EXPERIMENTAL CELL RESEARCH 203, 259-269 (1992) Nucleolin Is an AS-NOR Protein; This Property Is Determined by Its Amino-Terminal Domain Independently of Its Phosphorylation State PASCAL ROUSSEL,* PASCALE BELENGUER,t FRANCOIS AMALRIC,? AND DANIELE HERNANDEZ-VERDUN**’ *Institut Jacques Monad, 2, Place Jussieu, Tour 43, 75251 Paris Ceder 05, France; and tCmtre de Recherche de Biochimie et de Ggnne’tique Cellulaires du C. N. R. S. 118, route de Narbonne, 31062 Toulouse Cedex, France The Ag-NOR proteins are defined as markers of “ac- tive” ribosomal genes. They correspond to a set of pro- teins specifically located in the nucleolar organizer re- gions (NORs), but have not yet been clearly identified. We adapted the specific detection method of the Ag- NOR proteins to Western blots in order to identify these proteins. Using a purified protein, Western blots, and immunological characterization, the present study brings the first direct evidence leading to the identity of one Ag-NOR protein. We found that nucleolin is specifi- cally revealed by Ag-NOR staining. Using different nu- cleolin fragments generated by CNBr cleavage and by overexpression in Escherichia coli, we demonstrate that the amino-terminal domain of nucleolin and not the carboxy-part of the protein is involved in silver staining. Moreover, as the pattern of staining does not vary using casein kinase II- and cdc%phosphorylated nucleolin or dephosphorylated nucleolin, we conclude that the reduction of the silver ions is not linked to the phosphorylation state of the molecule. We propose that the concentration of acidic amino acids in the amino- terminal domain of nucleolin is responsible for Ag-NOR staining. This hypothesis is also supported by the find- ing that poly L-glutamic acid peptides are silver stained. These results provide data that can be used to explain the specificity of Ag-NOR staining. Furthermore, we clearly establish that proteolysis of the amino-terminal Ag-NOR-sensitive part of nucleolin occurs in vitro, leading to the accumulation of the carboxy-terminal Ag-NOR-negative part of the protein. We argue that this cleavage occurs in vivo as already proposed, bear- ing in mind that nucleolin is present in the fibrillar and in the granular component of the nucleolus, whereas no Ag-NOR staining is observed in the latter nucleolar component. 0 1992 Academic Press, Inc. INTRODUCTION One of the earliest events to occur following stimula- tion of cell growth is a change in the rate of ribosome ’ To whom correspondence and reprint requests should be ad- dressed. Fax: (33) 1 44 27 59 94. precursor synthesis, suggesting a close relationship be- tween the rate of cell proliferation and ribosome bio- genesis [l]. It is therefore of general interest to possess markers of the level of ribosome biogenesis correlated with cell proliferation. The best example of this correla- tion is provided by a set of proteins associated with the ribosomal genes whose amount is directly related to ri- bosomal gene transcription and/or cell duplication [2-4; for review see 51. These proteins, called Ag-NOR’ pro- teins, are defined as markers of “active” ribosomal genes. They correspond to a set of proteins specifically located in the NORs that are identified by their ability to reduce silver under acidic conditions in which most other cellular proteins remain unstained [6]. The interest in this set of proteins started when it was demonstrated that transcription of the ribosomal genes depends on the presence of Ag-NOR proteins [7]. More- over, the finding that the amount of these proteins re- veals the degree of nucleolar activity, presumably of transcriptional activity, explains the recent rush (362 references within the last 3 years) to detect Ag-NOR proteins in cancer cells [8-10; for review see 111. How- ever, even though the detection of Ag-NOR proteins is a selective and widely used marker of nucleolar activity, there is presently no clear identification of these pro- teins. Based on the size of the bands revealed on gels by Ag-NOR staining (104 and 37 kDa, and occasionally 190, 135, 78, and 29 kDa) [12-151, it has been proposed that nucleolin (100 kDa) and protein B23 (37 kDa) are the major Ag-NOR proteins. In the nucleoli, nucleolin and B23 have been located in the dense fibrillar compo- nent (DFC) and the granular component (GC) but ap- pear to be excluded from the fibrillar centers (FCs) [ 16- 201, whereas the Ag-NOR proteins are detected in the FCs and DFC exclusively and not in the GC [21, 221. ’ Abbreviations used: CKII, casein kinase II; DFC, dense fibrillar component; FC, fibrillar center; GC, granular component; MEM, minimum essential medium; NOR, nucleolar organizer region; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; rRNA, ribosomal RNA; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid. 259 0014-4827192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.