Large-scale analysis of phosphorylation site occupancy in eukaryotic proteins

Many recent high throughput technologies have enabled large-scale discoveries of new phosphorylation sites and phosphoproteins. Although they have provided a number of insights into protein phosphorylation and the related processes, an inclusive analysis on the nature of phosphorylated sites in proteins is currently lacking. We have therefore analyzed the occurrence and occupancy of phosphorylated sites (~100,281) in a large set of eukaryotic proteins (~22,995). Phosphorylation probability was found to be much higher in both the termini of protein sequences and this is much pronounced in transmembrane proteins. A large proportion (51.3%) of occupied sites had a nearby phosphorylation within a distance of 10 amino acids; however, this proportion is very high compared to the expected one (16.9%). The distribution of phosphorylated sites in proteins showed a strong deviation from the expected maximum randomness. An analysis of phosphorylation motifs indicated that just 40 motifs and a much lower number of associated kinases might account for nearly 50% of the known phosphorylations in eukaryotic proteins. Our results provide a broad picture of the phosphorylation sites in eukaryotic proteins.     

Activation of Helicobacter pylori CagA by tyrosine phosphorylation is essential for dephosphorylation of host cell proteins in gastric epithelial cells

Helicobacter pylori type I strains harbour the cag pathogenicity island (cag-PAI), a 37 kb sequence,which encodes the components of a type IV secretion system. CagA, the first identified effector protein of the cag-PAI, is translocated into eukaryotic cells and tyrosine phosphorylated (CagAP-tyr) by a host cell tyrosine kinase. Translocation of CagA induces the dephosphorylation of a set of phosphorylated host cell proteins of unknown identity. CagA proteins of independent H. pylori strains vary in sequence and thus in the number and composition of putative tyrosine phosphorylation motifs (TPMs). The CagA protein of H. pylori strain J99 (CagAJ99) does not carry any of three putative tyrosine phosphorylation motifs (TPM-A, TPM-B or TPM-C) predicted by the MOTIF algorithm in CagA proteins. CagA,n is not tyrosine phosphorylated and is inactive in the dephosphorylation of host cell proteins. By site-specific mutagenesis,we introduced a TPM-C into CagA,. by replacing a single lysine with a tyrosine. This slight modification resulted in tyrosine phosphorylation of CagAJ99 and host cell protein dephosphorylation. In contrast, the removal of the indigenous TPM-C from CagAP12 did not abolish its tyrosine phosphorylation, suggesting that further phosphorylated sites are present in CagAP12. By generation of hybrid CagA proteins, a phosphorylation of the most N-terminal TPM-A could be excluded. Our data suggest that tyrosine phosphorylation at TPM-C is sufficient, but not exclusive,to activate translocated CagA. Activated CagAPtr might either convert into a phosphatase itself or activate a cellular phosphatase to dephosphorylate cellular phosphoproteins and modulate cellular signalling cascades of the host.     

Proliferation and copy number variation of BEL-like long terminal repeat retrotransposons within the Diabrotica virgifera virgifera genome

The proliferation of retrotransposons within a genome can contribute to increased size and affect the function of eukaryotic genes. BEL/Pao-like long-terminal repeat (LTR) retrotransposons were annotated from the highly adaptable insect species Diabrotica virgifera virgifera, the Western corn rootworm, using survey sequences from bacterial artificial chromosome (BAC) inserts and contigs derived from a low coverage next-generation genome sequence assembly. Eleven unique D. v. virgifera BEL elements were identified that contained full-length gag–pol coding sequences, whereas 88 different partial coding regions were characterized from partially assembled elements. Estimated genome copy number for full and partial BEL-like elements ranged from ~ 8 to 1582 among individual contigs using a normalized depth of coverage (DOC) among Illumina HiSeq reads (total genome copy number ~ 8821). BEL element copy number was correlated among different D. v. virgifera populations (R² = 0.9846), but individual element numbers varied ≤ 1.68-fold and the total number varied by ~ 527 copies. These data indicate that BEL element proliferation likely contributed to a large genome size, and suggest that differences in copy number are a source of genetic variability among D. v. virgifera.     

