The multifunctional nuclear protein p54nrb is multiphosphorylated in mitosis and interacts with the mitotic regulator Pin1

The human protein p54(nrb) and its mouse homolog NonO have been implicated in a variety of nuclear processes including transcription, pre-mRNA processing, nuclear retention of edited RNA and DNA relaxation. We have identified p54(nrb) as an antigen of the phosphodependent monoclonal antibodies CC-3 and MPM-2 and shown that this protein is phosphorylated on multiple sites during mitosis. The use of the cyclin-dependent protein kinase inhibitor roscovitine and immunodepletion studies. with an anti-cyclin B1 antibody established that Cdk1 was responsible for the phosphorylation of the carboxy-terminal extremity of p54nrb whereas a different kinase appeared to be involved in the generation of CC-3 epitope(s) in the amino-terminal moiety of the protein. Like many CC-3 and MPM-2 antigens, we show that p54(nrb) is a target of the peptidylprolyl isomerase Pin1, suggesting that it may be regulated by phosphorylation-dependent conformational changes as many other nuclear proteins upon entry into mitosis. In addition, site-directed mutagenesis indicated that the interaction of Pin1 with p54(nrb) was mediated by three threonine residues located in the proline-rich carboxyterminal extremity of the protein. Our results also showed that Pin1 binding was favored when at least two of the three threonine residues were phosphorylated, suggesting a regulation mechanism based on multisite phosphorylation. (C) 2004 Elsevier Ltd. All rights reserved.