enoriega/keyword_pubmed · Datasets at Hugging Face

sentence stringlengths 1 5.95k	pmcid stringlengths 9 10	keyword_rank int32 -1 169k	keyword_indices sequence
Acquisition and FRET analyses were performed using the Metafluor 4.6 software ( Meta Imaging 4.6 ; Universal Imaging , Downingtown , PA ) .	PMC2568952	-1	[]
The live imaging was done with 4 × 4 binning to minimize exposure time , photobleaching and registration artifacts .	PMC2568952	-1	[]
To study agonist-induced changes in FRET : following the recording of a baseline , cells were first continuously superfused with the phosphodiesterase inhibitor IBMX ( 100 µ M ) and then treated with a mix of IBMX ( 100 µ M ) and the adenylate cyclase activator forskolin ( 25 µ M ) .	PMC2568952	-1	[]
The change in FRET signal due to mobilization of cAMP was detected by FRET / CFP ratiometric processing in both non stimulated and agonist-stimulated cells , in which both FRET ( CFP excitation – YFP emission ) and CFP ( CFP excitation – CFP emission ) images were first background subtracted .	PMC2568952	-1	[]
Cells were thresholded to discard any portions of the image with insufficient intensity to provide reasonable signal / noise .	PMC2568952	-1	[]
The resulting background-subtracted FRET image was divided by that of CFP image to obtain a pixel-to-pixel FRET / CFP ratio image Increasing cAMP levels result in a reduction in FRET ratio ( Em YFP / Em CFP ) .	PMC2568952	-1	[]
The final FRET images were displayed in pseudocolors scaled linearly from the lowest ( red ) to the highest ( blue ) signal to show relative increase in cAMP mobilization of levels within each cell at each treatment at both 3 and 24 hrs .	PMC2568952	154,268	[ 7 ]
Protein extraction and Western blotting .	PMC2568952	-1	[]
Western Blotting was performed as described [ 11 ] .	PMC2568952	-1	[]
Samples were extracted in sodium dodecyl sulfate ( SDS ) sample buffer ( 6.25 mM Tris-HCl , pH 6.8 , containing 10 % ( v / v ) glycerol , 3 mM SDS , 1 % ( v / v ) 2-mercaptoethanol and 0.75 mM of Bromophenol Blue ) , separated by 4 – 12 % SDS-PAGE and blotted to Immobilon P. Analysis of phospho - and total RhoA was performed by using an antibody produced against a peptide of RhoA phosphorylated at serine 188 by PRIMM ( Milan , Italy ) diluted 1 ∶ 1000 , against total RhoA ( sc-418 , Santa Cruz , CA ) diluted at 1 ∶ 1000 , against tubulin ( T5293 , Sigma ) and against the HA tag ( MMS-101R BabCo , diluted 1 ∶ 200 ) .	PMC2568952	-1	[]
Molecular weights standards were ‘ Biotinylated Protein Ladder Detection Pack ’ ( Cell Signaling Technology , MA ) .	PMC2568952	-1	[]
Phospho-Kinase Assays .	PMC2568952	-1	[]
For the kinase phosphorylation measurements total cellular protein was extracted in SDS-sample buffer ( 50 mM Tris-HCl pH 6.8 , 2 % SDS , 10 % glycerol and 0.1 % bromophenol blue ) and approximately 50 µ g was separated on 10 % SDS-PAGE and transfered to Immobilon P , ( Millipore ) .	PMC2568952	-1	[]
The relative amount of each phosphorylated kinase ( ERK , JNK and p38 ) to its total expression was determined by Western Blotting with antibodies specific to each obtained from Cell Signaling Technology ( MA , USA ) .	PMC2568952	-1	[]
p38 MAP kinase activity assay .	PMC2568952	-1	[]
To assay p38 MAP kinase activity , cells were grown to approximately 70 % confluence in 10 cm plates ( GIBCO ) and treated as decribed in figure legends .	PMC2568952	-1	[]
After treatment , cells were washed with ice-cold phosphate-buffered saline ( PBS ) and lysed by 5 minutes at 4 ° C in lysis buffer ( 150 mM NaCl , 1 mM EDTA , 1 mM EGTA , 1 % Triton X-100 , 2.5 mM sodium pyrophosphate , 1 mM ß - glycerophosphate , 1 mM Na VO , 1 µ g / ml leupeptin , 20 mM Tris , pH 7.4 ) plus 1 mM PMSF .	PMC2568952	-1	[]
The cells were scraped into Eppendorf tubes and triturated by sonification .	PMC2568952	-1	[]
The cell lysate was centifuged at 4 ° C for 10 min at 14,000 rpm and the supernatant collected .	PMC2568952	138,592	[ 4 ]
Protein levels were equalized by normalizing them to the protein levels measured before the assay.p38 MAP kinase activity was quantified using an immune complex kinase assay kit according to the manufacturer 's protocol ( New England Biolabs ) .	PMC2568952	-1	[]
