Absence Of Phosphorylation Research Articles

Protein phosphorylation and oxidative protein modification promote plant photosystem II disassembly for repair

The light-driven water-splitting reaction of photosystem II exposes its key reaction center core protein subunits to irreversible oxidative photodamage. A rapid repair cycle replaces the photodamaged core subunits in plants, but how the large antenna-core supercomplex structures of plant photosystem II disassemble for repair is not currently understood. We find a specific involvement of phosphorylation in removing the peripheral antenna from the core and monomerization of the dimeric cores. However, monomeric cores disassembled further into smaller subcomplexes even in the absence of phosphorylation as suggestive of other unknown mechanisms of disassembly. Here we show that the oxidative modifications of amino acids in core protein subunits of photosystem II are active mediators of the monomeric core disassembly. Oxidative modifications thus likely disassemble only the damaged monomeric cores, ensuring an economical photosystem disassembly process. Taken together, our results suggest phosphorylation and oxidative modification play distinct roles in photosystem II disassembly and repair.

The structures of protein kinase A in complex with CFTR: Mechanisms of phosphorylation and noncatalytic activation

Protein kinase A (PKA) is a key regulator of cellular functions by selectively phosphorylating numerous substrates, including ion channels, enzymes, and transcription factors. It has long served as a model system for understanding the eukaryotic kinases. Using cryoelectron microscopy, we present complex structures of the PKA catalytic subunit (PKA-C) bound to a full-length protein substrate, the cystic fibrosis transmembrane conductance regulator (CFTR)-an ion channel vital to human health. CFTR gating requires phosphorylation of its regulatory (R) domain. Unphosphorylated CFTR engages PKA-C at two locations, establishing two "catalytic stations" near to, but not directly involving, the R domain. This configuration, coupled with the conformational flexibility of the R domain, permits transient interactions of the eleven spatially separated phosphorylation sites. Furthermore, we determined two structures of the open-pore CFTR stabilized by PKA-C, providing a molecular basis to understand how PKA-C stimulates CFTR currents even in the absence of phosphorylation.

APCCdh1-mediated degradation of Cdh1 is necessary for faithful meiotic chromosome segregation in S. cerevisiae.

The Anaphase-Promoting Complex/Cyclosome (APC/C) is a ubiquitin ligase that promotes the ubiquitination and subsequent degradation of numerous cell cycle regulators during mitosis and in G1. Proteins are recruited to the APC/C by activator proteins such as Cdh1. During the cell cycle, Cdh1 is subject to precise regulation so that substrates are not degraded prematurely. We have explored the regulation of Cdh1 during the developmental transition into meiosis and sporulation in the budding yeast S. cerevisiae. Transition to sporulation medium triggers the degradation of Cdh1. Cdh1 degradation is mediated by the APC/C itself in a "trans" mechanism in which one molecule of Cdh1 recruits a second molecule of Cdh1 to the APC/C for ubiquitination. Degradation requires an intact glucose-sensing SNF1 protein kinase complex (orthologous to the mammalian AMPK nutritional sensor), which directly phosphorylates Cdh1 on Ser-200 within an unstructured N-terminal region. In the absence of phosphorylation, expression of a Cdh1-S200A mutant is fully stabilized, leading to chromosome instability and loss of viability. We hypothesize that Cdh1 degradation is necessary for the preservation of cell cycle regulators and chromosome cohesion proteins between the reductional and equational meiotic divisions, which occur without the intervening Gap or S phases found in mitotic cell cycles.

Proto-oncogene cSrc-mediated RBM10 phosphorylation arbitrates anti-hypertrophy gene program in the heart and controls cardiac hypertrophy