Distinct cellular pools of perilipin 5 point to roles in lipid trafficking

The PAT family of lipid storage droplet proteins comprised five members, each of which has become an established regulator of cellular neutral lipid metabolism. Perilipin 5 (also known as lsdp-5, MLDP, PAT-1, and OXPAT), the most recently discovered member of the family, has been shown to localize to two distinct intracellular pools: the lipid storage droplet (LD), and a poorly characterized cytosolic fraction. We have characterized the denser of these intracellular pools and find that a population of perilipin 5 not associated with large LDs resides in complexes with a discrete density (~ 1.15 g/ml) and size (~ 575 kDa). Using immunofluorescence, western blotting of isolated sucrose density fractions, native gradient gel electrophoresis, and co-immunoprecipitation, we have shown that these small (~ 15 nm), perilipin 5-encoated structures do not contain the PAT protein perilipin 2 (ADRP), but do contain perilipin 3 and several other as of yet uncharacterized proteins. The size and density of these particles as well as their susceptibility to degradation by lipases suggest that like larger LDs, they have a neutral lipid rich core. When treated with oleic acid to promote neutral lipid deposition, cells ectopically expressing perilipin 5 experienced a reorganization of LDs in the cell, resulting in fewer, larger droplets at the expense of smaller ones. Collectively, these data demonstrate that a portion of cytosolic perilipin 5 resides in high density lipid droplet complexes that participate in cellular neutral lipid accumulation.     

A wide-ranging cellular response to UV damage of DNA

The kinases ATM and ATR are central to proper function of the DNA damage response. These kinases phosphorylate proteins to coordinate cell cycle progression and DNA damage repair/bypass. We have recently reported a large-scale identification of ATM/ATR substrates phosphorylated in response to UV damage of DNA. Overall 231 sites of phosphorylation were induced by UV damage of DNA or dependent on proper function of ATR. The study expanded the number of phosphorylation sites from protein classes known to be involved in the DNA damage response. Further, many sites were identified from protein types not thought to have a role in damage signaling. This observation suggests that the DNA damage response affects a much wider range of cellular processes than was previously appreciated. This study has also extended the successful use of the PhosphoScan® proteomic method from phospho-tyrosine to serine/threonine motifs, providing a general blueprint to use the method to study signaling pathways underlying a wide range of diseases.     

Identification of Endogenously Phosphorylated KSP Sites in the High-Molecular-Weight Rat Neurofilament Protein

Abstract: The high-molecular-weight neurofilament protein (NF-H) is highly phosphorylated in vivo, with estimates as high as 16–51 mol of P_i/mol of protein. Most of the phosphorylation sites are thought to be located on Ser residues in multiple KSP repeats, in the carboxy-terminal tail region of the molecule. Because the extent and site-specific patterns of tail domain phosphorylation are believed to modulate neurofilament structure and function, it becomes essential to identify the endogenous sites of phosphorylation. In this study, we have used selective proteolytic cleavage procedures, P_i determinations, microsequencing, and mass-spectral analysis to determine the endogenously phosphorylated sites in the NF-H tail isolated from rat spinal cord. Twenty Ser residues in NF-H carboxy-terminal tail were analyzed; nine of these, all located in KSP repeats, were phosphorylated. No detectable phosphorylation could be identified in any of the 11 "non-KSP" Ser residues that were examined. KSPXKX, KSPXXX, and KSPXXK motifs were found to be phosphorylated. In addition, a 27-kDa KSP-rich domain, containing 43 virtually uninterrupted KSPXXX repeats, was isolated from the tail domain and found to contain between 30 and 35 mol of P_i/mol of protein. This domain appeared to be highly resistant to endoproteinase Glu-C digestion, although it contains a large number of glutamate residues. It could be proteolyzed, however, after dephosphorylation. This suggests that phosphorylation of the tail domain may contribute to neurofilament stability in vivo. A neuronal-derived protein kinase that specifically phosphorylates only KSPXKX motifs in neurofilaments has been reported. The presence of extensively phosphorylated KSPXXX repeats in NF-H in vivo suggests the existence of yet another, unidentified kinase(s) with specificity for KSPXXX motifs.     

Interactions of the NPXY microdomains of the low density lipoprotein receptor‐related protein 1

The low density lipoprotein receptor‐related protein 1 (LRP1) mediates internalization of a large number of proteins and protein–lipid complexes and is widely implicated in Alzheimer's disease. The cytoplasmic domain of LRP1 (LRP1‐CT) can be phosphorylated by activated protein‐tyrosine kinases at two NPXY motifs in LRP1‐CT; Tyr 4507 is readily phosphorylated and must be phosphorylated before phosphorylation of Tyr 4473 occurs. Pull‐down experiments from brain lysate revealed numerous proteins binding to LRP1‐CT, but the results were highly variable. To separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non‐phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC‐MS/MS, and confirmed by Western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY₄₅₀₇ (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine‐phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY₄₄₇₃ (membrane proximal) bound many fewer proteins and only to the phosphorylated form.     