Briefly , cleared lysates were immunoprecipitated overnight at 4 ° C with p38 MAPK antibodies conjugated to agarose ( New England BioLabs ) .	PMC2568952	-1	[]
Beads were washed three times with ice-cold lysis buffer and three times with kinase reaction buffer ( 25 mM Tris pH 7.5 , 5 mM ß - Glycerolphosphate , 2 mM DTT , 0.1 mM Na VO , 10 mM MgCl ) minus ATP and ATF-2 .	PMC2568952	-1	[]
The pellet was resuspended in 50 µ l kinase reaction buffer plus 200 µ M ATP and 10 µ g GST-ATF-2 as substrate and incubated at 30 ° C for 30 minutes .	PMC2568952	-1	[]
The reaction was stopped by addition of 2 × Laemmli buffer .	PMC2568952	-1	[]
The sample was run on 10 % SDS-PAGE and blotted onto polyvinylidene difluoride membranes ( Millipore ) for immunoanalysis .	PMC2568952	-1	[]
The amount of ATF-2 phosphorylated by p38 was analyzed by Western blotting with a Phospho-ATF-2 ( Thr71 ) antibody ( Cell Signaling ) that detects only catalytically activated ATF-2 .	PMC2568952	30,452	[ 16 ]
Total p38 expression measured by immunoblotting was not found to vary under any experimental conditions .	PMC2568952	-1	[]
Analysis of RhoA serine phosphorylation state .	PMC2568952	-1	[]
After treatment cell monolayers were washed twice with ice-cold PBS and lysed in ice-cold RIPA ( the above lysis buffer plus 0.1 % SDS and 0.2 % Na-deoxycholate ) .	PMC2568952	-1	[]
The cellular lysate was centrifuged at 14,000 rpm for 5 min at 4 ° C. Protein levels were equalized by normalizing them to the protein levels measured before the assay and the supernatent pre-cleared with protein A-agarose for 2 hrs at 4 ° C. Cleared lysates were immunoprecipitated overnight at 4 ° C with a phosphoserine antibody conjugated to agarose ( SIGMA ) .	PMC2568952	-1	[]
The agarose beads were washed four times with simple RIPA buffer .	PMC2568952	-1	[]
The pellet was resuspended in 50 µ l Laemmli buffer .	PMC2568952	-1	[]
The sample was run on 12 % SDS-PAGE and blotted onto Immobilon P ( Millipore ) for immunoanalysis of the amount of RhoA immunoprecipitated by antiphosphoserine with a RhoA antibody ( sc-418 , Santa Cruz ) .	PMC2568952	-1	[]
Analysis of the activity of RhoA .	PMC2568952	-1	[]
RhoA activity was assessed using the RhoA-binding domain of Rhotekin in a kit supplied from Upstate Biotechnology ( Lake Placid , NY ) .	PMC2568952	-1	[]
In brief , 3 × 10 cells were plated onto 10 cm cell culture dishes and after 24 hrs treated as indicated .	PMC2568952	-1	[]
After the indicated time , cells were extracted with RIPA buffer ( 50 mM Tris , pH 7.2 , 500 mM NaCl , 1 % Triton X-100 , 0.5 % sodium deoxycholate , 1 % SDS , 10 mM MgCl , 0.5 µ g / ml leupeptin , 0.7 µ g / ml pepstatin , 4 µ g / ml aprotinin , and 2 mM PMSF ) .	PMC2568952	-1	[]
After centrifugation at 14,000 g for 3 min , the extracts were incubated for 45 min at 4 ° C with glutathione beads coupled with GST – RBD ( Rho-Binding Domain of Rhotekin ) fusion protein ( Upstate Biotechnology , Lake Placid , NY ) , and then washed three times with Tris buffer , pH 7.2 , containing 1 % Triton X-100 , 150 mM NaCl , and 10 mM MgCl .	PMC2568952	-1	[]
The RhoA content in these samples or in 50 µ g protein of cell homogenate was determined by immunoblotting samples using anti-RhoA antibody from Santa Cruz ( sc-418 , Santa Cruz , CA ) .	PMC2568952	-1	[]
FRET assay for RhoA activity .	PMC2568952	-1	[]
For these experiments , endogenous RhoA activity was measured in FRET microscopy using the Raichu 1297 probe as previously described [ 11 ] .	PMC2568952	1,881	[ 14 ]
In this sensor , the Rho Binding Domain ( RBD ) of the RhoA effector protein , Rhotekin , is sandwiched by Venus-YFP and CFP .	PMC2568952	-1	[]
The binding of endogenous GTP-RhoA to RBD generates a conformational change that displaces YFP and CFP , thereby decreasing fluorescence resonance energy transfer ( FRET ) efficiency between the two fluorophores , while a reduction of intracellular active RhoA results in the opposite effect .	PMC2568952	-1	[]
The CFP channel images were divided by the YFP-FRET channel images .	PMC2568952	-1	[]
The activity of RhoA is monitored by measuring CFP ( 480 nm ) / YFP ( 545 ) fluorescence emission values upon excitation of the transfected cells at 430 nm .	PMC2568952	-1	[]
To eliminate the distracting data from regions outside of cells , the YFP channel is used as a saturation channel .	PMC2568952	-1	[]
