Page 175 - Haematologica June

Pin1 enhances TF expression and activity
with anti-Pin1 antibody resulted in co-precipitation of endogenous TF, while a pull-down with control IgG anti- body did not, thereby indicating that TF and Pin1 interact in these two cell types (Figure 2B). Next, we performed co-immunoprecipitation experiments in HEK293T cells over-expressing HA-tagged Pin1 and either wild-type TF or truncated TF lacking the cytoplasmic domain (TFDCD). These experiments showed that full-length TF interacts
with Pin1, while the TFDCD mutant does not, indicating that the TFCD is involved in the interaction with Pin1 (Figure 2C). Next, we performed pull-down assays with biotinylated peptides encoding the human TFCD, which confirmed our finding that Pin1 interacts with the TFCD, and showed that this interaction was completely disrupt- ed by mutating the Ser258 residue in the TFCD (Figure 2D). This result is consistent with the observation that this
A
BCD
Figure 3. Nuclear magnetic resonance (NMR) spectroscopy shows interaction between twenty-amino acid cytoplasmic domain (TFCD) and Pin1 requires phospho- rylation of Ser258 and trans-configuration of the pSer258-Pro259 peptide bond in the TFCD. (A) Superimposition of the assigned 1H/15N HSOC spectra of the Pin1 WW-domain with double phosphorylated TFCD (pSer253/pSer258) showing the chemical shift changes upon increasing amount of peptide to a 10x molar excess. A non-linear regression fit of the Ser18 (peak shift shown in black dotted box) was used to calculate the binding constant of the complex. (B) Bundle of 20 NMR con- formers sampling into two major conformers. Sidechains of Pro259 (TFCD), Arg21 and Trp34 (Pin1 WW-domain) are indicated with circles. (C) Representative models of the two lowest-energy conformations from (B) are shown in green and magenta. The polypeptide backbones are shown as ribbons. Pin1 WW-domain residues are underlined. (D) Contact of the TFCD pSer258-Pro259 motif with the Pin1 WW-domain loop1 and comparison of NMR and X-ray structures.
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