Ligand-bound and phosphorylated ErbB/HER heterodimers are potent signaling forms of this receptor family, and quantitative measurements of these active receptors may be predictive of patient response to targeted therapies. Using VeraTag™ technology, we developed and characterized quantitative assays measuring epidermal growth factor (EGF)-dependent increases in activated HER receptors in tumor cell line lysates and formalin-fixed, paraffin-embedded (FFPE) tumor sections. We demonstrated the ability of the assays to quantitatively measure changes in activated HER1 and HER2 receptor levels in cell lines following treatment with 2C4, erlotinib, and lapatinib. We utilized these assays to determine the prevalence and distribution of activated HER1, HER2, and HER1-HER2 heterodimers in 43 HER2-positive breast tumors.Assays for activated HER1 and HER2 receptors in FFPE and cell lysate formats were developed using VeraTag™ technology, which requires the proximity of an antibody pair for light-dependent release of a fluorescently labeled tag, followed by capillary electrophoresis-based quantitation.Ligand-dependent and independent HER1-HER2 heterodimer levels measured by lysate and FFPE VeraTag™ assays trended with HER1 and HER2 expression levels in tumor cell lines, which was confirmed by co-immunoprecipitation. The formation of EGF-dependent HER1-HER2 heterodimers were inhibited by the HER2-targeted monoclonal antibody 2C4 and stabilized by the HER1 tyrosine kinase inhibitor (TKI) erlotinib. EGF-dependent HER1 and HER2 phosphorylation was inhibited by lapatinib and erlotinib. Further, we observed that dominant receptor signaling patterns may switch between HER1-HER1 and HER1-HER2, depending on drug mechanism of action and relative levels of HER receptors. In FFPE breast tumors that expressed both HER1 and HER2, HER1-HER2 heterodimers were detected in 25 to 50% of tumors, depending on detection method. The levels of activated phospho-HER1-HER2 heterodimers correlated with HER1 or HER2 levels in an analysis of 43 HER2-positive breast tumors.®) in tumor samples may provide a more accurate prediction of clinical response to both HER1 and HER2 targeted therapies.VeraTag™ lysate assays can be used as a tool for understanding the mechanism of action of targeted HER-family inhibitors in the preclinical setting, while VeraTag™ FFPE assays of activated HER receptors combined with total HER2 measurements (HERmark Both the epidermal growth factor receptor EGFR/HER1) and HER2 are members of the ErbB family of the type I receptor tyrosine kinases, which also includes HER3 and HER4. These homologous receptors are comprised of an extracellular binding domain (ECD), a transmembrane domain, and an intracellular tyrosine kinase (TK) domain. Binding of ligand to the ECD induces structural reorganization allowing for functional homo- and heterodimerization and activation of the kinase domain  and HER2. A HER2 HER2 overexpression has been observed in several cancer types . From 15Several drugs that target HER1 and HER2 receptors have been utilized in both preclinical and clinical models of breast and other cancers. Treatment with the humanized monocolonal HER2 antibody trastuzumab is now standard of care for individuals with HER2-positive invasive breast cancer in both the metastatic and adjuvant settings. However, fewer than 50% of patients with metastatic HER2-positive breast tumors show initial benefit from trastuzumab treatment, and many of those eventually develop resistance -27. Thusin vitro and in xenograft models have demonstrated overexpression of HER1 and its ligands 39].Lysates  were pre-cleared by incubation with 40 to 80 μl protein G-sepharose beads  for one hour. Antibody was added  to pre-cleared lysates overnight at 4°C. Protein G-sepharose beads  were added and incubated 1.5 hours on a rotator at 4°C. Beads were retained after centrifugation  and washed twice with 1 × PBS and once with 50 mM Tris-HCL, pH 8.0. Beads were resuspended in Laemmli Sample Buffer  and incubated at 90°C for 10 minutes. Proteins were separated by electrophoresis using 5% or 7.5% Tris-HCl Ready Gels (Bio-Rad) and transferred to Immuno-Blot PVDF membranes (Bio-Rad). Membranes were blocked for two hours with 5% milk in 1 × PBS + 0.25% Tween 20 (PBS-T) at room temperature. Membranes were incubated overnight at 4°C with the detection antibody in 5% milk + PBS-T. Membranes were washed three times with PBS-T. Goat-anti-mouse-HRP  or anti-rabbit-HRP  was then added for two hours, and bands were detected using Supersignal West Pico or Femto substrate .HER1 IHC was performed by a variation of the Zymed/Invitrogen method, using pepsin  and 31G7 monoclonal antibody . 31G7 concentration was optimized on FFPE cell line controls and tumor sections and used at 0.4 ug/mL for one hour, following pepsin retrieval for 30 minutes at room temperature. All additional steps were performed per manufacturer's protocol.Cells were harvested by trypsinization and counted. Cells were plated in a 96-well plate and labeled with HER2-Ab4-biotin (Lab Vision) or HER1-Ab11 (Lab Vision) or isotype control  at a concentration of 4 μg/ml in 100 μl total volume. The cells were incubated with antibody on ice for 45 minutes and washed twice with 1X PBS, followed by labeling with R-Phycoerythrin-Avidin  at 2 μg/ml for 30 minutes on ice. The labeled cells were washed twice with 1X PBS and fixed with 1% paraformaldehyde in 1X PBS. Cells were analyzed on a FACSCalibur cytometer . PE fluorescence intensity of labeled cells was determined on a FL2 (585/42 nm band pass filter) detector. Determination of HER1 or HER2 receptor numbers were based on a calibrated standard curve using Quantum PE MESF Kit .In order to measure a HER1 and HER2 heterodimeric complex and associated receptor phosphorylation levels, assays of cell lysates were developed utilizing the VeraTag™ technology  FFPE assays [In order to evaluate HER1-HER2 activated forms as potential biomarkers of targeted drug response, cell lines characterized by flow cytometry and lysate assays just described were utilized to develop VeraTag™ assays that measure HER1-HER2 heterodimerization and activation in FFPE tumor tissue Figure . HER1 anE assays . HER1-HEIn order to discern active HER1-HER2 complexes, FFPE assays that detected phosphorylated forms of HER1, HER2, and the HER1-HER2 heterodimeric complex were developed. Based on target specificity, assay dynamic range, and effects of EGF stimulation, the optimal antibody pairing for measurement of activated HER1-HER2 heterodimers utilized a HER2-specific antibody together with a HER1 antibody specific for the kinase domain phosphotyrosine 1173 to produce a pHER1-HER2 assay Figure . An EGF-HER1 and HER2 may be phosphorylated due to actively signaling HER1-HER2 heterodimers, but other mechanisms such as HER1-HER1 dimerization, HER2 autophosphorylation, or via dimerization with other HER family members may also play a role. Thus, assays were designed that could independently assess phosphorylated HER1 and phosphorylated HER2 in FFPE tumor cells. Two phospho-HER1 (pHER1) assays were developed, both of which utilized a HER1 antibody that was matched with either an antibody to measure a specific HER1 phosphotyrosine site , or a pan-phosphotyrosine antibody to measure general HER1 and HER1-associated phosphorylation (pY: clone 4G10). Both the pHER1(1173) and the pHER1(pan) assay formats led to similar trending of pHER1 signal from the cell line panel, consistent with results from co-IP/Western blots Figure . The simTo measure the levels of activated HER2, a phosphorylated HER2 assay was developed by pairing a HER2 antibody with the pan-phosphotyrosine antibody 4G10 Figure . IntriguThe direct measurement of ligand-independent and -dependent HER dimers and phosphorylated forms by the VeraTag™ assays allowed the mechanistic characterization of the acute effects (time = 2 hours) of the HER-targeted drugs 2C4, lapatinib, and erlotinib. Cell stimulations were performed with 16 nM EGF instead of 100 nM EGF, which may more closely approximate physiologic conditions while ensuring the same level of dimerization response. Exposure of 2 or 20 ug/mL 2C4, a HER2 monoclonal antibody which inhibits HER2-HER3 heterodimerization , decreasAs expected, lapatinib, a dual HER1 and HER2 tyrosine kinase inhibitor, nearly eliminated both HER1 and HER2 phosphorylation in SKOV3 cells Figure . InteresThe dynamic changes in the levels of EGF-dependent HER1-HER2 heterodimers supported by 2C4 inhibition and erlotinib stabilization were utilized to establish the validity of these measurements in FFPE tumor cell lines. SKOV3 cells were exposed to either 2C4 or erlotinib and EGF as described above, fixed and made into FFPE blocks, and measurements were made by VeraTag™ FFPE assays. Consistent with results from the lysate assays, EGF-dependent HER1-HER2 heterodimerization was suppressed whether measured using the HER1-HER2 or the pHER1-HER2 assay. A decrease in HER2 phosphorylation was detected with the pHER2(pan) assay, while minimal change was observed in HER1 phosphorylation as detected by either the pHER1(pan) or pHER1(1173) assays Figure . Upon er® breast cancer assay [P = 0.0113; Figure S4A in Additional file P = 0.0068; Figure S4B in Additional file P < 0.0001; Figure S4C in Additional file To determine the prevalence of HER1 and HER1-HER2 dimeric and activated forms in HER2-positive breast cancer, 43 breast tumors that were pre-selected for high HER2 expression by IHC and subsequent scoring using the Allred system  , whereas 2 of 43 samples that had high HER1 levels by VeraTag™ assay and HER1 IHC were not HER2-positive by HERmarkTo evaluate the breast tumors with the HER1-HER2 heterodimer and activated assays, all samples were analyzed with a paired isotype control, and positives were classified as described in the Methods. Heterodimer signals in the tumors classified as positive were greater than or equal to that of cell line H1650 stimulated with 100 nM EGF. HER1-HER2 heterodimers were detected in 4 of the 16 tumors that were classified as positive for both HER1 and HER2, and pHER1-HER2 heterodimers were detected in 8 of these 16 tumors. The two heterodimer measurements were likely discrepant since the pHER1-HER2 assay appears to be more sensitive and may be able to detect lower levels of analyte.P = 0.001)  and HER2     Figure . However) Figure . HER1-HE) Figure . The cor) Figure . Similarin situ measurement of activated signal proteins in FFPE tumors toward biomarker identification in patient samples.Using VeraTag™ technology, we have developed multiple quantitative and sensitive assays for measurements of activated receptor species in the HER1-HER2 signaling axis in both breast tumor cell lysates and FFPE cell and tumor formats. Using the VeraTag™ assay for cell lysates, control cell lines were identified which expressed different levels of HER1 and HER2, and generated a range of ligand-dependent and independent levels of phosphorylated HER proteins and HER1-HER2 heterodimers. These findings were confirmed by co-immunoprecipitation analysis. The cell lines were then utilized in the development and characterization of VeraTag™ assays that measured activated HER1/HER2 forms in FFPE tumor cells. Both the VeraTag™ lysate and FFPE cell assays demonstrated utility in the study of drug action and in the evaluation of activated HER1/HER2 forms in HER2-positive breast tumor tissue. Taken together, these studies indicate that VeraTag™ assays can be utilized in preclinical studies of drug mechanism of action involving both protein phosphorylation and protein-protein modes of signal activation, and has the potential for use in the Several specific findings were made through the application of the VeraTag™ lysate assays. First, the levels of ligand-dependent and independent HER1-HER2 heterodimers appear to depend on the levels of HER1 and HER2. These results are consistent with recent studies indicating the existence of ligand independent HER1-HER2 and HER2-HER3 heterodimers -14,42,51Additional novel observations were made from VeraTag™ lysate analysis of drug effects of the 4-quinazoline tyrosine kinase inhibitors lapatinib and erlotinib. Lapatinib is a small molecule tyrosine kinase inhibitor (TKI) of both HER1 and HER2 , and erlin vivo drug studies. Further, our studies have suggested interesting biology regarding HER1 and HER2 receptor switching. While findings are preliminary, they are suggestive of the ability of cell lines to redistribute signaling patterns between HER1-HER1 homodimers and HER1-HER2 heterodimers depending on relative HER levels, acute drug exposure and mechanism of drug action.VeraTag™ assays on FFPE cell line sections utilizing 2C4 and erlotinib were consistent with results from lysate assays and with expectations from published results, supporting utility and extending the use of these assays in understanding receptor activation in cell lysate and FFPE tumor tissues. To the best of our knowledge, this is the first example of HER1-HER2 heterodimer measurements made in FFPE tissue that have been characterized using drug inhibition, and suggests application to both cell-based and In Situ Hybridization (FISH) and IHC, but showed disproportionately high pHER2 and downstream signaling [de novo or acquired Herceptin-resistant breast cancers as well as multiple additional cancer types, including squamous cell carcinoma of the head and neck, lung, gastric, and triple-negative breast cancers [Last, we utilized the HER1/HER2 FFPE cell assays to determine the prevalence of HER1-HER2 dimers and phosphorylated forms in 43 breast tumor samples that were pre-selected for HER2-positivity. Of the 16 tumors that co-expressed HER1, 25% had detectable HER1-HER2 heterodimers whereas 50% had detectable pHER1-HER2. Phosphorylation of HER2 was detected in more than 50% of the HER2-positive tumors. Both autophosphorylation and transphosphorylation due to heterodimerization may contribute to overall HER2 phosphorylation, and both may be relevant in determining patient treatment. Historically, there has been a reluctance to measure phosphorylation in FFPE tissues due to the potential for phosphatase activity during tumor excision and time to fixation. However, recent technical advances to stabilize phosphorylation, such as decreased time to fixation and addition of phosphatase inhibitors to the fixative solution, along with increased evidence for the utility of phosphoprotein measurements, support development of technologies to specifically and quantitatively measure phosphoproteins in FFPE tissues. Phosphorylated HER2 has been shown to be a predictor of poor response and survival in breast cancer patients in multiple studies -61. In aignaling . Ongoing cancers -71. StudIn our study, great care was taken to preserve phospho-protein epitopes. All tumor tissues were acquired from a single commercial vendor that utilized a rigorous protocol for tumor collection, fixation, and storage to promote preservation of epitopes, consistent with the ASCO/CAP guidelines for preparation of breast tumor tissue for HER2 testing (see Methods). Additionally, an independent study performed under similar conditions in tumor cell xenografts showed complete preservation of pHER . While iIn sum, the VeraTag™ lysate assays presented here may prove extremely useful when used in concordance with preclinical drug development, while FFPE assays may help fulfill the need for sensitive, quantitative methods for detection of activated HER1 and HER2 complexes in FFPE tumor tissues. With the advent of additional molecularly targeted therapies into the clinic, a thorough understanding of receptor activation and detection in patient samples has potential to enable the measurement of biomarkers that may be more predictive of drug response.We have developed novel, quantitative assays that measure HER1-HER2 dimerization and phosphorylation, HER1 phosphorylation, and HER2 phosphorylation in both lysate and FFPE tissue formats using VeraTag™ technology. The VeraTag™ assays were used to characterize the effects of 2C4, lapatinib, and erlotinib on cell lines, and receptor signaling switching between HER1-HER1 homodimers and HER1-HER2 heterodimers was observed under some conditions. Further, HER1-HER2 heterodimers were detected in a subset of HER2-positive breast tumors that co-expressed HER1. These VeraTag™ assays may have utility both in pre-clinical drug development and for predicting clinical response to targeted agents.CE: capillary electrophoresis; Co-IP: co-immunoprecipitation; DMSO: dimethyl sulfoxide; ECD: extracelluar domain; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; FACS: fluorescent activated cell sorting; FISH: fluorescence in situ hybridization; FFPE: formalin-fixed: paraffin-embedded; IB: illumination buffer; IHC: immunohistochemistry; ITC: isotype control; PBS: phosphate-buffered saline; PBS-T: phosphate-buffered saline-Tween 20; PLA: proximity ligation assay; RPA: relative peak area; SA-MB: streptavidin-methylene blue; TK: Tyrosine kinase; TKI: Tyrosine kinase inhibitor.® assays commercially for patient testing and uses VeraTag™ assays in collaborations with multiple private and academic institutions.All authors are or were previously employed by Monogram Biosciences, Inc., which offers HERmarkLDE designed and oversaw all experiments, carried out original co-immunoprecipitiations and some VeraTag™ FFPE assays, and drafted the manuscript. KS carried out the majority of the VeraTag™ FFPE and lysate assays depicted here. TDP designed and made the transfected 293 clones, carried out the co-immunoprecipitation experiments depicted here, performed the drug experiments on SKOV3 cells, and carried out some VeraTag™ FFPE assays. GP provided intellectual input and helped edit the manuscript. LG provided intellectual input, helped edit the manuscript, and selected tumor sets. JW provided intellectual input, selected tumor sets, oversaw tumor immunohistochemistry, and helped edit the manuscript.Supplementary Figures S1 to S6 Figure S1. Ligand-dependent and ligand-independent HER1-HER2 heterodimerization depends on receptor levels. A total of 293 cells were transfected with HER1 to generate 293H1_clone11. Clone11 was then co-transfected with HER2 to generate clones 12, 15, 16, and 19. Gray bars represent signal from mock-stimulated cells, and black bars represent signal from cells stimulated with 100 nM EGF. A) HER1 total lysate assay. All clones had the same amount of HER1. B) HER2 total lysate assay. Clones displayed different amounts of total HER2. C) HER1-HER2 heterodimer lysate assay. Ligand-dependent and ligand-independent HER1-HER2 heterodimer formation increased with increasing HER2 receptor number in clones with the same amount of HER1. Figure S2. Specificity of clone 53A5 for phospho-HER1 in FFPE assay. AU565 FFPE slides from cells were mock-stimulated or stimulated with 100 nM EGF for 10 minutes. The pHER1-HER2 FFPE assay was performed, but peptide was incubated with antibody in molar ratios 1:0, 1:1, 1:10, or 1:100 of antibody to peptide. Gray bars represent signal from mock-stimulated cells, and black bars represent signal from EGF-stimulated cells. Phospho-HER1-HER2 signal is competed to basal levels using the antigenic peptide H1pY1173 but not with the nonphosphorylated H1Y1173 nor the homologous HER2 peptides H2pY1248 nor H2Y1248. Figure S3. Effects of lapatinib and erlotinib measured by VeraTag™ lysate assays in H1650. H1650 cells were serum-starved overnight then treated with lapatinib or erlotinib for two hours at the indicated concentrations, in units of μM. A final concentration of 16 nM EGF was added or mock-added to the drug-containing media for 10 minutes prior to harvesting cells for lysate or FFPE. VeraTag™ lysate assays were used to examine A) effects of erlotinib and B) effects of lapatinib on HER1-HER2 heterodimerization (top row), phosphorylation of HER2 (second row), phosphorylation of HER1 (third row), and HER1-HER1 homodimerization (bottom row). Figure S4. Comparison of two different formats of a HER1 total protein VeraTag™ assay performed in HER2+ breast FFPE tumors. A) Comparison of a HER1 ICD-directed total protein assay with a HER1 ECD-directed total protein assay, both applied to intact FFPE tumor sections. B) Comparison of a HER1 ICD-directed total protein assay performed in intact FFPE tumor sections with a HER1 ECD-directed total protein assay having non-tumor elements macrodissected from the FFPE tumor sections. C) Comparison of HER1 values measured in intact versus macrodissected FFPE tumor sections assayed by the HER1 ECD-directed total protein assay. Figure S5. Comparison of HER1 ECD-directed total protein assay with HER1 IHC H-score. A) A subset of 30 HER2+ breast tumors was assayed by a HER1 ECD-directed total protein assay with non-tumor elements macrodissected away, and the HER1 levels were compared with the tumor cell HER1 IHC H-scores measured on adjacent sections. The IHC H-score spans 0 to 300. Tumors with an H-score of 0 were assigned a score of 0.1 to make plotting on a log scale possible. B) The percentage of the 30 tumors at each HER1 IHC test category. C-F) Micrographs of the HER1 IHC staining representing four separate invasive ductal carcinoma breast tumors that are representative of other tumors having similar staining intensity from IHC = 3+ to 0, respectively (4× magnification). G-J) Same tumor micrographs as in C-F but at 20× magnification. Figure S6. Western blot and co-IP/Western blots of lysed tumor tissue homogenates. Fresh frozen tumors matched with FFPE blocks of HER2+ breast tumors were lysed and homogenized with protease and phosphatase inhibitors, and analyzed by SDS-PAGE and Western blots. Sections of the matched FFPE blocks were assayed by the comparable VeraTag™ assay. A) HER2 Western blot comparison of tumor lysate with HER2 total protein VeraTag™ assay. B) HER1 Western blot of tumor lysate comparison with HER1 ECD-directed total protein VeraTag™ assay. C) Tumor lysate immunoprecipitated with a HER2 antibody and Western blot with a pan-phosphotyrosine antibody comparison with pHER2 VeraTag™ assay. D) Tumor lysate immunoprecipitated with pHER1(1173) antibody and Western blot with a HER2 antibody comparison with pHER1-HER2 VeraTag™ assay.Click here for file