The ratio images are presented in pseudocolor mode .	PMC2568952	-1	[]
Ratio intensity is displayed stretched between the low and high renormalization value , according to a temperature-based lookup table with blue ( cold ) and red ( hot ) indicating respectively high and low values of RhoA activity.Additionally , FRET was used to monitor RhoA activity due to the activity balance between endogenous guanine nucleotide exchange factors ( GEFs ) and GTPase-activating proteins ( GAPs ) : cells were transfected with a plasmid with the cDNAfor Raichu-RhoA-1293 , which consisted of truncated RhoA ( aa 1 – 189 ) , the RhoA-binding domain ( RBD ) of effectors , and a pair of GFP mutants , YFP and CFP .	PMC2568952	-1	[]
In these probes , the intramolecular binding of GTP-RhoA to the effector protein was expected to bring CFP in closer proximity to YFP , resulting in an increase in FRET from CFP to YFP.The set up of the microscope and the filters used for FRET excitation and emission were identical to those reported for the cAMP FRET measurements .	PMC2568952	-1	[]
Briefly , twenty-four hours after transfection , monolayers of cells were cultured an additional 24 h in the presence or absence of tetracycline and , images from filter sets dedicated for YFP , CFP , and FRET fluors were first captured , and then three to four random regions of interest within each cell , were chosen for analysis .	PMC2568952	-1	[]
Sensitized FRET measurements Off-line image analysis was performed using the Metafluor 4.6 software ( Meta Imaging 4.6 ; Universal Imaging , Downingtown , PA ) .	PMC2568952	-1	[]
Correction of FRET measurements for spectral bleedthrough and cross excitation was calculated on a pixel-by-pixel basis for the entire image by estimating net FRET ( nF ) as follows : nF = IFRET − ( IYFP × a ) − ( ICFP × b ) , where IFRET is sensitized YFP emission ( excitation 430 nm , emission 545 nm ) and IYFP and ICFP are YFP emission ( 545 nm ) upon excitation at 480 nm and CFP emission ( 480 nm ) upon excitation at 430 nm , respectively .	PMC2568952	33,363	[ 35, 62 ]
a is a norm of the percentage of CFP bleed-through , and b is a norm of the percentage of direct excitation of YFP at 430 nm .	PMC2568952	-1	[]
a and b were determined by analyzing images of cells expressing only CFP or YFP as described previously [ 11 ] , and for our system a and b values corresponded to 64 and 8 % , respectively .	PMC2568952	-1	[]
Corrected FRET ratio was calculated as ICFP / nF .	PMC2568952	-1	[]
Statistical Procedures .	PMC2568952	-1	[]
In the in vitro experiments , student 's t-test was applied to analyze the statistical significance between treatments .	PMC2568952	-1	[]
All comparisons were performed with InStat ( GraphPad Software ) .	PMC2568952	-1	[]
Results .	PMC2568952	-1	[]
Our goal was to study the signal transduction mechanisms involved in neoplastic transformation driven by a biologically relevant oncogene .	PMC2568952	-1	[]
The E7 oncoprotein of HPV16 is known to induce transformation both in vitro and in vivo and to fully transform immortalized rodent fibroblasts ( E7 / NIH3T3 cells ) or human keratinocytes which are tumorigenic in nude mice [ 48 ] , [ 50 ] .	PMC2568952	-1	[]
HPV16 E7-induced transformation induces down-regulation of p38 but not JNK or ERK MAPKinase activity .	PMC2568952	-1	[]
We first determined the effect of HPV16 E7 expression on MAP Kinase activity by transducing immortalized NIH3T3 cells or human foreskin kerotinocytes ( HFK ) , the natural host of the virus , with a recombinant retrovirus expressing non tagged , wild-type HPV16 E7 and measuring the phosphorylation state of ERK1 / 2 , JNK and p38 MAP kinase as described in Material and Methods .	PMC2568952	9,899	[ 21 ]
As seen in Figures 1A ( NIH3T3 ) and 1B ( HFK ) , E7 expression reduced the phosphorylation state of p38 MAP kinase ( p38 ) by approximately 90 % while having no significant effect on the phosphorylation level of either the ERK or JNK MAP kinases .	PMC2568952	-1	[]
The same results were observed in NIH3T3 cells infected with HA-tagged wild-type E7 ( data not shown ) .	PMC2568952	-1	[]
Further , two different protocols were used to determine whether it is simply E7 expression or E7-dependent transformation that leads to an inhibition of p38 .	PMC2568952	-1	[]
