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

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.

Abbreviations

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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Correspondence to Mia Horowitz.

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Fichtman, B., Ravid, L., Rapaport, D. et al. EHDS are serine phosphoproteins: EHD1 phosphorylation is enhanced by serum stimulation. Cell Mol Biol Lett 13, 632–648 (2008). https://doi.org/10.2478/s11658-008-0027-4

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  • DOI: https://doi.org/10.2478/s11658-008-0027-4

Key words

  • Endocytosis
  • EHD1
  • EH domain
  • Phosphorylation