enoriega/keyword_pubmed · Datasets at Hugging Face

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If this is occurring during transformation then we would expect RhoA to be phosphorylated by PKA and to be inhibited by E7-dependent transformation .	PMC2568952	-1	[]
To test this hypothesis , we first measured the effect of transformation on the phosphorylation state of RhoA via Western Blotting analysis with antibodies specific for phosphorylated Ser-188 of RhoA ( Fig. 5A ) or via co-immunoprecipitation with anti-phosphoserine followed by anti-RhoA Western Blotting ( Fig. 5B ) at different times of HPV16 E7 expression ( t0 , 1 hr , 3 hr , 6 hr , 12 hr ) .	PMC2568952	-1	[]
Indeed , transformation induced a significant increase in RhoA phosphorylation in 2BN11 cells .	PMC2568952	-1	[]
A similar increase in RhoA phosphorylation was observed in E7 expressing HFK cells and late passage HPK1A cells ( Figure S5 ) and blockage of NHE1 actvity with HOE642 had no effect on this process in either 2BN11 or HPK1A cells ( Figure S3 ) .	PMC2568952	-1	[]
To verify that this phosphorylation of RhoA observed upon transformation was due to PKA , we removed tet for 24 hrs in the absence or presence of a pharmacological PKA inhibitor ( H89 , 100 nM ) or activator ( forskolin , 10 µ M ) and we measured RhoA phosphorylation as above with the anti-phosphoSer188 .	PMC2568952	-1	[]
The induction of phosphorylation was blocked by incubation with H89 and potentiated by incubation with forskolin ( Fsk ) during the time of induction of E7 expression ( Fig. 5C ) .	PMC2568952	-1	[]
As PKA can inhibit RhoA activity via its phosphorylation of serine 188 , we next examined the effect of transformation on RhoA activity , first by a pulldown analysis of its binding to GST-fusion protein to the Rho binding domain of Rhotekin which associates preferentially with GTP-bound RhoA [ 59 ] .	PMC2568952	-1	[]
Figure 6A shows that the activity of RhoA was reduced upon E7-dependent transformation with a time course similar to that reported for the up-regulation of NHE1 activity by the same treatment [ 48 ] and for RhoA phosphorylation shown above and that H89 treatment blocked while forskolin ( Fsk ) treatment stimulated this reduction in RhoA activity .	PMC2568952	-1	[]
However , the biochemical detection of active RhoA via pull-down requires cell disruption and is performed in the presence of detergents which can lead to dissociation of preexisting complexes and , therefore , cause incorrect estimation of the extent of RhoA-GTP association to its down-stream effectors .	PMC2568952	-1	[]
In contrast , FRET microscopy permits the direct detection of the amount of active RhoA in intact living cells during E7-transformation .	PMC2568952	-1	[]
For this reason , we next measured RhoA activity state by using a a single-chain CFP / YFP FRET biosensor for RhoA ( pRaichu 1297 × ) [ 60 ] , which directly monitors the level of the endogenous RhoA-GTP by measuring FRET between the two pairs of GFP mutants fused to the Rho-Binding-Domain ( RBD ) of Rhotekin .	PMC2568952	-1	[]
Specifically , in this probe the binding of endogenous GTP-RhoA to RBD displaces YFP and CFP , thereby decreasing FRET efficiency .	PMC2568952	-1	[]
Using this FRET-based probe we verified that 24 hrs after tet removal , RhoA activity , which was assessed as the ratio of the CFP signal to the YFP signal , was significantly reduced ( Figure 6B ) .	PMC2568952	-1	[]
Further , treatment with H89 during the transformation period completely reversed this E7-dependent inhibition of RhoA , again indicating that E7-induced RhoA inhibition was dependent on PKA.In addition to phosphorylation by PKA , RhoA activity is controlled by the activity balance between other class of RhoA regulating proteins , the guanine nucleotide exchange factors ( GEFs ) and GTPase-activating proteins ( GAPs ) [ 29 ] .	PMC2568952	-1	[]
To assess if E7 alters the balance of GEFs and GAPs thus changing RhoA-GTP loading , we monitored the relative level of GEFs and GAPs in the presence and absence of tet for 24 hr , by using another FRET probe , pRaichu 1293 × , consisting of a chimera of RhoA and the RhoA binding domain ( RBD ) of PKN , sandwiched between YFP and CFP .	PMC2568952	-1	[]
The relative increase in GEF activity increases the amount of GTP-RhoA and the intramolecular binding of GTP-RhoA to RBD and brings CFP in close proximity to YFP , resulting in an increase in FRET from CFP to YFP [ 60 ] .	PMC2568952	-1	[]
With this probe , we did not find any difference in the FRET ratio during E7-dependent transformation ( 0.8 ± 0.031 vs 0.79 ± 0.030 , n = 20 , n.s ., for + tet and − tet , respectively ) demonstrating that transformation did not influence GEF or GAP activity .	PMC2568952	-1	[]
These data suggest that a PKA-dependent phosphorylation of RhoA is the critical mechanism of E7 induced-RhoA inhibition .	PMC2568952	-1	[]
HPV16 E7 expression activates NHE1 through a reduction in RhoA activity via its phosphorylation on serine 188 .	PMC2568952	-1	[]
While the preceding experiments indicate that RhoA is a substrate for PKA in these cells and that its phosphorylation at serine-188 is increased with E7-driven transformation , a critical question in the context of the current study is if the observed PKA-dependent phosphorylation and inhibition of RhoA is necessary for the transformation-dependent up-regulation of NHE1 activity .	PMC2568952	-1	[]
The phosphorylation of RhoA at serine 188 by PKA has been shown to block its action [ 46 ] , [ 61 ] , suggesting that this serine could be the PKA target also in our case .	PMC2568952	-1	[]
Therefore , as an approach to assess the role of PKA-dependent phosphorylation of RhoA in the activation of NHE1 , we mutated the PKA phosphorylation site , serine188 , to alanine to create a PKA phosphorylation dead ( pd ) RhoA mutant [ 11 ] , [ 12 ] .	PMC2568952	-1	[]
