Novel functional proteins interact with midkine in hepatic cancer cells.
BACKGROUND
Midkine is a heparin-binding growth factor that promotes the proliferation, survival, migration and differentiation of various target cells. Midkine plays an important role in tumorigenesis and tumor progression, and is overexpressed in many human malignant tumors. Patients with high tumor midkine expression frequently have a worse prognosis than those with low expression. The present study was designed to investigate the interaction network of midkine in hepatic cancer cells, and to elucidate its role in hepatocellular carcinoma.


METHODS
DNA encoding full-length midkine was cloned into pDBLeu vector to serve as bait in yeast two-hybrid screening to identify interacting proteins. Candidate proteins were examined on SC-Leu-Trp-His+3-AT (20 mmol/L) plates and assayed for X-gal activity, then sequenced and classified according to the GenBank. Finally, identified proteins were expressed by the in vitro expression system pCMVTnT, and protein interactions were confirmed by co-immunoprecipitation.


RESULTS
Using the yeast two-hybrid system, we found 6 proteins that interacted with midkine: NK-kappa-B inhibitor alpha (I-κ-B-alpha), Dvl-binding protein naked cuticle 2, granulin, latent active TGF-beta binding protein 3, latent active TGF-beta binding protein 4, and phospholipid scramblase 1. In vitro co-immunoprecipitation demonstrated that all identified proteins directly interacted with midkine.


CONCLUSIONS
The identification of midkine-interacting proteins in hepatic cancer cells indicates that midkine is a multifunctional factor that may participate in cell migration, differentiation, and proliferation, and is also associated with the multicellular response feedback during the development of hepatocellular carcinoma.
Zhejiang University School of Medicine, Hangzhou, China.
Qiang
Q
Yan
Hui-Lian
HL
Huang
Xing
X
Yao
Jing
J
Li
Li-Qin
LQ
Li
Jing
J
Zhong
Li-Shan
LS
Min
Li-Cheng
LC
Dai
Shu-Sen
SS
Zheng
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002352:Carrier Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D047468:Immunoprecipitation; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D053779:Latent TGF-beta Binding Proteins / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000078792:Midkine; D000072000:NF-KappaB Inhibitor alpha; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000378:metabolism; D000077153:Progranulins; D011485:Protein Binding; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; C492314:GRN protein, human; D020672:I-kappa B Proteins; D036341:Intercellular Signaling Peptides and Proteins; C509038:LTBP3 protein, human; C509040:LTBP4 protein, human; D053779:Latent TGF-beta Binding Proteins; C066254:MDK protein, human; C000606204:NFKBIA protein, human; C449092:NKD2 protein, human; D009414:Nerve Growth Factors; C493422:PLSCR1 protein, human; D049294:Phospholipid Transfer Proteins; D000077153:Progranulins; D000078792:Midkine; D000072000:NF-KappaB Inhibitor alpha