Abstract
BackgroundThe 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.ResultsAs 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 α and β, 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.ConclusionThese observations indicate that the mitotic phosphoproteins associated with Pin1 are phosphorylated on multiple sites, suggesting combinatorial regulation of substrate recognition and isomerization.
Highlights
The peptidyl-prolyl isomerase Pin1 recently revealed itself as a new player in the regulation of protein function by phosphorylation
MAb CC-3 is a mitotic marker CC-3 and MPM-2 are phosphodependent mouse monoclonal antibodies strongly reactive with mitotic cells [6,7]
The immunoblot reactivities of CC-3 and MPM-2 on mitotic extracts reveal that their mitotic antigens are much more numerous and the overall patterns are sufficiently distinct to expect that CC3 and MPM-2 epitopes are different [35]
Summary
The peptidyl-prolyl isomerase Pin recently revealed itself as a new player in the regulation of protein function by phosphorylation. 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 clear that many MPM-2 antigens are important mitotic regulators and effectors. They include the Cdc phosphatase [11], the Cdk1-inhibitory Wee and Myt kinases [12,13,14], the NIMA kinase [15], the microtubule associated-proteins MAP-1 and MAP-4 [16,17], DNA topoisomerase II α and β [18], p42mapk [19], and the Cdc component of the anaphase-promoting complex (APC) [20]. 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 [21]
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