The authors have declared that no competing interests exist.
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 (MNTGG
The three members of the cyclic AMP (cAMP)-regulated transcriptional coactivator (CRTC) family share a common domain structure consisting of a conserved N-terminal cAMP response element-binding protein (CREB) binding domain (CBD), a central regulatory domain, and a C-terminal transactivation domain (TA) [
Hormonal signals that stimulate intracellular cAMP concentrations increase gene expression via the phosphorylation of CREB and via the dephosphorylation of the CRTC family [
14-3-3 proteins bind as monomers or dimers to phosphorylated substrates that contain either MODE I (R[S/Ar][+/Ar]pS[L/E/A/M]P) or MODE II (Rx[Ar][+]pS[L/E/A/M]P; Ar = aromatic residue, + = basic residue, and x = any) recognition sites [
CRTCs contain three principal phosphorylation sites—(S70, S171, and S275 in CRTC2) that are regulated by cAMP and calcium signals, respectively–although the extent to which they and perhaps others modulate the activity of individual family members is unclear [
Small molecules were solubilized in DMSO (ACS, Sigma-Aldrich) at the indicated concentrations and stored until usage at -80°C (long term storage) or -20°C (working dilution): 20 mM Forskolin (Sigma-Aldrich) and 2 mM Carfilzomib (PR-171) (Selleck Chemicals).
The antibodies used in Western blot analysis were purchased from Santa Cruz Biotechnology (14-3-3 ε monoclonal & polyclonal), EMD Millipore (α-tubulin), Sigma-Aldrich (FLAG M2), Roche (Anti-HA-Peroxidase), Cell Signaling Technology (14-3-3 [pan], CRTC1, P-CREB S133, P-CRTC1 S151, P-CRTC2 S171, P-MARK AL [AL = activation loop], P-PKA substrate), and Covance (GFP, HA.11). See Antiserum Production for the P-CRTC3 S273 antiserum (PBL #7378).
All animal procedures were approved by the Institutional Animal Care and Use Committee of the Salk Institute and were conducted in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals (PHS Policy, 2015), the U.S. Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research and Training, the NRC Guide for Care and Use of Laboratory Animals (8th edition) and the USDA Animal Welfare Act and Regulations. Three 10 to 12-week old, female New Zealand white rabbits, weighing 3.0 to 3.2 kg at beginning of the study, were procured from Irish Farms (I.F.P.S. Inc., Norco, California, USA). All animals were housed in an AAALAC accredited facility in a climate controlled environment (65–72 degrees Fahrenheit, 30–70% humidity) under 12-hour light:12-hour dark cycles. Rabbits were provided with ad libitum feed (5326 Lab Diet High Fiber), micro-filtered water and weekly fruits, vegetables and alfalfa hay for enrichment. Upon arrival, animals were physically examined by veterinary staff for good health and acclimated for two weeks prior to initiation of antiserum production. Each animal was monitored daily by the veterinary staff for signs of complications and weighed every two weeks. Routine physical exams were also performed by the veterinarian quarterly on all rabbits.
The rabbits were injected with a peptide fragment encoding Cys268pSer273 CRTC3(268–282) coupled to keyhole limpet hemocyanin via maleimide. The peptide, <Hnt>CNTGGpSLPDLTNLHY<NH2>, was synthesized and purified by Jean E. Rivier (The Salk Institute). The antigen was delivered to host animals using multiple intradermal injections of peptide-KLH conjugate in Complete Freund's Adjuvant (initial inoculation) or incomplete Freund's adjuvant (booster inoculations) every three weeks. Rabbits were bled, <10% total blood volume, one week following booster injections and bleeds screened for titer and specificity. Animals were administered 1–2 mg/kg Acepromazine IM prior to injections of antigen or blood withdrawal. At the termination of study, animals were exsanguinated under anesthesia (ketamine 50 mg/kg and aceprozamine 1 mg/kg, IM) and euthanized with an overdose of pentobarbital sodium and phenytoin sodium (1 ml/4.5 kg of body weight IC to effect). After blood was collected death was confirmed. All animal procedures were conducted by experienced veterinary technicians, under the supervision of Salk Institute veterinarians.
