CRTC family members are phosphorylated at conserved sites that also mediate 14-3-3 binding.

<p>(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<u>S</u>XXXL; B being a basic residue) that might simultaneously serve as 14-3-3 binding sites (RXX<u>S</u>/<u>T</u>XP). Amino acids shown in red were assigned to be phosphorylated by mass spectrometry. Three conserved CRTC regions are highlighted with boxes: orange (region I), yellow (region II), and blue (region III). (<i>Mus musculus</i> CRTC = mCRTC). (D) Sequence alignment of conserved regions I-III of <i>M</i>. <i>musculus</i> CRTC1-3. (E) Tables list the relative phosphorylation stoichiometry of key sites in conserved region I-III (spectral counts for phosphorylated versus non-phosphorylated). (B,C,E): N- and C-terminally FLAG-tagged CRTC1-3 (N-/C-IP) were overexpressed in HEK293T cells and stimulated with Forskolin prior to the IP-MS protocol.</p

14-3-3 binding of CRTCs is modulated by cAMP.

<p>(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)</p

Characterization of 14-3-3 binding sites in the CRTC family.

<p>(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)</p

Multi-site phosphorylation of CRTCs by SIK and MARK kinases.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173013#pone.0173013.g004" target="_blank">Fig 4B</a>). (D) EVX-Luc reporter assay comparing the effect of SIK2 and MARK2 co-expression on the transcriptional activity of CRTC3 14-3-3 mutants (3A = S62A S329A S370A, 5A = S62A S162A S273A S329A S370A). (n = 10, ±s.e.m.)</p

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

Arginine residues inside a SIK/MARK phosphorylation site regulate 14-3-3 binding and transcriptional activity of CRTCs.

<p>(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.</p

HtrA1 Proteolysis of ApoE In Vitro Is Allele Selective

Apolipoprotein E (ApoE) belongs to a large class of proteins that solubilize lipids for physiological transport. Humans have three different <i>APOE</i> alleles, <i>APOE ε2</i>, <i>APOE ε3</i>, and <i>APOE ε4</i>, and genetic studies identified ApoE4 as the strongest genetic risk factor for Alzheimer’s disease (AD). People who are homozygous for ApoE4 (i.e., ApoE4/E4) are an order of magnitude more likely to develop late-onset AD (LOAD) than ApoE3/E3 carriers. Several differences between ApoE3 and ApoE4 may contribute to AD including the observation that ApoE4 is degraded to a greater extent than ApoE3 in the human brain. Experiments with high-temperature requirement serine peptidase A1 (HtrA1), which is found in the nervous system, demonstrate that HtrA1 is an allele-selective ApoE-degrading enzyme that degrades ApoE4 more quickly than ApoE3. This activity is specific to HtrA1, as similar assays with HtrA2 showed minimal ApoE4 proteolysis and trypsin had no preference between ApoE4 and ApoE3. HtrA1 has also been reported to cleave the tau protein (Tau) and the amyloid protein precursor (APP) to hinder the formation of toxic amyloid deposits associated with AD. Competition assays with ApoE4, ApoE3, and Tau revealed that ApoE4 inhibits Tau degradation. Thus, the identification of ApoE4 as an in vitro HtrA1 substrate suggests a potential biochemical mechanism that links ApoE4 regulation of AD proteins such as Tau

Expression and Functional Characterization of Membrane-Integrated Mammalian Corticotropin Releasing Factor Receptors 1 and 2 in <i>Escherichia coli</i>

<div><p>Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2β as well as fragments lacking the N-terminal extracellular domain, were overproduced in <i>E. coli</i>. The characteristics of different CRFR2β -PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1α and CRFR2β expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of <i>E. coli</i> bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2β product was obtained in comparison to CRFR1α. Following a detergent screen analysis, bacterial membranes containing CRFR1α and CRFR2β were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1α and CRFR2β membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our <i>E. coli</i> expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.</p></div