The CREB coactivator TORC2 functions as a calcium- and cAMP-sensitive coincidence detector

Elevations in circulating glucose and gut hormones during feeding promote pancreatic islet cell viability in part via the calcium- and cAMP-dependent activation of the transcription factor CREB. Here, we describe a signaling module that mediates the synergistic effects of these pathways on cellular gene expression by stimulating the dephosphorylation and nuclear entry of TORC2, a CREB coactivator. This module consists of the calcium-regulated phosphatase calcineurin and the Ser/Thr kinase SIK2, both of which associate with TORC2. Under resting conditions, TORC2 is sequestered in the cytoplasm via a phosphorylation-dependent interaction with 14-3-3 proteins. Triggering of the calcium and cAMP second messenger pathways by glucose and gut hormones disrupts TORC2:14-3-3 complexes via complementary effects on TORC2 dephosphorylation; calcium influx increases calcineurin activity, whereas cAMP inhibits SIK2 kinase activity. Our results illustrate how a phosphatase/kinase module connects two signaling pathways in response to nutrient and hormonal cues

CREB Is Activated by Muscle Injury and Promotes Muscle Regeneration

The cAMP response element binding protein (CREB) plays key roles in differentiation of embryonic skeletal muscle progenitors and survival of adult skeletal muscle. However, little is known about the physiologic signals that activate CREB in normal muscle. Here we show that CREB phosphorylation and target genes are induced after acute muscle injury and during regeneration due to genetic mutation. Activated CREB localizes to both myogenic precursor cells and newly regenerating myofibers within regenerating areas. Moreover, we found that signals from damaged skeletal muscle tissue induce CREB phosphorylation and target gene expression in primary mouse myoblasts. An activated CREB mutant (CREBY134F) potentiates myoblast proliferation as well as expression of early myogenic transcription factors in cultured primary myocytes. Consistently, activated CREB-YF promotes myoblast proliferation after acute muscle injury in vivo and enhances muscle regeneration in dystrophic mdx mice. Our findings reveal a new physiologic function for CREB in contributing to skeletal muscle regeneration

Analysis of a cAMP regulated coactivator family reveals an alternative phosphorylation motif for AMPK family members.

The second messenger cAMP stimulates cellular gene expression via the PKA-mediated phosphorylation of the transcription factor CREB and through dephosphorylation of the cAMP-responsive transcriptional coactivators (CRTCs). Under basal conditions, CRTCs are phosphorylated by members of the AMPK family of Ser/Thr kinases and sequestered in the cytoplasm via a phosphorylation-dependent association with 14-3-3 proteins. Increases in cAMP promote the dephosphorylation and nuclear translocation of CRTCs, where they bind to CREB and stimulate relevant target genes. Although they share considerable sequence homology, members of the CRTC family exert non-overlapping effects on cellular gene expression through as yet unidentified mechanisms. Here we show that the three CRTCs exhibit distinct patterns of 14-3-3 binding at three conserved sites corresponding to S70, S171, and S275 (in CRTC2). S171 functions as the gatekeeper site for 14-3-3 binding; it acts cooperatively with S275 in stabilizing this interaction following its phosphorylation by the cAMP-responsive SIK and the cAMP-nonresponsive MARK kinases. Although S171 contains a consensus recognition site for phosphorylation by AMPK family members, S70 and S275 carry variant motifs (MNTGGS275LPDL), lacking basic residues that are otherwise critical for SIK/MARK recognition as well as 14-3-3 binding. Correspondingly, the activity of these motifs differs between CRTC family members. As the variant (SLPDL) motif is present and apparently phosphorylated in other mammalian proteins, our studies suggest that the regulation of cellular targets by AMPK family members is more extensive than previously appreciated

Mitogenic Signals Stimulate the CREB Coactivator CRTC3 through PP2A Recruitment

Summary: The second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) stimulates gene expression via the cAMP-regulated transcriptional coactivator (CRTC) family of cAMP response element-binding protein coactivators. In the basal state, CRTCs are phosphorylated by salt-inducible kinases (SIKs) and sequestered in the cytoplasm by 14-3-3 proteins. cAMP signaling inhibits the SIKs, leading to CRTC dephosphorylation and nuclear translocation. Here we show that although all CRTCs are regulated by SIKs, their interactions with Ser/Thr-specific protein phosphatases are distinct. CRTC1 and CRTC2 associate selectively with the calcium-dependent phosphatase calcineurin, whereas CRTC3 interacts with B55 PP2A holoenzymes via a conserved PP2A-binding region (amino acids 380–401). CRTC3-PP2A complex formation was induced by phosphorylation of CRTC3 at S391, facilitating the subsequent activation of CRTC3 by dephosphorylation at 14-3-3 binding sites. As stimulation of mitogenic pathways promoted S391 phosphorylation via the activation of ERKs and CDKs, our results demonstrate how a ubiquitous phosphatase enables cross talk between growth factor and cAMP signaling pathways at the level of a transcriptional coactivator. : Biological Sciences; Biochemistry; Molecular Biology; Cell Biology Subject Areas: Biological Sciences, Biochemistry, Molecular Biology, Cell Biolog