The antiserum obtained from the rabbit (code PBL #7378) with the best characteristics of titer and specificity was used for all experiments. So that the same batch of serum could be used for this and future studies, a large volume of serum from a single bleed was depleted of antibodies recognizing the non-phosphorylated form of CRTC by passing over a column containing Cys268CRTC3(268–282)-agarose resin. Covalent attachment of peptide to resin (Sulfolink coupling resin, Thermo Fisher) was per manufacturer's instructions.
For overexpression studies we generated plasmids containing the
The cDNAs (h =
The plasmids used in this study were typically generated by restriction endonuclease based cloning, with final constructs coding for the following proteins (TV = transcript variant; in brackets UniProt identifier): mCRTC1 (Q68ED7-1), mCRTC2 (Q3U182-1), mCRTC3 (Q91X84-1), mNEK1(P51954-1), hMARK1 TV2 (Q9P0L2-1), hMARK2 TV3 (Q7KZI7-16), hMARK2 TV4 (Q7KZI7-1), hMARK3 TV3 (P27448-3), hMARK4 TV2 (Q96L34-2), mMARK4 (Q8CIP4-1), hMTFR1(Q15390-1), mSIK2 (Q8CFH6-1). All cDNAs derived plasmids, with the exception of hMTFR1, were N-terminally 3xFLAG-tagged; in case of mCRTC1-3 both N- and C-terminally tagged constructs were generated. In all experiments the N-terminal mCRTC1-3 plasmids were used, apart from IP-MS experiments were both the N- and C-terminally tagged CRTC proteins were subjected to the protocol.
Multi-step cloning was necessary for the following plasmids: mSIK2 (existing mutations in the ORF were reversed by site-directed mutagenesis to match Q8CFH6-1: T41I, R809Q) and hMARK2 TV4 (fusion PCR from two PCR products, template clones HsCD00331723 [TV3] and HsCD00323103 [incomplete TV4], assembled Q7KZI7-1).
All other mutations were generated via single or cumulative site-directed mutagenesis.
The luciferase reporter plasmid was generated by cloning the EVX promoter fragment (220 bp, 2x CRE half-sites; existing lab plasmid) into pGL4 (Promega).
Fusion PCR was performed as previously described [
HEK293T cells were purchased from ATCC (CRL-11268) and propagated in DMEM media (Gibco®, high glucose) supplemented with 10% Fetal Bovine Serum (Gemini Bio-Products) and 100 U/ml penicillin-streptomycin (Corning Inc.).
Experiments were performed in 6 well plates by reverse transfecting HEK293T cells (2.5 × 106 cells) with 2 μg plasmid DNA using Lipofectamine® 2000 (Invitrogen). 48 h post transfection cells were collected in PBS and resuspended in lysis buffer (50 mM Tris, 150 mM NaCl, 10% glycerol, 1% Igepal [Sigma-Aldrich], 1 mM DTT, EDTA-free cOmplete™ Protease Inhibitor Cocktail [Roche], Phosphatase Inhibitor Cocktail 2 and 3 [Sigma-Aldrich], 1 μM Carfilzomib; pH 8.0). The supernatant (= cell lysate) was either used in IP experiments or directly mixed with SDS-PAGE loading buffer. In all IP experiments, cells were pre-treated for 1 h with 1 μM Carfilzomib prior to cell lysis. Cell lysate was incubated with anti-FLAG ® M2 magnetic beads and 3xFLAG peptide was used for elution (both Sigma-Aldrich).
IP-MS was performed in 6 x 100 mm dishes by reverse transfecting HEK293T cells (1.5 × 107 cells) with 12 μg plasmid DNA using Lipofectamine® 2000 (Invitrogen). 48 h post transfection cells were treated for 1h with 1 μM Carfilzomib and 10 μM Forskolin. Cell collection and lysis was performed as described in the IP protocol.
Proteins were precipitated with 23% TCA and washed with acetone. Protein pellets were solubilized in 8 M urea, 100 mM Tris pH 8.5, reduced with 5 mM Tris(2-carboxyethyl)phosphine hydrochloride (Sigma-Aldrich), and alkylated with 55 mM 2-Chloroacetamide (Fluka Analytical). Proteins were digested for 18 h at 37°C in 2 M urea, 100 mM Tris pH 8.5, 1 mM CaCl2 with 2 ug trypsin (Promega). Five–step MudPIT analysis was performed using an Agilent 1200 G1311 quaternary pump and a Thermo LTQ Orbitrap Velos using an in-house built electrospray stage [
Protein and peptide identification and protein quantitation were done with Integrated Proteomics Pipeline—IP2 (Integrated Proteomics Applications). Tandem mass spectra were extracted from raw files using RawConverter [
HEK293T cells were plated in glass chamber slides (BD) and forward transfected with Lipofectamine® 2000 (Invitrogen) using mCRTC1 constructs in the pUbC-3xHA backbone. Roughly 24 h post-transfection cells were treated with either DMSO or 10 μM Forskolin for 30 min. Slides were fixed with 4% paraformaldehyde and incubated with primary antibodies (HA.11, CRTC1). Samples were incubated with secondary antibodies conjugated with Alexa Fluor® -488 (donkey anti-mouse) and -568 (donkey anti-rabbit) (Life Technologies) and subsequently counterstained with DAPI (Cayman Chemical Company) before images were acquired with a LSM 780 (Carl Zeiss).
Luciferase reporter assays were performed in 96 well plates by reverse transfecting HEK293T cells (100,000 cells). For each well 80 ng of DNA was used: 10 ng of EVX reporter plasmid, 10 ng of N-terminal FLAG-tagged CRTC1-3 plasmids, [20 ng of N-terminal FLAG-tagged kinase plasmid], 60 [
Amino acid sequences were aligned using MegAlign and Clustal W method (DNASTAR v7).
In mass spectrometry (MS) studies to identify proteins that modulate CREB-dependent transcription, we recovered large quantities of 14-3-3 proteins from immunoprecipitates (IPs) of epitope-tagged CRTCs prepared from HEK293T cells, even following exposure to the adenylyl cyclase activator Forskolin (Fsk) (
(A) Schematic representation depicting the inhibition of cAMP-regulated transcriptional coactivators (CRTCs) by AMPK family members (AMPK, SIK1-3, and MARK2). CRTC phosphorylation induces 14-3-3 binding and cytoplasmic sequestration. (CBD = CREB binding domain, TA = transactivation domain) (B) Table on the left shows the recovery of 14-3-3 protein family members (C-terminally tagged CRTCs) and table on the right recovered AMPK family members (arbitrary individual C-terminally tagged CRTC; SPCs = spectral counts). (C) List of the primary CRTC sequences of potential AMPK family phosphorylation sites (LXBS/TX
Binding of CRTCs to 14-3-3s sequesters them in the cytoplasm; and increases in cAMP promote the dephosphorylation and release of CRTCs from 14-3-3 proteins leading to their nuclear localization [
(A) and (B) Western blot analysis of the Co-IP of FLAG-tagged CRTC1-3. CRTCs were expressed from a plasmid containing the constitutive Ubiquitin C promoter (UbC), which drives the expression of EGFP via an internal ribosome entry site (IRES). In HEK293T cells the P-CREB S133 antibody recognizes two bands, the upper band being CREB1 and the lower band ATF1 (calculated molecular weight: ~ 37 and 29 kDa). (DMSO/Fsk treatment for 1h) (C) Immunofluorescence of HEK293T cells transfected with HA-tagged CRTC1. Cells were co-stained for HA & CRTC1 and counterstained with DAPI. (DMSO/Fsk treatment for 30 min)
We performed Ser/Ala mutagenesis of individual phospho-acceptor sites within each conserved region to determine their effects on CRTC transcriptional activity by transient assay with a luciferase reporter containing cAMP responsive elements (CREs) [