AimsRBM10 is a well-known RNA binding protein that regulates alternative splicing in various disease states. We have shown a splicing-independent function of RBM10 that regulates heart failure. This study aims to unravel a new biological function of RBM10 phosphorylation by proto-oncogene cSrc that enables anti-hypertrophy gene program and controls cardiac hypertrophy. Materials and methodsWe employ in vitro and in vivo approaches to characterise RBM10 phosphorylation at three-tyrosine residues (Y81, Y500, and Y971) by cSrc and target mRNA regulation. We also use isoproterenol induced rat heart and cellular hypertrophy model to determine role of cSrc-mediated RBM10 phosphorylation. Key findingsWe show that RBM10 phosphorylation is induced in cellular and animal heart model of cardiac hypertrophy and regulates target mRNA expression and 3′-end formation. Inhibition of cSrc kinase or mutation of the three-tyrosine phosphorylation sites to phenylalanine accentuates myocyte hypertrophy, and results in advancement and an early attainment of hypertrophy in the heart. RBM10 is down regulated in the hypertrophic myocyte and that its re-expression reverses cellular and molecular changes in the myocyte. However, in the absence of phosphorylation (cSrc inhibition or phospho-deficient mutation), restoration of endogenous RBM10 level in the hypertrophic heart or ectopic re-expression in vitro failed to reverse cardiomyocyte hypertrophy. Mechanistically, loss of RBM10 phosphorylation inhibits nuclear localisation and interaction with Star-PAP compromising anti-hypertrophy gene expression. SignificanceOur study establishes that cSrc-mediated RBM10 phosphorylation arbitrates anti-hypertrophy gene program. We also report a new functional regulation of RBM10 by phosphorylation that is poised to control heart failure.

Analysis of CenKR essentiality in Sinorhizobium meliloti and its activity at a target gene promoter in vivo.

The two-component regulatory system CenK-CenR has recently emerged as a regulator of cell envelope and cell division processes in the alpha-proteobacteria. In Sinorhizobium meliloti, CenK-CenR regulates the expression of SrlA, a thioredoxin-domain protein of unknown function. Deletion of srlA causes sensitivity to salt and oxidizing agents on solid growth medium. In this work, we report that the response regulator CenR, but not the histidine kinase CenK, is essential for cell viability in S. meliloti. We also demonstrate that phosphorylation of the target residue D55 is not required for viability, suggesting that the unphosphorylated transcription factor sufficiently regulates expression of one or more essential genes in the genome. Using transcription assays and phenotype testing we examine CenK-CenR-dependent activation of the srlA promoter and demonstrate its absolute dependence on phosphoryl-CenR for activity and that the CenR substitution D55E acts as a phosphomimetic that partially restores activity at the srlA promoter in the absence of phosphorylation by CenK. Finally, we report a mutational analysis of the CenR binding site in the srlA promoter required for transcriptional activation.

Structural and biochemical basis of FLS2-mediated signal activation and transduction in rice

The receptor-like kinase FLAGELLIN-SENSITIVE 2 (FLS2) functions as a bacterial flagellin receptor localized on the cell membrane of plants. In Arabidopsis, the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) cooperates with FLS2 to detect the flagellin epitope flg22, resulting in formation of a signaling complex that triggers plant defense responses. However, the co-receptor responsible for recognizing and signaling the flg22 epitope in rice remains to be determined, and the precise structural mechanism underlying FLS2-mediated signal activation and transduction has not been clarified. This study presents the structural characterization of a kinase-dead mutant of the intracellular kinase domain of OsFLS2 (OsFLS2-KDD1013A) in complex with ATP or ADP, resolved at resolutions of 1.98 Å and 2.09 Å, respectively. Structural analysis revealed that OsFLS2 can adopt an active conformation in the absence of phosphorylation, although it exhibits only weak basal catalytic activity for autophosphorylation. Subsequent investigations demonstrated that OsSERK2 effectively phosphorylates OsFLS2, which reciprocally phosphorylates OsSERK2, leading to complete activation of OsSERK2 and rapid phosphorylation of the downstream substrate receptor-like cytoplasmic kinases OsRLCK176 and OsRLCK185. Through mass spectrometry experiments, we successfully identified critical autophosphorylation sites on OsSERK2, as well as sites transphosphorylated by OsFLS2. Furthermore, we demonstrated the interaction between OsSERK2 and OsFLS2, which is enhanced in the presence of flg22. Genetic evidence suggests that OsRLCK176 and OsRLCK185 may function downstream of the OsFLS2-mediated signaling pathway. Our study reveals the molecular mechanism by which OsFLS2 mediates signal transduction pathways in rice and provides a valuable example for understanding RLK-mediated signaling pathways in plants.

111 (PB101) - IK930, a novel TEAD inhibitor, sensitizes KRAS and EGFR mutant tumors to oncogene targeted therapy

The Hippo signaling pathway plays a key role in the regulation of cell proliferation, survival, and tissue homeostasis. The transcription co-factors YAP and TAZ, the downstream effectors of this pathway, are phosphorylated by the LATS1/2 kinases in response to a signaling cascade initiated by the MST1/2 (Hippo) kinases. Upon phosphorylation, YAP/TAZ can be sequestered in the cytoplasm or targeted for degradation. In the absence of phosphorylation, YAP and TAZ translocate to the cell nucleus where they interact with members of the TEAD transcription factor family to activate target gene expression.

CAMP-dependent activation of the Rac guanine exchange factor P-REX1 by type I protein kinase A (PKA) regulatory subunits

Regulatory subunits of protein kinase A (PKA) inhibit its kinase subunits. Intriguingly, their potential as cAMP-dependent signal transducers remains uncharacterized. We recently reported that type I PKA regulatory subunits (RIα) interact with phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-REX1), a chemotactic Rac guanine exchange factor (RacGEF). Because P-REX1 is known to be phosphorylated and inhibited by PKA, its interaction with RIα suggests that PKA regulatory and catalytic subunits may fine-tune P-REX1 activity or those of its target pools. Here, we tested whether RIα acts as a cAMP-dependent factor promoting P-REX1-mediated Rac activation and cell migration. We observed that Gs-coupled EP2 receptors indeed promote endothelial cell migration via RIα-activated P-REX1. Expression of the P-REX1-PDZ1 domain prevented RIα/P-REX1 interaction, P-REX1 activation, and EP2-dependent cell migration, and P-REX1 silencing abrogated RIα-dependent Rac activation. RIα-specific cAMP analogs activated P-REX1, but lost this activity in RIα-knockdown cells, and cAMP pulldown assays revealed that P-REX1 preferentially interacts with free RIα. Moreover, purified RIα directly activated P-REX1 in vitro We also found that the RIα CNB-B domain is critical for the interaction with P-REX1, which was increased in RIα mutants, such as the acrodysostosis-associated mutant, that activate P-REX1 at basal cAMP levels. RIα and Cα PKA subunits targeted distinct P-REX1 molecules, indicated by an absence of phosphorylation in the active fraction of P-REX1. This was in contrast to the inactive fraction in which phosphorylated P-REX1 was present, suggesting co-existence of dual stimulatory and inhibitory effects. We conclude that PKA's regulatory subunits are cAMP-dependent signal transducers.

Phosphorylation of the phosphatase PTPROt at Tyr399 is a molecular switch that controls osteoclast activity and bone mass in vivo.

Bone resorption by osteoclasts is essential for bone homeostasis. The kinase Src promotes osteoclast activity and is activated in osteoclasts by the receptor-type tyrosine phosphatase PTPROt. In other contexts, however, PTPROt can inhibit Src activity. Through in vivo and in vitro experiments, we show that PTPROt is bifunctional and can dephosphorylate Src both at its inhibitory residue Tyr527 and its activating residue Tyr416 Whereas wild-type and PTPROt knockout mice exhibited similar bone masses, mice in which a putative C-terminal phosphorylation site, Tyr399, in endogenous PTPROt was replaced with phenylalanine had increased bone mass and reduced osteoclast activity. Osteoclasts from the knock-in mice also showed reduced Src activity. Experiments in cultured cells and in osteoclasts derived from both mouse strains demonstrated that the absence of phosphorylation at Tyr399 caused PTPROt to dephosphorylate Src at the activating site pTyr416 In contrast, phosphorylation of PTPROt at Tyr399 enabled PTPROt to recruit Src through Grb2 and to dephosphorylate Src at the inhibitory site Tyr527, thus stimulating Src activity. We conclude that reversible phosphorylation of PTPROt at Tyr399 is a molecular switch that selects between its opposing activities toward Src and maintains a coherent signaling output, and that blocking this phosphorylation event can induce physiological effects in vivo. Because most receptor-type tyrosine phosphatases contain potential phosphorylation sites at their C termini, we propose that preventing phosphorylation at these sites or its consequences may offer an alternative to inhibiting their catalytic activity to achieve therapeutic benefit.