Further, the CC2D1A protein, consisting of N-Terminal containing four DM14 domains and C-terminal containing C2 domain, was shown to regulate the cAMP-PKA pathway. A human deletion mutation lacking the fourth DM14 and the adjacent C2 domain results in Non Syndromic Intellectual Disability (NSID) also referred to as Non Syndromic Mental Retardation (NSMR).Cyclic adenosine 3CC2D1A mouse mutant cells, the absence of three of the four DM14 domains abolishes migration of the complex to the periphery and causes constitutive phosphorylation of PDE4D Serine 126 (S126) via the cAMP-dependent protein kinase A (PKA) resulting in PDE4D hyperactivity. Suppressing PDE4D activity with Rolipram in turn restores the down-stream phosphorylation of the “cAMP response element-binding protein” (CREB) that is defective in mouse mutant cells.Here we demonstrate that in Mouse Embryonic Fibroblasts (MEF) CC2D1A co-localizes with PDE4D in the cytosol before cAMP stimulation and on the periphery after stimulation, and that the movement to the periphery requires the full-length CC2D1A. In in vitro evidence we propose a model which links CC2D1A structure and function to cAMP homeostasis thereby affecting CREB phosphorylation. We speculate that CC2D1A and/or PDE4D may be promising targets for therapeutic interventions in many disorders with impaired PDE4D function such as NSID.Our findings suggest that CC2D1A is a novel regulator of PDE4D. CC2D1A interacts directly with PDE4D regulating its activity and thereby fine-tuning cAMP-dependent downstream signaling. Based on our  There2 sec–1) . In anim2 sec–1) . Upon st2 sec–1) . Activatet genes . Compartet genes . Tetheriet genes . A numbeet genes . Activatet genes ,16. ThisDrosophila melanogaster 14 (DM14) domains specific to this protein family with uncharacterized function(s) [133) phosphorylation [Recently, CC2D1A has been shown to be a novel regulator of the cAMP-PKA pathway . Amino action(s) . Mutant rylation .Interestingly, in NSID patients, the CC2D1A mutant protein has only the first three of the four DM14 domains and carriers have no physical defects but are intellectually disabled ,20, whilCC2D1A mutant cells and in brain tissue [in vitro pull-down experiments were performed  cells with forskolin, fixed them and co-stained them with anti-CC2D1A and anti-PDE4D antibodies. The results show that PDE4D and CC2D1A co-localize in the cytosol prior to stimulation and accumulate at the cell periphery after stimulation  causes activation [CC2D1A mutant MEF cells. When cells were stimulated with forskolin, lysed and western blotting was performed using anti-phospho-PDE4D and anti-PDE4D antibodies, we noted that the level of PDE4D phosphorylation was consistently increased in the mutant (n = 7) suggesting that PDE4D may be more active in the mutant even before stimulation which corresponds with CREB phosphorylation defect in the CC2D1A mutant cells on the same western blot  protein with PDE4D5 (IX) in the presence and absence of PKA. When CC2D1A was bound to PDE4D5 the activity was not affected by PKA suggesting that CC2D1A-binding PDE4D may prevent activation by PKA phosphorylation . The results show that PDE4D5 phosphorylation is dramatically reduced after binding to full-length CC2D1A (I)  displayed normal activity before and after activation by PKA  Figure A while P) Figure A and B. A Figure C and couA Figure C. In agrA Figure A, PKA doA Figure C. Althoun Figure C suggestn Figure C. The CCn Figure C.133) in CC2D1A mutant cells [CC2D1A mutant MEF cells with a PDE4 specific inhibitor Rolipram [133). Intriguingly, the results indicated that CREB (S133) phosphorylation in the CC2D1A mutant cells was restored to wt levels suggesting that PDE4-hyper activity in the mutant might be lowering the cAMP levels leading to defective PKA activity and thereby defective CREB (S133) phosphorylation , a reaction that is catalyzed by PKA. This hyper-phosphorylation leaves PDE4D constitutively activated and consequently disturbs cAMP homeostasis and cAMP-dependent downstream processes and notably CREB phosphorylation at (S133). The latter can be restored by the PDE4 inhibitor Rolipram suggesting that suppressing PDE4D activity may alleviate the effects of the defective phosphorylation of the PKA target CREB at (S133) in CC2D1A mutant cells. If CREB phosphorylation is disturbed, it is likely to lead to neural defects and abnormal brain development causing impairments in mental function [in vivo CC2D1A configuration prior to binding to the phospholipid. If this configuration is impaired it is likely to affect PDE4D5 regulation in vivo, and with it cellular cAMP homeostasis. However, the biological role and molecular mechanism of the fourth DM14 domain awaits further testing in vivo.While essential and sufficient for binding, the first DM14 of CC2D1A is not sufficient to confer wt function. Humans lacking the fourth DM14 domain of CC2D1A are intellectually disabled but have no other discernible symptoms suggesting that CC2D1A-dependent regulation is particularly critical in developing neural tissue. In turn, the mouse with a CC2D1A mutation that lacks all but the first DM14 has an even more severe phenotype causing death shortly after birth . The resfunction . The facfunction  is furthfunction . Given tfunction  at the m126) residue of PDE4D5 that in turn will be activated by the catalytic subunit of PKA that is released after cAMP activation , allowing PDE4D5 activation that then reduces cAMP levels and turns the signal “off”. We propose that CC2D1A regulates the cAMP/PKA pathway through fine-tuning the negative feedback loop that acts via PKA activation of PDE4D. When this pathway is defective, e.g. during neuronal development, it may lead to a state of compromised dendrite growth, synapse formation and neuronal survival [Here we propose a model Figure B that lisurvival  and thisCC2D1A mutant cells, we propose that CC2D1A has a role in preventing early PDE4D (hyper-) phosphorylation and activation which in turn would enable higher amplitude of cAMP and longer signal duration to exert the full function effects. Our finding revealed the uncharacterized function of the DM14 domain that, if proven in vivo, may be used in the future as a target for novel therapeutic agents in treatment of diseases caused by PDE4D5-linked disorders.Given that constitutive PDE4D phosphorylation at the PKA site leads to higher enzymatic activity in The findings presented here suggest that CC2D1A with its DM14 domains is a novel regulator of PDE4D and that specific and spatially correct binding of CC2D1A to PDE4D regulates the activity of the phosphodiesterase that is necessary for fine-tuning cAMP-dependent downstream signaling. We speculate that CC2D1A may be a promising target for therapeutic interventions in conditions with impaired PDE4D5 function such as NSID.E. coli DH5α and plated on agarose gel plates with Ampicillin for selection. For GST-fusion protein purification, overnight cultures of the pGEX-CC2D1A strains were grown in Luria-Bertani broth (LB) with 50 μg/ml of Ampicillin to an optical density of 0.45 at 600 nm. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM, and the mixture was placed in a shaker at 37°C for 4 h. Cells were pelleted and resuspended in 5 ml of phosphate-buffered saline (PBS) plus protease inhibitors  and the mixture was left on ice for 10 min. and then sonicated three times for 10 seconds each. Triton X-100 was then added to a final concentration of 0.1%, and the lysate was shaken gently at 4°C for 30 min. Cell debris was cleared by centrifugation at 15,000 rpm at 4°C for 30 min., and the supernatant was transferred to a new microcentrifuge tube with 100 μl of 5% slurry glutathione-conjugated beads. The lysate-GST bead slurry was incubated for 1 h at 4°C with gentle rocking. After that, the slurry was centrifuged at 1,000 rpm for 30 s, the supernatant was removed, and the beads were gently resuspended in 500 μl PBS. The centrifugation-resuspension was repeated three times to wash the beads free of most contaminating lysate proteins. The GST beads were then resuspended in 100 μl of PBS for further use. To cleave the GST tag from the proteins biotinylated Thrombin (Novagen) was used. First, 10 μl of thrombin was added -8000, 50% glycerol) to the 200 μl of the bead slurry (100 μl beads and 100 μl PBS), followed by 20 μl of 10X thrombin cleavage buffer  and the mixture was gently shaken at room temperature for 8 h. To capture the thrombin, 200 μl of streptavidin agarose beads  were added to each sample and incubated with gently shaking at room temperature for 30 min. After that, samples were centrifuged at 500 × g for 3 min. and the supernatant containing the cleaved protein was transferred to a new tube.A BamHI-EcoRI cDNA fragments encoding the proteins and protein fragments  for 5, 10, 15 and 30 min., washed in cold PBS and collected. For inhibition of PDE4 within cells, wild-type and CC2D1A mutant MEF cells were treated with 10 μM Rolipram for 20 min. then stimulated with 20 μM forskolin for 2, 7, and 30 min. Whole cell lysates (15 μg of protein) were separated by SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were incubated for 1 h at room temperature, or overnight at 4°C, in the corresponding primary and secondary antibodies membranes developed using the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences).Cells were cultured on poly-D-lysine-coated cover slips and after experimental treatments the cells were fixed with 4% paraformaldehyde in PBS for 10 min. at room temperature, washed 3 times with PBS, incubated for 20 min. at 37°C in PBS containing 5% goat serum, prior to adding the Anti-PDE4D and Anti-CC2D1A primary antibodies. Following three washes with PBS, the coverslips were incubated for 20 min. at 37°C with the Texas Red and Fluorescein isothiocyanate (FITC) conjugated secondary antibody . After three washes with PBS the cover slips were incubated for 5 min. on ice in PBS containing 50 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI), then washed three times with PBS and mounted