MAJOR CELL SURFACE-LOCATED PROTEIN SUBSTRATES OF AN ECTOPROTEIN KINASE ARE HOMOLOGS OF KNOWN NUCLEAR PROTEINS

Citation
P. Jordan et al., MAJOR CELL SURFACE-LOCATED PROTEIN SUBSTRATES OF AN ECTOPROTEIN KINASE ARE HOMOLOGS OF KNOWN NUCLEAR PROTEINS, Biochemistry, 33(49), 1994, pp. 14696-14706
Citations number
48
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
33
Issue
49
Year of publication
1994
Pages
14696 - 14706
Database
ISI
SICI code
0006-2960(1994)33:49<14696:MCSPSO>2.0.ZU;2-5
Abstract
Cell surface polypeptides serve as substrates for a casein kinase-like ecto-protein kinase activity which is demonstrable under stringent cr iteria with intact cells using micromolar levels of extracellular [gam ma-P-32]ATP. Two major P-32-labeled proteins, designated as pp100 and pp120 after their apparent molecular masses on SDS-PAGE under reducing and nonreducing conditions, have repeatedly appeared in the phosphopr otein spectra of different cell types. We have chosen HeLa cells as a source for the biochemical characterization and isolation of pp100 and pp120. Phosphorylation of pp100 and pp120 occurs in their extracellul ar domains at seryl residues of amino acid side chains. Several criter ia deduced from the heparin sensitivity of the ecto-protein kinase and its substrate-induced shedding into the cell supernatant indicated th at surface phosphorylation is a function of the ecto-protein kinase. T he radioactive phosphorylation of pp100 and pp120 which coincides with their biotinylation on 2D-blots can be reversed by mild trypsination of intact cells. Purification and enrichment of pp100 and pp120 were a chieved on the basis of radioactivity detection on and isolation from 1D- and 2D-gels. Amino acid sequence analysis performed on tryptic dig ests of purified ecto-phosphoproteins in most cases showed significant consensus sequences between pp100 and the nuclear RNA-binding protein nucleolin while pp120 sequences proved to be related to hnRNP U, a nu cleoplasmic pre-mRNA-binding protein. Immunochemical analysis using an ti-nucleolin and anti-hnRNP U antibodies combined with comparative pho sphorylation and characterization of the ecto-proteins with authentic nucleolin and hnRNP U further established the close relationship, sugg esting surface membrane versions of the nuclear proteins.