Phosphorylation-dependent protein design: design of a minimal protein kinase-inducible domain.

Protein kinases and phosphatases modulate protein structure and function, which in turn regulate cellular activities. The development of novel proteins and protein motifs that are responsive to protein phosphorylation provides new ways to probe the functions of individual protein kinases and the intracellular effects of their activation and downregulation. Herein we develop a minimal motif that is responsive to protein phosphorylation, termed a minimal protein kinase-inducible domain. The encodable protein motif comprises a 7- or 8-residue sequence (DKDADXW or DKDADXXW), derived from EF-Hand calcium-binding domains, that is necessary but not sufficient for binding terbium, combined with a protein phosphorylation site (Ser or Thr at residue 9) that, upon phosphorylation, completes the metal-binding motif. Thus, the motif binds metal poorly and exhibits weak terbium luminescence when not phosphorylated. Upon phosphorylation, the peptide binds metal with significantly higher affinity and exhibits robust terbium luminescence. Phosphorylation results in up to a 23× increase in terbium luminescence. Minimal phosphorylation-dependent motifs as small as 9 residues (DKDADGWIS) were developed. NMR spectroscopy on this lanthanum(iii)·phosphopeptide complex confirmed that binding occurs in a manner similar to that in an EF-Hand, despite the absence of the conserved Glu12 typically present in an EF-Hand. By combining molecular design with known protein kinase recognition sequences, minimal protein kinase-inducible domains were developed that were responsive to phosphorylation by Protein Kinase A (PKA: DKDADRRW(S/pS)IIAK), Protein Kinase C (PKC: DKDADGWI(T/pT)FRRKA), and Casein Kinase 1 (CK1: DKDADDWA(S/pS)I). Phosphorylation by PKA was quantified in HeLa cell extracts, with a 4.4× increase in fluorescence (terbium luminescence) observed at 544 nm. The optimized minimal motif includes alternating aspartate residues at positions 1, 3, and 5, plus binding through the main-chain carbonyl at position 7; a lysine at position 2 to provide electrostatic balance and reduce binding in the absence of phosphorylation; an alanine at residue 4 to promote the αL conformation observed at that position of the EF Hand; a tryptophan at residue 7 or 8 to sensitize terbium luminescence; and a phosphorylation site with serine or threonine at residue 9. Residues at positions 6; 7 or 8; and 10 or later may be changed to provide kinase specificity. In the CK1-responsive peptide, the acidic residues in the proto-terbium-binding motif are employed as part of the kinase recognition sequence. This work thus presents fundamental rules for the design of compact phosphorylation-responsive terbium-binding motifs, with potential further application to motifs responsive to other protein post-translational modifications.

Overexpression of thioredoxin m in tobacco chloroplasts inhibits the protein kinase STN7 and alters photosynthetic performance.

The activity of the protein kinase STN7, involved in phosphorylation of the light-harvesting complex II (LHCII) proteins, has been reported as being co-operatively regulated by the redox state of the plastoquinone pool and the ferredoxin-thioredoxin (Trx) system. The present study aims to investigate the role of plastid Trxs in STN7 regulation and their impact on photosynthesis. For this purpose, tobacco plants overexpressing Trx f or m from the plastid genome were characterized, demonstrating that only Trx m overexpression was associated with a complete loss of LHCII phosphorylation that did not correlate with decreased STN7 levels. The absence of phosphorylation in Trx m-overexpressing plants impeded migration of LHCII from PSII to PSI, with the concomitant loss of PSI-LHCII complex formation. Consequently, the thylakoid ultrastructure was altered, showing reduced grana stacking. Moreover, the electron transport rate was negatively affected, showing an impact on energy-demanding processes such as the Rubisco maximum carboxylation capacity and ribulose 1,5-bisphosphate regeneration rate values, which caused a strong depletion in net photosynthetic rates. Finally, tobacco plants overexpressing a Trx m mutant lacking the reactive redox site showed equivalent physiological performance to the wild type, indicating that the overexpressed Trx m deactivates STN7 in a redox-dependent way.

