Cell, Vol. 60, 791-601, March 9, 1990, Copyright 0 1990 by Cell Press Identification of Major Nucleolar Proteins As Candidate Mitotic Substrates of cdc2 Kinase M. Peter,’ J. Nakagawa,” M. Dor6e,t J. C. Labbe,+ and E. A. Nigg’ * Swiss institute for Experimental Cancer Research (ISREC) Chemin des Boveresses 155 CH-1066 Epalinges Switzerland tCNRS and INSERM B.P. 5051 F-34033 Montpellier Cedex France Summary Following the identification of the cdc2 kinase as a imajor element controlling entry of cells into mitosis, lit is important to define the physiological target range of this enzyme. Here, we demonstrate that two major nucleolar proteins, nucleolin and N038, are highly phosphorylated during mitosis. Importantly, the two nucleolar proteins are also phosphorylated by highly purified starfish cdc2 kinase in vitro, on sites that cor- respond to those observed specifically during mitosis iin vivo. A repeated motif (TPXKK) is identified as the likely mitotic phosphoacceptor site in nucleolin, in that a synthetic peptide mimicking this site functions as both a substrate and a competitive inhibitor of cdc2 kinase. These results identify two novel candidate substrates for cdc2 kinase, and they implicate protein phosphorylation in controlling mitotic changes in nu- cleolar structure and activity. Hntroduction During mitosis, cell nuclei undergo extensive changes in structural organization, and, concomitantly, most nuclear activities are transiently arrested. As indicated by the re- cent identification of the cdc2 kinase as a major compo- nent of the universal mitotic inducer MPF (M phase- promoting factor), many of these events are likely to be controlled by protein phosphorylation (for review see Dun- phy and Newport, 1988; Norbury and Nurse, 1989; Lohka, ‘1989; Hunt, 1989). Over the last few years, much effort has been dedicated to studying mitotic condensation of chro- matin, transient breakdown of the nuclear envelope, and rearrangements of the cytoskeleton (for review see Lohka and Mailer, 1987; Newport and Forbes, 1987; Mitchison, ‘1988; Nigg, 1988; Gerace and Burke, 1988; Lohka, 1989). In contrast, little information is currently available on the regulation of the mitotic reorganization of the structure of nucleoli. During interphase (I phase), nucleoli are compact or- ganelles functioning in both transcription and processing of ribosomal RNA as well as in the assembly of precursors for ribosomal subunits (for review see Hadjiolov, 1985; Sommerville, 1986). During mitosis, nucleolar structure undergoes extensive changes (Goessens, 1984) and nu- cleolar activities cease almost completely (Hadjiolov, 1985). lmmunocytochemical studies indicate that, beginning in prophase, most nucleolar proteins disperse throughout the mitotic cell, but a few proteins remain concentrated at the surface of mitotic chromosomes (reviewed in Som- merville, 1986; Nigg, 1988). Prominent among the nucleo- lar proteins that appear to disperse diffusely at the onset of mitosis is an abundant 38 kd protein implicated in pack- aging and transport of preribosomal particles (Busch, 1984; Schmidt-Zachmann et al., 1987; Borer et al., 1989). This protein has been given a number of different names: 823 (Busch, 1984), NO38 (Schmidt-Zachmann et al., 1987), numatrin (Feuerstein et al., 1987) and nucleophos- min (Chan et al., 1989). Hereafter it is referred to as N038. A representative of the nucleolar proteins that remain as- sociated, at least partially, with mitotic chromosomes is nucleolin, a 92 kd protein formerly called C23 (Ochs et al., 1983; Busch et al., 1984). Nucleolin is believed to function in the transcription and processing of ribosomal RNA (for review see Lapeyre et al., 1987; Jordan, 1987). Despite in- tensive study on the structure and properties of these nucleolar proteins, no information has so far been ob- tained on the mechanisms controlling cell cycle-depen- dent changes in their subcellular distribution and function. Following the identification of the cdc2 kinase as a ma- jor element controlling entry of cells into mitosis, much ef- fort is currently focused on identifying proteins that might represent direct and physiological substrates for this ki- nase (Langan et al., 1989; Morgan et al., 1989; Shenoy et al., 1989). Here we show that the major nucleolar proteins nucleolin and NO38 are highly phosphorylated during mi- tosis. In addition, we provide evidence that they are likely to be in vivo substrates of cdc2 kinase. These findings in- dicate that protein phosphorylation may play a major role in controlling nucleolar structure and activity during the cell cycle. Moreover, they suggest that cdc2 function is related not only to mitotic chromosome condensation, spindle formation, and nuclear envelope breakdown, but also to the control of the mitotic fate of nucleoli. Results Phosphorylation of Nucleolin and NO38 in Cell-Free Extracts To approach the question of whether phosphorylation might play a role in the mitotic reorganization of nucleoli, we first analyzed the phosphorylation of nucleolar pro- teins in a cell-free system suitable for studying mitotic events (Nakagawa et al., 1989). This in vitro system is based on incubating chicken embryonic nuclei in the presence of [Y-~~P]ATP in extracts prepared from either M phase-arrested or I phase chicken (DU249) cells. After in- cubation of nuclei in either M or I phase extracts, nucleolar proteins were immunoprecipitated by monoclonal anti- bodies (MAbs) (Borer et al., 1989) and their phosphoryla- tion state examined by gel electrophoresis. As shown in