2478/s11658-008-0027-4

EHDS are serine phosphoproteins: EHD1 phosphorylation is enhanced by serum stimulation

Boris Fichtman 1, Liat Ravid 2, Debora Rapaport 3, Mia Horowitz 3,✉
PMCID: PMC6275761  PMID: 18661112

Abstract

Endocytic processes are mediated by multiple protein-protein interacting modules and regulated by phosphorylation and dephosphorylation. The Eps15 homology domain containing protein 1 (EHD1) has been implicated in regulating recycling of proteins, internalized both in clathrin-dependent and clathrin-independent endocytic pathways, from the recycling compartment to the plasma membrane. EHD1 was found in a complex with clathrin, adaptor protein complex-2 (AP-2) and insulin-like growth factor-1 receptor (IGF-1R), and was shown to interact with Rabenosyn-5, SNAP29, EHBP1 (EH domain binding protein 1) and syndapin I and II. In this study, we show that EHD1, like the other human EHDs, undergoes serine-phosphorylation. Our results also indicate that EHD1 is a serum-inducible serine-phosphoprotein and that PKC (protein kinase C) is one of its kinases. In addition, we show that inhibitors of clathrin-mediated endocytosis decrease EHD1 phosphorylation, while inhibitors of caveolinmediated endocytosis do not affect EHD1 phosphorylation. The results of experiments in which inhibitors of endocytosis were employed strongly suggest that EHD1 phosphorylation occurs between early endosomes and the endocytic recycling compartment.

Key words: Endocytosis, EHD1, EH domain, Phosphorylation

Full Text

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Abbreviations used

AP-2

adaptor protein complex-2

BSA

bovine serum albumin

CHO

Chinese hamster ovary

cpm

counts per minute

dFCS

dialyzed FCS

DMEM

Dulbecco modified eagle medium

EHBP1

EH domain binding protein 1

FCS

fetal calf serum

FYVE

phenylalanine-tyrosine-valine-glutamate

IGF-1R

insulin-like growth factor-1 receptor

mDHFR

mouse dihydrofolate reductase

MEM-Alpha

minimal essential medium-alpha

ORF

open reading frame

PBS

phosphate buffered saline

PKC

protein kinase C

PVDF

polyvinidilen difluoride

TLC

thin-layer chromatography

TLE

thin-layer electrophoresis

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