Phosphorylation of Serine Induces Lysine p Ka Increases in Histone N-Termini and Signaling for Acetylation. Transcription Implications.

The mild acetylating agent, methyl acetyl phosphate, is used to estimate the p Ka values of some of the amine groups in peptides with sequences corresponding to a segment of the N-terminal tail of histone H4. When Ser-1 is not phosphorylated, the Lys ε amines have p Ka values in the range of 7.8-8.3, which are much lower than the currently assumed values. When Ser-1 is phosphorylated, the p Ka values of these Lys amines are elevated to the range of 8.8-10.3, thus providing the rationale for reports that they are then better substrates for acetyltransferases. Thus, reversal of suppressed p Ka values of Lys ε amines by Ser phosphorylation represents the basis for signaling in histone N-terminal tails to promote hyperacetylation, which is a hallmark of transcriptionally active euchromatin. In contrast, a state of hypoacetylation is present in the absence of phosphorylation as in transcriptionally inactive heterochromatin. A novel approach for estimating p Ka values based on a linkage between the Henderson-Hasselbalch and Michaelis-Menten equations indicates that the p Ka values of the Lys ε amines in H3 and H4 N-terminal tails have a highly variable charge gradient dependent on the location and proximity to the phosphorylation site.

Modulation of Cl- signaling and ion transport by recruitment of kinases and phosphatases mediated by the regulatory protein IRBIT.

IRBIT is a multifunctional protein that controls the activity of various epithelial ion transporters including NBCe1-B. Interaction with IRBIT increases NBCe1-B activity and exposes two cryptic Cl--sensing GXXXP sites that enable regulation of NBCe1-B by intracellular Cl- (Cl- in). Here, phosphoproteomic analysis revealed that IRBIT controlled five phosphorylation sites in NBCe1-B that determined both the active conformation of the transporter and its regulation by Cl- in Mutational analysis suggested that the phosphorylation status of Ser232, Ser233, and Ser235 was regulated by IRBIT and determined whether NBCe1 transporters are in active or inactive conformations. The absence of phosphorylation at Ser232, Ser233, or Ser235 produced NBCe1-B in the conformations pSer233/pSer235, pSer232/pSer235, or pSer232/pSer233, respectively. The activity of the pSer233/pSer235 form was similar to that of IRBIT-activated NBCe1-B, but it was insensitive to inhibition by Cl- in The properties of the pSer232/pSer235 form were similar to those of wild-type NBCe1-B, whereas the pSer232/pSer233 form was partially active, further activated by IRBIT, but retained inhibition by Cl- in Furthermore, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control phosphorylation of Ser65, which affected Cl- in sensing by the 32GXXXP36 motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser12, which affected Cl- in sensing by the 194GXXXP198 motif. Ser232, Ser233, and Ser235 are conserved in all NBCe1 variants and affect their activity. These findings reveal how multiple kinase and phosphatase pathways use phosphorylation sites to fine-tune a transporter, which have important implications for epithelial fluid and HCO3 - secretion.

Pyruvate dehydrogenase complex deficiency is linked to regulatory loop disorder in the αV138M variant of human pyruvate dehydrogenase

The pyruvate dehydrogenase multienzyme complex (PDHc) connects glycolysis to the tricarboxylic acid cycle by producing acetyl-CoA via the decarboxylation of pyruvate. Because of its pivotal role in glucose metabolism, this complex is closely regulated in mammals by reversible phosphorylation, the modulation of which is of interest in treating cancer, diabetes, and obesity. Mutations such as that leading to the αV138M variant in pyruvate dehydrogenase, the pyruvate-decarboxylating PDHc E1 component, can result in PDHc deficiency, an inborn error of metabolism that results in an array of symptoms such as lactic acidosis, progressive cognitive and neuromuscular deficits, and even death in infancy or childhood. Here we present an analysis of two X-ray crystal structures at 2.7-Å resolution, the first of the disease-associated human αV138M E1 variant and the second of human wildtype (WT) E1 with a bound adduct of its coenzyme thiamin diphosphate and the substrate analogue acetylphosphinate. The structures provide support for the role of regulatory loop disorder in E1 inactivation, and the αV138M variant structure also reveals that altered coenzyme binding can result in such disorder even in the absence of phosphorylation. Specifically, both E1 phosphorylation at αSer-264 and the αV138M substitution result in disordered loops that are not optimally oriented or available to efficiently bind the lipoyl domain of PDHc E2. Combined with an analysis of αV138M activity, these results underscore the general connection between regulatory loop disorder and loss of E1 catalytic efficiency.

