One of the biggest challenge in cell biology and cancer research is still to understand how cells divide and proceed to the equal partition of their genetic material in each daughter cell. The mechanisms underlying mitosis and cytokinesis are tightly controlled and reversible protein phosphorylation plays a major role in this regulation . Early and late mitotic events are dependent on protein phosphorylation by multiple serine and threonine kinases of the NIMA, Polo and Aurora families at the head of which sits the Cdk1/cyclin B complex [1–5]. The nature of most protein substrates of these kinases is still unknown so that the precise roles they play in the regulation of mitosis and cytokinesis remain to be clarified.
A few monoclonal antibodies (mAbs) have been raised in different laboratories against mitotic cell extracts and shown to react with subsets of proteins that are phosphorylated upon entry into mitosis [6–9]. The most characterized of these antibodies, mAb MPM-2, was selected for its preferential reactivity towards mitotic versus interphase cells  and shown to react with a phospho-epitope present on a set of proteins concentrated in the centrosomes, the kinetochores, the mitotic spindle and the midbody [6, 10], reinforcing the idea that the structural rearrangements observed during mitosis are controlled by phosphorylation events. After twenty years of continuous use as a mitotic marker in scores of laboratories -and insistent efforts to characterize the epitope(s) and the kinases involved- it is now clear that many MPM-2 antigens are important mitotic regulators and effectors. They include the Cdc25 phosphatase , the Cdk1-inhibitory Wee1 and Myt1 kinases [12–14], the NIMA kinase , the microtubule associated-proteins MAP-1 and MAP-4 [16, 17], DNA topoisomerase II α and β , p42mapk , and the Cdc27 component of the anaphase-promoting complex (APC) . Phosphorylation of the MPM-2 antigenic sites is thought to be functionally important as the MPM-2 antibody inhibits oocyte maturation upon microinjection and neutralizes mitosis promoting factor activity from M-phase extracts .
The biological relevance of MPM-2 phosphoepitopes was further reinforced when Shen and coworkers  showed that the peptidyl-prolyl isomerase (PPIase) Pin1 could bind and regulate many mitotic phosphoproteins also recognized by MPM-2. PPIases are ubiquitous enzymes catalyzing the cis-trans isomerization of the peptide bond preceding a proline residue and are thought to be involved in protein folding, protein assembly, protein trafficking or in the direct regulation of protein activity . Pin1 is unique among prolyl isomerases in that it specifically targets proline residues preceded by a phosphoserine (pS-P) or a phosphothreonine (pT-P) [24–26]. Pin1 is a ubiquitously expressed protein that is essential for cell cycle progression in yeast and in mammalian cells . Furthermore, Pin1 has been shown to interact with the essential mitotic kinase NIMA and to suppress its mitosis-promoting activity . It is now believed that Pin1 acts as an essential mitotic regulator since, in addition to NIMA, it binds other MPM-2-reactive proteins with important mitotic functions including Cdc25 [22, 28], Myt1, Wee1, Plk1 and Cdc27 . In the recent years, it was postulated that the analysis of the phosphorylated sites recognized by both Pin1 and MPM-2 might be a good starting point for a better understanding of the general role of phosphorylation in the mitotic processes. These efforts, mainly orchestrated by K.P. Lu and collaborators, have led to the elaboration of a novel post-phosphorylation regulatory mechanism, in which the isomerase Pin1 induces conformational changes into targeted proteins that have been first phosphorylated by proline-directed kinases [29, 30].
The regulative action of Pin1 may not be confined to mitotic processes. Recent results have shown that DNA damage induces an interaction between Pin1 and p53 depending on specific pS-P motifs in p53 [31–33], indicating that phosphorylation-dependent prolyl isomerization is a signalling mechanism also operating in the genotoxic response . Interestingly, it was shown that efficient binding of the p53 protein to Pin1 requires phosphorylation at all three sites of p53 involved in the interaction [32, 33].
Previous work from our laboratory has shown that, in human interphase cells, Pin1 interacts with two transcription-related proteins in a phosphorylation-dependent manner: the hyperphosphorylated form of RNA polymerase II largest subunit Rpb1 , and a phosphorylated form of the transcription elongation factor hSpt5 . Moreover, studies in yeast provided genetic evidence that Ess1, the yeast homolog of Pin1, interacts with the transcription machinery as most genetic suppressors of ess1 mutants encode proteins involved in transcription  consistent with the earlier observation that ess1 mutants are defective in mRNA processing [38, 39]. These results also indicate that Pin1 operates at multiple levels.
The discovery of the association of Pin1 with Rpb1 and hSpt5 was made possible thanks to the availability of a monoclonal antibody (mAb CC-3) selected for its strong phospho-dependent reactivity with mitotic cells and with nuclear speckles of interphase cells . The interphase CC-3 antigens located in the nuclear speckles were identified as Rpb1 and hSpt5 [35, 36] but the numerous CC-3 mitotic antigens have not been identified yet. As CC-3 behavior was somewhat reminescent of that of MPM-2, a comparison of both mAbs initially revealed that they share the hyperphosphorylated form of Rpb1 as their major interphase antigen . However, their epitope on Rpb1 appeared to be different since upon a heat shock, MPM-2 reactivity was increased whereas that of CC-3 was diminished, suggesting that both antibodies may discriminate between distinct functional forms of RNA polymerase II [35, 40].
In the present work, MALDI-TOF mass spectrometry and immunoblotting were combined to identify the major CC-3 mitotic antigens. They included a few proteins involved in transcription and/or mRNA maturation and most of them also appeared to be known or novel MPM-2 antigens. The fine CC-3- and MPM-2-epitope mapping of the carboxy-terminal domain (CTD) of Rpb1 confirmed that the epitopes are different and can be generated in vitro by distinct kinases. Pulldown experiments suggest that the CC-3 mitotic antigens are also Pin1 targets. Finally, incubation of HeLa cells with juglone, an irreversible Pin1 inhibitor , prevented the dephosphorylation of both mitotic MPM-2 and CC-3 epitopes. These observations suggest that Pin1 might promote M phase exit by stimulating the dephosphorylation rate of the mitotic regulators and/or effectors on multiple sites revealed by mAbs CC-3 and MPM-2.