Page 177 - Haematologica June

Pin1 enhances TF expression and activity
ing 10,000 steps of energy minimization, further demon- strating that our structures satisfy energy-of-motion (EOM) criteria with reasonable convergence parameters.
Taken together, our structural analyses show the inter- action between TFCD and Pin1 involves phosphorylation of Ser258 and trans-configuration of the pSer258-Pro259 peptide bond in the TFCD.
Pin1 extends TF protein half-life via the TFCD
Given our findings that Pin1 and TF interact and that Pin1 is known to enhance the protein half-life of several of
its substrates,18 we sought to determine the effect of Pin1 on TF protein half-life using full-length TF and TF lacking the cytoplasmic domain in cycloheximide-treated HEK293T cells. Pin1 significantly enhanced the protein half-life of full-length TF (Figure 4A), but not that of TFDCD (Figure 4B). Furthermore, Pin1 mutants with an inactive WW-domain (Pin1:W34A) or lacking isomerase activity (Pin1:K63A) were both still able to enhance the protein half-life of full-length TF (Figure 4A). Similar effects of Pin1 and Pin1 mutants on TF protein half-life were observed for endogenous TF protein in PMA-stimulated
A
B
C
Figure 4. Pin1 increases Tissue Factor (TF) protein half-life via the twenty-amino acid cytoplasmic domain (TFCD). (A) HEK293T cells over-expressing full-length TF with or without Pin1 or Pin1 mutants were treated with cycloheximide (CHX) for times indicated in hours (hrs). TF protein levels were determined by western blot. The graph shows the amount of TF protein remaining after CHX treatment as a percentage of the starting TF protein level. (B) HEK293T cells over-expressing TF∆CD with or without Pin1 were treated with CHX for times indicated. TF protein levels were determined by western blot. The graph shows the amount of TF protein remaining after CHX treatment as a percentage of the starting TF protein level. (C) PMA-stimulated smooth muscle cells (SMC) over-expressing Pin1 or Pin1 mutants were treat- ed with CHX for times indicated. Endogenous TF protein levels were determined by western blot. The graph shows the amount of TF protein remaining after CHX treat- ment as a percentage of the starting TF protein level. Data are shown as mean±Standard Error of Mean. P-values were calculated using two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001 versus Mock-transfected controls.
haematologica | 2018; 103(6)
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