Transfection of control 2BN11 cells with this pd RhoA mutant did not affect basal NHE1 activity levels of + tet cells , while it completely abrogated the increased NHE1 activity induced by expression of HPV16 E7 ( Figure 6C , blue cross-hatched bars ) , confirming the requirement for PKA-dependent phosphorylation of RhoA at serine 188 for the up-regulation of NHE1 activity by E7-dependent transformation .	PMC2568952	-1	[]
We next determined if it is the alteration in RhoA activity utilizing either dominant negative ( dn ) mutants , constitutively active ( ca ) mutants [ 11 ] and / or expression utilizing an siRNA against RhoA that underlies stimulated NHE1 activity in transformed cells .	PMC2568952	-1	[]
Transient transfection of these constructs or the siRNA had no effect on NHE1 activity in + tet cells .	PMC2568952	-1	[]
Both inactivation of RhoA with the dn N19RhoA mutant ( Figure 5C , green stippled bars ) or the knock-down of its expression ( Figure 6C , brown reverse stippled bars ) significantly potentiated the E7-induced stimulation of NHE1 activity while activating RhoA with the ca V14RhoA mutant reduced this stimulation by approximately 80 % ( Figure 6C , red striped bars ) .	PMC2568952	-1	[]
Altogether , these data demonstrate that the PKA-dependent phosphorylation of RhoA is a critical mechanism of HPV16 E7 induced-RhoA inhibition and it is an integral part of the PKA to p38alpha signal transduction module involved in the activation of the NHE1 during transformation .	PMC2568952	-1	[]
Discussion .	PMC2568952	-1	[]
There still is a dearth of data concerning the very early signal transduction events regulating neoplastic transformation - the first step of the carcinogenic process that is limited to the altered cell .	PMC2568952	-1	[]
The elucidation of the underlying alterations in signal transduction events mediating the initiation , development and regulation of transformation is a necessary prerequiste for understanding the origin and early development of cancer .	PMC2568952	-1	[]
As discussed in [ 17 ] , to accomplish this it is necessary to use an experimental model in which the transformation of a normal , immortalized cell by a single oncogene can be highly controlled , thus permitting the dissection of the sequence of events occuring after the initial alteration and the role of a particular gene or signal transduction pathway .	PMC2568952	-1	[]
These cells , unlike cancer-derived cell lines , have not aquired genomic instability nor unspecific genetic and epigenetic alterations that can mask the specific transformation-dependent alterations.We utilized this type of inducible cell model with the E7 oncogene of HPV16 to dissect the sequence of the very early steps in signal transduction underlying “ the moment ” in which a still normal cell becomes transformed .	PMC2568952	-1	[]
To accomplish this , NIH-3T3 cells were transfected with a construct in which E7 gene expression is under the control of a promoter that is negatively regulated by tetracycline so that E7 is expressed only after tetracycline removal [ 48 ] ( clone 2BN11 ) .	PMC2568952	-1	[]
Using this model system , we previously reported that the development of transformed phenotypes ( e. g. increased proliferative rate , anchorage-independent growth , serum independence and increased glycolytic metabolism ) are under the strict control of E7 expression in these cells and , further , that intracellular alkalinization driven by an up-regulation of the Na / H - exchanger ( NHE1 ) is a very early physiological event in transformation and which , in turn , is necessary for the development and maintenance of many of the cellular events occurring later in the transformation process such as serum independence , increased growth rate , anchorage-independent growth and in vivo tumour development in nude mice .	PMC2568952	-1	[]
For this reason , we here utilized changes in NHE1 activity as the readout for the analysis of the role of a particular signal transduction pathway in transformation in the dissection of the early signaling events occurring up-stream of NHE1 activation during E7-dependent transformation.This experimental model enabled us to recognise and follow a strictly defined sequential progression arising from the initial expression of E7 that rapidly transforms the cells .	PMC2568952	-1	[]
Our data demonstrate that an intracellular mobilization of cAMP is one of the first signaling events occurring during transformation ( Figure 3 ) .	PMC2568952	-1	[]
Further , we provide evidence that this initial rise in cAMP is followed by a PKA-dependent inhibition of RhoA activity ( Figures 6A and 6B ) which is necessary for the inactivation of p38alpha ( Figure 4B ) and which , in turn , regulates the subsequent E7-mediated transformation – dependent early stimulation of NHE1 activity ( Figures 4A and 6C ) .	PMC2568952	-1	[]
Although the individual importance of each of these signal systems in tumor induction both in vitro and in vivo is well documented , their interrelations in mediating transformation were heretofore still unknown.Indeed , while there is now much data demonstrating the tumor suppressor role of p38 MAP Kinase ( see introduction ) , to date it has not been demonstrated whether the down-regulation of p38alpha plays a role in the early induction of cellular events leading to transformation .	PMC2568952	-1	[]
Further , if it is involved in transformation , we asked at which point is it located in the transformation process , which up-stream mechanisms regulate its inhibition and what is its down-stream target .	PMC2568952	-1	[]
We first demonstrated in NIH3T3 cells and in human primary ( HPK1A ) and secondary ( HFK ) keratinocytes that E7-dependent transformation specifically reduces p38 phosphorylation .	PMC2568952	-1	[]
Then , using the inducible experimental model we show that p38alpha plays a key role in the acquisition of the increased activity of the NHE1 which sets the stage for the development of the other transformed phenotypes [ 48 ] .	PMC2568952	-1	[]