on glass slides. Antibody signals were visualized with a Delta Vision de-convolution microscope.We obtained a GST-PDE4D5 plasmid from Graeme B. Bolger (University of Alabama Comprehensive Cancer Center). GST-PDE4D5 (VIII) protein was expressed and purified and cleaved as described above. The four GST-tagged CC2D1A  proteins that were previously purified on GST beads, and the purified PDE4D5 (IX) protein, were used as follows. The PDE4D5 (IX) (20 μg) was added to the 200 μl of the GST bead solution with bound GST-CC2D1A (I), GST-CC2D1A (II), GST-CC2D1A (III), or GST-CC2D1A (IV) separately. After incubation at 4°C for 4h, samples were centrifuged at 500 x g for 1 min. and the supernatant was removed. Washing with PBS and centrifugation were repeated three times and the PBS from the last wash was totally removed. Samples were boiled at 95°C with 30 μl of protein loading buffer for 5 min. To assess binding, 20 μl of each sample was loaded on an SDS-PAGE gel and duplicate western blots were made and stained with anti-PDE4D affinity-purified rabbit antibody , or anti-GST mouse monoclonal antibody (GenScript) separately.The wt and CC2D1A mutant Mouse Embryonic Fibroblasts (MEF) cells were stimulated with Forskolin, processed and the broken up by sonication. Phosphorylation of the GST-PDE4D5 recombinant protein was assessed after PKA treatment and exposure to ATP (Assay Designs). PDE4 activity was measured using the PDE4 Enzymatic Assay Kit (FabGennix) according to the manufacture’s protocol. (The methods applied here are further detailed in the Additional file ′,5′-monophosphate; CC2D1A: Coiled-coil and C2 domain-containing 1A; CREB: cAMP response element-binding protein; DM14: Drosophila melanogaster 14 protein; GST: Glutathione S-transferase; MEF: Mouse embryonic fibroblasts; NSID: Non syndromic intellectual disability; PDE: Phosphodiesterase; PKA: cAMP-dependent protein kinase A.ACs: Adenylate cyclases; AKAP: A kinase anchoring protein; cAMP: Cyclic adenosine 3Both authors declare that they have no competing interests.AA has designed and performed the experiments; AA and CG have interpreted the data, conceived the model and written the manuscript. Both authors read and approved the final manuscript.Materials and Methods.Click here for file


CK1δ, a member of the casein kinase 1 family, is involved in the regulation of various cellular processes and has been associated with the pathophysiology of neurodegenerative diseases and cancer. Therefore recently, interest in generating highly specific inhibitors for personalized therapy has increased enormously. However, the efficacy of newly developed inhibitors is affected by the phosphorylation state of CK1δ. Cellular kinases phosphorylating CK1δ within its C-terminal domain have been identified but still more information regarding the role of site-specific phosphorylation in modulating the activity of CK1δ is required. Here we show that Chk1 phosphorylates rat CK1δ at serine residues 328, 331, 370, and threonine residue 397 as well as the human CK1δ transcription variants 1 and 2. CK1δ mutant proteins bearing one, two or three mutations at these identified phosphorylation sites exhibited significant differences in their kinetic properties compared to wild-type CK1δ. Additionally, CK1δ co-precipitates with Chk1 from HT1080 cell extracts and activation of cellular Chk1 resulted in a significant decrease in cellular CK1δ kinase activity. Taken together, these data point towards a possible regulatory relationship between Chk1 and CK1δ. The casein kinase 1 (CK1) family of evolutionary conserved serine/threonine specific kinases is ubiquitously expressed in all eukaryotic organisms Deregulation as well as the occurrence of mutations within the coding region of CK1 family members seem to play important roles in the development of neurological disorders like sleeping disorders, bipolar I disorder, Parkinsonism-dementia complex of Guam and Alzheimer’s disease In the cell CK1 expression and activity is tightly regulated. Processes regulating CK1 activity include stimulation of cells with insulin, viral transformation, treatment of cells with topoisomerase inhibitors or γ-irradiation, all resulting in elevated CK1 activity in vitro and in vivoS370A mutant enhanced the formation of an ectopic dorsal axis during Xenopus laevis development in vitro. CK1δ mutant proteins bearing one, two or three mutations at the identified phosphorylation sites exhibit altered kinetic characteristics compared to wild-type CK1δ. Furthermore we show an interaction of CK1δ and Chk1 by co-immunoprecipitation analyses and that activation of cellular Chk1 by hydroxyurea results in a significant decrease in the activity of CK1δ. Therefore our data suggest a regulatory effect of Chk1 on the activity of CK1δ.Differences in the activity of CK1δ in different tissues and cell lines have been suggested to be partially caused by site-specific phosphorylation by cellular kinases. One of these kinases has been identified as PKA which phosphorylates CK1δ preferentially at Ser-370 First, RNA was extracted from the human fibrosarcoma cell line HT1080 Full length CK1δ and C-terminal fragments of rat CK1δ  and the SDM primer pairs as indicated in 1–64 (FP267) encoding amino acids 1–64 of wild-type mouse p53 has been described elsewhere 1–181 (FP1355), coding for amino acids 1–181 of wild-type mouse β-catenin was generated according to The construction of pGEX-p53The production and purification of glutathione S-transferase (GST) fusion proteins were carried out as described previously S345) was detected using the rabbit monoclonal anti-phospho-Chk1 (Ser-345) antibody 133D3 . Furthermore, the goat polyclonal anti-CK1δ antibody R-19 , the mouse monoclonal anti-CK1δ antibody 128A , and the mouse monoclonal anti-β-actin antibody AC-15  were used. Secondary horseraddish peroxidase (HRP) conjugated antibodies for Western blot analyses were supplied by GE Healthcare, USA  and Rockland Immunochemicals, USA .For immunoprecipitation and the detection of total Chk1 in Western blot a mouse monoclonal anti-Chk1 antibody  was used. Phosphorylated Chk1  using 2 µCi [γ-32P]-ATP per reaction. Recombinant human Chk1 , GST-CK1δ , or single fractions of fractionated HT1080 cell extracts were used as source of enzyme and either GST-p531–64 . Results are shown as normalized graphs and are representative for measurements at least performed in triplicate. Error bars indicate the standard error of the mean. Data were normalized towards appropriate control measurements. Statistical analyses including the determination of the kinetic parameters Vmax  and Km (Michaelis constant) as well as the 50% inhibitory concentration (IC50) for SB-218078 were performed using GraphPad Prism 5 .32P]-ATP per reaction and either 1 µg CHKtide peptide substrate  or 0.5 µg GST-p531–64 (FP267) were used as substrate. Reactions were incubated for 30 min at 30°C and reactions using CHKtide peptide substrate were stopped by absorption of the complete reaction volume on P81 filter paper. Papers were washed three times with 75 mM orthophosphoric acid and once in acetone. Dried papers were used for Cherenkov counting to quantify phosphate incorporation. Reactions containing FP267 were separated by SDS-PAGE and phosphate incorporation into FP267 was determined by Cherenkov counting. For co-incubation experiments activated, precipitated Chk1 was incubated with GST-CK1δ for 10 min at 30°C prior to addition of GST-β-catenin1–181  and another incubation step at 30°C for 30 min.Using 2 µg of specific antibodies Chk1 or CK1δ were precipitated from 250 µg of HT1080 protein extracts after cells were treated with hydroxyurea. The phosphatase inhibitors sodium fluoride (50 mM) and sodium orthovanadate (0.2 mM) were added to the lysis buffer to protect proteins from getting dephosphorylated. Antibody complexes were captured using 30 µl of protein G sepharose (50% (v/v) protein G sepharose in PBS; GE Healthcare, USA). Sepharose-bound complexes were washed three times with lysis buffer and once with kinase buffer without ATP. Determination of Chk1- or CK1δ-speicific kinase activity was carried out in kinase buffer using 2 µCi  labeled amino acids were visible after autoradiography.Phosphoamino acid analysis was performed as described earlier 305–428 bands were pooled and in-gel digested with trypsin and GluC, respectively, as described elsewhere Samples were loaded on a NuPAGE Bis-Tris 4%–12% (w/v) gradient gel . Then, Coomassie-stained GST-CK1δ10% of the digest was directly analyzed by LC-MS/MS, acetonitrile was added to 90% of the peptide mixture to a final concentration of 30% and the pH was adjusted to 2–3. Enrichment of phosphopeptides by titanium dioxide chromatography was done as described previously Escherichia coli database containing 4163 forward protein sequences, the sequence of the GST-CK1δ305–428 fragment, and 262 frequently observed contaminants. Trypsin and GluC, respectively, were set as proteases in which two missed cleavage sites were allowed. Beside acetylation at the N-terminus and oxidation of methionine, phosphorylation of serine, threonine, and tyrosine were set as variable modifications. Carbamidomethylation of cysteine was set as fixed modification. Initial precursor mass tolerance was set to 7 parts per million (ppm) at the precursor ion and 0.5 Da at the fragment ion level. Spectra of modified peptides were manually validated. MASCOT scores are given for probabilistic evaluation of mass spectrometric results. Total obtained sequence coverage was 47.6%.Analyses of the peptides were done on a Proxeon Easy-LC system  coupled to a LTQ-Orbitrap-XL  equipped with a nanoelectrospray ion source  as described previously HT1080 cells For immunoprecipitation 250 µg whole cell protein extract was diluted to a final volume of 500 µl and incubated with 2 µg of Chk1-specific antibody or control serum overnight under slow rotation at 4°C. Antibodies were captured with 40 µl protein G sepharose (50% (v/v) protein G sepharose in PBS) under continued rotation at 4°C for 5 hours. Immunocomplexes were collected by pulse centrifugation and washed three times with 250 µl NP40 lysis buffer. Finally, collected complexes were mixed with 2× SDS sample buffer and incubated for 10 min at 37°C and 750 rpm in a thermal mixer. All samples were pulse-centrifuged, the supernatant was used for SDS-PAGE and subsequent Western blotting.S345, CK1δ, and β-actin membranes were probed with appropriate antibodies overnight. Immunocomplexes were detected using peroxidase conjugated anti-mouse or anti-rabbit IgG and chemiluminescence detection.Cell lysates used for Western blot analyses were prepared in NP40 lysis buffer. 