Rho mediates endocytosis of epidermal growth factor receptor through phosphorylation of endophilin A1 by Rho-kinase.
After binding of epidermal growth factor (EGF), the EGF receptor is activated, internalized by endocytosis, and subsequently degraded in the lysosomal pathway. Endocytotic trafficking of the activated EGF receptor is essential for controlling EGF signaling. Upon ligand-induced activation of EGF receptors, Cbl (ubiquitin ligase) binds to the activated receptor and leads to translocation of the CIN85 (Cbl-interacting protein of 85 kDa)/endophilin complex in the vicinity of the activated EGF receptors. Endophilin is known as a key regulator of clathrin-mediated endocytosis, and the translocation of endophilin in the vicinity of active EGF receptor is thought to promote receptor internalization. The constitutively active mutant of small GTPase Rho inhibits EGF receptor endocytosis. In this study, we found that this inhibitory effect was canceled by the dominant negative form of Rho-associated kinase (Rho-kinase), which is an effector of Rho. To clarify the molecular mechanisms of endocytosis downstream of Rho/Rho-kinase signal, we searched for and identified endophilin A1 as a novel substrate of Rho-kinase. We identified the phosphorylation site of endophilin A1 at Thr-14 and made endophilin T14D (substitution of Thr-14 by Asp), which is expected to mimic the phosphorylation state of endophilin A1. Endophilin T14D inhibited EGF receptor internalization. Furthermore, phosphorylation of endophilin by Rho-kinase inhibited the binding to CIN85. Taken together, these results suggest that Rho-kinase phosphorylates endophilin downstream of Rho and regulates EGF receptor endocytosis through the inhibition of binding between endophilin and CIN85.
Kaneko
Takako
T
Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine, 65 Tsurumai, Showaku, Nagoya, Aichi, 466-8550, Japan.
Maeda
Akio
A
Takefuji
Mikito
M
Aoyama
Hiroki
H
Nakayama
Masanori
M
Kawabata
Saeko
S
Kawano
Yoji
Y
Iwamatsu
Akihiro
A
Amano
Mutsuki
M
Kaibuchi
Kozo
K
D000217:Acyltransferases / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D019556:COS Cells; D002417:Cattle; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004705:Endocytosis / Q000502:physiology*; D066246:ErbB Receptors / Q000378:metabolism* / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D014162:Transfection; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D047908:Intracellular Signaling Peptides and Proteins; C433810:SH3KBP1 protein, human; D000217:Acyltransferases; C018764:2-acylglycerophosphate acyltransferase; D066246:ErbB Receptors; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein