In addition, as recent reports have indicated that heat shock proteins- (HSPs-) inducing drugs can help to ameliorate neurodegenerative diseases associated with tau pathology, we also discuss therapeutic strategies for PD focusing on inhibition of α-synuclein- and LRRK2-associated tau phosphorylation by HSPs.There is now a considerable body of experimental evidence that Parkinson's disease arises through physiological interaction of causative molecules, leading to tau pathology. In this review, we discuss the physiological role of  A histopathological study has detected the phosphorylated microtubule-associated protein tau in the LBs of some PD patients [Parkinson's disease (PD), a chronic and progressive movement disorder caused by dopaminergic neuron loss in the substantia nigra, is the second most common neurodegenerative disease after Alzheimer's disease (AD). Although several causative genes of PD have been identified, the molecular mechanism responsible is not yet fully understood. The neuropathological hallmark of PD is the appearance of intraneuronal protein aggregates known as Lewy bodies (LBs) consistent with filamentous inclusions composed of patients . Abnormapatients . Althoug2+/calmodulin-dependent protein kinase II (CaMKII), and protein kinase C (PKC) [β phosphorylates numerous sites on tau, and site-specific phosphorylation of tau by this kinase significantly reduces its tubulin-binding ability, resulting in neuronal cell degeneration [β as a potential therapeutic target for neurodegenerative diseases associated with tau pathology.Tau is one of the microtubule-associated proteins that promotes tubulin assembly and stability of microtubule and is predominantly expressed in the central nervous system , 4. Tau  C (PKC) . It has  C (PKC) , 17, andneration . Therefoα-Syn and leucine-rich repeat kinase-2 (LRRK2) with tau-related neuronal pathology. The presence of abnormally phosphorylated tau in LBs and the brain lesions of some PD patients with LRRK2 mutations strongly suggest that α-Syn and LRRK2 are closely associated with PD tau pathology. Our previous biochemical studies have indicated that α-Syn and LRRK2 interact directly with GSK-3β and enhance tau phosphorylation by the kinase [α-Syn and LRRK2 as PD-related molecules and the possibility of developing therapeutic targets.There is now accumulated pathological and biochemical evidence for an association of e kinase –21. Agaiα-Syn is a soluble protein composed of 140 amino acids and is abundantly detectable in the nucleus and presynapse of the neuronal cell [α-Syn is considered to be involved in functions such as the regulation of neuronal vesicular transmission and synapse plasticity [α-Syn comprises about 1% of total protein in the neuronal cytoplasm and is mainly expressed in the cerebral neocortex, hippocampus, substantia nigra, thalamus, and cerebellum [α-Syn is differentially expressed in different brain regions [α-Syn can be detected in both mitochondria and cytoplasm, whereas in the cerebral cortex and cerebellum it is abundant in cytoplasm but not in mitochondria [α-Syn interacts with the phospholipid plasma membrane and Golgi apparatus [α-Syn acts as molecular chaperone protein for formation of the soluble NSF attachment protein receptor (SNARE) protein complex involved in the fusion of synaptic vesicles with the cell membrane by assisting with the folding and refolding of synaptic proteins [α-Syn has a particularly important role in synaptic activation.nal cell . α-Syn iasticity . α-Syn crebellum . It has  regions . In the chondria , 27. It pparatus , 29. In proteins . Therefoα-Syn is known to be a major protein component of LBs [α-Syn, resulting in its excessive accumulation, may be responsible for the loss of dopaminergic neurons observed in PD. Braak et al. [α-Syn aggregation begins in the olfactory bulb, medulla oblongata, and gut and then gradually spreads to the midbrain, ultimately reaching the cerebral cortex. However, the molecular mechanism of α-Syn aggregation and spread is not yet fully understood. Interestingly, a recent study has revealed that intracellular accumulation of α-Syn in pathogenic brain regions is a common pathological feature of many neurological disorders [α-Syn, including PD, DLB, and multiple system atrophy, are collectively referred to as synucleinopathies [α-Syn share many common pathways and pathogenic mechanisms. Several studies focusing on the biological and physiological correlations between α-Syn and tau have noted that (i) polymorphisms of α-Syn and tau are commonly associated with sporadic PD [α-Syn and tau are colocalized in the same proteinaceous aggregates in some synucleinopathies and tauopathies, such as PD with dementia (PDD), DLB, and Lewy body variant of AD (LBVAD) [In the substantia nigra of PD patients, aggregates of the acidophilic protein, LBs, are evident, and t of LBs . Accordik et al. , 33 haveisorders . Accordiopathies . The curradic PD , 36 and  (LBVAD) , 37, 38.α-Syn and aggregation of abnormal phosphorylated tau are independent mechanisms that occur in different neurodegenerative diseases. However, a previous study has demonstrated that α-Syn and abnormal phosphorylated tau were colocalized in LBs in the brain tissue of patients with sporadic PD and DLB [α-Syn interacts directly with tau [+)/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) increases the expression of α-Syn and the phosphorylation level of tau at Ser262, Ser396, and Ser404 in cultured neuronal cells and wild-type mice, but not in α-Syn-knockout mice [β are the kinases responsible for α-Syn-dependent phosphorylation of tau [α-Syn-dependent phosphorylation of tau protein, we demonstrated that α-Syn directly stimulates the GSK-3β-mediated phosphorylation of tau at Ser396 and autophosphorylation of the kinase, without affecting the native kinase activity (phosphorylation of glycogen synthase) [β at Tyr216 in cultured cells is increased by MPP+ treatment [β is enhanced by phosphorylation at Tyr216 catalyzed by certain Tyr-kinases and autophosphorylation of GSK-3β [α-Syn does not induce Tyr216 phosphorylation of GSK-3β, it stimulates autophosphorylation of both the Ser and Thr sites in vitro [β alone is insufficient to induce hyperphosphorylation of tau, GSK-3β-related phospho-tau pathogenesis may require certain mediators that can coassociate with GSK-3β and tau molecules. Our previous study showed that α-Syn bound directly to GSK-3β and that α-Syn formed a tripartite complex with tau and GSK-3β in vitro [α-Syn-mediated formation of a tripartite complex with tau and GSK-3β initiates the phosphorylation of tau by GSK-3β.In general, it has been considered that formation of LBs containing  and DLB . More dewith tau , (ii) thn of tau , 42. In ynthase) . Anotherf GSK-3β . Althougα- and β-Syn isoforms equivalently stimulate GSK-3β-mediated phosphorylation of tau, the γ-Syn isoform was found to have no stimulatory effects on tau phosphorylation by GSK-3β [α- and β-Syn isoforms in tau phosphorylation by GSK-3β may be attributable to their structural features. α-Syn is an intrinsically unfolded protein with an N-terminal domain (residues 1–60) comprising seven imperfect KTKEGV sequence repeats, a middle hydrophobic domain (residues 61–95) termed the non-Abeta component (NAC) domain, and a C-terminal domain with a large proportion of acidic residues (residues 96–140) [γ-Syn does not match those of the other two isoforms. Although tau binds only to the acidic C-terminal domain of α-Syn, GSK-3β binds to the N-terminal region containing both the KTEGV repeat and the NAC domain [γ-Syn may not be able to form tripartite complexes with tau and GSK-3β, perhaps accounting for its lack of any stimulatory effect on tau phosphorylation by GSK-3β.The synuclein family comprises three different isoforms . Althougy GSK-3β . The com 96–140) . Among tC domain . Thus, γα-Syn and β-Syn exhibit similar levels of expression, although α-Syn mRNA decreases and β-Syn mRNA increases in an age-dependent manner [α-Syn mRNA and a decrease in the level of β-Syn mRNA in the substantia nigra of patients with PD, DLBD, and LBVAD [β-Syn is undetectable in LB, whereas the sequence homology and structure of β-Syn are similar to those of α-Syn [α-Syn- or β-Syn-mediated hyperphosphorylation of tau may occur in different neurodegenerative diseases. However, the specificity and physiopathological significance of α- and β-Syn for hyperphosphorylation of tau remain to be elucidated.Previously, it was revealed that t manner , 45. On nd LBVAD . Moreoveof α-Syn . From alα-Syn protein expression is upregulated by oxidative stress and mitochondrial dysfunction [α-Syn in neurons is estimated to be 70–140 μM [μM [α-Syn is 1 : 35–1 : 70. Our previous study has indicated that the optimum molar ratio of α-Syn and tau for inducing maximum phosphorylation by GSK-3β is 1 : 20 [α-Syn-mediated hyperphosphorylation of tau by GSK-3β may occur only when α-Syn is present at high concentrations induced by cellular stresses, such as oxidative stress and mitochondrial dysfunction.It seems likely that function . Under p0–140 μM , whereas0 μM [μM , 49. Thus 1 : 20 . Thus, αβ, defined by its Tyr216 phosphorylation, is insufficient to induce the hyperphosphorylation of tau. In our review, we hypothesize that a scaffolding molecule, such as α-Syn, capable of forming a complex between tau and GSK-3β, is absolutely essential for tau hyperphosphorylation by GSK-3β , and MAP Kinase Kinases (MKK) 4 and 7 [ in vitro and in vivo [LRRK2 is a 2527 amino acid protein with a molecular mass of approximately 250 kDa and unique domain architecture. LRRK2 is predicted to possess multiple functional domains, such as the armadillo repeat (ARM), ankyrin repeat (ANK), leucine-rich repeat (LRR), Ras of complex GTPase (Roc), the Roc C-terminal, a protein kinase, and WD40 (a tryptophan-aspartic acid repeat domain) . Basic b 4 and 7 , althoug in vivo . Thus it in vitro experiment using recombinant tau and LRRK2, we identified Thr181 of tau as the direct target site for LRRK2 phosphorylation [Our previous study has demonstrated that LRRK2 directly phosphorylates tubulin-associated tau but does not phosphorylate free tau . Furtherrylation . Anotherrylation . More rerylation . All of β [β-dependent mechanisms. Therefore, we performed a similar experiment with α-Syn to determine the physiological relationship between LRRK2 and GSK-3β in the tau phosphorylation mechanism. We found that LRRK2 directly interacts with GSK-3β, and this interaction enhances the kinase activity of GSK-3β in vitro [β by LRRK2. From these results, it is speculated that upon interaction with LRRK2, GSK-3β undergoes a conformational change, thus activating its intrinsic kinase activity. We also demonstrated that phosphorylation of tau at Ser396 was increased in LRRK2-overexpressing SH-SY5Y cells. Thus, our study suggests that LRRK2 may also contribute to tau phosphorylation through a GSK-3β-dependent mechanism [β.In the G2019S-LRRK2 transgenic mouse brain, an increased level of tau phosphorylation at Ser396/404 has been observed . Since pβ , we expe in vitro, whereas the R1441C mutation did not [β than the WT or the other two mutants and that only the G2019S mutant enhanced GSK-3β-dependent tau phosphorylation. Thus G2019S-LRRK2 may contribute to abnormal tau phosphorylation through both direct and GSK-3β-dependent mechanisms. This hypothesis is strongly supported by a previous report describing that (i) G2019S-LRRK2-transgenic Drosophila neurons exhibit GSK-3β-mediated hyperphosphorylation and mislocalization of tau [β, whereas R1441C does not bind to GSK-3β in the Drosophila model [β is specifically enhanced by G2019S mutation in vitro, whereas other mutations such as R1441C and I2020T have no such effect [β, LRRK2 mutants other than G2019S may contribute to tau phosphorylation through other tau kinase-dependent mechanisms. In fact, LRRK2 has been reported to interact with and phosphorylate Ste20 serine/threonine kinase family members , TAOK3 being a kinase with high sequence homology to MARKK [Several previous studies have reported tau-related neuronal pathology in the brains of PD patients with LRRK2 mutations and also in LRRK2-transgenic animal models –73. In p did not . We alson of tau , (ii) G2la model , and  are a group of proteins that act as molecular chaperones and are involved in the folding and stabilization of numerous signaling proteins. They have cytoprotective functions against many forms of cellular stress, such as heat shock and oxidative stress . In thisα-Syn and prevents aggregation of the latter [ in vitro study has also revealed that HSP70 inhibits the α-Syn-dependent phosphorylation of tau by GSK-3β, without affecting the α-Syn-independent normal phosphorylation of tau by the kinase [α-Syn-mediated complex formation between tau and GSK-3β by binding directly to the NAC domain of α-Syn [α-Syn-mediated abnormal tau phosphorylation, without any toxic effect on the normal function of tau regulated by GSK-3β-mediated phosphorylation. In our previous study, we found that HSP70 suppresses the phosphorylation of tau by GSK-3β both in vitro [α-Syn-overexpressing cultured neuronal cells (unpublished data). Therefore, we consider that an HSP70-inducing agent might be effective for treatment of PD by preventing α-Syn-associated tau phosphorylation.Several studies have shown that HSP70 binds to the NAC domain of e latter  and alsoe latter . Our pree kinase . Furtherof α-Syn . These fin vitro  and in αβ, which is required for activation of the latter. In addition, HSC70 shows over 90% sequence homology with HSP70 [β complex.HSP90 interacts with LRRK2, and inhibition of this association leads to proteasomal degradation of LRRK2, but not via the lysosomal pathway , 86. Furth HSP70 . HSP70 hth HSP70 , suggestβ reduces tau phosphorylation and aggregation in a mouse model [β inhibitors with the aim of developing therapies for AD [β have been developed using pharmacological approaches, but details of their possible side effects have yet to emerge. GSK-3β was first discovered as a protein kinase involved in the physiological regulation of glycogen metabolism, and a number of subsequent biochemical studies demonstrated that it has diverse physiological functions in the cell [β inhibitors may affect not only the abnormal signaling pathway implicated in neurodegeneration but also normal pathways required for maintaining physiological function. Indeed, it has been shown that lithium, a classical GSK-3β inhibitor, exerts toxic side effects in some elderly patients [α-Syn or LRRK2, which are capable of preventing the formation of α-Syn- or LRRK2-mediated tripartite complexes with tau and GSK-3β, may act as more specific inhibitors of abnormal tau phosphorylation, without affecting normal tau phosphorylation by GSK-3β. As described above, HSP interacts with α-Syn and LRRK2 [α-Syn- and LRRK2-associated tau phosphorylation catalyzed by GSK-3β.Previous pharmacological studies have shown that inhibition of GSK-3se model . Some phs for AD . Up to nthe cell . Therefopatients , and thend LRRK2 , 90 and α-Syn and LRRK2. Geldanamycin, a classical HSP90 inhibitor, may suppress LRRK2-mediated tau phosphorylation by reducing the protein expression of LRRK2, because inhibition of HSP90 binding with LRRK2 leads to proteasomal degradation of LRRK2. In addition, this drug increases the expression of HSP70 and HSP40 via activation of the HSF-1 transcriptional factor [α-Syn- and LRRK2-associated tau phosphorylation by inducing the expression of HSP70.Currently, the protective function of HSP70 against AD pathology has been highlighted by the fact that some HSP70-inducing agents such as estrogen, geranylgeranylacetone (GGA), curcumin, and celastrol affect AD pathology at different pathological levels . In partl factor . Therefoα-Syn or LRRK2 might be prevented by HSPs. Therefore, we propose that induction of HSPs expression might be able to prevent the pathogenesis of PD associated with abnormal tau phosphorylation. As a future task, discovery of a pharmacological modulator of HSPs capable of crossing the blood-brain barrier would be important. This might be applicable as an effective and safe therapeutic drug for PD, preventing the abnormal phosphorylation of tau mediated by α-Syn and LRRK2 (Finally, we conclude that the pathogenetic mechanisms of tau phosphorylation involving nd LRRK2 .


Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac+ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac+ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA) whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes.Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of  Listeria monocytogenes is a human pathogen linked to foodborne illness outbreaks involving dairy, poultry, and ready-to-eat (RTE) meat products. It has been a recurrent threat to RTE deli meats and hotdogs, frequently due to post-process contamination ) and able to diffuse into bacterial cells because of their net-neutral charge. When the pH is above the pKa of the acid, it is mostly in the dissociated form ([Ac−] + [H+]) and the charge on the lactate anion prevents its entry into cells . In the current study the pH of the Bac− spent broth (and five of six Bac+ preps) was at ~pH 4.37 and above the pKa of lactic acid and showed no inhibitory activity on agar when applied to lawns of L. monocytogenes 39-2 indicator cells  obtained against curvaticin FS47 was designated “R1” . A comparison of inhibitory bacteriocin spots on lawns of L. monocytogenes R0 and R1 demonstrated that the spontaneous resistance against Lb. curvatus bacteriocin FS47 gave cross-resistance to P. acidilactici bacteriocin Bac3 (Lb. curvatus bacteriocin Beef3 [L. monocytogenes 39-2 (R1) while others were still inhibitory to it  were classified as having the same MOA while those that were still inhibitory to R1  were considered to possess another MOA. This process was again repeated by isolating additional enterocin FS56-1 BacR mutants against R1 that possessed accumulated bacteriocin resistances, now designated “R2”  of still another MOA (L. monocytogenes (R0) and its two consecutively-derived BacR variants  were used as indicator organisms to screen for new Bac+ isolates as well as screen bacteriocin preparations in microplate inhibition assays [A bacteriocin-resistant variant of the wild-type cin Bac3  and Lb. in Beef3 . Some bary to it B. The dited “R2” C. Activither MOA C. The win assays . − preps in microplate growth inhibition assays in which L. monocytogenes 39-2 (R0) was treated with neutralized or non-neutralized cell-free culture supernatants (− control strain (Lb. delbrueckii 4797-2) were added to L. monocytogenes R0, some inhibition was observed whereby R0 did not grow as well as in the Bac− control assay while all the Bac+ preparations showed baseline-level inhibition  are presumed to have the same or highly similar MOA while those bacteriocins that were still inhibitory to R1 were considered to have a different MOA. The data obtained in microplate growth assays with L. monocytogenes 39-2 (R1) suggests that curvaticin FS47, curvaticin Beef3, and pediocin Bac3 belong to the same mode of action, MOA1.Microplate inhibition assays were also performed with the  variant B and gro variant . As obsehibition A wherebyhibition B. This ttrain R1 B. All ofL. monocytogenes 39-2 R0 and R1 performed with non-neutralized culture supernatants, we observed full growth of L. monocytogenes 39-2 (R2), partial inhibition of R2 by the Bac− supernatants, and full inhibition from the Bac+ supernatants  still showed baseline-level inhibition of R2  LAB obtained from our culture collection or from retail foods and animal sources [+ LAB included Lactobacillus curvatus FS47 and Beef3, Pediococcus acidilactici Bac3, Lactococcus lactis FLS1, Enterococcus faecium FS56-1 and En. thailandicus FS92. All cultures were propagated twice before use. Master cultures were maintained by resuspension of cell pellets in milk-based freezing media  after centrifugation  and stored frozen. L. monocytogenes 39-2  were grown in tryptic soy broth . The parent strain, L. monocytogenes 39-2 was isolated from packages of retail frankfurters [L. monocytogenes “cocktail” for inoculation of RTE meats evaluating meat surface pasteurization [Select bacteriocin-producing  at 30 °C and centrifuged at 8000 rpm (4 °C) for 10 min . The supernatants  were filter-sterilized with cellulose acetate syringe filters , aliquoted into 1 mL eppendorf tubes for use in experiments, and stored at 4 °C for short term experiments or at −20 °C for longer term storage.Cultures of BacThe possibility of inhibitory activity produced by other potential inhibitors  observed among some LAB was eliminated as described previously ,22.Culture supernatants were neutralized after centrifugation to remove the bulk of cells see . An Oakt+ preps were serially diluted by 2-fold dilutions in 0.1% buffered peptone water (BPW) in 96-well microtiter plates (Becton-Dickenson Labs) whereby the bacteriocin is diluted in half with each successive dilution. Indicator assay plates were made whereby L. monocytogenes 39-2 (8-log cfu/mL) was seeded at 1% dilution into soft TSA agar (0.75% agar), and then 5 mL of this seeded soft agar was overlaid onto normal TSA agar plates (1.5% agar). The indicator-seeded assay plates were marked in eight pie-section quadrants and 10 μL of each dilution was surface spotted onto the indicator lawns. Plates were allowed to incubate overnight at 30 °C and the titer end point was taken from highest dilution showing the last visible sign of inhibition. The titer was then designated as activity- or arbitrary-units (AU), and was determined as the reciprocal of the dilution × 100 (because 10 μL represents 1/100th of 1 mL) and reported as AU/mL. Filter-sterilized or pasteurized BacLAB cultures were propagated twice at 30 °C and centrifuged at 8000 rpm for 10 min at 4 °C . The centrifuged supernatant was transferred to sterile tubes and pasteurized at 80 °C for either 5-min (1 mL portions) suspended in a water-filled heating block. Pasteurized bacteriocins preparations were stored at 4 °C for near term experiments or frozen at −20 °C. Bacteriocin activity was determined as described previously and done in duplicate. Samples of pasteurized bacteriocin preparations were plated onto MRSA plates or inoculated into MRS broth to check the effectiveness of the pasteurization process.L. monocytogenes 39-2, bacteriocin-resistant colonies would appear at frequencies of approximately 10−5 to 10−7 . Colonies that arose against the bacteriocin were again streaked again on TSA + Bac+ (FS47) to insure isolation of a resistant phenotype. Bacteriocin-resistant (BacR) variants recovered in this manner were tested in spot tests with various cell-free bacteriocin preparations in comparison with the prior strain and was repeated with bacteriocins that still inhibited the BacRL. monocytogenes 39-2 variant. The use of bacteriocin-resistant variants as screening organisms was characterized by Macwana and Muriana [During prolonged incubation of inhibition zones during spot-on-lawn assays of bacteriocin preparations against  to 10−7 A. Bacter Muriana  as capabL. monocytogenes 39-2) and various culture supernatant preparations . Sterile MRS broth (not spent broth) was used as a control treatment and spent culture supernatant from bacteriocin-negative (Bac−) Lb. delbrueckii 4797 was used as a control to assess lactic acid effects. L. monocytogenes 39-2 was diluted and inoculated into double-strength TSB broth (~1 × 105 cfu/mL) from which 100 µL was distributed to various wells in a clear 96-well flat bottom microtiter plate (Becton Dickinson). Bacteriocin preparations (100 µL) were added and mixed by aspiration using a multi-channel pipette. Settings for the growth curve/turbidity analysis using a GENios microplate reader  were as follows: measurement mode: absorbance; measurement wavelength: 595 nm; number of flashes: 1; temperature range: 33–35 °C; shake duration : 10 s; kinetic interval: 1800 s; unit: optical density (OD); and total measurement time: 48 h. The 96-well plate was sealed with UltraClear film  to prevent evaporation of the liquid and well-to-well contamination. The OD595 values obtained were plotted against time and were used to illustrate the antilisterial activity of the bacteriocin preparations against L. monocytogenes.The microplate inhibition assay used a mixture of indicator organism  was used to perform one-way repeated measures analysis of variance (RM-ANOVA) to determine if significant difference exists between different treatments. For some assays, the Holm-Sidak one way ANOVA method was used to perform pair wise multiple comparisons with level of significance set at 0.05 .Microplate growth inhibition assays were repeated in triplicate and mean O.D. (595 nm) values were plotted L. monocytogenes 39-2 and to study the interactions of bacteriocins affecting different MOAs, a more reliable growth inhibition assay was developed. The comparison of non-neutralized and pH-neutralized preparations confirmed the participation of acid inhibition against L. monocytogenes, while evaluations performed with neutralized preparations allowed preferential evaluation of bacteriocin activities against L. monocytogenes. The comparison of wild-type L. monocytogenes 39-2 (R0) vs. bacteriocin-resistant variants  allowed a preferential selection of bacteriocins that are likely to work better together  and without confusion of whether activity is due to lactic acid. Although the primary interest was in evaluating only inhibition due to bacteriocin activity during in vitro assays, any additional inhibition by lactic acid during actual in-food applications would be an added and appreciable benefit. The use of such L. monocytogenes BacR variants allowed the differentiation of bacteriocins into different MOA’s  from which we may select an efficacious mixture of bacteriocin preparations for application as food preservatives, especially where recurrent problems with L. monocytogenes have been observed.Bacteriocinogenic LAB and their bacteriocin preparations are promising antimicrobials for application in food systems as effective biopreservatives, especially in RTE meat systems. However, crude screening methods such as agar diffusion assays alone would not be sufficient to forecast their effectiveness in meat systems that are highly complex. In this study we have employed agar diffusion assays (sandwich) only as a primitive approach to screen for new bacteriocinogenic LAB from different food samples. In order to obtain more detailed information on the effectiveness of the bacteriocin preparation on the inhibition of the


Background: The CFTR-Ser737 site has a phospho-dependent inhibitory effect on Cl− secretion.Results: LMTK2 phosphorylation of CFTR-Ser737 facilitates endocytosis, reduces cell surface density of CFTR, and inhibits Cl− secretion.Conclusion: Targeting LMTK2 regulates the cell surface density of CFTR Cl− channels.Significance: Targeting LMTK2 in CF patients may stabilize ΔF508-CFTR pharmacologically rescued to the cell surface. −-selective ion channel expressed in fluid-transporting epithelia. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser737 in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl− secretion mediated by the wild-type and rescued ΔF508-CFTR. Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-Ser737 mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl− channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl− channels and improve stability of pharmacologically rescued ΔF508-CFTR in patients with cystic fibrosis.Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl CFTR ithelia –, 5. CFTR surface –10. The surface –. Despitesurface ––15. Deleurface ––. Pharmacfunction . Severalfunction –19. Corr508-CFTR . The pre508-CFTR , 21. Hen508-CFTR –30.− channel is regulated by protein kinases including protein kinase PKA, PKC, and adenosine monophosphate-stimulated kinase (AMPK) (reviewed in Ref. 737 and Ser768 have a phospho-dependent inhibitory effect on the CFTR channel function (− secretion by maintaining the CFTR channels in a closed state (737 site in the recombinant CFTR regulatory domain can be phosphorylated by purified lemur tyrosine kinase 2 (LMTK2) in vitro  and whether the LMTK2-mediated phosphorylation regulates CFTR endocytic trafficking in human airway epithelial cells. We demonstrate that phosphorylation of CFTR-Ser737 mediated by LMTK2 facilitates endocytosis and reduces the cell surface density of CFTR Cl− channels in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl− secretion mediated by the wild-type (WT) and rΔF508-CFTR. Thus, interfering with LMTK2 phosphorylation of ΔF508-CFTR-Ser737 may serve as a novel strategy to improve the cell surface stability of pharmacologically rescued ΔF508-CFTR in CF patients.LMTK2 also known as kinase/phosphatase/inhibitor-2 (KPI2), brain-enriched kinase (BREK), apoptosis-associated tyrosine kinase (AATYK2), and cyclin-dependent kinase-5 (cdk5/p35) regulated kinase, is a member of the lemur family of membrane-anchored kinases –41. Desp–42–Fig. 1e domain , 41. Theactivity , 50. Theactivity , an actiactivity . At the activity . Some of2 at a density ∼7 × 105/cm2 as previously described and used for experimentation following 6–8 weeks of culture at an air-liquid interface  cells (WT-CFTR homozygous) were received from Dr. Joseph Pilewski  –53. HBE –2 at density ∼1 × 106) coated with plating medium containing Dulbecco's modified Eagle's medium  and 10% purified collagen  and used for experimentation following 3–4 weeks of culture at an air-liquid interface as described previously . Cells were seeded on Transwell permeable supports  coated with plating medium and used for experimentation following 7–9 days of culture at an air-liquid interface as described previously , were a generous gift from Dr. J. P. Clancy from the Department of Pediatrics, Cincinnati Children's Hospital, Cincinnati, OH , 55. CFBeviously , 57.HEK293 cells from the American Type Culture Collection were transfected with plasmid DNA, seeded on plastic tissue culture plates, and used 24 h later for kinase assays. HEK293 cells were also used as a well-accepted model to express recombinant proteins and conduct biochemical studies including the kinase assay.The following anti-human CFTR antibodies were used: mouse monoclonal, clone 596  and clone M3A7 , and rabbit polyclonal Ab-737 . Other antibodies used were rabbit anti-LMTK2 (Sigma-Aldrich) and anti-LMTK2 kinase domain , mouse anti-Na,K-ATPase (Millipore), anti-FLAG M2 (Sigma-Aldrich), and anti-ezrin , and horseradish peroxide-conjugated goat anti-mouse, goat anti-rabbit, secondary antibodies (Bio-Rad). All antibodies were used at the concentrations recommended by the manufacturer. The following reagents were used: Complete Protease Inhibitor Mixture and PhosSTOP phosphatase inhibitor mixture tablets , the adenylate cyclase activator forskolin and the cAMP phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) (Sigma-Aldrich), and the inhibitor of protein serine/threonine phosphatases calyculin A .m NaCl, 50 mm Tris, pH 7.4, 1% IGEPAL (Sigma-Aldrich), 5 mm MgCl2, 5 mm EDTA, 1 mm EGTA, 30 mm NaF, 1 mm Na3VO4, and Complete Protease Inhibitor mixture . After centrifugation at 14,000 × g for 15 min to pellet insoluble material, the soluble lysates were pre-cleared by incubation with protein G or protein A, as appropriate, conjugated to Sepharose beads  at 4 °C. The pre-cleared lysates were added to the protein G- or protein A-Sepharose beads antibody complexes. CFTR was immunoprecipitated by incubation with the mouse M3A7 antibody and LMTK2 was immunoprecipitated by incubation with the rabbit anti-LMTK2 kinase domain antibody. Non-immune mouse or rabbit IgGs  were used as controls. After washing the protein G- or protein A-Sepharose beads antibody complexes with the IP buffer, immunoprecipitated proteins were eluted by incubation at 85 °C for 5 min in sample buffer (Bio-Rad) containing 100 mm DTT. Immunoprecipitated proteins were separated by SDS-PAGE using 7.5% gels (Bio-Rad) and analyzed by Western blotting. The immunoreactive bands were visualized with Western Lightning Chemiluminescence Reagent Plus .Endogenous CFTR and LMTK2 were immunoprecipitated from Calu-3 or HBE cell lysates by methods described previously , 15, 27.6) were plated on collagen-coated tissue culture plates and incubated with the optimized transfection mixture containing 10 nm siRNA at 37 °C. The transfection medium was removed after 24 h and cells were cultured on the tissue culture plates until confluent. Under these conditions cells reached confluence at 96 h, and experiments were conducted at 96 h. Silencing the target genes resulted in the corresponding protein depletion by ∼70%. We aimed at such level of silencing to avoid off-target effects that may occur with more dramatic gene silencing.Transfection of CFBE41o- cells with siRNA targeting human LMTK2 gene  or the siRNA negative control  was conducted using HiPerFect Transfection Reagent (Qiagen) according to the manufacturer's instructions as we previously described , 10. For6) were plated on tissue culture plates and incubated with the optimized transfection mixture containing 50 nm siRNA at 37 °C. After 24 h, cells were trypsinized and plated on collagen-coated Snapwell permeable supports and cultured for an additional 6 days to establish polarized monolayers . All experiments were done under the same cell culture conditions to assure similar cellular polarization as well as protein expression and trafficking  or shRNAmir negative control (RHS4348) in the lentiviral vector pGIPZ with TURBO-GFP reporter  was carried at MOI 0.25 according to manufacturer's instructions. Cells transduced with shRNA were selected with puromycin for 5 days, subcultured to collagen-coated Snapwell filters at 1.0 × 10The WT-LMTK2Δ-FLAG plasmid was constructed by inserting part of the human LMTK2 sequence coding for the first 600 amino acid residues corresponding to the transmembrane and kinase domain with an engineered C-terminal FLAG into pcDNA3.1 vector (Invitrogen) as previously described . The humm Tris, pH 7.4, and 150 mm NaCl. LMTK2 was immunoprecipitated with the anti-FLAG M2 antibody (Sigma) and protein G-agarose beads. The beads-antibody-protein complexes were washed three times with RIPA buffer and once with 2× kinase buffer . The kinase assay was performed by incubating the beads for 2 h at 30 °C in a total reaction volume of 50 μl containing 1× kinase buffer, 10 μm ATP, and 20 μm of a peptide corresponding to CFTR residues 733–741 (ERRLSLVPD). A control reaction was performed as above with the exclusion of cell lysates. Extent of phosphorylation was determined using the Kinase-Glo Luminescent Assay (Promega) that assesses the amount of ATP remaining after the reaction and normalized for the amount of ATP in the control reaction.Kinase assays were performed as described previously . Brieflym HEPES, pH 8.0, 1% Triton, 10% glycerol, and Complete Protease Inhibitor Mixture (Roche Applied Science), as described previously in detail , followed by cell lysis in buffer containing 25 mn detail , 59. Biol-glutathione  at 4 °C. At this point in the protocol, biotinylated proteins reside within the endosomal compartment. Subsequently, cells were lysed, and biotinylated proteins were isolated by streptavidin-agarose beads, eluted into SDS-sample buffer, and separated by 7.5% SDS-PAGE. The amount of biotinylated CFTR at 4 °C and without the 37 °C warming was considered 100%. The amount of biotinylated CFTR remaining at the plasma membrane after GSH treatment at 4 °C and without the 37 °C warming was considered background (<7% compared with the amount of biotinylated CFTR at 4 °C without GSH treatment) and was subtracted from the CFTR biotinylated after warming to 37 °C at each time point. CFTR endocytosis was calculated after subtraction of the background and was expressed as the percent of biotinylated CFTR at each time point after warming to 37 °C compared with the amount of biotinylated CFTR present before warming to 37 °C. We have previously shown that the culture conditions and state of epithelial cell polarization affect CFTR endocytosis in human airway epithelial cells  were measured in Ussing-type chambers  as previously described (2/95% air. Short circuit current (Isc) was measured by voltage-clamping the transepithelial voltage across the monolayers to 0 mV with a voltage/current clamp . Transepithelial resistance was measured by periodically applying a 2.5-mV bipolar voltage pulse and was calculated using Ohm's law. The apical bath solution contained (in mm): 115 Na-gluconate, 5 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, 10 mannitol (pH 7.4). The basolateral bath solution contained (in mm): 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, 10.0 glucose (pH 7.4). A low Cl−, high-Na+, high-gluconate, apical bath solution was used to prevent cell swelling due to the increased apical Cl− permeability under these conditions, as previously described (SCI was recorded. Amiloride (10 μm) was added to the apical bath solution to inhibit Na+ absorption through ENaC. Subsequently, Isc was stimulated with forskolin (10 μm) and IBMX (50 μm) added to the apical and basolateral bath solutions followed by thiazolidonone CFTR inhibitor CFTRinh-172 (5 μm) added to the apical bath solution to inhibit CFTR-mediated Isc. Data are expressed as forskolin/IBMX stimulated Isc, calculated by subtracting the baseline Isc from the peak stimulated Isc.The short circuit currents . The means were compared by a two-tailed B). Next, we examined localization of LMTK2 in CFBE41o- cells, an immortalized human airway epithelial cell model because unlike HBE these cells are constantly available. Cells were cultured for 7–9 days in air-liquid interface as polarized monolayers. LMTK2 was detected at both the apical and basolateral membrane domain of similar to HBE cells .Little is known about the distribution of LMTK2 in human airway epithelial cells. If LMTK2 affects CFTR, it would be expected that it localized, like CFTR, at the apical plasma membrane. Thus, we first examined localization of LMTK2 in primary differentiated HBE cells by domain-selective cell surface biotinylation and Western blotting . HBE celFig. 1D and E). Taken together, the co-immunoprecipitation between endogenous CFTR and LMTK2 and their plasma membrane distribution in primary human airway epithelial cells suggest a physical association between CFTR and LMTK2 within the apical plasma membrane.We predicted that LMTK2 could interact with CFTR in polarized human airway epithelial cells. We used Calu-3 cells to study immunoprecipitation because these cells express both CFTR and LMTK2 endogenously, form polarized monolayers, and are constantly available as immortalized cells. Calu-3 cells were cultured for 3–4 weeks on semi-permeable growth supports as polarized monolayers. CFTR was immunoprecipitated with a monoclonal anti-CFTR antibody (M3A7), and LMTK2 was immunoprecipitated with a polyclonal anti-LMTK2 antibody. Western blot analysis demonstrated that endogenous CFTR and LMTK2 co-immunoprecipitate in a reciprocal fashion , D and EA). As determined by cell surface biotinylation and Western blotting, knockdown of LMTK2 increased the steady-state plasma membrane abundance of CFTR (A and B).We hypothesized that LMTK2 controls CFTR trafficking at the plasma membrane in human airway epithelial cells. To test this hypothesis, LMTK2 expression was knocked down in CFBE41o- cells stably expressing WT-CFTR by RNA interference. We used CFBE41o- cells because these immortalized cell lines localize LMTK2 at the plasma membrane similar to the primary HBE cells as shown in Fig. 2− secretion is determined by the activity and number of CFTR Cl− channels at the plasma membrane. Because LMTK2 knockdown increased the plasma membrane abundance of CFTR, we analyzed the functionality of the rescued fraction, predicting that it would also increase CFTR-mediated Cl− secretion. siLMTK2 increased the forskolin/IBMX-stimulated Isc across CFBE41o- monolayers  or with a siRNA control (siCTRL) and cultured on collagen-coated tissue culture plates for 96 h as monolayers. Endocytic assays were performed by the GSH protection assay , 60. As m) and forskolin (20 μm) added to the apical and basolateral medium at 37 °C for 10 min before experiments to raise the intracellular cAMP levels and promote PKA-mediated phosphorylation (m), added to the basolateral medium at 37 °C for 15 min before experiments . By contrast, CFTR phosphorylation was greatly enhanced after the forskolin/IBMX and calyculin A treatment . To demonstrate specificity of the phosphosite antibody, parental CFBE41o- cells were transiently transfected with WT-CFTR or CFTR with the S737A substitution (CFTR-S737A) inactivating the phosphorylation site . Together, these results demonstrate that the phosphosite antibody Ab-737 recognizes specifically the CFTR-pS737.To determine whether CFTR is an LMTK2 substrate in airway epithelial cells, CFBE41o- cells stably expressing WT-CFTR and cultured as polarized monolayers on permeable growth supports for 7–9 days were treated with IBMX . By contrast, ATP levels were similar in the reaction containing either the KM-LMTK2Δ-FLAG or vector control indicating that the K168M mutation impaired phosphorylation of the CFTR-derived peptide (A and B). These results confirm the prediction that the residue Lys168 located in the catalytic domain of LMTK2 is critical for catalysis.To ascertain that the K168M mutation impairs LMTK2 kinase activity, we transfected HEK293 cells with the WT-LMTK2Δ-FLAG and KM-LMTK2Δ-FLAG or empty pcDNA3.1 vector and the resulting protein fragments were immunoprecipitated with anti-FLAG M2 antibody. The immunoprecipitated protein complexes were used to  peptide , A and B peptide , A and B737 using the phosphosite antibody Ab-737. CFBE41o- cells stably expressing WT-CFTR were transfected with WT-LMTK2Δ-FLAG, KM-LMTK2Δ-FLAG, or vector control (V). Forty-eight hours after transfection cells were treated with calyculin A (50 nm) added to the basolateral medium at 37 °C for 15 min before experiments. CFTR-pS737 detected by immunoblotting with antibody Ab-737 was normalized for total CFTR detected with antibody CFF596. CFTR-pS737 abundance was decreased in cells expressing KM-LMTK2Δ-FLAG compared with WT-LMTK2Δ-FLAG or vector control (C and D). Our conclusion was that the LMTK2 fragment with inactive kinase KM-LMTK2Δ-FLAG attenuated phosphorylation of CFTR-Ser737 because it competed with endogenous LMTK2 for phospohorylation of the CFTR-Ser737 site in CFBE41o- cells.Next, we assessed phosphorylation of CFTR-Ser control , C and D737 in human airway epithelial cells. Studies were conducted to determine whether CFTR endocytosis is regulated by the LMTK2 phosphorylation of CFTR-Ser737. Endocytic assays were performed to examine the effects of WT-LMTK2Δ-FLAG and KM-LMTK2Δ-FLAG on CFTR endocytosis. We determined the time course of CFTR endocytosis in CFBE41o- cells stably expressing WT-CFTR cultured for 48 h as monolayers on collagen-coated tissue culture plates. CFTR endocytosis reached maximum at 7.5 min (A and B). Thus, in subsequent experiments we examined CFTR endocytosis at the 7.5-min time point. Differences in the magnitude and maximum of CFTR endocytosis in CFBE41o- cells cultured as polarized monolayers on permeable growth supports in 65–C and D). Moreover, the KM-LMTK2Δ-FLAG increased the steady-state plasma membrane CFTR abundance when compared with the WT-LMTK2Δ-FLAG or vector control (C and E). Inhibition of CFTR endocytosis is consistent with increased CFTR abundance at plasma membrane in the KM-LMTK2Δ-FLAG-transfected cells. Because expression of the WT-LMTK2Δ-FLAG did not increase phosphorylation of CFTR (C and D) these conditions were not expected to alter endocytosis or the plasma membrane abundance of CFTR compared with vector control. Indeed, the WT-LMTK2Δ-FLAG had no effect on endocytosis or the plasma membrane abundance of CFTR compared with vector control (C--E). Together, the above results demonstrate that the LMTK2 phosphorylation of CFTR-Ser737 facilitates CFTR endocytosis and reduces CFTR abundance at the plasma membrane.The above data have shown that LMTK2 facilitates CFTR endocytosis and that LMTK2 phosphorylates CFTR-Ser 7.5 min , A and B control , C and E of CFTR , C and D control , C--E. TC and F). By contrast, internalization of the KM-LMTK2Δ-FLAG was decreased by 50%. Compared with the WT-LMTK2Δ-FLAG, the KM-LMTK2Δ-FLAG fragment was more abundant at the plasma membrane due to decreased endocytosis (C and F). These data demonstrate for the first time the dynamic, kinase domain-dependent, spatial, and temporal regulation of LMTK2 distribution between the cell surface and endocytic compartments, consistent with the role of LMTK2 in endocytic trafficking in human airway epithelial cells.Nothing is known about membrane trafficking of LMTK2 in human airway epithelial cells. Thus, to gain insight, we examined the plasma membrane abundance and endocytic uptake of the LMTK2 fragments in CFBE41o- cells. Approximately 70% of the WT-LMTK2Δ-FLAG was internalized in 7.5 min , C and Focytosis , C and F737 induces CFTR endocytosis, eliminating the phosphorylation site by the S737A substitution should reduce CFTR endocytosis. Parental CFBE41o- cells were transfected with WT-CFTR or the CFTR-S737A mutant. We determined the time course of WT-CFTR endocytosis in parental CFBE41o- cells cultured for 48 h as monolayers on collagen-coated tissue culture plates. CFTR endocytosis reached maximum at 5 min (data not shown). Thus, in subsequent experiments we examined CFTR endocytosis at the 5-min time point. The S737A mutation attenuated CFTR endocytosis and increased the plasma membrane abundance of CFTR . VX-809 partially rescued the biosynthetic processing defect of ΔF508-CFTR demonstrated by appearance of the mature, fully glycosylated CFTR band C (C). Compared with control (shCTRL), LMTK2 knockdown increased band C abundance and the forskolin/IBMX-stimulated Isc across CFBE41o- monolayers . When compared with the LMTK2 knockdown, the kinase deficient LMTK2 fragment produced lesser effect on the cell surface abundance and endocytosis of CFTR . Our interpretation is that endogenous LMTK2 diminished the effects of the kinase deficient fragment when compared with LMTK2 knockdown. Increased AMPK activity was proposed to facilitate phosphorylation of Ser737 and inhibition of CFTR in non-stimulated epithelia . These data emphasize that the effects of LMTK2 knockdown on CFTR were mediated via alterations of post-maturational trafficking of CFTR during endocytic uptake and not during the biosynthetic processing of CFTR.LMTK2 knockdown increased CFTR mediated Cl508-CFTR  and 10. 508-CFTR  and 10. 508-CFTR , 61, 71.FTR .Using the primary differentiated human bronchial epithelial cells and a human polarized airway epithelial cell model CFBE41o- we have shown that LMTK2 targets to the apical and basolateral plasma membrane. Moreover, we provide novel information that the kinase domain dynamically regulated the endocytic uptake of LMTK2 in human airway epithelial cells , C and FIn summary our data provide direct evidence that CFTR endocytosis is regulated by phosphorylation mediated by the LMTK2 kinase domain. Because rescued ΔF508-CFTR has decreased plasma membrane stability we propose that targeting LMTK2 phosphorylation of CFTR may offer a novel approach to investigate the stability defect and to design pharmacological approaches to stabilize rescued ΔF508-CFTR. Our findings may also point to a new direction in elucidating the mechanisms of decreased CFTR function in lung diseases resulting from cigarette smoke inhalation.