80 µg of cleared extracts were separated on 12.5% (v/v) gels in SDS-PAGE and transferred to a PVDF membrane . For immunodetection of Chk1, pChk1For subsequent fractionation cells were lysed in sucrose lysis buffer. Cell lysates were passed through a 0.4 µm filter and equal amounts of total protein were applied to an anion exchange column  attached to an Ettan LC purifier . Bound proteins were eluted with a linear ascending gradient between 0 and 1000 mM NaCl in 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 5% (v/v) glycerol, 0.04% (v/v) Brij, 1 mM benzamidine, 25 µg/ml aprotinin, and 0.1% (v/v) β-mercaptoethanol.305–428 (FP1181), which was phosphorylated by Chk1 in vitro, revealed that CK1δ can be phosphorylated by Chk1 at positions Ser-328, Thr-329, Ser-331, Ser-361, and Ser-382 , GST-CK1δ353–375 (FP1022), and GST-CK1δ375–428 (FP1183) as well as corresponding GST-mutant CK1δ proteins. Expectedly, one phosphopeptide is missing in the phosphopeptide analysis of GST-CK1δ305–375 S328A (FP1269) compared to that of GST-CK1δ305–375 (FP1006) (305–375 T329A (FP1344), GST-CK1δ305–375 S331A (FP1340), and GST-CK1δ305–375 S361A (FP1295) (data not shown). Phosphorylation of Ser-370 could clearly be verified by phosphopeptide analysis of GST-CK1δ353–375 S370A (FP1021) which compared to GST-CK1δ353–375 (FP1022) is lacking one phosphopeptide most probably due to mutation of Ser-370 in FP1021 (375–428 T397A (FP1221) with that of GST-CK1δ375–428 (FP1183) revealed a weakened signal at the indicated position, thereby confirming that Thr-397 can be phosphorylated by Chk1 in vitro (375–428 S382A (FP1347), GST-CK1δ375–428 T392A (FP1322), and GST-CK1δ375–428 T427A (FP1262) could not give any new information compared to GST-CK1δ375–428 (data not shown).Additionally, the significance of these findings was improved by phosphopeptide analyses using the GST-wt CK1δ fusion proteins GST-CK1δ(FP1006) . Phospho(FP1006) . No new n FP1021 . Compariin vitro . Phosphoin vitro.In conclusion, combined data of mass spectrometric and classical biochemical approaches provide evidence for the CK1δ C-terminal residues Ser-328, Ser-331, Ser-370, and Thr-397 to be targeted by Chk1 The GST-CK1δ fusion proteins used in the previous section all originate from the rat CK1δ sequence FP449, . Howevermax) and Km values of all analyzed phosphorylation-site mutants increased compared to those of wild-type GST-CK1δ. GST-CK1δS370A and GST-CK1δS328A, S370A, T397A presented the highest Km values in this test , indicating lowest substrate affinity. Among the tested single-site mutants the most significant change in Vmax could be obtained for GST-CK1δS370A.In order to investigate the importance of the so far identified target sites for Chk1 within the C-terminal domain of CK1δ, residues in GST-CK1δ (FP449) which have been identified as targets for Chk1-mediated phosphorylation were exchanged to alanine by site-directed mutagenesis and the kinetic parameters of wild-type CK1δ and the resulting CK1δ single and double phosphorylation-site mutants or the triple mutant were experimentally determined using α-casein as substrate. Data shown in 1–181 (FP1355). Chk1(IP) alone shows no notable phosphorylation of this substrate. In contrast, GST-β-catenin1–181 can be intensely phosphorylated by GST-CK1δ. When GST-CK1δ is pre-incubated with Chk1(IP) the GST-CK1δ-mediated phosphorylation of GST-β-catenin1–181 is markedly decreased by about 30% ) prior to addition of the non-canonical CK1δ substrate GST-β-cateninbout 30% . However1781–64 (FP267) as substrate and GST-wt CK1δ or GST-CK1δS328A, S370A, T397A as enzymes. Both wild-type and mutant GST-CK1δ were inhibited to a similar extend by compounds 17 and 8. However, slightly stronger inhibitory effects for both small molecules could be detected for GST-CK1δS328A, S370A, T397A , or compound 8 (20 nM) using GST-p531–64 (FP267) as substrate and GST-wt CK1δ alone or in combination with Chk1 as enzymes. Whereas Chk1-mediated phosphorylation of CK1δ significantly enhanced the ability of compound 17 to inhibit CK1δ kinase activity, the efficacy of D4476 was decreased in reactions containing Chk1 and CK1δ. The ability of compound 8 to inhibit CK1δ was only modestly affected in the presence of Chk1 . According to Walker and co-workers In order to analyze the relevance of the Chk1/CK1δ interplay in cultured cells, the fibrosarcoma cell line HT1080 was treated with hydroxyurea (HU) to activate Chk1 S345 level after four hours of treatment  as substrate for in vitro kinase reactions. Interestingly, after two hours of HU treatment CK1-specific kinase activity dramatically decreased by more than 60% compared to that of control cells  peptides resulting from tryptic digestion  are too small to be resolved by Nano LC-MS/MS, (ii) GST-CK1δ305–428 used as substrate was incompletely phosphorylated, or (iii) loss of phosphorylation at these sites occurred during sample processing. Additional analysis of phosphopeptides derived from GluC digestion of GST-CK1δ305–428 also failed in confirming phosphorylation of Ser-370 and Thr-397 but revealed that aa 340 to 366 did not contain any modified residue. This is in line with our phosphorylation analyses showing that Chk1 does not phosphorylate any amino acid of CK1δ between Ser-332 and Ser-369.The use of state of the art mass spectrometry, CK1δ phosphorylation site mutants, and classical biochemical methods revealed that Chk1 phosphorylates Ser-328, Ser-331, Ser-370, and Thr-397 within the C-terminal domain of rat CK1δ. These findings highlight the central role of Ser-370, which can be phosphorylated by a number of cellular kinases, including PKA, AKT, Clk2, and PKC-α in vivo phosphorylation of CK1δ at Ser-370, a site clearly identified as target for several cellular kinases using classical methods  was compared to GST-CK1δ305–375 (FP1006) (data not shown). Therefore this site only was considered as phosphorylation target with pending validation.Although Ser-328 could be identified as a target for Chk1 by all of the employed experimental approaches, the entirety of in vitro data for rat CK1δ can easily be applied to the human CK1δ TV1 and 2 as well. Beyond aa 399 the primary sequences of human CK1δ TV1 and 2 differ significantly from the rat sequence but also contain residues, which can be phosphorylated by cellular kinases 375–415 TV1 and GST-CK1δ375–409 TV2.We furthermore provide evidence that in addition to rat CK1δ also human CK1δ TV1 and 2 are phosphorylated by Chk1. Up to aa 399 all three proteins are identical and our in vitro, the kinetic constants of the phosphorylation of α-casein by GST-CK1δS328A, GST-CK1δS370A, GST-CK1δT397A, GST-CK1δS328A, S370A, GST-CK1δS328A, T397A, and GST-CK1δS328A, S370A, T397A were calculated experimentally. Especially GST-CK1δS370A and the tested multi-site mutants  differed remarkably in their kinetic parameters compared to those of wild-type CK1δ. The maximum catalytic velocity, as expressed by Vmax, is nearly doubled for these mutants. However, the Km values are also increasing, indicating decreased substrate affinity for these CK1δ mutants. These findings illustrate a major influence of Ser-370 and Thr-397 on CK1δ activity. The potential phosphosite Ser-331 was not taken into account in these experiments since its exact role has not finally been validated yet.In order to examine the importance of Ser-328, Ser-370, and Thr-397 for the catalytic activity of CK1δ 1–1811–181 in contrast to GST-p531–64 cannot be phosphorylated by Chk1 and the use of activated Chk1 furthermore allows to compare this result to previously published data generated using the active subunit of PKA In a first approach to establish a direct functional connection between Chk1 and CK1δ, phosphorylation of the non-canonical substrate GST-β-cateninIn further analyses we demonstrate that mutation of Chk1 target sites in GST-CK1δ as well as co-incubation of recombinant Chk1 and GST-CK1δ results in modification of the effects of CK1-specific inhibitors. These differences are specific for the inhibition of CK1δ because selectivity screening approaches revealed that the tested classes of inhibitors did not show any effects on the activity of Chk1 in vivo. Actually, CK1δ activity in HT1080 cells dramatically decreases after hydroxyurea treatment, which induces the depletion of dNTP pools resulting in S-phase arrest and Chk1 activation in vitro, which may falsify the presented result. Without this effect SB-218078 may potentially lead to complete reversal of the HU-induced effects.In addition we provide evidence that Chk1 influences CK1δ activity in the cellular context. Results from co-immunoprecipitation and co-incubation experiments lead to the assumption, that a regulatory interaction between CK1δ and Chk1 might also be observed in vitro, are also phosphorylated in vivo and that phosphorylation of these sites can be observed in response to cell cycle related signaling Our cell culture results are further supported by recent phosphorylation analyses using large-scale mass spectrometry approaches showing that Ser-328, Ser-331, and Thr-397, which can be phosphorylated by Chk1 A tight physiological interaction between Chk1 and CK1 isoforms is furthermore indicated by the fact that both kinases are implicated in regulation of DNA damage response and replication by modulating the activity of various regulatory proteins by site-specific phosphorylation in concert. For example, phosphorylation of Cdc25A (cell division cycle 25 A) by both, CK1 (isoforms α and ε) and Chk1, contributes to Cdc25A degradation and precise control of the cell cycle in vitro and most likely also within cellular signal transduction regulation. However, detailed information about this interaction remains to be determined by future work.In summary, we clearly identified phosphorylation sites within the C-terminal domain of CK1δ which can be targeted by Chk1. Furthermore, our results show for the first time that site-specific phosphorylation of CK1δ by Chk1 plays an important role in modulating the activity of CK1δ Figure S1Chk1- and CK1δ-specific kinase activity in HT1080 cells is altered after treatment with hydroxyurea. Cellular Chk1 was activated by treating HT1080 cells with 2.5 mM hydroxyurea (HU) for the indicated periods of time. Immunoprecipitated Chk1 and CK1δ were used to phosphorylate CHKtide substrate peptide (Chk1) or GST-p531–64 . Substrate phosphorylation was quantified by Cherenkov counting. Data are presented as normalized bar graph.(TIF)Click here for additional data file.Figure S2CK1δ is inhibited by SB-218078 in vitro. Kinase activity of rat CK1δ (FP449) was assayed in presence of increasing concentrations of the Chk1-specific inhibitor SB-218078. Phosphorylation intensity of the substrate GST-p531–64 (FP267) was quantified by Cherenkov counting. Dose-response data were processed using GraphPad Prism 5.(TIF)Click here for additional data file.