In-silico analysis of simple and imperfect microsatellites in diverse tobamovirus genomes

An in-silico analysis of simple sequence repeats (SSRs) in 30 species of tobamoviruses was done. SSRs (mono to hexa) were present with variant frequency across species. Compound microsatellites, primarily of variant motifs accounted for up to 11.43% of the SSRs. Motif duplications were observed for A, T, AT, and ACA repeats. (AG)–(TC) was the most prevalent SSR-couple. SSRs were differentially localized in the coding region with ~ 54% on the 128 kDa protein while 20.37% was exclusive to 186 kDa protein. Characterization of such variations is important for elucidating the origin, sequence variations, and structure of these widely used, but incompletely understood sequences.     

Specific motifs of the V-ATPase a2-subunit isoform interact with catalytic and regulatory domains of ARNO

We have previously shown that the V-ATPase a2-subunit isoform interacts specifically, and in an intra-endosomal acidification-dependent manner, with the Arf-GEF ARNO. In the present study, we examined the molecular mechanism of this interaction using synthetic peptides and purified recombinant proteins in protein-association assays. In these experiments, we revealed the involvement of multiple sites on the N-terminus of the V-ATPase a2-subunit (a2N) in the association with ARNO. While six a2N-derived peptides interact with wild-type ARNO, only two of them (named a2N-01 and a2N-03) bind to its catalytic Sec7-domain. However, of these, only the a2N-01 peptide (MGSLFRSESMCLAQLFL) showed specificity towards the Sec7-domain compared to other domains of the ARNO protein. Surface plasmon resonance kinetic analysis revealed a very strong binding affinity between this a2N-01 peptide and the Sec7-domain of ARNO, with dissociation constant K_D = 3.44 × 10⁻⁷ M, similar to the K_D = 3.13 × 10⁻⁷ M binding affinity between wild-type a2N and the full-length ARNO protein. In further pull-down experiments, we also revealed the involvement of multiple sites on ARNO itself in the association with a2N. However, while its catalytic Sec7-domain has the strongest interaction, the PH-, and PB-domains show much weaker binding to a2N. Interestingly, an interaction of the a2N to a peptide corresponding to ARNO's PB-domain was abolished by phosphorylation of ARNO residue Ser₃₉₂. The 3D-structures of the non-phosphorylated and phosphorylated peptides were resolved by NMR spectroscopy, and we have identified rearrangements resulting from Ser₃₉₂ phosphorylation. Homology modeling suggests that these alterations may modulate the access of the a2N to its interaction pocket on ARNO that is formed by the Sec7 and PB-domains. Overall, our data indicate that the interaction between the a2-subunit of V-ATPase and ARNO is a complex process involving various binding sites on both proteins. Importantly, the binding affinity between the a2-subunit and ARNO is in the same range as those previously reported for the intramolecular association of subunits within V-ATPase complex itself, indicating an important cell biological role for the interaction between the V-ATPase and small GTPase regulatory proteins.     

Monophosphothreonyl extracellular signal-regulated kinases 1 and 2 (ERK1/2) are formed endogenously in intact cardiac myocytes and are enzymically active

ERK1 and ERK2 (ERK1/2) are central to the regulation of cell division, growth and survival. They are activated by phosphorylation of the Thr- and the Tyr- residues in their Thr-Glu-Tyr activation loops. The dogma is that dually-phosphorylated ERK1/2 constitute the principal activities in intact cells. We previously showed that, in neonatal rat cardiac myocytes, endothelin-1 and phorbol 12-myristate 13-acetate (PMA) powerfully and rapidly (maximal at ~ 5 min) activate ERK1/2. Here, we show that dually-phosphorylated ERK1/2 rapidly (< 2 min) appear in the nucleus following stimulation with endothelin-1. We characterized the active ERK1/2 species in myocytes exposed to endothelin-1 or PMA using MonoQ FPLC. Unexpectedly, two peaks of ERK1 and two peaks of ERK2 activity were resolved using in vitro kinase assays. One of each of these represented the dually-phosphorylated species. The other two represented activities for ERK1 or ERK2 which were phosphorylated solely on the Thr- residue. Monophosphothreonyl ERK1/2 represented maximally ~ 30% of total ERK1/2 activity after stimulation with endothelin-1 or PMA, and their k_cat values were estimated to be minimally ~ 30% of the dually-phosphorylated species. Appearance of monophosphothreonyl ERK1/2 was rapid but delayed in comparison with dually-phosphorylated ERK1/2. Of 10 agonists studied, endothelin-1 and PMA were most effective in terms of ERK1/2 activation and in stimulating the appearance of monophosphothreonyl and dually-phosphorylated ERK1/2. Thus, enzymically active monophosphothreonyl ERK1/2 are formed endogenously following activation of the ERK1/2 cascade and we suggest that monophosphothreonyl ERK1/2 arise by protein tyrosine phosphatase-mediated dephosphorylation of dually-phosphorylated ERK1/2.     