First , we used human primary keratinocytes ( HPKIA cells ) immortalized by transfection of the entire HPV16 genome and which become spontaneously transformed at high passage number and are tumorigenic in nude mice [ 48 ] , [ 50 ] .	PMC2568952	-1	[]
As shown in Figure 1C , p38 was much less phosphorylated in the transformed ( late passage ) keratinocytes than in the immortalized ( early passage ) cells while , also here , having no significant effect on the phosphorylation levels of either the ERK1 / 2 or JNK MAP kinases .	PMC2568952	-1	[]
To further confirm this dependance of p38 dephosphorylation upon HPV-induced transformation , we then extended our study to NIH3T3 cells infected with recombinant retroviruses expressing either wild-type HPV16 E7 or transformation-deficient mutants [ 53 ] , [ 54 ] .	PMC2568952	-1	[]
As seen in Fig. 1D , the wild-type E7 reduced p38 phosphorylation as above while the transformation negative mutants had no effect on p38 phosphorylation .	PMC2568952	-1	[]
Altogether , these results demonstrate that HPV16-induced transformation reduces the p38 activity through E7 without affecting either the ERK or the JNK MAP kinases and are consistent with the reported specificity of p38 in oncogenic processes .	PMC2568952	-1	[]
HPV16 E7-dependent down-regulation of p38 is an early event in transformation .	PMC2568952	-1	[]
The above experimental systems do not permit the determination of the dynamic processes occurring during transformation .	PMC2568952	-1	[]
To determine whether inhibition of p38 is an early event in transformation , a cell model was constructed in which the transformation of normal cells can be rapidly induced and early events subsequently monitored .	PMC2568952	-1	[]
NIH3T3 cells were infected with a recombinant retrovirus in which HPV16 E7 gene expression is under the control of a promoter that is negatively regulated by tetracycline , clone 2BN11 [ 48 ] .	PMC2568952	-1	[]
The temporal sequence of the increase in E7 message ( Fig. 2A ) and the change in p38 phosphorylation state ( Fig. 2B ) and in activity , measured as the phosphorylation of its substrate , ATF2 , ( Fig. 2C ) was monitored after induction of E7 expression .	PMC2568952	-1	[]
These experiments showed that the increase in E7 mRNA and reduction of both p38 phosphorylation and activity levels started approximately 2 – 3 hrs after tet removal , which is in agreement with the previously observed time course for the appearance of E7 mRNA and protein [ 48 ] .	PMC2568952	-1	[]
HPV16 E7 expression activates the cAMP pathway .	PMC2568952	-1	[]
As the cAMP / PKA system has been demonstrated to be involved in both transformation / tumor progression [ 12 ] , [ 35 ] , [ 55 ] , [ 56 ] and regulation of p38 [ 57 ] , we next measured the effect of E7-driven transformation on the levels of cellular cAMP at both early ( 3 hrs ) and late ( 24 hrs ) time points after tet removal .	PMC2568952	-1	[]
We started with a biochemical assay in which the bioluminescence intensity level inversely correlates with cAMP intracellular mobilization ( see Materials and Methods ) .	PMC2568952	-1	[]
Cells were first treated with 100 µ M of the phosphodiesterase inhibitor , IBMX , in order to observe the activity level of endogenous adenylate cyclase and then were treated with IBMX together with 20 µ M of the pharmacologic adenylate cyclase activator , forskolin , to determine the dynamic range of increasing activity of the remaining non E7-stimulated enzyme .	PMC2568952	-1	[]
The left panel of Figure 3A shows a much greater decrease of bioluminescence after inhibition of intracellular phosphodiesterases by IBMX in the E7-transformed cells ( − tet ) while the subsequent decrease of bioluminescence after addition of the adenylate cyclase stimulator , forskolin , was similar in control and transformed cells .	PMC2568952	-1	[]
Calculation of cAMP concentration with a standard curve showed that transformation increased cellular cAMP levels ( measured in the presence of IBMX ) , by approximately 2.5-fold at 3 hrs and 5-fold at 24 hrs without increasing total potential adenylate cyclase activity ( Figure 3A right panel ) .	PMC2568952	-1	[]