Interestingly , p38alpha has also been found to have a negative role in regulating both migration and invasion of pancreatic and breast carcinoma cells [ 11 ] – [ 12 ] , [ 32 ] and its activity is reduced in hepatocellular carcinomas in comparison to adjacent normal tissue [ 62 ] , suggesting that its down-regulation is not limited to just transformation and early tumorigenesis but also in later metastatic / aggressive stages.The involvement of the cAMP / PKA pathway in mediating tumor progression [ 35 ] together with the demonstrated integration of p38 with cAMP / PKA signaling in different cell systems [ 43 ] , [ 57 ] , [ 63 ] , [ 64 ] focused our attention on the possibility that E7 transformation-dependent down-regulation of p38 involves the cAMP / PKA system .	PMC2568952	-1	[]
Transformation induced substantial increase in adenylate cyclase-dependent cellular cAMP mobilization ( Fig. 2 ) and incubation with the PKA selective inhibitor , H89 , during E7-dependent transformation blocked the down-regulation of p38 phosphorylation while stimulation of the cAMP / PKA system by forskolin ( Fsk ) enhanced this E7-dependent down-regulation of p38 phosphorylation ( Fig. 4B and 4C ) .	PMC2568952	-1	[]
Further , inhibition of p38 with either SB203580 or via transient expression of a dominant negative p38alpha mutant ( dn p38alpha ) blocked the H89-dependent abrogation of the transformation-dependent activation of NHE1 ( Figure 3A ) , further supporting the suggestion that PKA and p38alpha belong both physically and functionally to a common signalling unit , in which PKA acts up-stream to p38 in regulating the transformation-dependent stimulation of NHE1 activity.As RhoA is known be a PKA-signalling effector in a plethora of cell responses [ 11 ] , [ 58 ] , [ 44 ] and to play an important role in regulating p38 kinase activity in several cellular regulatory contexts [ 11 ] , [ 32 ] , [ 63 ] , [ 64 ] , we analysed its involvement in underlying the down-regulation of p38 by PKA and in their regulation of E7-dependent activation of NHE1 .	PMC2568952	-1	[]
We observed that RhoA was rapidly phosphorylated at Ser-188 upon E7 expression ( Figs .	PMC2568952	-1	[]
4A & B ) and PKA is involved in this phosphorylation since inhibition of the kinase with H89 or its stimulation by forskolin ( Fsk ) during the time of induction of E7 expression respectively blocked or potentiated the E7-induced phosphorylation ( Fig. 4C ) .	PMC2568952	-1	[]
Further , RhoA was inhibited upon E7 expression with a time course parallel to its E7-induced phosphorylation ( Figures 6A & B ) and inhibition of PKA by H89 reversed the decrease in RhoA activity only in transformed cells ( Figure 6B ) .	PMC2568952	-1	[]
Interestingly , FRET measurements of Rho-GEFs / GAP activity revealed that the inhibition of RhoA via changes in their activity could be excluded during E7 transformation .	PMC2568952	-1	[]
Altogether these data suggest that the PKA that is activated in HPV16 E7 transformed cells inhibits RhoA activity through its phosphorylation of RhoA at serine 188 .	PMC2568952	-1	[]
RhoA has been recently reported to be inhibited as a result of transformation driven by TGFbeta [ 67 ] or , importantly , HPV16 E7 expression [ 33 ] suggesting that this might be a common mechanism .	PMC2568952	-1	[]
An important question was if the observed PKA-dependent phosphorylation and inhibition of RhoA is necessary for the transformation-dependent up-regulation of NHE1 activity .	PMC2568952	-1	[]
Analysis of NHE1 activity after transfection of a dn RhoA mutant or siRNA to block RhoA function / expression or a ca RhoA mutant to enhance RhoA function ( Figure 6C ) demonstrated the importance of the inhibition of RhoA activity in E7 transformation-induced NHE1 activity while transfection of the phosphodead ( pd ) RhoA mutant revealed the important role for its PKA-dependent phosphorylation in E7 transformation-induced NHE1 activity.The activity of many enzymes is strictly controlled by intracellular pH ( pHi ) and , therefore , an important question is what extent this E7-induced signal cascade described herein might be altered via feedback by the changes in pHi driven by the stimulated NHE1 [ 48 ] .	PMC2568952	-1	[]
To address this question , we measured cAMP production , phosphorylation of RhoA and the inhibition of p38 in the presence and absence of 2 µ M HOE642 , a potent and specific inhibitor of the NHE1 , in both 2BN11 cells and in early and late passages of HPK1A cells .	PMC2568952	-1	[]
This treatment has been previously shown to block the development of phenotypes down-stream of the NHE1 in both cell lines [ 48 ] .	PMC2568952	-1	[]
As can be seen in Figure S3 , this treatment had no effect on any of these process in either of the cell lines suggesting a strict unidirectionality of this signal cascade both in the rat fibroblast and human keratinocyte models.In conclusion , in this study we have recognised a strictly defined sequential progression arising from the initial expression of E7 that rapidly transforms the cells .	PMC2568952	-1	[]
Our data demonstrate that an intracellular mobilization of cAMP is one of the first signaling events occurring during transformation and that this initial rise in cAMP is followed by a PKA-dependent inhibition of RhoA activity which is necessary for the inactivation of p38alpha which , in turn , regulates the subsequent E7-mediated transformation – dependent early stimulation of NHE1 activity .	PMC2568952	-1	[]
An important question is whether this signal cascade is engaged by simple E7 expression or is transformation dependent .	PMC2568952	-1	[]
The data in Figure 1D shows that only transformation competent E7 is able to inhibit p38 and the use of the HPK1A model system in this study , in which the original viral infection ( early passage ) simply immortalizes the cells and with time they become transformed ( late passage ) , further validated the concept of transformation specific effects .	PMC2568952	-1	[]
Importantly , finding the same signal transduction cascade turned on in the late passage HPK1A cells also demonstrated that this signal cascade is engaged during the natural progression of primary human keratinocyte cells that were infected with the actual HPV16 virus and is not just a consequence of the expression of a single viral gene as was also observed previously for NHE1 activity [ 48 ] .	PMC2568952	-1	[]
The elucidation of these signal transduction systems and , more importantly , their interrelations in the early stages of the HPV-dependent transformation processes could provide indications for novel therapeutic strategies and / or a potential marker test for the clinical determination of pre-cancer , HPV-transformed cells in the cervix .	PMC2568952	-1	[]
Supporting Information .	PMC2568952	-1	[]
Figure S1Effect of pharmacological treatment and transfection of cDNA plasmids on E7 message expression by RT-PCR analysis .	PMC2568952	-1	[]
2BN11 cells were treated with each of the pharmacological agents or transfected with the indicated cDNA encoding vectors as described in Results .	PMC2568952	-1	[]
RNA was extracted and 500 ng of each sample total RNA were subjected to a semi quantitative RT-PCR for HPV16 E7 expression analysis .	PMC2568952	-1	[]
GAPDH : loading control .	PMC2568952	-1	[]
( 0.07 MB TIF ) Click here for additional data file.Figure S2Analysis by immunofluorescence microscopy of the decrease in RhoA expression by RNAi in 2BN11 cells .	PMC2568952	36,775	[ 11 ]
A. Cells were transfected with either control , non targeting siRNA ( left panel ) or RhoA specific siRNA ( right panel ) as described in Materials and Methods .	PMC2568952	-1	[]
RhoA was visualized with Alexa Fluor 488 ( green ) and the nuclei with DAPI ( blue ) .	PMC2568952	-1	[]
B. Quantification of the intensity of RhoA signals through the cell area normalized to cell number .	PMC2568952	-1	[]
Data represent mean ± SE of three independent experiments .	PMC2568952	-1	[]
( 1.35 MB TIF ) Click here for additional data file.Figure S3Monolayers of either 2BN11 or HPK1A were treated or not with 2 µ M of the specific NHE1 inhibitor , HOE642 , and analyzed for cAMP , phospho-RhoA or phospho-p38 levels as described in Materials and Methods .	PMC2568952	-1	[]
In 2BN11 cells the measurements were made in the presence of tetracycline ( + tet ) or 3 or 24 hours after its removal while in HPK1A cells the comparison was between early and late passage cells .	PMC2568952	-1	[]
( 0.21 MB TIF ) Click here for additional data file.Figure S4Non transformed ( + tet ) and transformed ( − tet ) cells were not treated ( Cont ) or treated with either 10 − 7 M H89 or 10 − 5 M FSK for 24 hrs and cells were homogenized as described in Materials and Methods .	PMC2568952	-1	[]
Aliquots containing 50 µ g of protein were subjected to 10 % SDS-PAGE and total and phosphorylated JNK ( upper blot ) or ERK1 / 2 ( lower blot ) was determined in Western Blot as in Figure 1 .	PMC2568952	-1	[]
A representative immunoblot is shown for each MAP kinase .	PMC2568952	-1	[]
( 0.39 MB TIF ) Click here for additional data file.Figure S5Western Blotting analysis of the phosphorylation state of RhoA in HFK or HPK1A cells with antibodies specific for phosphorylated Ser-188 of RhoA ( phospho RhoA ) followed by polyclonal anti-RhoA antibody ( total RhoA ) .	PMC2568952	-1	[]
A representative immunoblot is shown for each .	PMC2568952	-1	[]
HFK cells were infected with empty pLXSN vector or pLXSN vector containing wild-type , non tagged E7 while HPK1A early passage cells were compared with late passage cells .	PMC2568952	-1	[]
( 0.13 MB DOC ) Click here for additional data file .	PMC2568952	-1	[]
Detecting Nitrous Oxide Reductase ( nosZ ) Genes in Soil Metagenomes : Method Development and Implications for the Nitrogen Cycle .	PMC4049103	-1	[]
ABSTRACT .	PMC4049103	-1	[]
Microbial activities in soils , such as ( incomplete ) denitrification , represent major sources of nitrous oxide ( N 2 O ) , a potent greenhouse gas .	PMC4049103	-1	[]
The key enzyme for mitigating N 2 O emissions is NosZ , which catalyzes N 2 O reduction to N 2 .	PMC4049103	-1	[]
We recently described " atypical " functional NosZ proteins encoded by both denitrifiers and nondenitrifiers , which were missed in previous environmental surveys ( R.	PMC4049103	-1	[]
A. Sanford et al ., Proc .	PMC4049103	-1	[]
Natl .	PMC4049103	-1	[]
Acad .	PMC4049103	-1	[]
Sci .	PMC4049103	-1	[]
U. S.	PMC4049103	-1	[]
A. 109:19709 -19714 , 2012 , doi :10.1073 / pnas.1211238109 ) .	PMC4049103	-1	[]
Here , we analyzed the abundance and diversity of both nosZ types in whole-genome shotgun metagenomes from sandy and silty loam agricultural soils that typify the U.S. Midwest corn belt .	PMC4049103	-1	[]
First , different search algorithms and parameters for detecting nosZ metagenomic reads were evaluated based on in silico-generated ( mock ) metagenomes .	PMC4049103	-1	[]
Using the derived cutoffs , 71 distinct alleles ( 95 % amino acid identity level ) encoding typical or atypical NosZ proteins were detected in both soil types .	PMC4049103	-1	[]
Remarkably , more than 70 % of the total nosZ reads in both soils were classified as atypical , emphasizing that prior surveys underestimated nosZ abundance .	PMC4049103	-1	[]
Approximately 15 % of the total nosZ reads were taxonomically related to Anaeromyxobacter , which was the most abundant genus encoding atypical NosZ-type proteins in both soil types .	PMC4049103	28,139	[ 12 ]
Further analyses revealed that atypical nosZ genes outnumbered typical nosZ genes in most publicly available soil metagenomes , underscoring their potential role in mediating N 2 O consumption in soils .	PMC4049103	-1	[]
Therefore , this study provides a bioinformatics strategy to reliably detect target genes in complex short-read metagenomes and suggests that the analysis of both typical and atypical nosZ sequences is required to understand and predict N 2 O flux in soils .	PMC4049103	-1	[]
INTRODUCTION .	PMC4049103	-1	[]
In recent years , anthropogenic emissions of greenhouse gases have received increasing attention because of their contribution to global warming ( 1 , 2 ) .	PMC4049103	-1	[]
Prominent among these gases is nitrous oxide ( N O ) ( 3 ) , which also contributes to ozone depletion ( 4 , 5 ) .	PMC4049103	-1	[]

