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Glucose Controls CREB Activity in Islet Cells Via Regulated Phosphorylation of TORC2

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Glucose Controls CREB Activity in Islet Cells Via Regulated Phosphorylation of TORC2 Glucose controls CREB activity in islet cells via regulated phosphorylation of TORC2 Deidre Jansson, Andy Cheuk-Him Ng, Accalia Fu, Chantal Depatie, Mufida Al Azzabi, and Robert A. Screaton* Apoptosis Research Centre, Children’s Hospital of Eastern Ontario Research Institute, 401 Smyth Road, Ottawa, ON, Canada K1H 8L1 Edited by Anthony J. Pawson, University of Toronto, Toronto, ON, Canada, and approved May 1, 2008 (received for review January 25, 2008) CREB is a cAMP- and calcium-responsive transcriptional activator that apoptosis; glucose intolerance; and, eventually, diabetes in the is required for islet beta cell proliferation and survival. Glucose and mouse (16–18). incretin hormones elicit beta cell insulin secretion and promote A distinct set of CREB coactivators, Transducers of Regulated synergistic CREB activity by inducing the nuclear relocalization of CREB Activity (TORCs), are also activated by PKA; however, by TORC2 (also known as Crtc2), a coactivator for CREB. In islet cells under a distinct mechanism whereby cAMP-PKA signaling promotes basal conditions when CREB activity is low, TORC2 is phosphorylated TORC relocalization from the cytoplasm to the nucleus where they and sequestered in the cytoplasm by 14-3-3 proteins. In response to bind to the DNA-binding/dimerization domain of CREB (19, 20). feeding stimuli, TORC2 is dephosphorylated, enters the nucleus, and A growing body of evidence indicates that TORCs play a central binds to CREB located at target gene promoters. The dephosphory- role in signal integration to activate CREB in response to glucose lation of TORC2 at Ser-171 in response to cAMP is insufficient to and hormonal cues (21–23). TORC2 is negatively regulated by account for the dynamics of TORC2 localization and CREB activity in phosphorylation at Ser-171, which is a substrate for the salt- islet cells. Here, we identify Ser-275 of TORC2 as a 14-3-3 binding site inducible kinases (SIKs) and AMP-activated protein kinase that is phosphorylated under low glucose conditions and which (AMPK) of the AMPK family (21, 23). Phospho-Ser-171 forms becomes dephosphorylated by calcineurin in response to glucose docking site for 14-3-3 proteins, phosphorylation-dependent allo- influx. Dephosphorylation of Ser-275 is essential for both glucose and steric ‘‘chaperones’’ (24, 25) that, upon binding, mask an adjacent cAMP-mediated activation of CREB in beta cells and islets. Using a nuclear localization signal and thereby promote cytoplasmic accu- cell-based screen of 180 human protein kinases, we identified mulation of TORC2 (23). Upon receipt of extracellular cues that MARK2, a member of the AMPK family of Ser/Thr kinases, as a Ser-275 increase intracellular cAMP, and in some contexts calcium, TORC2 is released from 14-3-3 proteins and relocalizes to the nucleus (21, kinase that blocks TORC2:CREB activity. Taken together, these data 23, 26). Although mutation of Ser-171 to Ala is sufficient to reduce provide the mechanistic underpinning for how cAMP and glucose 14-3-3-mediated cytoplasmic retention of TORC2, lowering the cooperatively promote a transcriptional program critical for islet cell threshold for TORC2 nuclear entry and for TORC-dependent survival, and identifies MARK2 as a potential target for diabetes CREB target gene activation, dephosphorylation of Ser-171 alone treatment. in response to cAMP signals does not fully activate TORC2 in all cell types. In this study, we identify the remaining sites on TORC2 ͉ ͉ ͉ ͉ beta cell kinase screening MARK2 cAMP kinome that mediate binding to 14-3-3 proteins, identify MARK2 as a TORC2 kinase, and delineate how glucose and cAMP signals oss of pancreatic beta cell function is the central feature of all converge on two of these sites to control TORC2 activity in islet Lforms of diabetes mellitus (1). Glucose is a critical stimulus for cells. beta cell proliferation, and the capacity of the islet cell mass to expand (islet hyperplasia) in response to chronically elevated blood Results glucose, together with functional adaptive responses, permits se- Identification of a Second Regulatory Phosphorylation Site on TORC2 cretion of sufficient insulin to meet demand in prediabetic patients in Islet Cells. CREB activity in islet cells is synergistically enhanced (2). Failure of beta cells to proliferate and increase insulin output by costimulation with glucose and cAMP (23). In agreement with to meet increasing demand leads to type 2 diabetes. Although these data, we observed that treatment of glucose-starved MIN6 growth factor signaling pathways have been implicated in the insulinoma cells with glucose and the incretin