Recent analyses have shown that significant changes have occurred in patterns of sea ice seasonality in West Antarctica since 1979, with wide-ranging climatic, biological and biogeochemical consequences. Here, we provide the first detailed report on long-term change and variability in annual timings of sea ice advance, retreat and resultant ice season duration in East Antarctica. These were calculated from satellite-derived ice concentration data for the period 1979/80 to 2009/10. The pattern of change in sea ice seasonality off East Antarctica comprises mixed signals on regional to local scales, with pockets of strongly positive and negative trends occurring in near juxtaposition in certain regions e.g., Prydz Bay. This pattern strongly reflects change and variability in different elements of the marine “icescape”, including fast ice, polynyas and the marginal ice zone. A trend towards shorter sea-ice duration (of 1 to 3 days per annum) occurs in fairly isolated pockets in the outer pack from∼95–110°E, and in various near-coastal areas that include an area of particularly strong and persistent change near Australia's Davis Station and between the Amery and West Ice Shelves. These areas are largely associated with coastal polynyas that are important as sites of enhanced sea ice production/melt. Areas of positive trend in ice season duration are more extensive, and include an extensive zone from 160–170°E  and the near-coastal zone between 40–100°E. The East Antarctic pattern is considerably more complex than the well-documented trends in West Antarctica e.g., in the Antarctic Peninsula-Bellingshausen Sea and western Ross Sea sectors. Better identification, quantification and understanding of change and variability in global sea ice coverage are increasingly recognised as a high priority in climate and ecological research. Sea ice plays a crucially-important role as a key modulator of the Earth's climate system, a sensitive bellwether of climate variability/change  and the Antarctic Peninsula and Bellingshausen Sea region (>3 month lengthening of open water conditions). The magnitude of the latter is in fact even greater than the loss that has occurred in regions of greatest decline in the Arctic i.e., the western Beaufort, East Siberian and Chukchi seas By contrast, relatively little is known about patterns of change and variability in sea ice seasonality across East Antarctica, where sea ice coverage is strongly (though not exclusively) seasonal and occurs in a relatively narrow yet complex zone. This represents a major knowledge gap, and one that has severely compromised our ability to gauge and understand circum-Antarctic climate change and variability and their biological and physical impacts. The crucial need for improved knowledge of change and variability in East Antarctic sea ice seasonality is underpinned by the fact that seasonal loss, or alternatively gain, of sea ice in this region can have global consequences, given that the coupling between sea ice, oceanic and atmospheric circulation and temperature and biogeochemical cycles can result in positive feedbacks that drive climate change 2 and thus potentially ocean acidification Biological ramifications of change in East Antarctic sea ice seasonality are also likely to be profound http://nsidc.org). The dataset used is the NASA Bootstrap SMMR-SSM/I combined dataset (http://nsidc.org/data/docs/daac/nsidc0079_bootstrap_seaice.gd.html), which offers complete coverage of the Antarctic sea ice zone on a daily basis after July 1987 and every other day prior to that (back to October 1978), at a spatial resolution of 25×25 km. Following Maps of annual days of advance and retreat, and resultant ice season duration, for the sector 30–170°E were computed from daily sea ice concentration data obtained from the US National Snow and Ice Data Centre , anomalies in advance, retreat and season length were calculated relative to the long-term means for 1979/80–2009/10. Variability was also assessed more generally  in terms of the standard deviation in day of advance/retreat and ice season duration.Analysis of change and variability in ice conditions (concentration) along the 110°E and 140°E meridian. These were chosen in order to examine seasonality variability within a given ice regime, and as they are long-term biological monitoring transects for Australia and Japan i.e. to provide physical input to studies analysing biological change and variability. Two transects at 90°E and 100°E were also examined for comparison, in that they intersected regions that exhibited different sea ice seasonality patterns. Taken togetherCalculation of trends in sea ice seasonality over the entire time series (1979/80–2009/10). These form the basis of identification and detailed analysis of spatio-temporal patterns of sea ice change, including identification of sea ice-mediated “hot-spots” , on the assumption that the trends are linear.Information on major features of oceanic circulation across East Antarctica, for comparison with observed climatological (mean) patterns of sea ice seasonality derived by this study, was obtained from Unless otherwise specified, “sea ice” refers here to the moving pack ice and stationary landfast sea ice (fast ice) combined. Fast ice forms a narrow zone  around the Antarctic coastal margin, where its distribution is associated with (and is governed by) coastal promontories, sheltered embayments and groups of icebergs grounded in waters shallower than approximately 350–400 m To provide context (the background setting) for interpretation of observed change and variability presented in subsequent sections, we first present mean patterns of annual sea ice advance, retreat and duration for the period 1979/80–2009/10 in The maps presented in As depicted in Based on the spatial characteristics of sea ice seasonality in As shown in previous studies e.g., , the disTo the west of∼70°E, the mean pattern of sea ice advance is roughly from southeast to northwest for the first half of the year, after which time it becomes more zonal. Comparison of In contrast, mean advance in the region east of Kerguelen Plateau (from∼90 to∼145°E) is largely near-zonal early in the growth season, trending more from SE to NW as the season progresses. Rates of advance are proportionately similar to the SE Indian Ocean zone relative to the overall width of the sea ice zone at maximum extent, which is very narrow at these longitudes. The narrowness of the seasonal sea ice zone (zone of advance and retreat) in the region 115–145°E  is the rClimatological patterns of sea ice advance east of 145°E are distinctively different, but again mirror the geographical setting. Sea ice advance in this sector is multi-directional across a bulge centred on∼160°E. This appears to relate to strong topographic influence on ocean circulation, including a northward deflection of the ACC at∼140°E, and ice build-up of the SE Indian Ridge and Balleny Islands . MoreoveAlthough the relative rapidity of overall Antarctic sea ice retreat in late spring–summer is well known The mean patterns of ice season duration shown in Correlation maps of mean  ice season duration versus annual advance and retreat for the 31-year time series are shown in Regions of strong polynya activity are also where correlations between retreat and duration  are highA fundamental feature of East Antarctic sea ice seasonality is large year-to-year variability. This is illustrated in In The high degree of year-to-year variability is also reflected in Large seasonal differences between years are also apparent along the 140°E meridian, which depicts a significantly different ice regime , right. Variability was investigated by mapping standard deviations (in days) of annual days of advance and retreat and ice season duration, and results are shown in Maps of trends in annual timings of East Antarctic sea ice advance, retreat, and duration are shown in In the outer half of the pack between∼60°E and 110°E, a general pattern of later advance and earlier retreat results in a general overall shortening of the ice season by 1 to 2 days per year, but this trend only reaches significance in the area between∼95–110°E. Along the coast there are other localized areas of change as well (in addition to the “hot spots” mentioned above). Ice season shortening is apparent between∼90°E and 150°E: to the east and west of the Mertz Glacier tongue (∼148°E), to the west of the Dibble Iceberg Tongue (∼135°E), in a corridor from 120–130°E), and adjacent to parts of the Shackleton Ice Shelf (centred on∼100°E). These areas correspond to locations of recurrent coastal polynyas Further insight into the regional variability in observed trends in East Antarctic sea ice seasonality comes from analysis of coincident trends in daily ice concentration along four meridional transects that cut across different regimes as indicated in the trend maps shown in Along the 110°E transect, there is a contrasting decrease in sea ice concentration in the sea ice edge zone during winter maximum, albeit rather patchy . This isThe pattern of trends along 100°E  shows inAlthough attribution is beyond the scope of this paper, we next carried out an initial analysis of possible relationships between trends in sea ice seasonality and sea surface temperatures (SSTs). Monthly SST trends (1982–2010) equatorward of the sea ice zone are shown in Broadly speaking, mean patterns in sea ice seasonality off East Antarctica can be split into three different regimes: west of∼90°E,∼90–145°E, and east of∼145°E, although boundaries are somewhat indistinct. Within each regime, net patterns of seasonality are strongly related to patterns of oceanic circulation, which are in turn linked to bathymetry. These regional differences in seasonality in fact correspond to satellite-derived patterns of mean sea ice drift and ocean currents presented in In the SE Indian Ocean sector (30–90°E), rapid and extensive annual sea ice advance  is largeMoving east, seasonal change in the relatively narrow sea ice zone from 90–150°E) reflects not only the patterns of ocean currents linked to bathymetry, but also the delicate, seasonally-varying balance between sea ice dynamics and thermodynamics. Mean patterns of ice edge advance observed across this sector in Finally and in the sector east of 145°E, the pattern of ice edge advance shown in As shown in this study, the pattern of change in sea ice seasonality across the East Antarctic sector is considerably more complex than the well-documented trends from West Antarctica shown by In East Antarctica, regions of significant shortening of the sea ice season, by 1 to 3 days per year over the 31-year period, are limited to relatively small pockets along the ice sheet coastal margin between∼75°E and 150°E and a more extensive sector of the outer pack offshore from Wilkes Land (between 95°E and 110°E) . The latAlthough geographically limited, “hot spots” of negative trends in sea ice seasonality along the coast observed between∼75–150°E are of considerable interest for a number of reasons. For example, the near-coastal “hot spots” appear to coincide with the location of certain important coastal polynyas e.g., the Barrier Polynya in eastern Prydz Bay and the Mertz Glacier Polynya centred on∼145°E. Such polynyas are important as sites of enhanced sea ice production By contrast, areas of positive trend in sea ice duration are more extensive in East Antarctica than areas of negative duration for the overall (31-year) period, and appear to be associated with both earlier advance and later retreat. In addition to the extensive area of lengthening in the Western Ross Sea regime, other key regions of ice season lengthening occur in the near-coastal zone to the west of∼105°E and particularly between∼40°E and 90°E, in the vicinity of Cape Ann. This change may relate to change in behaviour of the Cosmonaut Polynya, an offshore sensible heat polynya that has an average location centred on 56°E and 65°S In terms of annual sea ice advance and retreat, there is a predominant trend towards earlier sea ice advance through much of the central pack ice region from 30–150°E, although this is statistically significant in only a few localized pockets . The outSome insight into the regional variability in observed trends in East sea ice seasonality in the W Pacific sector comes from statistically-significant trends towards increasing mid-pack sea ice concentration along 110°E and 140°E shown in The broader trend towards later sea ice advance and earlier sea ice retreat in the outer pack is consistent with an observed trend for increased SSTs to the north of the ice edge from August to October, which would inhibit maximum winter ice edge extent and initiate early retreat (melt back). This area of late advance was also recently highlighted in a study describing wind-driven trends in Antarctic sea ice drift Where there is shown