Biomedical Section, Department of Biological Sciences, University of Hull, Cottingham Road, Hull HU6 7RX, UK.
Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing, Glenfield General Hospital, Leicester LE3 9QP, UK.
Biochim Biophys Acta Mol Cell Res. 2018 Jan;1865(1):12-24. doi: 10.1016/j.bbamcr.2017.09.016. Epub 2017 Sep 28.
The exposure and release of TF is regulated by post-translational modifications of its cytoplasmic domain. Here, the potential of Pin1 to interact with the cytoplasmic domain of TF, and the outcome on TF function was examined. MDA-MB-231 and transfected-primary endothelial cells were incubated with either Pin1 deactivator Juglone, or its control Plumbagin, as well as transfected with Pin1-specific or control siRNA. TF release into microvesicles following activation, and also phosphorylation and ubiquitination states of cellular-TF were then assessed. Furthermore, the ability of Pin1 to bind wild-type and mutant forms of overexpressed TF-tGFP was investigated by co-immunoprecipitation. Additionally, the ability of recombinant or cellular Pin1 to bind to peptides of the C-terminus of TF, synthesised in different phosphorylation states was examined by binding assays and spectroscopically. Finally, the influence of recombinant Pin1 on the ubiquitination and dephosphorylation of the TF-peptides was examined. Pre-incubation of Pin1 with Juglone but not Plumbagin, reduced TF release as microvesicles and was also achievable following transfection with Pin1-siRNA. This was concurrent with early ubiquitination and dephosphorylation of cellular TF at Ser253. Pin1 co-immunoprecipitated with overexpressed wild-type TF-tGFP but not Ser258→Ala or Pro259→Ala substituted mutants. Pin1 did interact with Ser258-phosphorylated and double-phosphorylated TF-peptides, with the former having higher affinity. Finally, recombinant Pin1 was capable of interfering with the ubiquitination and dephosphorylation of TF-derived peptides. In conclusion, Pin1 is a fast-acting enzyme which may be utilised by cells to protect the phosphorylation state of TF in activated cells prolonging TF activity and release, and therefore ensuring adequate haemostasis.
TF 的暴露和释放受其细胞质结构域的翻译后修饰调节。在此，研究了 Pin1 与 TF 细胞质结构域相互作用的潜力及其对 TF 功能的影响。MDA-MB-231 和转染的原代内皮细胞用 Pin1 失活剂 Juglone 或其对照物 Plumbagin 孵育，并用 Pin1 特异性或对照 siRNA 转染。然后评估 TF 在激活后释放到微泡中，以及细胞 TF 的磷酸化和泛素化状态。此外，通过免疫共沉淀研究了 Pin1 结合过表达 TF-tGFP 的野生型和突变型的能力。此外，通过结合测定和光谱法研究了重组或细胞 Pin1 结合 TF C 末端不同磷酸化状态合成的肽的能力。最后，研究了重组 Pin1 对 TF 肽的泛素化和去磷酸化的影响。用 Juglone 而非 Plumbagin 预孵育 Pin1 可减少 TF 作为微泡的释放，并且在用 Pin1-siRNA 转染后也可实现。这与细胞 TF 在 Ser253 处的早期泛素化和去磷酸化一致。Pin1 与过表达的野生型 TF-tGFP 共免疫沉淀，但不与 Ser258→Ala 或 Pro259→Ala 取代突变体共免疫沉淀。Pin1 与 Ser258 磷酸化和双磷酸化的 TF 肽相互作用，前者具有更高的亲和力。最后，重组 Pin1 能够干扰 TF 衍生肽的泛素化和去磷酸化。总之，Pin1 是一种快速作用的酶，细胞可能会利用它来保护激活细胞中 TF 的磷酸化状态，从而延长 TF 的活性和释放，从而确保充分的止血。