Archaeoglobus fulgidus (Afu Pol-D). The C-terminal region of DP2 contains two conserved cysteine clusters, and their roles are investigated using site-directed mutagenesis. The cluster nearest the C terminus is essential for polymerase activity, and the cysteines are shown to serve as ligands for a single, critical Zn2 + ion. The cysteines farthest from the C terminal were not required for activity, and a role for these amino acids has yet to be defined. Additionally, it is shown that Afu Pol-D activity is slowed by the template strand hypoxanthine, extending previous results that demonstrated inhibition by uracil. Hypoxanthine was a weaker inhibitor than uracil. Investigations with isolated DP2, which has a measurable polymerase activity, localised the deaminated base binding site to this subunit. Uracil and hypoxanthine slowed Afu Pol-D “in trans”, that is, a copied DNA strand could be inhibited by a deaminated base in the alternate strand of a replication fork. The error rate of Afu Pol-D, measured in vitro, was 0.24 × 10− 5, typical for a polymerase that has been proposed to carry out genome replication in the Archaea. Deleting the 3′–5′ proofreading exonuclease activity reduced fidelity twofold. The results presented in this publication considerably increase our knowledge of Pol-D.Archaeal family-D DNA polymerases (Pol-D) comprise a small (DP1) proofreading subunit and a large (DP2) polymerase subunit. Pol-D is one of the least studied polymerase families, and this publication investigates the enzyme from   •Archaeal DNA Polymerase-D binds Zn, this is essential for activity.•Archaeal DNA Polymerase-D is inhibited by uracil and hypoxanthine.•cis and in trans.Inhibition by deaminated bases occurs both in •Deaminated bases are sensed by the large (polymerase) subunit.•Polymerase-D has high fidelity, compatible with genome replication. Saccharomyces cerevisiae, initially implicated Pol δ in lagging strand copying and Pol ε in leading Thermococcus kodakarensis and Methanococcus maripaludis, Pol-B is not essential, inferring that Pol-D constitutes the single replicative polymerase 2 + and an Fe–S cluster Archaeoglobus fulgidus (Afu), Methanopyrus kandleri, Methanothermobacter thermoautotrophicus, and Thermococcus gammatolerans in Escherichia coli. Our group has previously produced Pyrococcus furiosus (Pfu) Pol-D in this bacterium Accurate transmission of the data encoded within the genome requires faithful replication, a function performed by the replisome, a multiprotein complex of which DNA polymerases form a key component 2CX8CX2CX9CX2CX6-18CX2C and a reduced version CX2CX8CX2C, which seems restricted to the Methanococcales, Methanobacteriales, Thermoplasmatales, and Nanoarchaeales. The second set, closer to the C terminus has the consensus CX2CX12-14CX2C. In some instances, CX2C is compacted to CXC, this shorter version seems unrelated to any particular Archaeal group and is distributed randomly. We have used site-directed mutagenesis to probe the role of these residues in Afu Pol-D, and three mutants, each having two closely spaced cysteines replaced by alanine, were prepared: mut2, C663A/C666A; mut4, C688A/C691A; and mut5, C1062A/C1065A . Clearly, this cysteine cluster, and any putative metals it binds, is not required for polymerase activity. Additionally, these thiols do not play any role in the previously observed inhibition of Pol-D by template strand uracil Alignment of the amino acid sequence of the DP2 subunit of Pol-D sequence reveals two cysteine clusters . The firA/C1065A a. PrimerA/C1065A a. In conA/C1065A b. These trans” inhibition of Pfu Pol-D; the presence of uracil in the lagging template not only slows its copying but also retards the extension of the leading strand and vice versa trans” inhibition ability. An initial experiment showed, as expected, that Afu Pol-D itself behaved in a similar fashion to Pfu Pol-D, and a single uracil in either the lagging or the leading template inhibits the copying of both strands . With Afu DP2, the control fork mimic, lacking any uracil, can be extended to full length in both branches, although less efficiently than observed with Afu Pol-D , and Afu DP2. The results are given in − 5. A previous investigation, using a PCR-based method, with Pol-D from the archaeon Thermococcus species 9°N reported a higher error rate of 95 × 10− 5− 5 is much more accurate and, as an additional control, the fidelities of Pfu Pol-B and Taq Pol were measured and found to agree with previous results, strengthening confidence in the data seen with Pol-D in vitro assay, have been reported as ≤ 1.3 × 10− 5 and ≤ 0.2 × 10− 5, respectively in vitro, is compatible with it functioning as a genome copying polymerase. Using a single amino acid substitution in DP1 to disable the proofreading exonuclease increased the error rate by a factor of two. Interestingly, Afu-DP2, in which the entire DP1 subunit is absent, demonstrated the same fidelity as the H325A variant, suggesting that DP1 subunit plays a straightforward role in fidelity by simply supplying the proofreading exonuclease active site. With eukaryotic Pol δ, the proofreading exonuclease makes a similarly small contribution, although its influence is much more pronounced with Pol ε lacZα gene was completely sequenced for the 52 white (mutant) colonies observed, and the results are summarised in Supplementary Fig. S3. Although the mutations are distributed across the lacZα sequence, a hotspot is apparent at a run of four adenines, with 18% to 28% of the changes occurring in this region. At this site, Afu Pol-D, Afu Pol-D exo−, and Afu DP2 are prone to cause insertions or deletions, probably due to polymerase slippage during elongation . Base substitutions are also overrepresented at or near a run of three guanines and one run of two guanines . Base transitions are the most frequently observed change followed by frameshifts, with transversions being least abundant  and in trans . Trans-inhibition is never seen with Pol-B, where deaminated bases stall replication only at the strand on which they are located, emphasising a fundamental difference in the mechanism by which Pol-B and Pol-D respond to the uracil/hypoxanthine. Third, the fidelity of Pol-D has been accurately determined and shown to be compatible with a genome copying enzyme. The proofreading exonuclease activity has only a modest, about twofold, influence on accuracy. Probably, the outstanding question concerns the function of the cysteine cluster away for the C terminus. These amino acids are not required for polymerase activity or deaminated base sensing. It may be that these cysteines support an Fe–S cluster, which, in the recent years, have been demonstrated in many nucleic acid processing enzymes such as glycosylases, primases, helicases, nucleases, transcription factors, RNA polymerases, and RNA methyltransferases E. coli under aerobic conditions, proved negative; and UV/visible spectroscopy, ICP-MS, and inorganic sulphide analysis gave no indication for the presence of such a system (data not shown). Attempted expression under anaerobic conditions, with iron and sulphur supplementation and using E. coli strains enhanced for Fe–S biosynthesis E. coli. This is also the case with eukaryotic polymerases, where assembly of the Fe–S centre requires expression yeast rather than E. coliThe Pol-D was first observed in the archaea in the late 1990s UK BBSRC (grant number: BB/K005359/1). We thank Dr. Patrik Jones  for E. coli BL21(DE3)∆ iscR and Professor Dennis Dean  for E. coli BL21(DE3) (pDB1282).This work was supported by the


Two isoforms of Rho-associated protein kinase (ROCK), ROCKI and ROCKII, play a pivotal role in regulation of cytoskeleton and are involved in multiple cellular processes in mammalian cells. Knockout mice experiments have indicated that the functions of ROCKI and II are probably non-redundant in physiology. However, it is difficult to differentiate the activation status of ROCKI and ROCKII in biological samples. Previously, we have identified phosphorylation site of ROCKII at Ser1366 residue sensitive to ROCK inhibition. We further investigated the activity-dependent phosphorylation site in ROCKI to establish the reagents that can be used to detect their individual activation.The phosphorylation site of ROCKI sensitive to its inhibition was identified to be the Ser1333 residue. The ROCKI pSer1333-specific antibody does not cross-react with phosphorylated ROCKII. The extent of S1333 phosphorylation of ROCKI correlates with myosin II light chain phosphorylation in cells in response to RhoA stimulation.Active ROCKI is phosphorylated at Ser1333 site. Antibodies that recognize phospho-Ser1333 of ROCKI and phospho-S1366 residues of ROCKII offer a means to discriminate their individual active status in cells and tissues. They −/− and ROCKII−/− mice have been generated ATP at 30°C for 20 min. The reaction was stopped and products were separated by SDS-PAGE, transferred to a PVDF membrane. The phosphorylation status and amounts of the proteins were detected by autoradiography and Western blotting with anti-ROCKI antibody, respectively.Flag-ROCKI-expressing cells were harvested in an IP buffer  as an antigen to immunize rabbits. Anti-sera were collected and sequentially affinity purified by phosphopeptide- and non-phosphopeptide-conjugated columns .in vitro kinase reaction using immunoprecipitated flag-tagged ROCKI protein in the presence of [γ-32P]ATP. Autoradiography detected the phosphorylation signal, which was abolished by including ROCK inhibitor Y27632 in the in vitro kinase reaction  that were treated with nocodazole to activate the RhoA signal via GEF-H1 released from microtubules disassembly . As showIt is known that ROCKs form dimer -32. In tSmall-molecule inhibitors against ROCK, such as Y27632 and Fasudil, have been developed to have potential in clinical implication ,36,37. ISer1333 phosphorylation can indicate the active status of ROCKI in response to RhoA signaling. Thus, antibodies that recognize phosphorylation at Ser1333 and S1366 residues of ROCKI and II, respectively, are capable of probing their corresponding activation in biological samples. Also, these antibodies might be very useful reagents for drug screening of inhibitors specific against ROCKI and ROCKII isoform.The authors declare that they have no competing interests.HHC and SWL performed experiments. HHL and ZFC designed the study and wrote the paper. All authors read and approved the final paper.