to be a decrease in SSTs to the north of the ice edge (at around 40°E for example), there is an earlier advance and a later retreat  and 8. TThe apparently close relationship between “hotspots” of changing sea ice seasonality and polynyas noted above points to the importance of accounting for change and variability in the different elements comprising the sea ice zone, and their interaction. This steps beyond simply treating the sea ice zone as an amorphous mass that is delineated by an ice edge. These “icescape elements” include the outer pack or marginal ice zone, the inner pack and the near-coastal zone including areas of fast ice and polynyas  Complex patterns of change and variability in sea ice seasonality are observed on the continental shelf and within the Antarctic Coastal Current. Here, sea ice distribution and characteristics are strongly affected by interaction with the ice sheet margin and icebergs Within this coastal zone, the largest variability in days of retreat and ice season duration coincides with polynya locations. The Mertz Glacier polynya regime, for example, has a dominant effect on regional sea ice retreat and its variability around 140–155°E . As showIn contrast to polynyas, areas of low sea ice variability in the coastal zone correspond to regions of fast ice, particularly during the retreat phase . InformaIn general, regions of thick consolidated sea ice occurring in regions of net convergence  or fast ice in more sheltered locations have a higher likelihood of surviving longer than regions of predominantly diffuse and thinner ice e.g. within the marginal ice zone. This is illustrated by the occurrence off the George V and Oates Land coasts of the largest area of multi-year sea ice in East Antarctica . Persist2. Another important offshore element is the so-called West Ice Shelf Tongue (WIST), a prominent meridionally-trending sea ice tongue which recurs and persists annually in the vicinity of 85°E The WIST illustrates the influence of complex interactions between large-scale ocean and atmosphere circulation patterns on the seasonal development and maintenance of the East Antarctic sea ice cover. Sea ice advance, retreat and concentration are affected not only by thermodynamic but also by dynamic processes


Abnormal hyperphosphorylation of microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD). The aggregation of hyperphosphorylated tau into neurofibrillary tangles is also a hallmark brain lesion of AD. Tau phosphorylation is regulated by tau kinases, tau phosphatases, and O-GlcNAcylation, a posttranslational modification of proteins on the serine or threonine residues with β-N-acetylglucosamine (GlcNAc). O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase, the enzyme catalyzing the transfer of GlcNAc to proteins, and N-acetylglucosaminidase (OGA), the enzyme catalyzing the removal of GlcNAc from proteins. Thiamet-G is a recently synthesized potent OGA inhibitor, and initial studies suggest it can influence O-GlcNAc levels in the brain, allowing OGA inhibition to be a potential route to altering disease progression in AD. In this study, we injected thiamet-G into the lateral ventricle of mice to increase O-GlcNAcylation of proteins and investigated the resulting effects on site-specific tau phosphorylation. We found that acute thiamet-G treatment led to a decrease in tau phosphorylation at Thr181, Thr212, Ser214, Ser262/Ser356, Ser404 and Ser409, and an increase in tau phosphorylation at Ser199, Ser202, Ser396 and Ser422 in the mouse brain. Investigation of the major tau kinases showed that acute delivery of a high dose of thiamet-G into the brain also led to a marked activation of glycogen synthase kinase-3β (GSK-3β), possibly as a consequence of down-regulation of its upstream regulating kinase, AKT. However, the elevation of tau phosphorylation at the sites above was not observed and GSK-3β was not activated in cultured adult hippocampal progenitor cells or in PC12 cells after thiamet-G treatment. These results suggest that acute high-dose thiamet-G injection can not only directly antagonize tau phosphorylation, but also stimulate GSK-3β activity, with the downstream consequence being site-specific, bi-directional regulation of tau phosphorylation in the mammalian brain. Microtubule-associated protein tau is a cytosolic protein that stimulates microtubule assembly and stabilizes microtubule structure. The integrity of the microtubule system is essential for the transport of materials between the cell body and synaptic terminals of neurons. The microtubule system is disrupted and replaced by the accumulation of highly phosphorylated tau as neurofibrillary tangles in affected neurons in the brains of individuals with Alzheimer disease (AD) and other neurodegenerative disorders collectively called tauopathies. Neurofibrillary tangles are also one of the hallmark histopathological lesions of AD brain. Many studies have demonstrated the critical role of hyperphosphorylation and aggregation of tau in neurodegeneration in AD and other tauopathies. The abnormal hyperphosphorylation may cause dissociation of tau from microtubules and, consequently, raise intracellular tau concentration enough to initiate its polymerization into neurofibrillary tangles The mechanisms by which tau becomes abnormally hyperphosphorylated in AD and other tauopathies are not well understood. Many studies have demonstrated that in the brain, tau phosphorylation is mainly controlled by the kinases glycogen synthase kinase-3β (GSK-3β) and cyclin-dependent protein kinase 5 (cdk5) It was demonstrated recently that tau phosphorylation is negatively regulated by O-GlcNAcylation, a posttranslational modification of proteins with β-N-acetylglucosamine (GlcNAc) In this study, we injected a highly selective OGA inhibitor, thiamet-G, into the lateral ventricle of mice to increase O-GlcNAcylation of proteins and investigated alterations of site-specific tau phosphorylation. We found that acute high-dose thiamet-G treatment led to decreased phosphorylation at some sites but increased phosphorylation at other sites of tau in the brain. We further investigated possible underlying mechanisms for these differential effects.The primary antibodies used in this study are listed in 441, were used in this study. The transgenic mice Thirty transgenic (Tg) mice  that express the largest isoform of wild-type human tau, tauMice were first anesthetized by an intraperitoneal injection of 0.3% sodium pentobarbital (40 mg/kg). Thiamet-G (2.5 µl of 35 µg/µl) dissolved in 0.9% NaCl was injected bilaterally into the lateral ventricles of the brains at a dose of 175 µg/mouse, which correlated to a final concentration of approximately 700 µM in the brain, assuming a combined brain and spinal cord volume of 900 µL. The same volume of 0.9% NaCl alone was injected in control mice. The stereotaxic coordinates for the icv injection were: 0.4 mm anterior to posterior Bregma, 1.0 mm mid to lateral, and 2.2 mm dorsal to ventral dura. Mice were euthanized by cervical dislocation 4.5 h, 9 h or 24 h after icv injection . Anesthet-test, and the differences between groups were regarded to be significant when p<0.05.The left hemispheres of the mouse brains were homogenized in pre-chilled buffer containing 50 mM tris-HCl (pH7.4), 8.5% sucrose, 10 mM β- mercaptoethanol, 1.0 mM EGTA, 1.0 mM phenyl-methylsulfonyl fluoride, 2.0 µg/ml aprotinin, 25 µg/ml leupeptin, and 1.0 µg/ml pepstatin. Protein concentrations of the homogenates were determined by modified Lowry method 2 atmosphere, as described previously 3VO4, 1.0 mM phenyl-methylsulfonyl fluoride, 2.0 µg/ml aprotinin, 25 µg/ml leupeptin and 1.0 µg/ml pepstatin.Neuronal progenitor cells were isolated from the hippocampus of adult Wistar rats  and cultured in neurobasal A medium consisting of 2% B27, 0.5 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10 ng/ml fibroblast growth factor-2 (FGF-2) at 37°C in a humidified 5% CODifferentiated AHP cells were also used in this study. The AHP cells were differentiated by replacing FGF-2 with 5 µM retinoic acid and 10% fetal calf serum when the cells reached approximately 80% confluency. The neuronal cultures were treated with 20 nM thiamet-G after 5–7 days of growth in the differentiation medium. At this stage, approximately 80% of the cells expressed the neuronal markers βIII-tubulin and microtubule-associated protein 2, and less than 5% expressed the astroglial marker Glial fibrillary acidic protein or the oligodendrocyte marker O4 (data not shown).441, were generated as described PC12 cells that stably express the largest isoform of human brain tau, taup<0.05 was considered to be statistically significant.Where appropriate, the data are presented as mean ± SEM. One-way ANOVA and post hoc analyses were performed using the scientific statistic software Graph Pad Prism 4 . To investigate the effects of thiamet-G on tau phosphorylation at multiple phosphorylation sites in vivo, we first confirmed the elevation of protein O-GlcNAcylation level of the brains of tau Tg mice after icv injection of the drug. We observed a time-dependent elevation of O-GlcNAcylation after thiamet-G treatment, as determined by Western blots developed with monoclonal antibody RL2 toward O-GlcNAc . A markeWe then studied alterations of tau phosphorylation at individual phosphorylation sites in the mouse brains after treatment with thiamet-G by using Western blots developed with a battery of phosphorylation-dependent and site-specific tau antibodies. Antibody 92e toward total tau in a phosphorylation-independent manner was also included to check whether there was any change in the total tau level. We found that thiamet-G did not alter tau level in the mouse brain, but led to site-specific, bi-directional changes of tau phosphorylation . Along wNegative regulation of phosphorylation by O-GlcNAcylation of tau has been observed previously CDK5 is the second most important tau kinase in the brain To elucidate the complex regulation of site-specific phosphorylation induced by thiamet-G, we employed cultured cells, because cell cultures are more easily manipulated. We first selected AHP cells because they are more close to brain neurons than tumor cell lines. As expected, treatment of AHP cells with 20 nM thiamet-G increased protein O-GlcNAcylation . While wTo learn whether the phenomena we observed in undifferentiated AHP cells were specific to these cells, we also performed similar experiments in differentiated AHP cells and differentiated PC12 cells. As seen in proliferating AHP cells, we did not observe any marked elevation of tau phosphorylation at any phosphorylation sites or changes of tau kinases upon thiamet-G treatments in these two types of cells (data not shown).Thiamet-G is a highly specific OGA inhibitor that was synthesized based on rationale design We and other groups have previously shown that O-GlcNAcylation regulates phosphorylation of tau in a reciprocal manner overall Our results indicated marked differences in the effects of thiamet-G on tau phosphorylation between the mouse brains and the cultured neurons. Further experiments indicated that thiamet-G–induced increase of tau phosphorylation at several sites resulted from activation of GSK-3β, a major tau kinase, but this activation did not occur in cultured cells. Different regulations of tau phosphorylation by extracellular signaling between the brains and the cultured neurons might also contribute to the different results we observed. A previous study has demonstrated that tau phosphorylation is regulated by FGF-2 through GSK-3β Our studies on the upstream regulating kinases of GSK-3β suggest that thiamet-G led to marked GSK-3β activation as the result of an inhibition of AKT by reducing its phosphorylation at Ser473 and Thr 308, which regulates its activity positively Tau is abnormally hyperphosphorylated and aggregated in AD and other tauopathies. Previous studies from our and other groups have demonstrated differential roles of tau phosphorylation at various phosphorylation sites. A quantitative in vitro study demonstrated that phosphorylation of tau at Ser262, Thr231, and Ser235 inhibits its binding to microtubules by ∼35%, ∼25%, and ∼10%, respectively In conclusion, thiamet-G is a specific OGA inhibitor and is very effective in elevating protein O-GlcNAcylation level in the mammalian brain. Because thiamet-G not only directly modulated tau phosphorylation inversely, but also stimulated GSK-3β activity likely via inhibition of AKT, the final consequences were a decrease in tau phosphorylation at Thr181, Thr212, Ser214, Ser262/Ser356, Ser404 and Ser409, and an increased in tau phosphorylation at Ser199, Ser202, Ser396 and Ser422 in the brain after thiamet-G treatment. The site-specific, bi-directional regulation of tau phosphorylation warrants further studies on evaluation of dose and time dependent effects on OGA inhibition.


