Related Experiment Videos
Ecto-protein kinase substrate p120 revealed as the cell-surface-expressed nucleolar phosphoprotein Nopp140: a
1German Cancer Research Center, Division of Pathochemistry B0100, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. d.kuebler@dkfz.de
Abstract:
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 (32)P-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 Ca(2+)-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.
Insights
Researchers identified a cell-surface protein, ecto-nucleolar phosphoprotein p140 (ecto-Nopp140), a homolog of a human nucleolar protein. Its phosphorylation is regulated by extracellular calcium levels, indicating a new role for this protein outside the cell nucleus.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cell surface protein kinases (ecto-PKs) phosphorylate ecto-domains of membrane proteins across various cell types.
- The identification and function of specific ecto-PK substrates are crucial for understanding cell surface signaling.
- Human nucleolar phosphoprotein p140 (hNopp140) is a known intracellular protein involved in nucleolar functions.
Purpose of the Study:
- To isolate and identify a frequent ecto-PK substrate from HeLa cells.
- To characterize the identified substrate and its relationship to known proteins.
- To investigate the regulation of this ecto-PK substrate's phosphorylation.
Main Methods:
- Mass spectrometry (MS) for protein identification.
- Biochemical and immunochemical techniques for characterization.
- Phosphorylation assays using radiolabeled proteins and calcium concentration manipulation.
Main Results:
- A frequent ecto-PK substrate, ecto-p120, was isolated and identified as a cell-surface homolog of hNopp140, named ecto-hNopp140.
- Ecto-hNopp140 was found in a wide range of cell lines, suggesting near-universal occurrence.
- Phosphorylation of ecto-hNopp140 was rapidly suppressed by increased extracellular calcium and restored by lowering calcium, without affecting other ecto-PK substrates.
Conclusions:
- A nucleolar protein, hNopp140, previously considered strictly intracellular, is identified as a cell-surface phosphoprotein (ecto-hNopp140).
- Ecto-hNopp140 phosphorylation is specifically and reversibly regulated by extracellular calcium concentrations.
- This discovery expands the known functions of nucleolar proteins and highlights a novel cell surface signaling mechanism.