A variety of cell membrane proteins become phosphorylated in their ecto-domains by cell-surface protein kinase (ecto-PK) activities, as detected in a broad spectrum of cell types. This study reports the isolation and identification of a frequent ecto-PK substrate, ecto-p120, using HeLa cells as a model. Data from MS and further biochemical and immunochemical means identified ecto-p120 as a cell-surface homologue of human nucleolar phosphoprotein p140 (hNopp140), which belongs to the family of argyrophilic (AgNOR-stainable) proteins. The superposition of 32P-labelled ecto-nucleolar phosphoprotein p140 (ecto-Nopp140) with anti-Nopp140 immunostaining could be demonstrated in a wide range of cell lines without any exceptions, suggesting a nearly universal occurrence of cell-surface Nopp140. A previous, tentative association of ecto-p120 with the nucleoplasmic pre-mRNA-binding protein hnRNP U has thus been supplanted, since improved purification techniques have allowed unambiguous identification of this ecto-PK cell-surface substrate. Furthermore, we have shown that rapid suppression of ecto-hNopp140 phosphorylation resulted upon a rise in the free extracellular calcium, while lowering the calcium concentrations returned ecto-Nopp140 phosphorylation to the original level. It is important to note that these Ca2+-dependent effects on ecto-Nopp140 phosphorylation are not accompanied by alterations in the phosphorylation of other ecto-PK substrates. Our results indicate that, in addition to nucleolin, a further nucleolar protein, which was considered initially to be strictly intracellular, is identified as a cell-surface phosphoprotein.
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December 2001
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Research Article|
December 10 2001
Ecto-protein kinase substrate p120 revealed as the cell-surface-expressed nucleolar phosphoprotein Nopp140: a candidate protein for extracellular Ca2+-sensing
Dieter KÜBLER
Dieter KÜBLER
1
German Cancer Research Center, Division of Pathochemistry B0100, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
1e-mail d.kuebler@dkfz.de
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Publisher: Portland Press Ltd
Received:
March 23 2001
Revision Received:
September 27 2001
Accepted:
October 10 2001
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2001
2001
Biochem J (2001) 360 (3): 579–587.
Article history
Received:
March 23 2001
Revision Received:
September 27 2001
Accepted:
October 10 2001
Citation
Dieter KÜBLER; Ecto-protein kinase substrate p120 revealed as the cell-surface-expressed nucleolar phosphoprotein Nopp140: a candidate protein for extracellular Ca2+-sensing. Biochem J 15 December 2001; 360 (3): 579–587. doi: https://doi.org/10.1042/bj3600579
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