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

The human protein p54nrb 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 p54nrb 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 p54nrb 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 p54nrb was mediated by three threonine residues located in the proline-rich carboxy-terminal 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.

Multisite phosphorylation of Pin1-associated mitotic phosphoproteins revealed by monoclonal antibodies MPM-2 and CC-3.

BACKGROUND: The peptidyl-prolyl isomerase Pin1 recently revealed itself as a new player in the regulation of protein function by phosphorylation. Pin1 isomerizes the peptide bond of specific phosphorylated serine or threonine residues preceding proline in several proteins involved in various cellular events including mitosis, transcription, differentiation and DNA damage response. Many Pin1 substrates are antigens of the phosphodependent monoclonal antibody MPM-2, which reacts with a subset of proteins phosphorylated at the G2/M transition. RESULTS: As MPM-2 is not a general marker of mitotic phosphoproteins, and as most mitotic substrates are phosphorylated more than once, we used a different phosphodependent antibody, mAb CC-3, to identify additional mitotic phosphoproteins and eventual Pin1 substrates by combining affinity purification, MALDI-TOF mass spectrometry and immunoblotting. Most CC-3-reactive phosphoproteins appeared to be known or novel MPM-2 antigens and included the RNA-binding protein p54nrb/nmt55, the spliceosomal protein SAP155, the Ki-67 antigen, MAP-1B, DNA topoisomerases II alpha and beta, the elongation factor hSpt5 and the largest subunit of RNA polymerase II. The CC-3 mitotic antigens were also shown to be Pin1 targets. The fine CC-3- and MPM-2-epitope mapping of the RNA polymerase II carboxy-terminal domain confirmed that the epitopes were different and could be generated in vitro by distinct kinases. Finally, the post-mitotic dephosphorylation of both CC-3 and MPM-2 antigens was prevented when cellular Pin1 activity was blocked by the selective inhibitor juglone. CONCLUSION: These observations indicate that the mitotic phosphoproteins associated with Pin1 are phosphorylated on multiple sites, suggesting combinatorial regulation of substrate recognition and isomerization.

[Unexpected roles of the peptidyl-prolyl cis/trans isomerase Pin1]. / Pin1: une peptidyl-prolyl cis/trans isomérase aux rôles insoupçonnés.

Peptidyl-prolyl isomerases (PPIases) are chaperone enzymes which alter the peptide bond between a given amino acid and a proline, changing it from the cis to the trans conformation and vice versa. This modification can cause dramatic structural modifications which can affect the properties of targeted proteins. The ubiquitous PPIase Pin1, conserved from yeast to human, has been shown to be necessary for entry into mitosis. The yeast homologue, Ess1, is essential for cell survival. Pin1 possesses a WW domain which specifically recognizes pSer-Pro and pThr-Pro motifs in which the first amino acid is phosphorylated. Pin1 binds to many proteins implicated in cell cycle regulation (e.g. p53, Myt1, Wee1, and Cdc25C). Pin1 also targets tau, a protein forming part of hte neuronal cytoskeleton which is hyper-phosphorylated in patients suffering from Alzheimer's disease (AD). Pin1 could, therefore, be involved in the pathogenesis of Ad. Furthermore, Pin1 also binds two proteins involved in transcription: Rpb1, the largest subunit of RNA polymerase II and Spt5, a regulator of the elongation of transcription. Both theses proteins possess domains rich in S/T-P motifs which can be targeted by Pin1 when phosphorylated. Recent studies show that Pin1 modulates the dephosphorylation of some proteins by allowing trans-specific phosphatases to recognize their target after isomerization. This unexpected role might allow protein regulation via peptidyl-prolyl isomerase activity.