Diel vertical migration (DVM) of zooplankton is a global phenomenon, characteristic of both marine and limnic environments. At high latitudes, patterns of DVM have been documented, but rather little knowledge exists regarding which species perform this ecologically important behaviour. Also, in the Arctic, the vertically migrating components of the zooplankton community are usually regarded as a single sound scattering layer (SSL) performing synchronized patterns of migration directly controlled by ambient light. Here, we present evidence for hitherto unknown complexity of Arctic marine systems, where zooplankton form multiple aggregations through the water column seen via acoustics as distinct SSLs. We show that while the initiation of DVM during the autumnal equinox is light mediated, the vertical positioning of the migrants during day is linked more to the thermal characteristics of water masses than to irradiance. During night, phytoplankton biomass is shown to be the most important factor determining the vertical positioning of all migrating taxa. Further, we develop a novel way of representing acoustic data in the form of a Sound Image (SI) that enables a direct comparison of the relative importance of each potential scatterer based upon the theoretical contribution of their backscatter. Based on our comparison of locations with contrasting hydrography, we conclude that a continued warming of the Arctic is likely to result in more complex ecotones across the Arctic marine system. Diel vertical migration (DVM) of zooplankton is a characteristic feature of the world's oceans and lakes, and is suggested to be the largest synchronized movement of biomass on the planet . Researcet al. . We present the results from a study of DVM in the Arctic collected from four contrasting sites around the high Arctic archipelago of Svalbard (78–80°N) during the autumn equinox, combining net-based zooplankton collection with moored acoustic instruments at a time of strong diel change in illumination. Based on this, we address our two main research questions. First, to what extent is there uniformity in the patterns of DVM occurring in arctic locations with contrasting hydrography? Second, can variations in DVM patterns be related to the physical environment? When observing DVM with acoustics, it is inevitable that we are also interested in the species composition of any SSL. Therefore, net samples were used both to detect organisms present at different depth layers in the water column and as a basis for calculating the theoretical backscatter of organisms that Helmer Hanssen between 16th and 28th of September 2010. The period for observations was centred on the equinox to ensure the maximum diel cycle in solar irradiance. Data were collected at four sites in the Svalbard archipelago: Kongsfjorden, Rijpfjorden, Billefjorden and an Ice Station north of Spitsbergen  profiles as a measure of phytoplankton biomass, measured as emitted fluorescence at 685 nm. Additionally, a Seabird Electronics 19+ CTD unit fitted with a Biospherical Instruments scalar irradiance sensor, Eo , was placed on the moorings.At each site, an instrumented mooring was deployed for a minimum of 36 h, thereby ensuring that the nets and acoustics covered at least one full DVM cycle. The moorings in the shallower fjord sites were anchored to the seabed and retrieved using an acoustic release. At the deeper Ice Station, the mooring was suspended from the ship, which then followed the path of the ice floe to which it was anchored; drifting ∼4.5 nautical miles during the 2-day sampling period. At each site, a Seabird Electronics 911 CTD unit was used to obtain vertical profiles of temperature and salinity. The unit was fitted with a calibrated Seapoint fluorometer to provide Chl Supplementary data, Fig. S1. Swimming speeds of the constituent zooplankton are inferred from the inclination of the SSLs (Each mooring was fitted with 307.2 kHz RDI Acoustic Doppler Current Profilers (ADCP) . The sampling depths were standardized between sites and are presented in Table Calanus spp. from each sample were identified to stage and then measured according to Kwasniewski et al.  in the upper 50 m (100 m in Kongsfjorden). At the Ice Station, with a high concentration of sea ice, the larger MIK net was replaced by the smaller WP3 net . For all nets, the counts were converted to individuals per unit volume (ind. m−3) and are presented in Table Macrozooplankton was collected during one LSN and one LM with a MIK net , s the (standard deviation of L)/L, D the mean body width (m), Dβ = L/D, f the acoustic frequency in Hz = 307.2 × 103, and c the speed of sound in water, defined here as 1500 m s−1. The theoretical backscatter  of each taxonomic group of zooplankton present in the water column is then estimated by . The theoretical volume backscatter at abundance level of 1 ind. m−3 was calculated, which is numerically identical to the TS and is referred to here as Sv(1). Of the species treated in the present study, the chaetognaths and the ctenophore Mertensia ovum are most likely the two taxa least suited to the bent fluid model used for calculating target strength  and small (mean length 8 mm) krill. All groups examined are hereafter referred to as “species”. To enable a viable comparison between each of the species' individual contributions to the measured backscatter levels, both the numerical difference (Δ Sv) in Sv(1) values between the species and the linear transformation (according to (dB/10) are presented in Table From the theoretical estimate of backscatter for each zooplankton group, we are then able to identify those organisms that will dominate the measured acoustic signals. TS was calculated for each of the most common species from the region, with the lower limit in size being the older (≥CIV) copepodite stages of n is the estimated Sv value (Eq. 2) for species n.We then calculate a total estimated backscatter for each net haul, which we term the Sound Image (SI). The SI (using Equation 3) is the sum over all theoretical backscatter values for each species at each site and is based on the abundance data from the net hauls presented in Table The SI reflects the relative contribution (in dB) from each species to the total estimated backscatter, enabling a more direct comparison of backscatter contribution than from single species dB values alone.The relative contribution to the SI of each species is presented as sectors within a pie chart, where the entire pie represents the entire SI value. All calculations for the SI are based on MIK nets (or WP3 at the Ice Station), except the values for calanoid copepods which are based on MPS samples in the equivalent depth intervals.As the MIK net used to sample the macrozooplankton could not be closed, the SI value can only be determined unequivocally for the upper part of the water column at LSN and LM for each site. We make the assumption that zooplankton species performing DVM will have different abundances in the upper layer during LM compared with LSN due to DVM behaviour. Calculations for the theoretical Sv values also assume a homogenous distribution of scatterers, but by definition an SSL is an aggregation of organisms. Therefore, the calculated Sv values will under-estimate backscatter values compared with the measured backscatter within an SSL. Taking this into consideration, and the unavoidable problem of calibration  from one fixed depth is overlain on the backscatter distribution. The right-hand panel in each sub-figure shows the mean Sv profile at LSN with the SSLs identified as the peaks in backscatter relative to the mean Sv value for the deployment. These SSLs are numbered for reference, with the circle indicating the position of maximum Sv value within each SSL  distributions detected by the ADCPs at each site are presented in Fig. SSL Fig. .There is a difference in the number and vertical distribution of SSLs between each of the sample sites Fig. . Focusina (μg L−1) in the upper 100 m and the position of the surface SSL at midnight (LM) at each of the four sites. The Chl a profiles are comparable at all three fjord localities with maximum values measured around 20. At each of these sites, the peak of the SSL at LM is also located at ∼20 m, coincident with the peak in Chl a. At the Ice Station, the surface SSL also shows peak backscatter at 20 m, but the highest Chl a is found immediately under the ice. In most sites, there appears to be a clear association between the vertical position of the zooplankton and the location of the Chl a maximum (Chl amax).In Fig. The peak backscatter in the surface at LM is located precisely at the depth of maximum density gradient, the pycnocline Fig.  with theCalanus spp. dominating within the fjords and Metridia longa at the Ice Station. The second most dominant group  was chaetognaths, followed by Mertensia ovum, krill and Themisto spp.  to be present at the Ice Station under the sea ice during daytime.The zooplankton community in the upper 50 m (100 m in Kongsfjorden) was sampled at all four sites during both LSN and LM  and their linear transformations are presented for all taxa examined in Table Calanus spp. and T. libellula were the two most dominant taxa at all the sites, with the exception of krill during LM in Rijpfjorden and Billefjorden and Metridia longa during LM at the Ice Station. In Kongsfjorden . Importantly, for all calculations of SI, the contribution by Mertensia ovum was disregarded, as this species is poorly suited to the bent fluid model used for calculating target strength  or little auks  are known to be common at all of the study sites (Gadus morhua) or capelin  are more dominant in Kongsfjorden compared with the other study sites . It is also a contribution to the Natural Environment Research Council program Oceans 2025. Funding to pay the Open Access publication charges for this article was provided by the CircA project.This work is a contribution to the NFR funded project CircA; Circadian rhythms of Arctic zooplankton from polar twilight to polar night—patterns, processes and ecosystem implications (project number


