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TFCD having a lower affinity for Pin1 (K of 1.53 mM)
than double phosphorylated TFCD (K d of 137 mM). d
Similarly, our pull-down assays showed that unphospho- rylated TF peptides can still pull-down Pin1 protein (Figure 2E). These two results suggest that Pin1 can interact with TF in both phosphorylation-specific and phosphorylation- independent manners. It has previously been shown that pre-attachment of Pin1 to an unphosphorylated “dynamic anchoring” region of c-Myc that is distant from its pSer- Pro motif is functionally important for the interaction between these two proteins.33 It could, therefore, be spec- ulated that a similar distal, phosphorylation-independent interaction occurs between Pin1 and TF. Further studies are needed to confirm whether or not this is indeed the case.
It is also important to emphasize that intermolecular stacking of TFCD trans-Pro259 and Pin1 WW-domain Trp34 residues is highly orthologous to the intramolecular stacking between Trp8 and Pro37 of the Pin1 WW- domain. Given that mutational studies have shown that Trp8-Pro37 stacking can stabilize the WW-domain fold,32 we think that a similar, but intermolecular trans-Pro259 to Trp34 interaction-mechanism is utilized to promote the observed affinity between Pin1 and TF even when TFCD is unphosphorylated at Ser258. Indeed, complex forma- tion due to the hydrophobic Pro-Trp stacking with unphosphorylated Ser-Pro or Ser-Thr motifs is consistent with previous studies in mammalian cell lines34 and in vitro studies of the WW-domain binding to unphosphorylated Ser-Pro motifs such as the cytoplasmic polyadenylation element binding (CPEB) protein.35,36
In our peptide pull-down and protein half-life experi- ments, we found that full-length Pin1 as well as Pin1 mutants with a disrupted WW-domain (Pin1:W34A) or lacking isomerase activity (Pin1:K63A) could bind the TFCD and stabilize TF protein. The discrepancy between these findings and our NMR data showing the importance of the Pin1 Trp34 residue in binding the TFCD can be
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haematologica | 2018; 103(6)
Pin1 enhances TF expression and activity
ray structures, as well as to the Cdc25 pThr peptide com- plex NMR structure, suggesting that loop1 undergoes a thermodynamic switch between ligand-bound and ligand- free states (Figure 3D).
Our calorimetric experiments revealed a weak interac-
tion between the unphosphorylated TFCD and the Pin1
WW-domain (data not shown), with unphosphorylated
explained by the fact that purified Pin1 WW-domain was used in our NMR studies, and that both domains of Pin1 can recognize and bind pSer-Pro motifs independently of each other.37 Therefore, it is possible that the Pin1 WW- domain mutant (Pin1:W34A) interacts with and stabilizes TF via its isomerase domain. However, whether the inter- action between TF and Pin1 involves sequential and syn- ergistic action of the Pin1 WW-domain and isomerase domain remains to be determined.
Several human and animal studies have shown that TF plays a crucial role in thrombosis development in vivo. We demonstrated that Pin1 has a regulatory role in FXa gener- ation in both activated ECs and SMCs in vitro. Additional studies with Pin1-deficient mice may further delineate the role of Pin1 in TF gene expression, TF activity, and coagu- lation in vivo. However, Pin1-deficient mice already suffer from decreased body weight, testicular and retinal atro- phies, and deficiencies in breast proliferative changes dur- ing pregnancy, which may interfere with in vivo coagula- tion experiments.38
In summary, our results demonstrate that Pin1 enhances TF gene expression, interacts with TF via the TFCD, and positively modulates TF half-life and pro-coagulant activi- ty in vascular cells. Our findings suggest that Pin1 con- tributes to a hypercoagulable state in the local and sys- temic circulation. Specific Pin1 inhibitors could, therefore, be considered as potential novel therapeutic agents for the prevention of coagulation-driven pathologies, such as thrombosis and atherosclerosis.
Funding
This work was supported by the research program of the BioMedical Materials institute, co-funded by the Dutch Ministry of Economic Affairs as a part of Project P1.02 NEXTREAM. This work was also supported by the Rembrandt Institute for Cardiovascular Research (RICS-grant 2013), the Netherlands CardioVascular Research Initiative (CVON: 2012-08), and the National Institute of Health (grants HL-60742 and P01- HL16411).
Acknowledgments
We would like to thank Dr. Youlin Xia and the UH NMR facility-DOR for NMR data acquisition, CACDS for the struc- ture calculations, and Amr Elnashai, Amy Sater, and Glen Legge for their support of this research.
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