Identification of nucleolin as a binding protein for midkine (MK) and heparin-binding growth associated molecule (HB-GAM).
Midkine (MK) is a heparin-binding growth/differentiation factor with a molecular weight of 13 kDa, and is structurally unrelated to fibroblast growth factors (FGF). We studied MK-binding proteins in order to clarify the action mechanism of MK. A 100-kDa protein was identified in PYS-2, 3T3, and L cells as an MK-binding protein by a ligand blot experiment. This MK-binding protein was purified by affinity chromatography on an MK-agarose column followed by SDS polyacrylamide gel electrophoresis. Sequence determination of N-terminal 23 amino acid residues revealed that the MK-binding protein was nucleolin, a major nucleolar protein, which functions as a shuttle protein between the nucleus and cytoplasm and is located also on the cell surface. Heparin-binding growth associated molecule (HB-GAM), which has 50% sequence identity with MK, fused to maltose-binding protein also bound to nucleolin. On the other hand, basic FGF (bFGF) scarcely bound to nucleolin in the absence of heparin, while both MK and bFGF bound weakly to nucleolin in the presence of heparin. Nuclear localization of MK was shown in hemangioma cells by immunohistochemical staining. These findings supported the hypothesis that parts of the MK and HB-GAM are translocated to the nucleus after binding with nucleolin.
Take
M
M
Department of Internal Medicine, Faculty of Medicine, Kagoshima University.
Tsutsui
J
J
Obama
H
H
Ozawa
M
M
Nakayama
T
T
Maruyama
I
I
Arima
T
T
Muramatsu
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D000078792:Midkine; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D016601:RNA-Binding Proteins; D017385:Sequence Homology; D000097796:Nucleolin
D002352:Carrier Proteins; D016207:Cytokines; D016222:Fibroblast Growth Factor 2; D000078792:Midkine; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D000097796:Nucleolin; C066845:pleiotrophin