Background: γ-Tubulin moderates gene expression by accumulating in the nucleus during early cell division.Results: SadB-mediated phosphorylation of Ser385-γ-tubulin exposes the nuclear localization signal of γ-tubulin.Conclusion: SadB kinases regulate the cellular location of γ-tubulin and in this way control cell growth.Significance: All knowledge on tubulins can aid the design of more efficient chemotherapeutic agents. 385 formed chromatin-associated γ-tubulin complexes that moderate gene expression. In this way, the C-terminal region of γ-tubulin regulates S-phase progression. In addition, chromatin levels of γ-tubulin were decreased by the reduction of SadB levels or expression of a non-phosphorylatable Ala385-γ-tubulin but were enhanced by expression of SadB, wild-type γ-tubulin, or a phosphomimetic Asp385-γ-tubulin mutant. Our results demonstrate that SadB kinases regulate the cellular localization of γ-tubulin and thereby control S-phase progression.γ-Tubulin is an important cell division regulator that arranges microtubule assembly and mitotic spindle formation. Cytosolic γ-tubulin nucleates α- and β-tubulin in a growing microtubule by forming the ring-shaped protein complex γTuRC. Nuclear γ-tubulin also regulates S-phase progression by moderating the activities of E2 promoter-binding factors. The mechanism that regulates localization of γ-tubulin is currently unknown. Here, we demonstrate that the human Ser/Thr kinase SadB short localizes to chromatin and centrosomes. We found that SadB-mediated phosphorylation of γ-tubulin on Ser During symmetric cell division, a cell becomes two identical daughter cells in a process that is highly regulated. An important regulator of cell division is γ-tubulin, which orchestrates microtubule assembly and ubiquitin-mediated processes during cell growth –7. In th–4–8–131 in cytosolic γ-tubulin by SADB kinases /BRSK1), γ-tubulin regulates centrosomal duplication and function (L (long), SADBS (short), and SADBS1 (L) have been described  that regulates its nuclear accumulation remains elusive. In the present study, we found that there are two pools of γ-tubulin, one cytosolic and the other nuclear. Using mammalian cell lines , we demoL (pcDNA3-FLAG-SADBL) was a gift from Dr. J. Sanes ), Ser385-Cγ-tubGFP (γ-tubulin(334-452)), His6-γ-tubulin, His6-Ala131-γ-tubulin, and GST-γ-tubulin were prepared as reported previously  or into the mammalian expression vector pcDNA3.1 (Invitrogen) using the following primers: 5′-CGCGGATCCACCATGTCGTCCGGGGCCAAGGA-3′ and 5′-CGCGAATTCCCTCCTCACTGCGCAGCTC-3′; 5′-GCGAAGCTTACCATGGATTATAAAGATGATGATGATAAAATGTCGTCCGGGGCCAAGGA-3′and 5′-CGCGAATTCTTACTCCTCACTGCGCAGCT-3′.Human γ-tubulin fragments and His6-Asp131-γ-tubulin were obtained by PCR from γ-tubulin/pcDNA3-GFP and Asp131-γ-tubulin/pcDNA3-GFP (131-γ-tubulin). Ser385 and Ser383 were replaced in the various constructs using a QuikChange site-directed mutagenesis kit (Stratagene) and the following primers (mutated bases underlined): 5′-GATGGCCAACCACACCAGCATCGATTCGCTCTTCGAGAGAACCTGTCG-3′ and 5′-CGACAGGTTCTCTCGAAGAGCGAATCGATGCTGGTGTGGTTGGCCATC3′ (S385D); 5′-GGCCAACCACACCAGCATCGCCTCGCTCTTCGAGAGAAC-3′ and 5′-GTTCTCTCGAAGAGCGAGGCGATGCTGGTGTGGTTGGCC-3′ (S385A); and 5′-CATGATGGCCAACCACACCGGCATCTCCTCGCTCTTCG-3′ and 5′-GCAAGAGCGAGGAGATGCCGGTGTGGTTGGCCATCATG-3′ (S383G). Each mutation was verified by sequence analysis.Human C-terminal GFP-tagged γ-tubulin/pcDNA3 was provided by Dr. J. Bartek , pcDNA3-J. Sanes . SADB shlin334-45), His6-γlin334-45), His6-γ32P]ATP ; and SDS-PAGE reagents (Bio-Rad). All other reagents were obtained from Sigma.The following antibodies and reagents were used: anti-GCP2 (Atlas Antibodies); anti-histone (Chemicon/Millipore); anti-GCP3 anti-GFP, anti-cyclin E, anti-cyclin A, anti-E2F1, and anti-cyclin B ; anti-γ-tubulin  and anti-FLAG (Sigma); anti-α-tubulin ; anti-cyclin D ; anti-phospho-histone H1 ; protein G PLUS-agarose, protein A PLUS-Sepharose, and [γ-m Tris, (pH 7.5), 150 mm NaCl, 1 mm dithiothreitol, 1 mm EGTA, 1 mm MgCl2, 0.1 mm GTP, 0.5% Triton X-100, 0.1 mm Na3VO4, 2 μg/ml aprotinin, 10 μm leupeptin, 1 μg/ml pepstatin, and 1 mm phenylmethylsulfonyl fluoride) and immunoprecipitated, as described  or washed and incubated in normal growth medium for various times 385)-γ-tubulin levels in the centrosomes or nucleus was calculated.NIH3T3 or U2OS cells were cultured and fixed as reported (S: 5′-TCGGGCCGGGACCAAGGGCACCATGT-3′ and 5′-TCGGGCCTCCTTGGGCGTCAGTCTCCC-3′ (N terminus); 5′-CCTGGTTCTGGAGCACGTCTCGGG-3′ and 5′-CGGGTGCAGGGGTCTTGGGGTCTTACTC-3′ (C terminus). The entire SadBS sequence was then cloned into the HindIII/EcoRI sites of pcDNA3 (Invitrogen) with the FLAG epitope introduced 5′ of hSadBS or into BamHI/EcoRI sites of pGEX2T. We encountered difficulties in cloning SADBS, because Escherichia coli DH5α frequently deleted the kinase domain, thereby causing a frameshift mutation creating a nonsense codon. One of the recovered mutants was SadBSΔ61–198, which was cloned into pET21d using the primers 5′-CGCGAATTCACCATGTCGTCCGGGGCCAA-3′ and 5′-GCGAAGCTTCTCCTCACTGCGCAGCTC-3′. The gene carried a nonsense codon that was removed using the QuikChange site-directed mutagenesis kit and these primers (inserted bases underlined): 5′-CCCATTATGCGTGGCTCCAGAGGTGATTAAG-3′ and 5′-CTTAATCACCTCTGGAGCCACGCATAATGGG-3′.Total RNA was isolated from U2OS cells as described elsewhere  and was purified as described (A rabbit anti-Ser(P)escribed .Total lysates from cells, SadB kinases, and HA- and FLAG-tagged immunoprecipitates were prepared as reported , 8. To iS was expressed in DH5α, and exponentially growing bacteria were induced with 0.2 mm isopropyl-1-thio-β-d-galactopyranoside at room temperature overnight. GST-γ-tubulin, C-terminal His6-tagged human SadBSΔ61–198, WT-γ-tubulin, Ala131-γ-tubulin, Asp131-γ-tubulin, WT-γ-tubulin(222-334), WT-γ-tubulin(335-451), Gly-383-γ-tubulin(335-451), or Ala385-γ-tubulin(335-451) was purified as described (S and γ-tubulin were excised from GST using thrombin (Amersham Biosciences).The SADB kinase assay and λ-phosphatase treatment were conducted as described . The N-tn < 4) or S.E. (n ≥ 4), and Student's paired t test was used to analyze differences. Cell cycle profiles were assessed using FlowJo . Western blotting bands were quantitated with ImageJ software.All data are expressed as mean ± S.D. (A and B). Upon S phase entry, γ-tubulin accumulated in the nucleus (A). During G2 phase, a successive decrease of chromatin-bound γ-tubulin was observed, reaching the lowest level in mitotic chromosomes (A). Finally, at early mitosis, γ-tubulin was found in the cytosol and centrosomes (A and B). In most eukaryotic cells, γ-tubulin links a growing microtubule to a γTuRC by interacting with GCP2 and GCP3 (B) and of GCP2 and α-tubulin by Western blotting (C). Although the localization of γ-tubulin varied (A and B), GCP2 and GCP3 occurred only in the cytosol, centrosomes, and mitotic spindles (B). Analysis of biochemical fractionations (C). These results indicate that the composition of chromatin-associated γ-tubulin complexes differs from γTuRC.To visualize the cellular localization of γ-tubulin during cell cycle , 10, 11, nucleus , 10, 11  nucleus A. Duringomosomes A. Finalltrosomes , A and Band GCP3 . To studand GCP3 B and of blotting C. Althoun varied , A and Bspindles B. Analysonations  confirmeonations C. These TUBULIN shRNA accumulated in G1 phase , an effect that was rescued by ectopic expression of either an sh-resistant γ-TUBULIN gene  or an sh-resistant C γ-tubulin terminus  but not by expression of the sh-resistant γ-tubulin N terminus (Nγ-tubGFP) (A).To identify the domain in γ-tubulin important for its effect on S-phase entry , we test-tubGFP) , suggest-tubGFP) A.399, Lys400, Arg409, and Lys410 (B) (X-Ser (Ser383-Ile384-Ser385) 14 amino acids upstream of Arg399 (B) (ShortSADB (SSADB) (GenBankTM accession number HQ830199) from U2OS cells. Expression of the SADB gene product, SadBS, was decreased in U2OS cells expressing SADB shRNA (C) . We have previously demonstrated that SADB kinases localized to centrosomes, but the exact cellular localization of the various SADB isoforms has not yet been elucidated. Biochemical fractionations of U2OS cells showed disparate localization of SadBL and SadBS. Endogenous SadBL and recombinant FLAG-SADBL localized to cytosolic and chromatin fractions (S and recombinant FLAG-SadBS were found mainly in chromatin fractions (E). Notably, elevated expression of mouse SADBL or hSadBS increased the pool of chromatin-bound γ-tubulin (E). A densitometric analysis of Western blots containing the different cellular fractions showed that increase protein levels of SadBS caused a 67.2%  rise of the amount of endogenous γ-tubulin in the chromatin (E). Immunofluorescence analysis of endogenous SadB and FLAG-SadBS and HA-SADBS confirmed that SadBS localized not only to centrosomes (F). Furthermore, compared with control cells, increased expression of SadBS raised the nuclear γ-tubulin content by 84% in U2OS (n = 8) and with 72% (n = 8) in NIH3T3 cells, creating distinct γ-tubulin domains in the nuclei of these cells (F). Thus, augmented nuclear γ-tubulin levels coincided with an accumulation of cells in S phase  ) encompasses the DNA-binding domain and the NLS, the latter of which includes residues Argd Lys410 B 1, 8)..399, Lyss410 (B) . Applyinf Arg399 B (8). ToDB shRNA C (2). Hu) encompae latter D. We havractions , whereasractions E. Notabl-tubulin E. A denshromatin E. Immunotrosomes F. Furthese cells F. Thus, 385 in the motif Ser383-Ile384-Ser385 (B), we investigated SADB molecules in kinase assays using various recombinant proteins as substrates. Immunopurified endogenous SADB and recombinant FLAG-SADBL and HA-SADBS phosphorylated bacterially produced human γ-tubulin(335-451) but not the corresponding fragment γ-tubulin(222-334), which lacks Ser385 (A). Moreover, SADBS phosphorylated the Gly383-γ-tubulin(335-451) but not the Ala385-γ-tubulin(335-451) substitution mutant (B). Finally, bacterially produced hSadBS phosphorylated the γ-tubulin(335-451) fragment (C). We noticed that the motif surrounding Ser385 is conserved in vertebrates and most invertebrates but not in the fission yeast Schizosaccharomyces pombe, which has an Ala instead of Ser385 but nonetheless highly conserved Ser383, Ser386, and motifs surrounding Ala385 (B). These findings suggest that the functions of this region in γ-tubulin are conserved among species.To detect whether SADB phosphorylates Ser4-Ser385 B, we invs Ser385 A. Moreovn mutant B. Finallfragment C. We notg Ala385 B. These 385-γ-tubulin levels and their potential dependence on SadB in cells, we prepared an anti-Ser(P)385 antibody that recognized purified bacterially produced full-length human γ-tubulin that had been phosphorylated in in vitro by purified bacterially produced SadBS, and notably, this signal was reduced by phosphatase treatment or incubation with the kinase-dead mutant SadBSΔ61–198 (D). To study a possible link between phosphorylation of Ser131-γ-tubulin and of Ser385, we analyzed the effect of phosphorylation on Ser385 in a non-phosphorylatable Ser131 → Ala γ-tubulin (E) and a phosphomimetic Ser131 → Asp γ-tubulin (F). We found that phosphorylation of Ser385 was reduced in the Ala131-γ-tubulin mutant but restored in the Asp131-γ-tubulin mutant, suggesting that phosphorylation of Ser131 is a prerequisite for the phosphorylation of Ser385. However, the band detected by the anti-Ser(P)385 antibody had a higher molecular mass than expected  and was not detected by total anti-γ-tubulin antibody (D–F) but was recognized by anti-Ser(P)131 antibody (E) (in vitro SadB kinases phosphorylate γ-tubulin on Ser385 and on Ser131.To examine the endogenous Ser(P)SΔ61–198 D. To stu-tubulin E and a p-tubulin F. We fouantibody , D–F butantibody E 2). Al. Al385-γ385-γ-tubulin, we varied their protein levels in U2OS cells. Analysis of U2OS cellular extracts using the anti-Ser(P)385 antibody showed an ∼60,000 Da band, which levels were affected upon increased recombinant SadBS levels (G) or reduced endogenous SadB (H) or γ-tubulin expression (I). These data show that the 60 kDa band is γ-tubulin and that SadBS regulates the cellular levels of Ser(P)385-γ-tubulin.To determine whether the expression levels of SadB or γ-tubulin affected the phosphorylation levels of SerS levels G or reduous SadB H or γ-tupression I. These 385-γ-tubulin levels in synchronized NIH3T3 cells. Western blotting of chromatin fractions of NIH3T3 cells with Ser(P)385 antibody revealed accumulation of endogenous Ser(P)385-γ-tubulin during early S phase (A). The 60 kDa band recognized by anti-Ser(P)385 antibody was phosphatase-sensitive (B), and increased Ser(P)385-γ-tubulin levels coincided with a rise in nuclear γ-tubulin and E2F1 proteins (A). In contrast, decreased SADB levels reduced the transient increase in nuclear γ-tubulin and Ser(P)385-γ-tubulin and also delayed S-phase entry (C) (A) showed that Ser(P)385-γ-tubulin localized to centrosomes  and chromatin . Together, our data indicate that the transient increase in