Interactive structural analysis of βTrCP1 and PER2 phosphoswitch binding through dynamics simulation assay

Circadian rhythm is rhythmic gene expression that is involved in various processes of life over a day and night cycle. The rhythmic sleep disorders arise due to misalignment of sleep-wake cycle influenced by phosphorylation of PERIOD2 (PER2) phosphodegron (SSGYGS), the conserved interaction site of β-transducin repeat-containing protein (βTrCP1). Here, we employed in silico approach to study the interaction pattern of βTrCP1 with PER2WT, PER2SER480ALA and PER2SER484ALA phosphodegron peptides. Substitution of phosphorylatable SER480 or SER484 into ALA resulted in the shifting of PER2 phosphodegron binding at the lower face of β-propeller, by involvement of both SER residues. PER2 binding at the shallow cavity of βTrCP1 induced conformational readjustment in ARG524 residue that connected the upper hemisphere base (10.5 Å) with the roof of lower hemisphere (6.6 Å) to create a uniform tunnel-like structure. In the absence of phosphorylation, PER2 and βTrCP1 binding stability may be compromised resulting in the enhancement of PER2 level in the cytoplasm that may disrupt circadian clock and aging. Taken together, this study will help in understanding the structural basis of conserved phosphoswitch mechanism in the mammalian circadian oscillation.

Phosphorylation of GPCR Kinase 2 in Intact Cells: A Proteomic Approach

G protein‐coupled receptor (GPCR) kinases (GRKs) were first discovered by their ability to phosphorylate GPCRs in an agonist‐dependent manner. GRKs belong to the AGC kinase family of kinases. Activation of AGC kinases is often accompanied by phosphorylation of activation loop residues as well as other conformational changes in catalytically important residues. Crystal structures of GRK2 suggest that the activation segment region is in a relatively “active” conformation in the absence of phosphorylation. Nonetheless, a phosphoproteomic study (Oppermann et al., 2009) indicated that GRK2 Ser350 and Thr353 can be phosphorylated in intact cancer cells. Ser350 and Thr353 reside in the activation loop and P+1 site of the peptide‐binding site, respectively. To test the role of Ser350 and Thr353 phosphorylation in GRK2 function, we substituted alanine and aspartate at these two positions and tested the ability to phosphorylate the beta‐adrenergic receptor. We found that Thr353 is particularly important for phosphorylation of beta‐adrenergic receptor by GRK2 in COS‐7 cells, since conversion to Ala or Asp prevented substrate phosphorylation. In cells transfected with both beta‐adrenergic receptor and GRK2, multiple forms of GRK2 were detected in cells treated with agonist. The multiple forms are consistent with various phosphorylation states. Our goal is to assess Ser350/Thr353 phosphorylation in intact cells using phosphoproteomic approaches. To this end, we engineered a hexahistidine tag on GRK2 expressed from a mammalian cell vector to facilitate its purification and have demonstrated that the tagged kinase is fully functional in agonist‐dependent receptor phosphorylation. We are further developing methods to assess GRK2 phosphorylation by isoelectric focusing and mass spectrometry.Support or Funding InformationThis work was funded by the National Science Foundation MCB0744739 and Siena College Center for Undergraduate Research and Creative Activity (CURCA).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