(A) EVX-Luc reporter assay of putative 14-3-3 site mutants inside CRTC1-3. (n = 10, ±s.e.m.) (B) Western blot analysis of the Co-IP of FLAG-tagged CRTC 14-3-3 mutants. (C) Immunofluorescence of HEK293T cells transfected with HA-tagged CRTC1 14-3-3 mutants. Cells were co-stained for HA & CRTC1 and counterstained with DAPI. (DMSO/Fsk treatment for 30 min). (A-C): Ser/Ala mutations of the three conserved CRTC sites are highlighted (region I = orange, region II = yellow, blue = region III, and double mutant of site II and III in red)
Having seen that mutation of region II in CRTC1 (S151A) has a stronger effect on 14-3-3 binding and CRE reporter activity compared to the corresponding mutant in CRTC3 (S162A), we examined the potential role of residues flanking this phospho-acceptor site in modulating the transcriptional activity of individual family members. S151 in CRTC1 is preceded by two arginine residues at positions (-4, -3), whereas CRTC2 and CRTC3 contain only a single arginine (
(A) EVX-Luc reporter assay comparing arginine mutants inside region II of CRTC1 and CRTC3. Phosphorylated region II serines (yellow), arginine residues (underlined), and mutated amino acids (red) are highlighted. (n = 10, ±s.e.m.) (B) Western blot analysis of the Co-IP of corresponding FLAG-tagged arginine CRTC1 and CRTC3 mutants.
Although members of the AMPK family have been shown to modulate CREB target gene expression through inhibitory phosphorylation of the CRTCs, their relative activities have not been examined [
(A) Western blot analysis of the overexpression of FLAG-tagged wild type (WT) versus kinase dead (KD) SIK2 and MARK2 (SIK2 KD = T175A, MARK2 KD = T208A S212A). The P-CRTC3 S273 antibody recognizes two bands in HEK293T cells, the upper band being CRTC2 and the lower band CRTC3 (calculated molecular weight: ~ 73 and 67 kDa). (DMSO/Fsk treatment for 1h) (B) EVX-Luc reporter assay comparing the effect of AMPK family members SIK2 and MARK2 co-expression on the transcriptional activity of CRTC1-3. EVX-Luc activity was measured after 4 h of DMSO or Fsk treatment. (n = 5, ±s.e.m.) (C) Graph depicting the corresponding fold changes in reporter activity upon Fsk treatment in three independent experiments (A-C, A = results depicted in
cAMP has been reported to inhibit SIKs and other AMPK family members through PKA-mediated phosphorylation [
Knowing that AMPK family members inhibit CRTCs primarily through phosphorylation at sites II and III, we reasoned that mutant CRTC proteins containing alanine substitutions at these sites should be resistant to repressive effects of SIKs and MARKs. Although it disrupted the activities of individual site II and III CRTC3 mutants, SIK2 over-expression had only modest effects on site II/III double mutants and no effect on the activity of a mutant with alanine substitutions at both conserved and non-conserved sites (WT ~3%, S162A S273A ~50%, and 5A = S62A S162A S273A S329A S370A ~200% activity) (
Based on the ability of MARK2 to repress CRTC activity by multi-site phosphorylation, we examined the roles of other MARK family members in this process. Each family member was competent to inhibit CRE reporter activity, with MARK2 and MARK4 appeared to be most potent. Overexpression of MARK 2/4 inhibited the activities of both CRTC2 and to a lesser extent CRTC3 (
(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
Although SIKs and MARKs appear to phosphorylate CRTCs comparably at regions II and III, region III lacks a basic residue within the LXBS/TX
(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)
cAMP has been shown to stimulate CREB target gene expression through the dephosphorylation of CRTCs at sites that otherwise mediate 14-3-3 protein binding and cytoplasmic sequestration (
(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
Members of the AMPK family are basophilic kinases that phosphorylate substrates containing LXBS/TX
The key functional feature of region III is the
We thank J. Rivier, C. Miller, and J. Erchegyi for peptide synthesis and purification.