If this is occurring during transformation then we would expect RhoA to be phosphorylated by PKA and to be inhibited by E7-dependent transformation .

PMC2568952

-1

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To test this hypothesis , we first measured the effect of transformation on the phosphorylation state of RhoA via Western Blotting analysis with antibodies specific for phosphorylated Ser-188 of RhoA ( Fig. 5A ) or via co-immunoprecipitation with anti-phosphoserine followed by anti-RhoA Western Blotting ( Fig. 5B ) at different times of HPV16 E7 expression ( t0 , 1 hr , 3 hr , 6 hr , 12 hr ) .

PMC2568952

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Indeed , transformation induced a significant increase in RhoA phosphorylation in 2BN11 cells .

PMC2568952

-1

[]

A similar increase in RhoA phosphorylation was observed in E7 expressing HFK cells and late passage HPK1A cells ( Figure S5 ) and blockage of NHE1 actvity with HOE642 had no effect on this process in either 2BN11 or HPK1A cells ( Figure S3 ) .

PMC2568952

-1

[]

To verify that this phosphorylation of RhoA observed upon transformation was due to PKA , we removed tet for 24 hrs in the absence or presence of a pharmacological PKA inhibitor ( H89 , 100 nM ) or activator ( forskolin , 10 µ M ) and we measured RhoA phosphorylation as above with the anti-phosphoSer188 .

PMC2568952

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The induction of phosphorylation was blocked by incubation with H89 and potentiated by incubation with forskolin ( Fsk ) during the time of induction of E7 expression ( Fig. 5C ) .

PMC2568952

-1

[]

As PKA can inhibit RhoA activity via its phosphorylation of serine 188 , we next examined the effect of transformation on RhoA activity , first by a pulldown analysis of its binding to GST-fusion protein to the Rho binding domain of Rhotekin which associates preferentially with GTP-bound RhoA [ 59 ] .

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[]

Figure 6A shows that the activity of RhoA was reduced upon E7-dependent transformation with a time course similar to that reported for the up-regulation of NHE1 activity by the same treatment [ 48 ] and for RhoA phosphorylation shown above and that H89 treatment blocked while forskolin ( Fsk ) treatment stimulated this reduction in RhoA activity .

PMC2568952

-1

[]

However , the biochemical detection of active RhoA via pull-down requires cell disruption and is performed in the presence of detergents which can lead to dissociation of preexisting complexes and , therefore , cause incorrect estimation of the extent of RhoA-GTP association to its down-stream effectors .

PMC2568952

-1

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In contrast , FRET microscopy permits the direct detection of the amount of active RhoA in intact living cells during E7-transformation .

PMC2568952

-1

[]

For this reason , we next measured RhoA activity state by using a a single-chain CFP / YFP FRET biosensor for RhoA ( pRaichu 1297 × ) [ 60 ] , which directly monitors the level of the endogenous RhoA-GTP by measuring FRET between the two pairs of GFP mutants fused to the Rho-Binding-Domain ( RBD ) of Rhotekin .

PMC2568952

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Specifically , in this probe the binding of endogenous GTP-RhoA to RBD displaces YFP and CFP , thereby decreasing FRET efficiency .

PMC2568952

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Using this FRET-based probe we verified that 24 hrs after tet removal , RhoA activity , which was assessed as the ratio of the CFP signal to the YFP signal , was significantly reduced ( Figure 6B ) .

PMC2568952

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Further , treatment with H89 during the transformation period completely reversed this E7-dependent inhibition of RhoA , again indicating that E7-induced RhoA inhibition was dependent on PKA.In addition to phosphorylation by PKA , RhoA activity is controlled by the activity balance between other class of RhoA regulating proteins , the guanine nucleotide exchange factors ( GEFs ) and GTPase-activating proteins ( GAPs ) [ 29 ] .

PMC2568952

-1

[]

To assess if E7 alters the balance of GEFs and GAPs thus changing RhoA-GTP loading , we monitored the relative level of GEFs and GAPs in the presence and absence of tet for 24 hr , by using another FRET probe , pRaichu 1293 × , consisting of a chimera of RhoA and the RhoA binding domain ( RBD ) of PKN , sandwiched between YFP and CFP .

PMC2568952

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The relative increase in GEF activity increases the amount of GTP-RhoA and the intramolecular binding of GTP-RhoA to RBD and brings CFP in close proximity to YFP , resulting in an increase in FRET from CFP to YFP [ 60 ] .