Interestingly , a similar rise in cAMP was observed in late passage compared to early HPK1A cells and , further , blockage of NHE1 activity with 2 µ M of its specific inhibitor , HOE642 , had no effect on this E7-induced increase in cAMP in either 2BN11 or HPK1A cells ( Figure S3 ) .	PMC2568952	-1	[]
To further verify this specific stimulation of cAMP mobilization in response to HPV16 E7 expression and visualize cAMP dynamics in vivo , we transfected 2BN11 cells with a PKA-based FRET construct composed of green fluorescent protein variants bound to either the PKA catalytic subunit ( Cat-YFP ) or the PKA regulatory II subunit ( RII-CFP ) and recorded FRET images with a dual-emission CCD camera .	PMC2568952	-1	[]
Increased levels of cAMP produce a decrease in FRET signal , measured as YFP-to-CFP emissions ratio , due to cAMP binding to the chimeric PKA reporter and the resultant separation of the catalytic and regulatory subunits .	PMC2568952	-1	[]
In time course experiments , when the cAMP signal was stable , cells were first treated with IBMX in order to observe the activity level of endogenous adenylate cyclase and then were perfused with IBMX together with forskolin to determine the dynamic range of increasing activity of the remaining non E7-stimulated enzyme .	PMC2568952	-1	[]
As can be seen in Figure 3B , IBMX superfusion stimulated cAMP production approximately 2-fold at 3 hrs and 4.5-fold at 24 hrs in the E7 transformed cells compared to the control cells , while the further cAMP elevation after forskolin ( FSK ) treatment was similar in both conditions .	PMC2568952	-1	[]
Altogether , these data suggest that , indeed , transformation of the cells by E7 stimulates adenylate cyclase-dependent production of cAMP above very low basal levels in the control cells .	PMC2568952	-1	[]
Figure 3C shows the relative increase in cAMP mobilization as pseudocolor changes in typical FRET experiments .	PMC2568952	-1	[]
Involvement of PKA and p38 in HPV16 E7-mediated transformation .	PMC2568952	-1	[]
To assess the role and dynamics of PKA and p38 in mediating transformation , we utilized changes in NHE1 activity as the transformation readout because we have previously demonstrated that stimulation of NHE1 occurs early in transformation and is necessary and sufficient for the further development of transformed phenotypes [ 48 ] .	PMC2568952	-1	[]
As seen in Figure 4 , the induction of cellular E7 expression and transformation by removal of tetracycline for 24 hrs stimulated NHE1 activity by 69.5 ± 6.2 % , n = 15 , P < 0.02 ( blue cross-hatched bar ) and this stimulation was blocked by 2 µ M of the specific inhibitor of the NHE1 , HOE642 ( data not shown ) .	PMC2568952	-1	[]
Inhibition of PKA by its specific inhibitor , H89 , completely abrogated the transformation-dependent increase in NHE1 activity ( green stippled bar ) while inhibition of p38 activity by either its pharmacological inhibitor , SB203580 , or by the transient expression of the dominant negative ( dn ) mutant for p38alpha ( p38AF ) further potentiated the transformation-dependent increase in NHE1 activity by approximately 2-fold ( black stripped bars ) .	PMC2568952	-1	[]
Furthermore , inhibition of p38 either with SB203580 or via transient expression of dnp38alpha blocked the abrogation of the transformation-dependent activation of NHE1 by H89 ( red stripped bars ) .	PMC2568952	-1	[]
These data strongly suggest that PKA and p38alpha are part of the same pathway in regulating the transformation-dependent stimulation of NHE1 and that PKA is up-stream of p38 .	PMC2568952	-1	[]
This conclusion is further supported by the ability of H89 to block and forskolin ( Fsk ) to potentiate the E7-dependent down-regulation of p38 phosphorylation ( Figures 4B & C ) while having no effect on the phosphorylation state of JNK and ERK ( Figure S4 ) .	PMC2568952	-1	[]
HPV16 E7-dependent transformation induces PKA-dependent phosphorylation and down-regulation of RhoA .	PMC2568952	-1	[]
However , the steps that mediate the down-regulation of p38 by PKA still need to be identified .	PMC2568952	-1	[]
RhoA is a potential candidate since it has been shown to be an upstream regulator of p38 in enhancing migration and invasion of breast cancer [ 11 ] and pancreatic carcinoma cells [ 32 ] and to be inhibited by PKA-dependent phosphorylation on serine 188 [ 11 ] , [ 58 ] .	PMC2568952	-1	[]

