SIRT1 Regulates TSH Release Figure 2. SIRT1 upregulates exocytosis of TSH. (A) Western blot analysis of SIRT1 in primary pituitary cells and in cells overexpressing (O/E) SIRT1 (left panel) and SIRT1 RNAi cells (right panel) levels. (B) Amounts of TSH secreted by SIRT1-O/E and SIRT1-RNAi pituitary cells over 24h (n = 10). Statistical analyses were performed using Fisher’s PLSD test. *P,0.05 versus control. (C) A TIRF image of an anterior pituitary cell. Several GFP-TSH granules that disappeared within 5 s are circled in red. Scale bar, 20 mm. (D) Number of expelled TSH granules were morphologically identified using the TIRF images (n = 5). Statistical analyses were performed using Fisher’s PLSD test. *P,0.05 versus control. Values represent mean6SEM (% of control: B and D). doi:10.1371/journal.pone.0011755.g002 (NAM), a SIRT1 inhibitor [35], increased the lysine acetylation of PIP5Kc, but did not alter the acetylation levels of PIP5Ka and PIP5Kb (Fig. 4A). The SIRT1-binding site of PIP5Kc has 2 lysine residues (residues 265 and 268; shaded in Fig. 4B) besides the 5 identical lysine residues conserved in all the 3 PIP5Ks (residues 255, 260, 266, 270 and 276) (Fig. 4B) [34]. Within light of the preferential interaction between SIRT1 and PIP5Kc, we hypothesized that SIRT1 mainly deacetylates lysines 265 and/or 268 of PIP5Kc. To test this hypothesis, we constructed a PIP5Kc plasmid in which 2 lysine residues were substituted with arginine residues (K265/ 268R). As expected, the K265/268R mutant was not further acetylated, regardless of treatment with NAM (Fig. 4C). This indicates that SIRT1 deacetylates the residues 265 and/or 268 of PIP5Kc. To prove that SIRT1 directly deacetylates PIP5Kc, we performed an in vitro deacetylase activity assay. PIP5Kc deacetyla- tion occurred upon the addition of recombinant wild-type SIRT1 but not the inactive form of SIRT1 (H355A) (Fig. 4D). In contrast, PIP5Ka and PIP5Kb were not deacetylated by SIRT1 (Fig. 4D). Taken together, our findings indicate that SIRT1 selectively deacetylates PIP5Kc among all the PIP5Ks. We next asked whether the acetylation state affects the enzymatic activity of PIP5Kc. We measured the in vitro kinase activity of PIP5Kc with some modifications of the assay system of previous reports [36,37]. We preincubated PIP5Kc with recom- binant wild-type SIRT1 or the inactive form of SIRT1 (H355A) for 3 h at 30uC, and then examined the activity of PIP5Kc. The production of PIP2 was significantly increased when PIP5Kc was deacetylated with wild-type SIRT1, whereas no increase occured when PIP5Kc was incubated with the inactive SIRT1 (H355A) (Fig. 4E). These results indicate that SIRT1 deacetylates PIP5Kc, thereby increasing the latter’s activity. Consistent with this, PIP2 production was significantly decreased in HEK293T cells treated with SIRT1-RNAi (Fig. 4F and 4G). Our findings demonstrate that SIRT1 increases the kinase activity of PIP5Kc by deacetylat- ing this kinase. Involvement of PIP5Kc in SIRT1-mediated TSH Secretion PIP5Kc is the major PI(4,5)P2 synthesizing enzyme in the brain [38]. In addition, PIP5Kc KO mice show defects in vesicle trafficking [33]. We examined whether PIP5Kc was expressed and physically interacted with SIRT1 in the pituitary gland, before testing whether PIP5Kc is involved in the SIRT1-mediated TSH secretion in pituitary cells. PIP5Kc is expressed in pituitary gland in addition to other regions of the mouse brain (Fig. 5A). In the pituitary gland, SIRT1 and PIP5Kc were detected in the cytosolic fraction (Fig. 5B). Further, they colocalized in the cytoplasm of many cells in the anterior pituitary gland (Fig. 5C). These two proteins were coimmunoprecipitated with each other from pituitary cytosolic lysates by antibodies specific for each protein (Fig. 5D). Thus, SIRT1 and PIP5Kc are physically associated in pituitary cells. To determine whether PIP5Kc is involved in SIRT1-mediated TSH secretion in pituitary cells, we knocked down PIP5Kc in PLoS ONE | www.plosone.org 4 July 2010 | Volume 5 | Issue 7 | e11755