Regulation of cell differentiation by hNUDC via a Mpl-dependent mechanism in NIH 3T3 cells.
Thrombopoietin receptor (Mpl) belongs to the cytokine receptor surperfamily with a large extracellular N-terminal portion responsible for cytokine recognition and binding. Thrombopoietin (TPO) has so far been the only widely studied cytokine for Mpl. However we have recently identified human NUDC (hNUDC), previously described as a human homolog of a fungal nuclear migration protein, as another putative binding partner of Mpl. The purpose of this study is to test the extent of the functioning of hNUDC by identifying protein-protein interactions with Mpl in mammalian cells. The full-length cDNAs encoding Mpl and hNUDC were cloned into pEGFP-N1 and pDsRed2-N1 respectively which were subsequently expressed as Mpl-EGFP (green) and hNUDC-DsRed (red) fusion proteins. Using ELISA and immunofluorescence studies, we have demonstrated the direct binding of hNUDC to cell surface-captured Mpl. We also observed that hNUDC induced significant changes in cellular morphology in NIH 3T3 cells stably transfected with pMpl-EGFP. Interestingly, these morphological changes were characteristic of cells undergoing megakaryocyte differentiation. Extracellular-signal-regulated protein kinases 1 and 2 (ERK1/2) have been shown to mediate such megakaryocyte-like differentiation. In addition, co-expression of Mpl-EGFP and hNUDC-DsRed led to the release of hNUDC-DsRed into the culture medium.
Zhang
Yu Ping
YP
State Key Laboratory of Biocontrol, Zhongshan University, Guangzhou 510275, People's Republic of China.
Tang
Yong Song
YS
Chen
Xu Shen
XS
Xu
Peilin
P
D000818:Animals; D018797:Cell Cycle Proteins / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000502:physiology*; D011485:Protein Binding; D053628:Receptors, Thrombopoietin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013926:Thrombopoietin / Q000502:physiology*
D018797:Cell Cycle Proteins; C512648:NUDC protein, human; D009687:Nuclear Proteins; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; C082218:MPL protein, human; D049452:Green Fluorescent Proteins; D013926:Thrombopoietin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3