The target of rapamycin (TOR) pathway regulates cell growth and aging in various organisms. In Saccharomyces cerevisiae, silent information regulator 2 (Sir2) modulates cellular senescence. Moreover, Sir2 plays a crucial role in promoting ribosomal DNA (rDNA) stability and longevity under TOR inhibition. Here we review the implication of rDNA stabilizers in longevity, discuss how Sir2 stabilizes rDNA under TOR inhibition and speculate on the link between sumoylation and Sir2-related pro-aging pathways. Saccharomyces cerevisiae provides valuable information on understanding aging process and identifying the molecular mechanisms that regulate longevity in eukaryotic organisms. Two types of aging, replicative and chronological, have been described in S. cerevisiae. Replicative aging is an aging model of mitotically active cells in which the lifespan of a mother cell is measured by the number of daughter cells produced before death [S. cerevisiae, a well-established mechanism for regulating longevity is the alteration of ribosomal DNA (rDNA) stability by the recombination between rDNA repeats and the accumulation of extrachromosomal rDNA circles (ERCs) [S. cerevisiae [. In this research perspective, we review the implication of rDNA stabilizers in longevity regulation, discuss how Sir2 stabilizes rDNA under TOR inhibition and speculate on the link between sumoylation and Sir2-related aging pathway.Aging and longevity have always been an important issue in biological sciences. Several reports have indicated that calorie restriction leads to lifespan extension from yeast to humans -3. Longere death , 15. Chrre death . In S. cs (ERCs) . Recentlrevisiae . In this+-dependent deacetylases that are involved in the regulation of lifespan from yeast to humans [S. cerevisiae, Sir2 is a functional component of epigenetic complexes required for the establishment of silenced chromatin regions in rDNA locus, telomeres and silent mating-type loci, HML and HMR [S. cerevisiae rDNA consists of a 9.1-kb unit that contains RNA polymerase (Pol) I-transcribed 35S ribosomal RNA (rRNA) and Pol III-transcribed 5S rRNA gene, separated by a non-transcribed spacer, and is repeated 100-200 times on chromosome XII [S. cerevisiae is known to be the homologous recombination between rDNA repeats, which leads to the formation of ERCs that accumulate to toxic levels in mother cells [Sir2 proteins, or sirtuins, are a well-known, highly conserved, family of NADo humans -22. Manyo humans -31. Sirto humans , 33. In  and HMR -38. S. csome XII . Becauseer cells . Sir2 prer cells .A well-known epigenetic regulator of yeast rDNA locus is the regulator of nucleolar silencing and telophase exit (RENT) complex that is composed of Sir2, Net1 and Cdc14 . Net1, tS. cerevisiae, Caenorhabditis elegans and Drosophila melanogaster [TOR kinase is a nutrient-responsive phosphatidylinositol kinase-related protein kinase structurally and functionally conserved from yeast to humans and plays critical roles in cell growth in response to nutrient availability by regulating transcription, translation, ribosome biogenesis and autophagy , 57-59. nogaster -66. Recenogaster , 68.et al. reported that the inhibition of TORC1 signaling increases lifespan but has no effect on Sir2 activity [et al. indicated that TORC1 inhibition activates Sir2 by increasing the expression of PNC1 encoding a nicotinamidase that depletes cellular nicotinamide, a physiological inhibitor of Sir2, thereby suppressing the formation of ERCs and leading to the extension of replicative lifespan [How the inhibition of TORC1 signaling extends lifespan is poorly understood. Meanwhile, whether Sir2 functions in rDNA stability and lifespan extension in yeast during TORC1 inhibition has been a matter of debate. Kaeberlein activity , whereaslifespan . Corrobolifespan . These fRecent studies have reported that Sir2 inhibits replicative senescence by additional non-rDNA mechanisms. Sir2 is required for the asymmetric inheritance of oxidatively damaged proteins during cytokinesis, resulting in an enhanced capacity to respond to oxidative stress in daughter cells -72. In aSmall ubiquitin-like modifier (SUMO) modification is an important posttranslational mechanism for regulating protein function, especially in the nucleus and nucleolus. Sumoylation is involved in various nuclear functions such as transcription, DNA repair and nuclear domain organization , 75. In Recent studies have shown that sumoylation is involved in the regulation of Sir2 function in the maintenance of chromatin structure at rDNA. Esc2, a member of a conserved family of proteins that contain SUMO-like domains, interacts with Sir2 through a SUMO-binding motif and is required for the maintenance of silent chromatin structure at rDNA . A SUMO-Sirtuins are a highly conserved family of proteins that have been implicated in regulating diverse functions including longevity in a variety of species. Many studies suggest that Sir2 regulates rDNA recombination, DNA repair, chromosomal stability and longevity. In this research perspective, we briefly reviewed how Sir2 and other epigenetic regulators function as the stabilizer of rDNA and regulate longevity. Sir2 is part of a pathway mediating the longevity effect of TORC1 inhibition. Based on recent findings, we also propose that sumoylation may be involved in the regulation of Sir2 function in rDNA maintenance and thus longevity (Figure


Post-translational modifications (PTMs) regulate multiple biological functions of the promyelocytic leukemia (PML) protein and also the fission, disassembly, and rebuilding of PML nuclear bodies (PML-NBs) during the cell cycle. Pathway-specific PML modification patterns ensure proper signal output from PML-NBs that suit the specific functional requirements. Here we comprehensively review the signaling pathways and enzymes that modify PML and also the oncogenic PML-RARα fusion protein. Many PTMs occur in a hierarchical and timely organized fashion. Phosphorylation or acetylation constitutes typical starting points for many PML modifying events, while degradative ubiquitination is an irreversible end point of the modification cascade. As this hierarchical organization of PTMs frequently turns phosphorylation events as primordial events, kinases or phosphatases regulating PML phosphorylation may be interesting drug targets to manipulate the downstream modifications and thus the stability and function of PML or PML-RARα. The analysis of a manually curated PML-NB interactome . PML recruits the AKT phosphatase PP2A and the phosphorylated AKT  protein into PML-NBs, thus ensuring that the majority of AKT is present in the unphosphorylated, inactive form , thus resulting in its ubiquitination which in turn allows its proteasomal degradation. Stimulation of cells with interferon leads to the enrichment of KLHL20 in PML-NBs, thereby separating KLHL20 from DAPK which stops ubiquitination and degradation of this kinase  PML-NBs are dynamically regulated during the cell cycle by a fission mechanism during S phase . This phosphorylation is associated with increased SUMOylation of PML and elevated As2O3-induced cell death  phosphorylates PML at an unknown site(s) which in turn results in the nucleolar translocation of PML and the sequestration of MDM2 . Similarly, PML acetylation was also induced by overexpression of the lysine acetyl transferases p300 or GCN5  receptor α (Rarα) gene by the t chromosomal translocation generates a fusion protein that preserves the N-terminal part of PML including its phosphorylation sites (De The et al., While our knowledge on PML phosphorylation is steadily growing, less is known about this modification in the oncogenic PML-RARα fusion protein. Fusion of the Presently only indirect evidence suggests a functional role of PML-RARα acetylation for disease progression, as revealed by pharmacological evidence in a transgenic mouse model. PML-RARα expressing mice develop leukemia which is responsive to RA treatment. This mouse model recapitulates the function of PML-RARα as a transcriptional repressor and the repressor activity is lost upon treatment of cells with RA in combination with TsA (He et al., The PML-RARα fusion protein still contains two of the three PML SUMOylation sites at Lys65 and Lys160. Data obtained in cell models and transgenic animals show the necessity of the Lys160 SUMOylation site in the PML moiety of the fusion protein for the development of acute PML (Zhu et al., Of special pathophysiological interest is the ubiquitin-dependent degradation of PML-RARα by two clinically active drugs, arsenic, and RA. A SUMO-ubiquitin-cascade has been identified by several groups as the molecular mechanism leading to the arsenic-mediated degradation of PML-RARα (Lallemand-Breitenbach et al., Apparently, many of the PML modifications occur in hierarchical and timely organized fashion, as schematically summarized in Figure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.