2290

OBJECTIVES/SPECIFIC AIMS: Accumulation of cholesterol-laden macrophages in arterial walls leads to atherosclerosis. LXRs induce expression of genes that are atheroprotective in macrophages including CCR7, a chemokine receptor that promotes their emigration from the plaque. CCR7 expression has been shown to be negatively regulated by phosphorylation of LXRα at S198 and is reduced in diabetic mice that show impaired plaque regression. I hypothesized that LXRα phosphorylation at S198 diminishes macrophage emigration from atherosclerotic plaque and contributes to impaired regression in diabetes. METHODS/STUDY POPULATION: Inducible LXRα S198A phosphorylation deficient knock in mouse were used as donors for bone marrow transplantation into mice prone to develop atherosclerosis. Plaques were developed by placing mice on western diet; and regression was induced by lowering their lipid levels. Aortic plaques were then analyzed by using morphometric, histological, and molecular analyses in control and diabetic mice expressing either LXRα WT or LXRα S198A during regression. RESULTS/ANTICIPATED RESULTS: Surprisingly, lack of phosphorylation increased plaque macrophage content and impaired regression under normoglycemic condition; however, it did not exacerbate diabetic regression. Plaques in diabetic mice were associated with increased LXRα S198 phosphorylation. Consistent with this, LXRα phosphorylation is enhanced in macrophages cultured under hyperglycemic conditions indicating glucose-dependent regulation of LXRα phosphorylation. Monocyte trafficking studies reveal that lack of phosphorylation and diabetes independently increase recruitment of monocytes in the plaque that might contribute to increased macrophage content. Importantly, I found that diabetes also increases macrophage retention in the plaque, which is reversed in the absence of phosphorylation. We predict that this increased retention results from inhibition of emigration of plaque macrophages through enhanced phosphorylation in diabetes. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings suggest that inhibiting LXRα phosphorylation could be beneficial in diabetic atherosclerosis to reverse the accumulation of macrophages in the plaque. This study imparts insight on regulation of plaque macrophage trafficking through LXRα S198 phosphorylation.

Exploring the CK2 Paradox: Restless, Dangerous, Dispensable

The history of protein kinase CK2 is crowded with paradoxes and unanticipated findings. Named after a protein (casein) that is not among its physiological substrates, CK2 remained in search of its targets for more than two decades after its discovery in 1954, but it later came to be one of the most pleiotropic protein kinases. Being active in the absence of phosphorylation and/or specific stimuli, it looks unsuitable to participate in signaling cascades, but its “lateral” implication in a variety of signaling pathways is now soundly documented. At variance with many “onco-kinases”, CK2 is constitutively active, and no oncogenic CK2 mutant is known; still high CK2 activity correlates to neoplasia. Its pleiotropy and essential role may cast doubts on the actual “druggability” of CK2; however, a CK2 inhibitor is now in Phase II clinical trials for the treatment of cancer, and cell clones viable in the absence of CK2 are providing information about the mechanism by which cancer becomes addicted to high CK2 levels. A phosphoproteomics analysis of these CK2 null cells suggests that CK2 pleiotropy may be less pronounced than expected and supports the idea that the phosphoproteome generated by this kinase is flexible and not rigidly pre-determined.

Flagellin FliC Phosphorylation Affects Type 2 Protease Secretion and Biofilm Dispersal in Pseudomonas aeruginosa PAO1.

Protein phosphorylation has a major role in controlling the life-cycle and infection stages of bacteria. Proteome-wide occurrence of S/T/Y phosphorylation has been reported for many prokaryotic systems. Previously, we reported the phosphoproteome of Pseudomonas aeruginosa and Pseudomonas putida. In this study, we show the role of S/T phosphorylation of one motility protein, FliC, in regulating multiple surface-associated phenomena of P. aeruginosa PAO1. This is the first report of occurrence of phosphorylation in the flagellar protein, flagellin FliC in its highly conserved N-terminal NDO domain across several Gram negative bacteria. This phosphorylation is likely a well-regulated phenomenon as it is growth phase dependent in planktonic cells. The absence of phosphorylation in the conserved T27 and S28 residues of FliC, interestingly, did not affect swimming motility, but affected the secretome of type 2 secretion system (T2SS) and biofilm formation of PAO1. FliC phosphomutants had increased levels and activities of type 2 secretome proteins. The secretion efficiency of T2SS machinery is associated with flagellin phosphorylation. FliC phosphomutants also formed reduced biofilms at 24 h under static conditions and had delayed biofilm dispersal under dynamic flow conditions, respectively. The levels of type 2 secretome and biofilm formation under static conditions had an inverse correlation. Hence, increase in type 2 secretome levels was accompanied by reduced biofilm formation in the FliC phosphomutants. As T2SS is involved in nutrient acquisition and biofilm dispersal during survival and spread of P. aeruginosa, we propose that FliC phosphorylation has a role in ecological adaptation of this opportunistic environmental pathogen. Altogether, we found a system of phosphorylation that affects key surface related processes such as proteases secretion by T2SS, biofilm formation and dispersal.