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With this probe , we did not find any difference in the FRET ratio during E7-dependent transformation ( 0.8 ± 0.031 vs 0.79 ± 0.030 , n = 20 , n.s ., for + tet and − tet , respectively ) demonstrating that transformation did not influence GEF or GAP activity .

PMC2568952

-1

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These data suggest that a PKA-dependent phosphorylation of RhoA is the critical mechanism of E7 induced-RhoA inhibition .

PMC2568952

-1

[]

HPV16 E7 expression activates NHE1 through a reduction in RhoA activity via its phosphorylation on serine 188 .

PMC2568952

-1

[]

While the preceding experiments indicate that RhoA is a substrate for PKA in these cells and that its phosphorylation at serine-188 is increased with E7-driven transformation , a critical question in the context of the current study is if the observed PKA-dependent phosphorylation and inhibition of RhoA is necessary for the transformation-dependent up-regulation of NHE1 activity .

PMC2568952

-1

[]

The phosphorylation of RhoA at serine 188 by PKA has been shown to block its action [ 46 ] , [ 61 ] , suggesting that this serine could be the PKA target also in our case .

PMC2568952

-1

[]

Therefore , as an approach to assess the role of PKA-dependent phosphorylation of RhoA in the activation of NHE1 , we mutated the PKA phosphorylation site , serine188 , to alanine to create a PKA phosphorylation dead ( pd ) RhoA mutant [ 11 ] , [ 12 ] .

PMC2568952

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Transfection of control 2BN11 cells with this pd RhoA mutant did not affect basal NHE1 activity levels of + tet cells , while it completely abrogated the increased NHE1 activity induced by expression of HPV16 E7 ( Figure 6C , blue cross-hatched bars ) , confirming the requirement for PKA-dependent phosphorylation of RhoA at serine 188 for the up-regulation of NHE1 activity by E7-dependent transformation .

PMC2568952

-1

[]

We next determined if it is the alteration in RhoA activity utilizing either dominant negative ( dn ) mutants , constitutively active ( ca ) mutants [ 11 ] and / or expression utilizing an siRNA against RhoA that underlies stimulated NHE1 activity in transformed cells .

PMC2568952

-1

[]

Transient transfection of these constructs or the siRNA had no effect on NHE1 activity in + tet cells .

PMC2568952

-1

[]

Both inactivation of RhoA with the dn N19RhoA mutant ( Figure 5C , green stippled bars ) or the knock-down of its expression ( Figure 6C , brown reverse stippled bars ) significantly potentiated the E7-induced stimulation of NHE1 activity while activating RhoA with the ca V14RhoA mutant reduced this stimulation by approximately 80 % ( Figure 6C , red striped bars ) .

PMC2568952

-1

[]

Altogether , these data demonstrate that the PKA-dependent phosphorylation of RhoA is a critical mechanism of HPV16 E7 induced-RhoA inhibition and it is an integral part of the PKA to p38alpha signal transduction module involved in the activation of the NHE1 during transformation .

PMC2568952

-1

[]

Discussion .

PMC2568952

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[]

There still is a dearth of data concerning the very early signal transduction events regulating neoplastic transformation - the first step of the carcinogenic process that is limited to the altered cell .

PMC2568952

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The elucidation of the underlying alterations in signal transduction events mediating the initiation , development and regulation of transformation is a necessary prerequiste for understanding the origin and early development of cancer .

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As discussed in [ 17 ] , to accomplish this it is necessary to use an experimental model in which the transformation of a normal , immortalized cell by a single oncogene can be highly controlled , thus permitting the dissection of the sequence of events occuring after the initial alteration and the role of a particular gene or signal transduction pathway .

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These cells , unlike cancer-derived cell lines , have not aquired genomic instability nor unspecific genetic and epigenetic alterations that can mask the specific transformation-dependent alterations.We utilized this type of inducible cell model with the E7 oncogene of HPV16 to dissect the sequence of the very early steps in signal transduction underlying “ the moment ” in which a still normal cell becomes transformed .

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To accomplish this , NIH-3T3 cells were transfected with a construct in which E7 gene expression is under the control of a promoter that is negatively regulated by tetracycline so that E7 is expressed only after tetracycline removal [ 48 ] ( clone 2BN11 ) .

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Using this model system , we previously reported that the development of transformed phenotypes ( e. g. increased proliferative rate , anchorage-independent growth , serum independence and increased glycolytic metabolism ) are under the strict control of E7 expression in these cells and , further , that intracellular alkalinization driven by an up-regulation of the Na / H - exchanger ( NHE1 ) is a very early physiological event in transformation and which , in turn , is necessary for the development and maintenance of many of the cellular events occurring later in the transformation process such as serum independence , increased growth rate , anchorage-independent growth and in vivo tumour development in nude mice .

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For this reason , we here utilized changes in NHE1 activity as the readout for the analysis of the role of a particular signal transduction pathway in transformation in the dissection of the early signaling events occurring up-stream of NHE1 activation during E7-dependent transformation.This experimental model enabled us to recognise and follow a strictly defined sequential progression arising from the initial expression of E7 that rapidly transforms the cells .

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Our data demonstrate that an intracellular mobilization of cAMP is one of the first signaling events occurring during transformation ( Figure 3 ) .

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Further , we provide evidence that this initial rise in cAMP is followed by a PKA-dependent inhibition of RhoA activity ( Figures 6A and 6B ) which is necessary for the inactivation of p38alpha ( Figure 4B ) and which , in turn , regulates the subsequent E7-mediated transformation – dependent early stimulation of NHE1 activity ( Figures 4A and 6C ) .

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Although the individual importance of each of these signal systems in tumor induction both in vitro and in vivo is well documented , their interrelations in mediating transformation were heretofore still unknown.Indeed , while there is now much data demonstrating the tumor suppressor role of p38 MAP Kinase ( see introduction ) , to date it has not been demonstrated whether the down-regulation of p38alpha plays a role in the early induction of cellular events leading to transformation .