nuclear γ-tubulin during early S phase is caused by SadB-mediated phosphorylation of Ser385.We subsequently examined endogenous Ser(P) S phase A. The 60ensitive B, and inproteins A. In conse entry C 2). Fi. Fi385-γntry (C) A showed 131 131-γ-tubulin levels in synchronized NIH3T3 cells (F). Western blot analysis of Ser(P)131 immunoprecipitates (131 in early S and G2/M phases (F), a pattern that coincided with the replication of centrosomes and the formation of the mitotic spindle , implying a possible interrelationship. In support of this view, in vitro phosphorylation in Ser385 was altered in an Ala131-γ-tubulin and Asp131-γ-tubulin mutants (E and F), and endogenous Ser(P)385-γ-tubulin had a centrosomal localization (D). Based on our results, we postulate that phosphorylation of Ser131 (385-γ-tubulin (A and C).During cell division, the activity of SadB follows the replication of centrosomes and chromosomes by phosphorylation of γ-tubulin on Ser131 . In this131 , 16. To T3 cells F. Westeripitates  revealedM phases F, a patt spindle , suggest at 14 h , A and F mutants , E and Flization D. Based -tubulin , A and C385-γ-tubulin phosphorylation regulates the cellular location of γ-tubulin, we analyzed expression of a non-phosphorylatable Ala385-γ-tubulin or a phosphomimetic mutant Asp385-γ-tubulin tagged with green fluorescence protein  in U2OS cells. Biochemical fractionation analysis of U2OS cells transiently expressing the non-phosphorylatable Ala385-γ-tubGFP mutant exhibited a reduced amount of chromatin-associated endogenous γ-tubulin and recombinant Ala385-γ-tubGFP (A). In contrast, ectopic expression of Asp385-γ-tubGFP increased the levels of endogenous nuclear γ-tubulin  in the studied cell populations (A). Considering the opposite effects of Ala385-γ-tubGFP and Asp385-γ-tubGFP mutants on the accumulation of endogenous γ-tubulin in chromatin fractions, we propose that phosphorylation of Ser385 initiates a signal cascade that mediates translocation of phosphorylated and non-phosphorylated γ-tubulin molecules to the chromatin.To determine whether Serγ-tubGFP A. In conulations A. Consid385-γ-tubGFP was not detectable by Western blotting of lysates of 1 × 106 cells (A), and immunofluorescence evaluation showed a very weak nuclear signal (B). To ensure the chromatin localization of Asp385-γ-tubGFP, we immunoprecipitated it from fractions of 6 × 106 U2OS cells (A). Asp385-γ-tubGFP occurred mainly in the chromatin fraction, suggesting that the phosphorylation levels of Ser385 modulate the nuclear function of γ-tubulin. Accordingly, immunofluorescence analysis (B) showed that γ-tubGFP was found in the nucleus of a larger number of U2OS cells  than the Ala385-γ-tubGFP mutant .Unfortunately, expression of the Asp06 cells A, and imr signal B. To ensOS cells A. Asp385analysis B showed 385-γ-tubGFP, we analyzed the effect of a proteosomal inhibitor, MG132, on the expression of the mutant protein. In comparison with the γ-tubGFP expression, the expression levels of Asp385-γ-tubGFP were not affected by MG132 (A), implying that premature proteosomal degradation is not the cause of the low expression. To further elucidate the reason of the low protein expression of Asp385-γ-tubGFP, we evaluated the effect of the various γ-tubGFP mutants on the activity of the nuclear γ-tubulin downstream target E2F1 by performing an assay using luciferase reporter plasmids containing E2F-binding sites . Considering these findings, we hypothesized that Ser385-γ-tubulin may have a regulatory impact on transcription depending on its phosphorylation status.To identify the underlying cause of the low expression levels of Aspby MG132 A, implying sites . The lucng sites  was reduγ-tubGFP B. Consid385-γ-tubulin mutants, an mRNA expression array was performed on control GFP-transfected and the various γ-tubGFP-mutant-transfected cells, and the impact of γ-tubulin on RNA expression was examined. By comparison with control cells (GFP-transfected), expression of Asp385-γ-tubGFP correlated with the up-regulated expression of 162 genes, but the expression of 251 genes was down-regulated  in an expression pattern that differed from the expression profile found in cells expressing γ-tubGFP or Ala385-γ-tubGFP . However, we identified the two γ-tubulin isoforms, TUBG1 and TUBG2 . Indeed, the expressions of genes such as RB1, DUSP2, and RBM38 are affected by the phosphorylation levels of Ser385-γ-tubulin, demonstrating that the various recombinant γ-tubulin proteins alter gene expression differently (C and D).To further visualize the effect of the various mutants on known γ-tubulin downstream targets , the expFig. 61 and mitosis (A and B) . Immunofluorescence analysis showed that localization of WT-γ-tubGFP and Ala385-γ-tubGFP differed, being clearly nuclear for the former but mostly cytosolic for the latter. Moreover, chromatin-bound WT-γ-tubGFP accumulated in defined nuclear sites (B). Despite the low expression of Asp385-γ-tubGFP, the phosphomimetic mutant increased the chromatin-bound levels of endogenous γ-tubulin and of the G2-M markers phosphorylated histone H1 and cyclin B (A). Together, these findings support the involvement of Ser385-γ-tubulin in regulation of cell cycle progression  . Consequstone H1 A. Immunoar sites B. Despitcyclin B A. Togeth385-γ-tubulin in cell division, we examined the cell cycle profile of a population of U2OS cells expressing the various γ-tubGFP mutants (A). Compared with cells expressing GFP, those expressing WT-γ-tubGFP showed increased numbers in S phase (A) (S (G). In contrast, expression of the Ala385-γ-tubGFP mutant led to accumulation of cells in G1 phase , whereas Asp385-γ-tubGFP expression increased the number of cells in phases S and G2-M .To further characterize the involvement of Ser mutants A. Compar S phase A 8), si, si385-γe (A) , which supports the idea that the N terminus of γ-tubulin masks the NLS. However, Ala385-Cγ-tubGFP and Ser385-Cγ-tubGFP localized to the nucleus, the centrosomes, and the microtubules, but Asp385-Cγ-tubGFP was mostly in the nucleus and in the centrosomes (B). Although the presence of Asp385-Cγ-tubGFP was detected in single cells by immunofluorescence, neither anti-GFP nor anti-γ-tubulin antibodies detected the C-terminally tagged GFP Asp385-Cγ-tubulin mutant by immunostaining or Western blotting analysis (C and D). Our findings indicate that the conformation of nuclear γ-tubulin differs from the cytosolic pool, in a similar way as described previously for nuclear actin , altogether suggesting that the cellular phosphorylation levels of Ser385-γ-tubulin play a role in determining the cellular localization of γ-tubulin.Moreover, we found that the total cytosolic amount of Cγ-tubGFP increased by 16% when the Alaxpressed C, altogeC and 8A). Western blot analysis of GFP immunoprecipitates with anti-γ-tubulin or -GFP antibodies detected two distinct bands. The expected relative molecular mass of Cγ-tubGFP is 43,200 Da, but the observed protein size varied from 43 to 60 kDa. Both Ser385-Cγ-tubGFP and Ala385-Cγ-tubGFP mutants were detected as a 43 and a 60 kDa band, whereas the phosphomimetic mutant, Asp385-Cγ-tubGFP, was only detected as a single 60 kDa band (A). In addition, analysis of the various Cγ-tubGFP immunoprecipitates disclosed a Ser385-dependent association with α-tubulin and GCP2 (A), which provides a potential explanation for the observed location of Ser385-Cγ-tubGFP and Ala385-Cγ-tubGFP in microtubules (B). In contrast, the phosphomimetic mutant, Asp385-Cγ-tubGFP, neither formed tubular structures nor associated with microtubule components (B), indicating that Ser385 regulates the binding of γ-tubulin to microtubules. However, both Ser385-Cγ-tubGFP and Ala385-Cγ-tubGFP are found in the nuclear compartment and are detected by Western blotting as a 60 kDa band, indicating that the conformation state of Asp385-Cγ-tubGFP can transiently be induced by environmental factors  is a consequence of mitotic failure due to an aberrant association of the Cγ-tubGFP mutants with mitotic chromosomes, we analyzed the effect of constitutive chromatin-bound γ-tubulin in mitotic cells by immunofluorescence studies (B). We found that upon mitosis entry, the Ser385-Cγ-tubGFP and the Ala385-Cγ-tubGFP mutant proteins were detached from the chromatin, whereas the Asp385-Cγ-tubGFP remained chromatin-bound during metaphase (B and C); only cells with non-chromatin-bound Asp385-Cγ-tubGFP progressed through mitosis . However, cells with chromatin-bound Asp385-Cγ-tubGFP were unable to divide. The cells remained in metaphase during several h, and with time, the amount of chromatin-bound Asp385-Cγ-tubGFP decreased, and cytosolic aggregates were formed. Finally, cells formed blebs and died . Mitotic cells expressing the Ser385-Cγ-tubGFP divided normally . Together, these results demonstrate that the cellular localization of γ-tubulin controls mitotic progression.To ascertain whether the accumulation of cells in G2/M E is a co studies B. We fouetaphase , B and CS and SadBL 385-γ-tubulin in membranes containing cell lysates with higher concentrations of the phosphorylated protein. Furthermore, we can with an anti-Ser(P)385-γ-tubulin antibody detect, in a phosphatase-dependent manner, endogenous levels of Ser(P)385-γ-tubulin or in vitro phosphorylated Ser385-γ-tubulin. Second, Ser(P)385-γ-tubulin and the γ-tubulin C terminus undergo a size shift in SDS gels. Finally, only Ser385-Cγ-tubGFP and Ala385-Cγ-tubGFP form tubular structures and associate with α-tubulin and GCP2. Altogether, the results reported here support the existence of different γ-tubulin conformational states that may aid γ-tubulin to bind structurally distinct proteins and in this way provide γ-tubulin with the observed functional properties (–Microtubule formation requires longitudinal stabilization by nucleation onto γTuRC and a GTP-dependent conformational change of αβ-tubulin dimers , 4, 6. Aoperties –14, 27.1-to-S, G2-to-M, and M-to-G1 (385 at early S phase ends the first E2F-mediated transcriptional wave facilitating S phase execution (385-γ-tubulin affects the mediated transcriptional waves of the following E2Fs (Regarding the mechanism by which γ-tubulin regulates cell cycle progression, we think that the presence of nuclear γ-tubulin at early S phase turns off the mediated gene transcription of E2Fs , 9. Cell M-to-G1 . Interfeing E2Fs  and thus1 and S regulate the phosphorylation levels of γ-tubulin at Ser131. In this way, SadB enhances γ-tubulin polymerization at the nascent centriole and inhibits acentriolar formation of centrosomes elsewhere in the cell. However, phosphorylated Ser131-γ-tubulin also reduced astral microtubule nucleation at the centrosomes (385 at the G1 to S phase transition. Phosphorylation of Ser385-γ-tubulin in the centrosomes then triggers a conformational change in γ-tubulin that releases this protein from GCP2, GCP3, and microtubules. In the nucleus, γ-tubulin puts an end to the activities of E2Fs, ensuring that the centrosomes and the chromosomes are replicated only once per cell cycle (We propose that the fluctuating activities of SadB during Gtrosomes , 4, whicll cycle , 30.385, which induces a conformational change in γ-tubulin that leads to its nuclear accumulation during S phase. This identifies SadB as a multifunctional cell cycle regulator that triggers centrosome replication and S phase progression in mammals by controlling phosphorylation of Ser131 (385 in γ-tubulin.Together, our data indicate that the transient increase in nuclear γ-tubulin during S phase is caused by SadB-mediated phosphorylation of Serf Ser131  and Ser3