Cytoplasmic domain phosphorylation of heparin-binding EGF-like growth factor.
Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a transmembrane precursor protein that is anchored to the plasma membrane. The extracellular EGF-like domain acts as a mitogen and motogen upon ectodomain shedding, but the functional roles of the transmembrane and cytoplasmic domains are largely unknown. We demonstrate here that cytoplasmic domain of HB-EGF is phosphorylated by external stimuli, and that the phosphorylation site is involved in HB-EGF-dependent tumorigenesis. Treatment of Vero cells overexpressing human HB-EGF with 12-O-tetradecanoylphorbol-13-acetate (TPA) caused ectodomain shedding of HB-EGF and generated two carboxyl (C)-terminal fragments with distinct electrophoretic mobilities. Mutation analysis showed that Ser207 in the cytoplasmic domain of HB-EGF is phosphorylated upon TPA stimulation, generating two C-terminal fragments with distinct phosphorylation states. Treatment of cells with lysophosphatidic acid, anisomycin, and calcium ionophore, all of which are known to induce ectodomain shedding, also caused phosphorylation of HB-EGF. Although ectodomain shedding and phosphorylation of HB-EGF occurred coordinately, Ala substitution of Ser207 had no effect on TPA-induced or constitutive ectodomain shedding. Injection of cells overexpressing HB-EGF into nude mice showed that Ala substitution of Ser207 reduced the tumorigenic activity of HB-EGF, even though the cell surface level and ectodomain shedding of HB-EGF were not affected by the mutation. Moreover, we found that the cytoplasmic domain of another EGFR ligand, transforming growth factor-alpha, is phosphorylated upon TPA stimulation. Thus, the present results suggest a novel role for the cytoplasmic domain of HB-EGF and other EGF family growth factors that is regulated by phosphorylation.
Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.
Xiaobiao
X
Wang
Hiroto
H
Mizushima
Satoshi
S
Adachi
Minako
M
Ohishi
Ryo
R
Iwamoto
Eisuke
E
Mekada
D000409:Alanine / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D000841:Anisomycin / Q000494:pharmacology; D002460:Cell Line; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000737:chemistry* / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007476:Ionophores / Q000494:pharmacology; D008246:Lysophospholipids / Q000494:pharmacology; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009154:Mutation; D009374:Neoplasms, Experimental / Q000209:etiology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000032:analysis / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D016211:Transforming Growth Factor alpha / Q000378:metabolism; D014709:Vero Cells
C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007476:Ionophores; D008246:Lysophospholipids; D010446:Peptide Fragments; D016211:Transforming Growth Factor alpha; D012694:Serine; D004815:Epidermal Growth Factor; D000841:Anisomycin; D013755:Tetradecanoylphorbol Acetate; D000409:Alanine; C032881:lysophosphatidic acid