Proteomic analysis of in vivo phosphorylated synaptic proteins

In the nervous system, protein phosphorylation is an essential feature of synaptic function. Although protein phosphorylation is known to be important for many synaptic processes and in disease, little is known about global phosphorylation of synaptic proteins. Heterogeneity and low abundance make protein phosphorylation analysis difficult, particularly for mammalian tissue samples. Using a new approach, combining both protein and peptide immobilized metal affinity chromatography and mass spectrometry data acquisition strategies, we have produced the first large scale map of the mouse synapse phosphoproteome. We report over 650 phosphorylation events corresponding to 331 sites (289 have been unambiguously assigned), 92% of which are novel. These represent 79 proteins, half of which are novel phosphoproteins, and include several highly phosphorylated proteins such as MAP1B (33 sites) and Bassoon (30 sites). An additional 149 candidate phosphoproteins were identified by profiling the composition of the protein immobilized metal affinity chromatography enrichment. All major synaptic protein classes were observed, including components of important pre- and postsynaptic complexes as well as low abundance signaling proteins. Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest that a small number of kinases phosphorylate many proteins and that each substrate is phosphorylated by many kinases. These data substantially increase existing knowledge of synapse protein phosphorylation and support a model where the synapse phosphoproteome is functionally organized into a highly interconnected signaling network.     

The Versatility of Helicobacter pylori CagA Effector Protein Functions: The Master Key Hypothesis

Several bacterial pathogens inject virulence proteins into host target cells that are substrates of eukaryotic tyrosine kinases. One of the key examples is the Helicobacter pylori CagA effector protein which is translocated by a type‐IV secretion system. Injected CagA becomes tyrosine‐phosphorylated on EPIYA sequence motifs by Src and Abl family kinases. CagA then binds to and activates/inactivates multiple signaling proteins in a phosphorylation‐dependent and phosphorylation‐independent manner. A recent proteomic screen systematically identified eukaryotic binding partners of the EPIYA phosphorylation sites of CagA and similar sites in other bacterial effectors by high‐resolution mass spectrometry. Individual phosphorylation sites recruited a surprisingly high number of interaction partners suggesting that each phosphorylation site can interfere with many downstream pathways. We now count 20 reported cellular binding partners of CagA, which represents the highest quantitiy among all yet known virulence‐associated effector proteins in the microbial world. This complexity generates a highly remarkable and puzzling scenario. In addition, the first crystal structure of CagA provided us with new information on the function of this important virulence determinant. Here we review the recent advances in characterizing the multiple binding signaling activities of CagA. Injected CagA can act as a ‘master key’ that evolved the ability to highjack multiple host cell signalling cascades, which include the induction of membrane dynamics, actin‐cytoskeletal rearrangements and the disruption of cell‐to‐cell junctions as well as proliferative, pro‐inflammatory and anti‐apoptotic nuclear responses. The discovery that different pathogens use this common strategy to subvert host cell functions suggests that more examples will emerge soon.     

Identification of phosphorylation sites in mammalian mitochondrial ribosomal protein DAP3