Acquisition and FRET analyses were performed using the Metafluor 4.6 software ( Meta Imaging 4.6 ; Universal Imaging , Downingtown , PA ) .

PMC2568952

-1

[]

The live imaging was done with 4 × 4 binning to minimize exposure time , photobleaching and registration artifacts .

PMC2568952

-1

[]

To study agonist-induced changes in FRET : following the recording of a baseline , cells were first continuously superfused with the phosphodiesterase inhibitor IBMX ( 100 µ M ) and then treated with a mix of IBMX ( 100 µ M ) and the adenylate cyclase activator forskolin ( 25 µ M ) .

PMC2568952

-1

[]

The change in FRET signal due to mobilization of cAMP was detected by FRET / CFP ratiometric processing in both non stimulated and agonist-stimulated cells , in which both FRET ( CFP excitation – YFP emission ) and CFP ( CFP excitation – CFP emission ) images were first background subtracted .

PMC2568952

-1

[]

Cells were thresholded to discard any portions of the image with insufficient intensity to provide reasonable signal / noise .

PMC2568952

-1

[]

The resulting background-subtracted FRET image was divided by that of CFP image to obtain a pixel-to-pixel FRET / CFP ratio image Increasing cAMP levels result in a reduction in FRET ratio ( Em YFP / Em CFP ) .

PMC2568952

-1

[]

The final FRET images were displayed in pseudocolors scaled linearly from the lowest ( red ) to the highest ( blue ) signal to show relative increase in cAMP mobilization of levels within each cell at each treatment at both 3 and 24 hrs .

PMC2568952

154,268

[ 7 ]

Protein extraction and Western blotting .

PMC2568952

-1

[]

Western Blotting was performed as described [ 11 ] .

PMC2568952

-1

[]

Samples were extracted in sodium dodecyl sulfate ( SDS ) sample buffer ( 6.25 mM Tris-HCl , pH 6.8 , containing 10 % ( v / v ) glycerol , 3 mM SDS , 1 % ( v / v ) 2-mercaptoethanol and 0.75 mM of Bromophenol Blue ) , separated by 4 – 12 % SDS-PAGE and blotted to Immobilon P. Analysis of phospho - and total RhoA was performed by using an antibody produced against a peptide of RhoA phosphorylated at serine 188 by PRIMM ( Milan , Italy ) diluted 1 ∶ 1000 , against total RhoA ( sc-418 , Santa Cruz , CA ) diluted at 1 ∶ 1000 , against tubulin ( T5293 , Sigma ) and against the HA tag ( MMS-101R BabCo , diluted 1 ∶ 200 ) .

PMC2568952

-1

[]

Molecular weights standards were ‘ Biotinylated Protein Ladder Detection Pack ’ ( Cell Signaling Technology , MA ) .

PMC2568952

-1

[]

Phospho-Kinase Assays .

PMC2568952

-1

[]

For the kinase phosphorylation measurements total cellular protein was extracted in SDS-sample buffer ( 50 mM Tris-HCl pH 6.8 , 2 % SDS , 10 % glycerol and 0.1 % bromophenol blue ) and approximately 50 µ g was separated on 10 % SDS-PAGE and transfered to Immobilon P , ( Millipore ) .

PMC2568952

-1

[]

The relative amount of each phosphorylated kinase ( ERK , JNK and p38 ) to its total expression was determined by Western Blotting with antibodies specific to each obtained from Cell Signaling Technology ( MA , USA ) .

PMC2568952

-1

[]

p38 MAP kinase activity assay .

PMC2568952

-1

[]

To assay p38 MAP kinase activity , cells were grown to approximately 70 % confluence in 10 cm plates ( GIBCO ) and treated as decribed in figure legends .

