Ecto-protein kinase substrate p120 revealed as the cell-surface-expressed nucleolar phosphoprotein Nopp140: a candidate protein for extracellular Ca2+-sensing.
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.
Kübler
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German Cancer Research Center, Division of Pathochemistry B0100, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. d.kuebler@dkfz.de
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004533:Egtazic Acid / Q000494:pharmacology; D005110:Extracellular Space / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D010446:Peptide Fragments; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; C075564:NOLC1 protein, human; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D004533:Egtazic Acid; D011494:Protein Kinases; C034454:ectoprotein kinase; D002118:Calcium