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Further , if it is involved in transformation , we asked at which point is it located in the transformation process , which up-stream mechanisms regulate its inhibition and what is its down-stream target .

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We first demonstrated in NIH3T3 cells and in human primary ( HPK1A ) and secondary ( HFK ) keratinocytes that E7-dependent transformation specifically reduces p38 phosphorylation .

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Then , using the inducible experimental model we show that p38alpha plays a key role in the acquisition of the increased activity of the NHE1 which sets the stage for the development of the other transformed phenotypes [ 48 ] .

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Interestingly , p38alpha has also been found to have a negative role in regulating both migration and invasion of pancreatic and breast carcinoma cells [ 11 ] – [ 12 ] , [ 32 ] and its activity is reduced in hepatocellular carcinomas in comparison to adjacent normal tissue [ 62 ] , suggesting that its down-regulation is not limited to just transformation and early tumorigenesis but also in later metastatic / aggressive stages.The involvement of the cAMP / PKA pathway in mediating tumor progression [ 35 ] together with the demonstrated integration of p38 with cAMP / PKA signaling in different cell systems [ 43 ] , [ 57 ] , [ 63 ] , [ 64 ] focused our attention on the possibility that E7 transformation-dependent down-regulation of p38 involves the cAMP / PKA system .

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Transformation induced substantial increase in adenylate cyclase-dependent cellular cAMP mobilization ( Fig. 2 ) and incubation with the PKA selective inhibitor , H89 , during E7-dependent transformation blocked the down-regulation of p38 phosphorylation while stimulation of the cAMP / PKA system by forskolin ( Fsk ) enhanced this E7-dependent down-regulation of p38 phosphorylation ( Fig. 4B and 4C ) .

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Further , inhibition of p38 with either SB203580 or via transient expression of a dominant negative p38alpha mutant ( dn p38alpha ) blocked the H89-dependent abrogation of the transformation-dependent activation of NHE1 ( Figure 3A ) , further supporting the suggestion that PKA and p38alpha belong both physically and functionally to a common signalling unit , in which PKA acts up-stream to p38 in regulating the transformation-dependent stimulation of NHE1 activity.As RhoA is known be a PKA-signalling effector in a plethora of cell responses [ 11 ] , [ 58 ] , [ 44 ] and to play an important role in regulating p38 kinase activity in several cellular regulatory contexts [ 11 ] , [ 32 ] , [ 63 ] , [ 64 ] , we analysed its involvement in underlying the down-regulation of p38 by PKA and in their regulation of E7-dependent activation of NHE1 .

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We observed that RhoA was rapidly phosphorylated at Ser-188 upon E7 expression ( Figs .

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4A & B ) and PKA is involved in this phosphorylation since inhibition of the kinase with H89 or its stimulation by forskolin ( Fsk ) during the time of induction of E7 expression respectively blocked or potentiated the E7-induced phosphorylation ( Fig. 4C ) .

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Further , RhoA was inhibited upon E7 expression with a time course parallel to its E7-induced phosphorylation ( Figures 6A & B ) and inhibition of PKA by H89 reversed the decrease in RhoA activity only in transformed cells ( Figure 6B ) .

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Interestingly , FRET measurements of Rho-GEFs / GAP activity revealed that the inhibition of RhoA via changes in their activity could be excluded during E7 transformation .

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Altogether these data suggest that the PKA that is activated in HPV16 E7 transformed cells inhibits RhoA activity through its phosphorylation of RhoA at serine 188 .

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RhoA has been recently reported to be inhibited as a result of transformation driven by TGFbeta [ 67 ] or , importantly , HPV16 E7 expression [ 33 ] suggesting that this might be a common mechanism .

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An important question was if the observed PKA-dependent phosphorylation and inhibition of RhoA is necessary for the transformation-dependent up-regulation of NHE1 activity .

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Analysis of NHE1 activity after transfection of a dn RhoA mutant or siRNA to block RhoA function / expression or a ca RhoA mutant to enhance RhoA function ( Figure 6C ) demonstrated the importance of the inhibition of RhoA activity in E7 transformation-induced NHE1 activity while transfection of the phosphodead ( pd ) RhoA mutant revealed the important role for its PKA-dependent phosphorylation in E7 transformation-induced NHE1 activity.The activity of many enzymes is strictly controlled by intracellular pH ( pHi ) and , therefore , an important question is what extent this E7-induced signal cascade described herein might be altered via feedback by the changes in pHi driven by the stimulated NHE1 [ 48 ] .

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To address this question , we measured cAMP production , phosphorylation of RhoA and the inhibition of p38 in the presence and absence of 2 µ M HOE642 , a potent and specific inhibitor of the NHE1 , in both 2BN11 cells and in early and late passages of HPK1A cells .

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This treatment has been previously shown to block the development of phenotypes down-stream of the NHE1 in both cell lines [ 48 ] .

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As can be seen in Figure S3 , this treatment had no effect on any of these process in either of the cell lines suggesting a strict unidirectionality of this signal cascade both in the rat fibroblast and human keratinocyte models.In conclusion , in this study we have recognised a strictly defined sequential progression arising from the initial expression of E7 that rapidly transforms the cells .

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Our data demonstrate that an intracellular mobilization of cAMP is one of the first signaling events occurring during transformation and that this initial rise in cAMP is followed by a PKA-dependent inhibition of RhoA activity which is necessary for the inactivation of p38alpha which , in turn , regulates the subsequent E7-mediated transformation – dependent early stimulation of NHE1 activity .

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An important question is whether this signal cascade is engaged by simple E7 expression or is transformation dependent .