Mammalian mitochondrial ribosomes synthesize 13 proteins that are essential for oxidative phosphorylation. In addition to their role in protein synthesis, some of the mitochondrial ribosomal proteins have acquired functions in other cellular processes such as apoptosis. Death-associated protein 3 (DAP3), also referred to as mitochondrial ribosomal protein S29 (MRP-S29), is a GTP-binding pro-apoptotic protein located in the small subunit of the ribosome. Previous studies have shown that phosphorylation is one of the most likely regulatory mechanisms for DAP3 function in apoptosis and may be in protein synthesis; however, no phosphorylation sites were identified. In this study, we have investigated the phosphorylation status of ribosomal DAP3 and mapped the phosphorylation sites by tandem mass spectrometry. Mitochondrial ribosomal DAP3 is phosphorylated at Ser215 or Thr216, Ser220, Ser251 or Ser252, and Ser280. In addition, phosphorylation of recombinant DAP3 by Protein kinase A and Protein kinase Cdelta at residues that are endogenously phosphorylated in ribosomal DAP3 suggests both of these kinases as potential candidates responsible for the in vivo phosphorylation of DAP3 in mammalian mitochondria. Interestingly, the majority of the phosphorylation sites detected in our study are clustered around the highly conserved GTP-binding motifs, speculating on the significance of these residues on protein conformation and activity. Site-directed mutagenesis studies on selected phosphorylation sites were performed to determine the effect of phosphorylation on cell proliferation and PARP cleavage as indication of caspase activation. Overall, our findings suggest DAP3, a mitochondrial ribosomal small subunit protein, is a novel phosphorylated target.     

N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT)

The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~ 55 kDa), higher than predicted (~ 50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 °C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~ 35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~ 47 kDa protein; substitution of both sites gave a smaller (~ 35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH₂ or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.     

Differential processing of mammalian l-histidine decarboxylase enzymes

In the mammalian species studied so far, the l-histidine decarboxylase (HDC) enzyme responsible for histamine biosynthesis has been shown to undergo post-translational processing. The processing is best characterized for the mouse enzyme, where di-asparate DD motifs mediate the production of active ∼55 and ∼60 kDa isoforms from the ∼74 kDa precursor in a caspase-9 dependent manner. The identification of conserved di-aspartate motifs at similar locations in the rat and human HDC protein sequences has led to proposals that these may represent important processing sites in these species also. Here we used transfected Cos7 cells to demonstrate that the rat and human HDC proteins undergo differential processing compared to each other, and found no evidence to suggest that conserved di-aspartate motifs are required absolutely for processing in this cell type. Instead we identified SKD and EEAPD motifs that are important for caspase-6 dependent production of ∼54 and ∼59 kDa isoforms in the rat and human proteins, respectively. The addition of staurosporine, which is known to pharmacologically activate caspase enzymes, increased processing of the human HDC protein. We propose that caspase-dependent processing is a conserved feature of mammalian HDC enzymes, but that proteolysis may involve different enzymes and occur at diverse sites and sequences.     

Prokaryotic overexpression of TEV–rhGH and characterization of its polyclonal antibody

Recombinant protein technology represents one of the best solutions to achieve rapid, efficient, and cost-effective protein expression and purification of therapeutic proteins. Growth hormone (GH) is an excellent example of these proteins used in the therapy of hormone deficiencies. In this work, a plasmid, pRSET–TEV–rhGH, has been constructed to overexpress recombinant human GH (rhGH) by cloning its gene downstream of an N-terminal 6 × His-tagged polypeptide (43 aa) in the T7 promoter-plasmid pRSET. This polypeptide was cleavable by means of the integrated recognition site for the tobaccos etch virus (TEV) protease, resulting in an rhGH protein at an exact length and sequence. After IPTG induction, this plasmid effectively expressed TEV–rhGH protein (27 kDa) in the cytoplasm of Escherichia coli, which accumulated in the form of inclusion bodies. The 6 × His-tagged protein, with a yield of ~ 150 mg/L of culture, was purified from the cell extract using metal affinity chromatography, as shown after SDS-PAGE blue staining, and was confirmed by immunoblotting using specific commercial monoclonal antibodies. In order to detect TEV–rhGH, in ELISA and immunoblotting, specific polyclonal antibody, with high titer (~ 10^− 5 fold dilution), was produced in a rabbit and purified using affinity chromatography. Preliminary tests have proved that TEV–rhGH protein and its specific purified IgG antibody could provide valuable tools for rhGH productive and diagnostic purposes.     