PMC2568952

-1

[]

After treatment , cells were washed with ice-cold phosphate-buffered saline ( PBS ) and lysed by 5 minutes at 4 ° C in lysis buffer ( 150 mM NaCl , 1 mM EDTA , 1 mM EGTA , 1 % Triton X-100 , 2.5 mM sodium pyrophosphate , 1 mM ß - glycerophosphate , 1 mM Na VO , 1 µ g / ml leupeptin , 20 mM Tris , pH 7.4 ) plus 1 mM PMSF .

PMC2568952

-1

[]

The cells were scraped into Eppendorf tubes and triturated by sonification .

PMC2568952

-1

[]

The cell lysate was centifuged at 4 ° C for 10 min at 14,000 rpm and the supernatant collected .

PMC2568952

138,592

[ 4 ]

Protein levels were equalized by normalizing them to the protein levels measured before the assay.p38 MAP kinase activity was quantified using an immune complex kinase assay kit according to the manufacturer 's protocol ( New England Biolabs ) .

PMC2568952

-1

[]

Briefly , cleared lysates were immunoprecipitated overnight at 4 ° C with p38 MAPK antibodies conjugated to agarose ( New England BioLabs ) .

PMC2568952

-1

[]

Beads were washed three times with ice-cold lysis buffer and three times with kinase reaction buffer ( 25 mM Tris pH 7.5 , 5 mM ß - Glycerolphosphate , 2 mM DTT , 0.1 mM Na VO , 10 mM MgCl ) minus ATP and ATF-2 .

PMC2568952

-1

[]

The pellet was resuspended in 50 µ l kinase reaction buffer plus 200 µ M ATP and 10 µ g GST-ATF-2 as substrate and incubated at 30 ° C for 30 minutes .

PMC2568952

-1

[]

The reaction was stopped by addition of 2 × Laemmli buffer .

PMC2568952

-1

[]

The sample was run on 10 % SDS-PAGE and blotted onto polyvinylidene difluoride membranes ( Millipore ) for immunoanalysis .

PMC2568952

-1

[]

The amount of ATF-2 phosphorylated by p38 was analyzed by Western blotting with a Phospho-ATF-2 ( Thr71 ) antibody ( Cell Signaling ) that detects only catalytically activated ATF-2 .

PMC2568952

30,452

[ 16 ]

Total p38 expression measured by immunoblotting was not found to vary under any experimental conditions .

PMC2568952

-1

[]

Analysis of RhoA serine phosphorylation state .

PMC2568952

-1

[]

After treatment cell monolayers were washed twice with ice-cold PBS and lysed in ice-cold RIPA ( the above lysis buffer plus 0.1 % SDS and 0.2 % Na-deoxycholate ) .

PMC2568952

-1

[]

The cellular lysate was centrifuged at 14,000 rpm for 5 min at 4 ° C. Protein levels were equalized by normalizing them to the protein levels measured before the assay and the supernatent pre-cleared with protein A-agarose for 2 hrs at 4 ° C. Cleared lysates were immunoprecipitated overnight at 4 ° C with a phosphoserine antibody conjugated to agarose ( SIGMA ) .

PMC2568952

-1

[]

The agarose beads were washed four times with simple RIPA buffer .

PMC2568952

-1

[]

The pellet was resuspended in 50 µ l Laemmli buffer .

PMC2568952

-1

[]

The sample was run on 12 % SDS-PAGE and blotted onto Immobilon P ( Millipore ) for immunoanalysis of the amount of RhoA immunoprecipitated by antiphosphoserine with a RhoA antibody ( sc-418 , Santa Cruz ) .

PMC2568952

-1

[]

Analysis of the activity of RhoA .

PMC2568952

-1

[]

RhoA activity was assessed using the RhoA-binding domain of Rhotekin in a kit supplied from Upstate Biotechnology ( Lake Placid , NY ) .

PMC2568952

-1

[]

In brief , 3 × 10 cells were plated onto 10 cm cell culture dishes and after 24 hrs treated as indicated .

PMC2568952

-1

[]

After the indicated time , cells were extracted with RIPA buffer ( 50 mM Tris , pH 7.2 , 500 mM NaCl , 1 % Triton X-100 , 0.5 % sodium deoxycholate , 1 % SDS , 10 mM MgCl , 0.5 µ g / ml leupeptin , 0.7 µ g / ml pepstatin , 4 µ g / ml aprotinin , and 2 mM PMSF ) .

PMC2568952

-1

[]

After centrifugation at 14,000 g for 3 min , the extracts were incubated for 45 min at 4 ° C with glutathione beads coupled with GST – RBD ( Rho-Binding Domain of Rhotekin ) fusion protein ( Upstate Biotechnology , Lake Placid , NY ) , and then washed three times with Tris buffer , pH 7.2 , containing 1 % Triton X-100 , 150 mM NaCl , and 10 mM MgCl .

