The role of the prolyl-isomerase Pin1 in regulating the p53 protein family network: A fine tuner or a dangerous amplifier?

MANTOVANI F.; GIRARDINI J.E.; TIBERI L.; TOCCO F.; RUSTIGHI A.; GUIDA E.; BISSO A.; DEL SAL G.

Ein Gedi, Israel

Workshop; 2nd International workshop on mutant p53; 2005

<!-- /* Font Definitions */ @font-face {font-family:Times; panose-1:2 2 6 3 6 4 5 2 3 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:7 0 0 0 147 0;} @font-face {font-family:"American Typewriter Condensed"; mso-font-alt:"Courier New"; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:50331648 0 0 0 1 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:"American Typewriter Condensed"; mso-ascii-font-family:"American Typewriter Condensed"; mso-fareast-font-family:Times; mso-hansi-font-family:"American Typewriter Condensed"; mso-bidi-font-family:"Times New Roman"; mso-ansi-language:EN-US;} @page Section1 {size:612.0pt 792.0pt; margin:72.0pt 90.0pt 72.0pt 90.0pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> The overwhelming complexity of signaling networks present in a living cell poses a challenging question: How the signaling events and its functional consequences on different pathways are coordinated in order to achieve a viable equilibrium? Since different signaling networks could provoke completely opposite effects in response to several stimuli, it seems clear that precise and effective mechanisms, able to sense and regulate this delicate interplay, should exist. One of this mechanisms, emerging as an important fine tuner of signaling networks, is phosphorylation-directed prolyl isomerization. This mechanism is based on the action of the prolyl isomerase Pin1, which recognizes specific phosphorylated motifs on its target proteins and catalizes its isomerization. Among Pin1 substrates there are proteins belonging to different pathways, which could transmit proliferative or anti-proliferative signals. Pin1 levels are also regulated by transcriptional and postranscriptional mechanisms. In the context of a tumor cell, this situation becomes dramatically changed. In particular, when p53 has been mutated, the cell loses an efficient tumor suppressor and gains a protein with pro-oncogenic activitiy. We have previously shown that Pin1 interacts with mutp53 in tumor derived cell lines that endogenously express p53 mutant forms. Pin1 is an important interactor of wt p53, able to modulate its function as a tumor suppressor. Pin1 may also regulate the pro-oncogenic activity of mut p53, adding a further level of complexity to the oncogenic process. We are interested in studying the functional link between Pin1 and mut p53 and the consequences of the outcome of this interaction in tumor cells. In order to further characterize this interaction we altered Pin1 recognition sites on some hot spot p53 mutants (referred to as 4M p53 mutants). The analysis of the ability of these mutants to bind Pin1 allowed us to understand which sites are relevant for the interaction. Since this interaction is dependent on phosphorylation on Pin1 biding sites we analyzed the phosphorylation status of these mutants.  In mut p53 the are six S/T-P motifs, that may be recognized by Pin1: S33, S46, T81, S127, T150 and S315. The results obtained indicate that S127 and T150 are not phosphorylated when ectopically expressed, even upon stimulation with cisplatinum. Residues S33 and S46 were found to be phosphorylated in mutants R175H, R248W and R273H. The influence of the polymorphism on codon 72 on the interaction between Pin1 and p53 R175H was studied. The isoform R72 showed a strongest binding with Pin1. In agreement with the differential affinity for Pin1 presented by the two isoforms, we observed that residues S33 and S46 are less phosphorylated in the P72 isoform of R175H. We were interested in studying if Pin1 could influence the ability of mutp53 to transactivate specific target genes. To this end, luciferase assays using a reporter for the EGR1 promoter were conducted. We observed that 4M p53 hot spot mutants, unable to bind Pin1, stimulated transcription as well as the original mutants from which they are derived. Pin1 overexpression did not enhanced transactivation activity of the p53 mutant forms analyzed. Even if these results suggest that Pin1 does not influence the transactivation activity of mut p53 on EGR1 promoter, it can not be excluded that it may do influence transcription on other target promoters of mutp53. The interaction between p73 and mtp53 was also studied. Preliminary results indicated that 4M mutants bind strongly to p73 than mutant p53 forms with intact Pin1 binding sites. At present the effect of Pin1 on this interaction is being studied.