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The data in Figure 1D shows that only transformation competent E7 is able to inhibit p38 and the use of the HPK1A model system in this study , in which the original viral infection ( early passage ) simply immortalizes the cells and with time they become transformed ( late passage ) , further validated the concept of transformation specific effects .

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Importantly , finding the same signal transduction cascade turned on in the late passage HPK1A cells also demonstrated that this signal cascade is engaged during the natural progression of primary human keratinocyte cells that were infected with the actual HPV16 virus and is not just a consequence of the expression of a single viral gene as was also observed previously for NHE1 activity [ 48 ] .

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The elucidation of these signal transduction systems and , more importantly , their interrelations in the early stages of the HPV-dependent transformation processes could provide indications for novel therapeutic strategies and / or a potential marker test for the clinical determination of pre-cancer , HPV-transformed cells in the cervix .

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Supporting Information .

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Figure S1Effect of pharmacological treatment and transfection of cDNA plasmids on E7 message expression by RT-PCR analysis .

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2BN11 cells were treated with each of the pharmacological agents or transfected with the indicated cDNA encoding vectors as described in Results .

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RNA was extracted and 500 ng of each sample total RNA were subjected to a semi quantitative RT-PCR for HPV16 E7 expression analysis .

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GAPDH : loading control .

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( 0.07 MB TIF ) Click here for additional data file.Figure S2Analysis by immunofluorescence microscopy of the decrease in RhoA expression by RNAi in 2BN11 cells .

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A. Cells were transfected with either control , non targeting siRNA ( left panel ) or RhoA specific siRNA ( right panel ) as described in Materials and Methods .

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RhoA was visualized with Alexa Fluor 488 ( green ) and the nuclei with DAPI ( blue ) .

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B. Quantification of the intensity of RhoA signals through the cell area normalized to cell number .

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Data represent mean ± SE of three independent experiments .

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( 1.35 MB TIF ) Click here for additional data file.Figure S3Monolayers of either 2BN11 or HPK1A were treated or not with 2 µ M of the specific NHE1 inhibitor , HOE642 , and analyzed for cAMP , phospho-RhoA or phospho-p38 levels as described in Materials and Methods .

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In 2BN11 cells the measurements were made in the presence of tetracycline ( + tet ) or 3 or 24 hours after its removal while in HPK1A cells the comparison was between early and late passage cells .

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( 0.21 MB TIF ) Click here for additional data file.Figure S4Non transformed ( + tet ) and transformed ( − tet ) cells were not treated ( Cont ) or treated with either 10 − 7 M H89 or 10 − 5 M FSK for 24 hrs and cells were homogenized as described in Materials and Methods .

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Aliquots containing 50 µ g of protein were subjected to 10 % SDS-PAGE and total and phosphorylated JNK ( upper blot ) or ERK1 / 2 ( lower blot ) was determined in Western Blot as in Figure 1 .

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A representative immunoblot is shown for each MAP kinase .

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( 0.39 MB TIF ) Click here for additional data file.Figure S5Western Blotting analysis of the phosphorylation state of RhoA in HFK or HPK1A cells with antibodies specific for phosphorylated Ser-188 of RhoA ( phospho RhoA ) followed by polyclonal anti-RhoA antibody ( total RhoA ) .

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A representative immunoblot is shown for each .

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HFK cells were infected with empty pLXSN vector or pLXSN vector containing wild-type , non tagged E7 while HPK1A early passage cells were compared with late passage cells .

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( 0.13 MB DOC ) Click here for additional data file .

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Detecting Nitrous Oxide Reductase ( nosZ ) Genes in Soil Metagenomes : Method Development and Implications for the Nitrogen Cycle .

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ABSTRACT .

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Microbial activities in soils , such as ( incomplete ) denitrification , represent major sources of nitrous oxide ( N 2 O ) , a potent greenhouse gas .

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The key enzyme for mitigating N 2 O emissions is NosZ , which catalyzes N 2 O reduction to N 2 .

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We recently described " atypical " functional NosZ proteins encoded by both denitrifiers and nondenitrifiers , which were missed in previous environmental surveys ( R.

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A. Sanford et al ., Proc .

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Natl .

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Acad .

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Sci .

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U. S.

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A. 109:19709 -19714 , 2012 , doi :10.1073 / pnas.1211238109 ) .

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Here , we analyzed the abundance and diversity of both nosZ types in whole-genome shotgun metagenomes from sandy and silty loam agricultural soils that typify the U.S. Midwest corn belt .

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First , different search algorithms and parameters for detecting nosZ metagenomic reads were evaluated based on in silico-generated ( mock ) metagenomes .

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Using the derived cutoffs , 71 distinct alleles ( 95 % amino acid identity level ) encoding typical or atypical NosZ proteins were detected in both soil types .

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Remarkably , more than 70 % of the total nosZ reads in both soils were classified as atypical , emphasizing that prior surveys underestimated nosZ abundance .

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Approximately 15 % of the total nosZ reads were taxonomically related to Anaeromyxobacter , which was the most abundant genus encoding atypical NosZ-type proteins in both soil types .

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Further analyses revealed that atypical nosZ genes outnumbered typical nosZ genes in most publicly available soil metagenomes , underscoring their potential role in mediating N 2 O consumption in soils .

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Therefore , this study provides a bioinformatics strategy to reliably detect target genes in complex short-read metagenomes and suggests that the analysis of both typical and atypical nosZ sequences is required to understand and predict N 2 O flux in soils .

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INTRODUCTION .

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In recent years , anthropogenic emissions of greenhouse gases have received increasing attention because of their contribution to global warming ( 1 , 2 ) .

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Prominent among these gases is nitrous oxide ( N O ) ( 3 ) , which also contributes to ozone depletion ( 4 , 5 ) .

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