Comparative analysis of Leishmania exoproteomes: Implication for host–pathogen interactions

Leishmaniasis is a vector-borne disease caused by the protozoa Leishmania. We have analyzed and compared the sequences of three experimental exoproteomes of Leishmania promastigotes from different species to determine their specific features and to identify new candidate proteins involved in interactions of Leishmania with the host. The exoproteomes differ from the proteomes by a decrease in the average molecular weight per protein, in disordered amino acid residues and in basic proteins. The exoproteome of the visceral species is significantly enriched in sites predicted to be phosphorylated as well as in features frequently associated with molecular interactions (intrinsic disorder, number of disordered binding regions per protein, interaction and/or trafficking motifs) compared to the other species. The visceral species might thus have a larger interaction repertoire with the host than the other species. Less than 10% of the exoproteomes contain heparin-binding and RGD sequences, and ~ 30% the host targeting signal RXLXE/D/Q. These latter proteins might thus be exported inside the host cell during the intracellular stage of the infection. Furthermore we have identified nine protein families conserved in the three exoproteomes with specific combinations of Pfam domains and selected eleven proteins containing at least three interaction and/or trafficking motifs including two splicing factors, phosphomannomutase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, the paraflagellar rod protein-1D and a putative helicase. Their role in host–Leishmania interactions warrants further investigation but the putative ATP-dependent DEAD/H RNA helicase, which contains numerous interaction motifs, a host targeting signal and two disordered regions, is a very promising candidate.     

Cooperative KaiA–KaiB–KaiC Interactions Affect KaiB/SasA Competition in the Circadian Clock of Cyanobacteria

The circadian oscillator of cyanobacteria is composed of only three proteins, KaiA, KaiB, and KaiC. Together, they generate an autonomous ~ 24-h biochemical rhythm of phosphorylation of KaiC. KaiA stimulates KaiC phosphorylation by binding to the so-called A-loops of KaiC, whereas KaiB sequesters KaiA in a KaiABC complex far away from the A-loops, thereby inducing KaiC dephosphorylation. The switch from KaiC phosphorylation to dephosphorylation is initiated by the formation of the KaiB–KaiC complex, which occurs upon phosphorylation of the S431 residues of KaiC. We show here that formation of the KaiB–KaiC complex is promoted by KaiA, suggesting cooperativity in the initiation of the dephosphorylation complex. In the KaiA–KaiB interaction, one monomeric subunit of KaiB likely binds to one face of a KaiA dimer, leaving the other face unoccupied. We also show that the A-loops of KaiC exist in a dynamic equilibrium between KaiA-accessible exposed and KaiA-inaccessible buried positions. Phosphorylation at the S431 residues of KaiC shift the A-loops toward the buried position, thereby weakening the KaiA–KaiC interaction, which is expected to be an additional mechanism promoting formation of the KaiABC complex. We also show that KaiB and the clock-output protein SasA compete for overlapping binding sites, which include the B-loops on the CI ring of KaiC. KaiA strongly shifts the competition in KaiB's favor. Thus, in addition to stimulating KaiC phosphorylation, it is likely that KaiA plays roles in switching KaiC from phosphorylation to dephosphorylation, as well as regulating clock output.     

Endoplasmic reticulum stress-mediated inhibition of NSMase2 elevates plasma membrane cholesterol and attenuates NO production in endothelial cells

Chronic exposure of blood vessels to cardiovascular risk factors such as free fatty acids, LDL-cholesterol, homocysteine and hyperglycemia can give rise to endothelial dysfunction, partially due to decreased synthesis and bioavailability of nitric oxide (NO). Many of these same risk factors have been shown to induce endoplasmic reticulum (ER) stress in endothelial cells. The objective of this study was to examine the mechanisms responsible for endothelial dysfunction mediated by ER stress. ER stress elevated both intracellular and plasma membrane (PM) cholesterols in BAEC by ~ 3-fold, indicated by epifluorescence and cholesterol oxidase methods. Increases in cholesterol levels inversely correlated with neutral sphingomyelinase 2 (NSMase2) activity, endothelial nitric oxide synthase (eNOS) phospho-activation and NO-production. To confirm that ER stress-induced effects on PM cholesterol were a direct consequence of decreased NSMase2 activity, enzyme expression was either enhanced or knocked down in BAEC. NSMase2 over-expression did not significantly affect cholesterol levels or NO-production, but increased eNOS phosphorylation by ~ 1.7-fold. Molecular knock down of NSMase2 decreased eNOS phosphorylation and NO-production by 50% and 40%, respectively while increasing PM cholesterol by 1.7-fold and intracellular cholesterol by 2.7-fold. Furthermore, over-expression of NSMase2 in ER-stressed BAEC lowered cholesterol levels to within control levels as well as nearly doubled the NO production, restoring it to ~ 74% and 68% of controls using tunicamycin and palmitate, respectively. This study establishes NSMase2 as a pivotal enzyme in the onset of endothelial ER stress-mediated vascular dysfunction as its inactivation leads to the attenuation of NO production and the elevation of cellular cholesterol.