SIRT1 Regulates TSH Release Figure 1. Effect of SIRT1 on hormone secretion. (A, B) Western blot analysis of SIRT1 in the systemic organs (A) and nerve tissues (B) of mice. (C) Immunohistochemical staining of SIRT1 in the rat brain. The upper right panel shows the anterior pituitary area at higher magnification. Scale bar, 1 cm. (D) SIRT1 immunoreactivity is absent in the pituitary gland of SIRT1 KO mouse. (E, F) Double immunostaining with SIRT1 (red) and pituitary hormones (green) in the pituitary gland of a 12-weeks old mouse (E) and a 64-years old human (F). Scale bar, 100 mm. doi:10.1371/journal.pone.0011755.g001 kinase [28]. Thus we used the catalytically inactive form (H355A) of SIRT1 [29,30], to screen for binding partners. The Flag-tagged SIRT1 and SIRT1 (H355A) constructs were expressed in HEK293T cells and immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were digested and then subjected to a nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS) system [31]. Proteins identified as candidates of SIRT1 binding partners included phosphatidylino- sitol-4-phosphate 5-kinase (PIP5Ks) (Fig. 3A). The enzymes PIP5Ks (PIP5Ka, PIP5Kb, and PIP5Kc) are enzymes that catalyze the synthesis of PI(4,5)P2 mainly through the cellular route [32]. We focused on PIP5Ks since PIP5Kc is reported to play an important role in the exocytosis of large dense-core vesicles by inducing the synthesis of PI(4,5)P2 in endocrine cells [33]. The common regions of all PIP5Ks were pulled down with SIRT1 in the fist screening with LC-MS/MS. To examine if SIRT1 more specially interacts with PIP5Kc than with the other PIP5Ks, we performed immunoprecipitation analysis against endogenous proteins with antibodies to SIRT1 and to PIP5Ks. Amongst the PIP5Ks examined, PIP5Kc was selectively immunoprecipitated with SIRT1 (Fig. 3B). Further investigation with deletion mutants of PIP5Kc revealed that the interaction between SIRT1 and PIP5Kc was mediated through a region of the kinase core domain [34] (amino acid residues 247—281) of PIP5Kc (Fig. 3C). SIRT1-mediated deacetylation of PIP5Kc The binding of SIRT1 with PIP5Kc suggests that SIRT1 deacetylates acetylated PIP5Kc. Treatment with nicotinamide PLoS ONE | www.plosone.org 3 July 2010 | Volume 5 | Issue 7 | e11755