hormone exendin-4 process of beta cell expansion, the molecular details of how glucose (EX4, a cAMP agonist) provides 11-fold and 7-fold increases in promotes islet cell proliferation are still unclear (3, 4). mRNA for the CREB target genes NR4A2 and IRS2, respectively, Insulin secretion is triggered by numerous metabolites, principal compared with a 4-fold for EX4 alone (Fig. 1A). Given that TORC2 among which are glucose and incretin hormones (5). Beta cell nuclear relocalization underlies this synergy, and a phosphorylation glucose oxidation and consequent ATP production results in mem- defective Ser171Ala mutant of TORC2 isolated from HIT-T15 brane depolarization, influx of extracellular calcium, and insulin insulinoma cells retains 14-3-3 binding capacity (Fig. 1B and ref. release (6). Incretins promote the insulin secretory response pri- 23), we sought to identify additional regulatory phosphorylation marily through activation of cAMP-dependent protein kinase A sites on TORC2 that bind to 14-3-3 proteins. To identify these (PKA), promote cellular proliferation, increased beta cell mass, and site(s), we used a far-Western approach, using GST-tagged 14-3-3 resistance to apoptosis (7–14). In addition to insulin secretion, protein to screen a series of N- and C-terminal deletion mutants of calcium and cAMP signaling events also synergistically elicit TORC2 purified from HIT-T15 cells for their capacity to bind through cAMP response element binding protein (CREB) a tran- scriptional program that is thought to restore the beta cell to a Author contributions: R.A.S. designed research; D.J., A.C.-H.N., A.F., C.D., M.A.A., and R.A.S. metabolically fit state in preparation for the next round of feeding performed research; C.D. and R.A.S. contributed new reagents/analytic tools; D.J., A.C.- (15). CREB (and the related proteins CREM and ATF1) is the H.N., A.F., C.D., and R.A.S. analyzed data; and R.A.S. wrote the paper. critical transcriptional activator that mediates cellular gene regu- The authors declare no conflict of interest. lation in response to cAMP. CREB-dependent gene regulation is This article is a PNAS Direct Submission. critical for governing islet cell proliferation and survival in vivo, *To whom correspondence should be addressed. E-mail: [email protected]. because promoting CREB activity in islets via stabilization of the This article contains supporting information online at www.pnas.org/cgi/content/full/ CREB:CBP complex increases beta cell mass and, conversely, 0800796105/DCSupplemental. MEDICAL SCIENCES disrupting CREB function in insulin-producing cells promotes © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800796105 PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 ͉ 10161–10166 Downloaded by guest on September 26, 2021 A 14 (CIP) before SDS/PAGE abolished 14-3-3 binding (Fig. 1B, lanes 12 8 and 9). Importantly, whereas CIP treatment abolished 14-3- NR4A2 3:TORC2 complex formation, mutation on Ser-171 to Ala reduced 10 IRS2 but did not prevent 14-3-3 interaction with the N terminus of 8 TORC2 (Fig. 1B, lane 7). Thus, we conclude that an additional 6 TORC2 phosphorylation site for 14-3-3 interaction lies within amino acids 1–321. 4 Because Ser-171 is highly conserved from human to zebrafish in Relatvie mRNA levels Relatvie mRNA 2 TORCs1–3 (19, 20, 23), we assumed that the additional regulatory 0 site that mediates 14-3-3 binding would also be well conserved. CON GLU EX4 GLU+EX4 TORC2 is exclusively phosphorylated on serine residues in HIT- T15 cells (23), so we selected five additional serine residues in TORC2 (Ser-70, Ser-127, Ser-238, Ser-245, and Ser-275) to eval- B 1-321 1-3 98 uate as possible 14-3-3 interaction sites [supporting information (SI) 389 389-692 1- FLAG T :2 C NOWT 1-389 171 WT 171 171 171 Fig. S1], because they fit the following criteria: (i)resideinamino C PI : - - -- -- - + + acids 1–321, (ii) conform to a consensus or near-consensus 14-3-3 100 binding site [mode 1 (R/KXXpSXP) and mode 2 (R/KXXX- pSXP)], in which pS is the phosphorylated serine, and X is any 75 FAR-WESTERN 14-3-3 amino acid (24)], and (iii) highly conserved between human and 50 mouse TORCs 1 and 2. Given that TORC2:14-3-3 binding requires TORC2 to be phosphorylated, we expected to see that loss of 100 critical phosphorylation sites would lead to a corresponding loss of 75 14-3-3 binding in the far-Western screen. To test this, these five Ser PI : FLAG TORC residues were mutated to Ala in the 1–321 and 1–389 deletion 50 BLOT:FLAG constructs, and these new mutants were screened for 14-3-3 binding 1 2 3 4 5 6 7 8 9 by far-Western analysis. Whereas mutation of either Ser 171 or S171 S275 692 Ser-275 significantly reduced 14-3-3 binding (Fig. 1C, lanes 3, 4, 7, WT 1 3- 89 and 8), mutation of these residues together abolished binding in the 389-692 context of both 1–321 and 1–389 (Fig.
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