Abstract 485: Chronic exposure to a low dose of interferon-β increases levels of DNA damage resistance signature genes through unphosphorylated ISGF3

Abstract Ionizing radiation and DNA damaging chemotherapeutic agents are used widely to treat many cancers, but the presence of cells that resist DNA damage often leads to failure to cure. One of the specific features of therapy-resistance is high expression of Interferon-Related DNA damage resistance Signature (IRDS) genes. The IRDS genes are a subset of (about 30 genes) Interferon Stimulated Genes (ISGs) including IFI27, OAS2, Mx1, and IFIT1. However, pro-apoptotic and anti-proliferative ISGs, such as IRF1, caspases, and FASL, are not included in this signature. In this study, we elucidated the molecular mechanism of how the expression of IRDS genes are increased, with no increase of other ISGs, in therapy-resistant cancer cells. When cells expressed high levels of IRF9 and STATs 1 and 2 in the absence of phosphorylation of Tyr-701-STAT1 and Tyr-690-STAT2, the expression of IRDS genes was increased, but that of other ISGs was not. The un-phosphorylated STAT1, STAT2, and IRF9 proteins formed a complex resembling Interferon Stimulated Gene Factor 3 (ISGF3), a major transcription factor in type I interferon signaling. While ISGF3, consisting of phosphorylated STATs 1 and 2 and IRF9, mediates the expression of whole set of ISGs, the unphosphorylated ISGF3 (U-ISGF3) induces only IRDS genes. ChIP assay revealed that U-ISGF3 selectively bound to the promoters of IRDS genes (IFI27, OAS2, and Mx1), but not to those of other ISGs (MyD88, IFI16, and IRF1). Continuous exposure of cells to a low level of interferonβ, which might be produced by cancer cells or surrounding immune cells, lead to steady-state increases in the expression of only U-ISGF3 and IRDS genes with no increase of phosphorylated ISGF3 and non-IRDS ISGs. We examined the correlation between the U-ISGF3 levels and DNA damage resistance in various small cell lung carcinoma (SCLC) cell lines. The expression of IRF9 and STATs 1 and 2 proteins varied in different SCLC cell lines, and high levels of U-ISGF3 correlated with resistance to DNA damage. H82, H1048, and H146 cells, which express low levels of U-ISGF3, were sensitive to DNA damage induced by radiation, etoposide, and doxorubicin, while H196, H1688, and H2195 cells expressing high levels of U-ISGF3 were resistant to DNA damage. In response to radiation, γH2A.X levels were significantly increased and remained high up to 48 hrs in H1048 cells, but the increased γH2A.X levels returned to lower levels in H196 cells, suggesting that U-ISGF3-induced gene products may increase DNA damage repair. When STAT1 was knocked down to reduce U-ISGF3 levels in H196 cells, γH2A.X levels increased by radiation remained high. Knocking IRF9 down also decreased the resistance to DNA damage. In conclusion, U-ISGF3, which is increased by a low dose of interferons, plays an important role in inducing DNA damage resistance, and its targeting sensitizes cancer cells to DNA damage induced by various cancer therapies. Citation Format: HyeonJoo Cheon, Elise G. Holvey-Bates, George R. Stark. Chronic exposure to a low dose of interferon-β increases levels of DNA damage resistance signature genes through unphosphorylated ISGF3. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 485. doi:10.1158/1538-7445.AM2014-485