Signaling pathways and genes that inhibit pathogen-induced macrophage apoptosis - CREB and NF-κB as key regulators

SummaryCertain microbes evade host innate immunity by killing activated macrophages with the help of virulence factors that target prosurvival pathways. For instance, infection of macrophages with the TLR4-activating bacterium Bacillus anthracis triggers an apoptotic response due to inhibition of p38 MAP kinase activation by the bacterial-produced lethal toxin. Other pathogens induce macrophage apoptosis by preventing activation of NF-κB, which depends on IκB kinase β (IKKβ). To better understand how p38 and NF-κB maintain macrophage survival, we searched for target genes whose products prevent TLR4-induced apoptosis and a p38-dependent transcription factor required for their induction. Here we describe key roles for transcription factor CREB, a target for p38 signaling, and the plasminogen activator 2 (PAI-2) gene, a target for CREB, in maintenance of macrophage survival

A variant SIK2/MARK2 recognition site modulates CRTC activity.

<p>(A) Sequence alignment of CRTC conserved region III. The phosphorylated region III serines are shown in blue. (B) EVX-Luc reporter assay comparing the transcriptional activity of CRTC3 carrying mutations in region III. (n = 10, ±s.e.m.) (C) EVX-Luc reporter assay measuring the effect of SIK2 or MARK2 co-expression on the transcriptional activity of CRTC3 region III mutants (red). In case of SIK2 the mutants were assayed in the background of the S162A mutant. (n = 5, ±s.e.m.) (D) & (E) Western blot analysis of the Co-IP of FLAG-tagged CRTC3 region III mutants upon co-expression of HA-tagged SIK2. F) Western blot analysis of the Co-IP of FLAG-tagged CRTC3 mutants upon co-expression of HA-tagged MARK2. (cont = control / CRTC3 S162A, LNT = L268A N269A T270A, TGG = T270A G271A G272A, PDL = P275A D276A L277A)</p

Effects of MARK family members on CRTC phosphorylation and cAMP dependent gene expression.

<p>(A) EVX-Luc reporter assay comparing the effect of MARK kinase family co-expression on the transcriptional activity of CRTC2 and CRTC3 (TV = transcript variant). EVX-Luc activity was measured after 4 h of DMSO/Fsk treatment. (n = 5, s.e.m.) (B) Primary sequences of potential PKA sites in <i>H</i>. <i>sapiens</i> MARK2 (TV3 = 1–724; TV4 1–788). Phosphorylated residues are shown in red. (C) Western blot analysis of the overexpression of FLAG-tagged MARK kinase family members and MARK2 (TV3) mutants. (P-MARK (AL) = P-MARK activation loop, DMSO/Fsk treatment for 1h) (D) Western blot analysis of the Co-IP of FLAG-tagged MARK2 (TV3) mutants. (DMSO/Fsk treatment for 1h) (E) EVX-Luc reporter assay measuring the effect of MARK2 S409 (TV4) mutants upon the transcriptional activity of co-expressed CRTC2 and CRTC3. As a control, a EVX-Luc reporter assay with SIK2 and known PKA-site mutant (S587A) with co-expressed CRTC3 was performed. EVX-Luc activity was measured after 4 h of DMSO/Fsk treatment. (n = 5, ±s.e.m.)</p

Role of the SLPDL motif in the SIK and MARK kinase-mediated phosphorylation of cellular substrates?

<p>(A) Schematic representation depicting the CRTC activation/inactivation mechanism. SIK and MARK mediated phosphorylation at multiple conserved sites leads to 14-3-3 binding and cytoplasmic sequestration. Highlighted amino acids are either phosphorylated 14-3-3 sites II (yellow) and III (blue) or essential for phosphorylation and 14-3-3 functionality (red). The second messenger cAMP strongly inhibits SIKs via PKA-mediated phosphorylation, while MARKs CRTC directed activity remains unaffected. (B) Tables list <i>H</i>. <i>sapiens</i> proteins harboring the <u>S</u>LPDL motif. Serines shown in blue were found to be phosphorylated in the PhosphoSitePlus database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173013#pone.0173013.ref037" target="_blank">37</a>]. (C) Western blot analysis of the Co-IP of FLAG-tagged CRTC3, MTRF1 (<i>H</i>. <i>sapiens</i>, isoform 1), and NEK1 (<i>M</i>. <i>musculus</i>). Correspondingly, wild type (WT) and <u>S</u>LPDL motif phospho-acceptor site mutants (mCRTC3 S273A, hMTFR1 S119A, and mNEK1 S997A) were compared with each other (NEK1: DMSO/Fsk treatment for 1h).</p