PMC2568952

-1

[]

The RhoA content in these samples or in 50 µ g protein of cell homogenate was determined by immunoblotting samples using anti-RhoA antibody from Santa Cruz ( sc-418 , Santa Cruz , CA ) .

PMC2568952

-1

[]

FRET assay for RhoA activity .

PMC2568952

-1

[]

For these experiments , endogenous RhoA activity was measured in FRET microscopy using the Raichu 1297 probe as previously described [ 11 ] .

PMC2568952

1,881

[ 14 ]

In this sensor , the Rho Binding Domain ( RBD ) of the RhoA effector protein , Rhotekin , is sandwiched by Venus-YFP and CFP .

PMC2568952

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The binding of endogenous GTP-RhoA to RBD generates a conformational change that displaces YFP and CFP , thereby decreasing fluorescence resonance energy transfer ( FRET ) efficiency between the two fluorophores , while a reduction of intracellular active RhoA results in the opposite effect .

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The CFP channel images were divided by the YFP-FRET channel images .

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The activity of RhoA is monitored by measuring CFP ( 480 nm ) / YFP ( 545 ) fluorescence emission values upon excitation of the transfected cells at 430 nm .

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To eliminate the distracting data from regions outside of cells , the YFP channel is used as a saturation channel .

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The ratio images are presented in pseudocolor mode .

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Ratio intensity is displayed stretched between the low and high renormalization value , according to a temperature-based lookup table with blue ( cold ) and red ( hot ) indicating respectively high and low values of RhoA activity.Additionally , FRET was used to monitor RhoA activity due to the activity balance between endogenous guanine nucleotide exchange factors ( GEFs ) and GTPase-activating proteins ( GAPs ) : cells were transfected with a plasmid with the cDNAfor Raichu-RhoA-1293 , which consisted of truncated RhoA ( aa 1 – 189 ) , the RhoA-binding domain ( RBD ) of effectors , and a pair of GFP mutants , YFP and CFP .

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In these probes , the intramolecular binding of GTP-RhoA to the effector protein was expected to bring CFP in closer proximity to YFP , resulting in an increase in FRET from CFP to YFP.The set up of the microscope and the filters used for FRET excitation and emission were identical to those reported for the cAMP FRET measurements .

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Briefly , twenty-four hours after transfection , monolayers of cells were cultured an additional 24 h in the presence or absence of tetracycline and , images from filter sets dedicated for YFP , CFP , and FRET fluors were first captured , and then three to four random regions of interest within each cell , were chosen for analysis .

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Sensitized FRET measurements Off-line image analysis was performed using the Metafluor 4.6 software ( Meta Imaging 4.6 ; Universal Imaging , Downingtown , PA ) .

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Correction of FRET measurements for spectral bleedthrough and cross excitation was calculated on a pixel-by-pixel basis for the entire image by estimating net FRET ( nF ) as follows : nF = IFRET − ( IYFP × a ) − ( ICFP × b ) , where IFRET is sensitized YFP emission ( excitation 430 nm , emission 545 nm ) and IYFP and ICFP are YFP emission ( 545 nm ) upon excitation at 480 nm and CFP emission ( 480 nm ) upon excitation at 430 nm , respectively .

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a is a norm of the percentage of CFP bleed-through , and b is a norm of the percentage of direct excitation of YFP at 430 nm .

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a and b were determined by analyzing images of cells expressing only CFP or YFP as described previously [ 11 ] , and for our system a and b values corresponded to 64 and 8 % , respectively .

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Corrected FRET ratio was calculated as ICFP / nF .

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Statistical Procedures .

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In the in vitro experiments , student 's t-test was applied to analyze the statistical significance between treatments .

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All comparisons were performed with InStat ( GraphPad Software ) .

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Results .

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Our goal was to study the signal transduction mechanisms involved in neoplastic transformation driven by a biologically relevant oncogene .

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The E7 oncoprotein of HPV16 is known to induce transformation both in vitro and in vivo and to fully transform immortalized rodent fibroblasts ( E7 / NIH3T3 cells ) or human keratinocytes which are tumorigenic in nude mice [ 48 ] , [ 50 ] .

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HPV16 E7-induced transformation induces down-regulation of p38 but not JNK or ERK MAPKinase activity .

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We first determined the effect of HPV16 E7 expression on MAP Kinase activity by transducing immortalized NIH3T3 cells or human foreskin kerotinocytes ( HFK ) , the natural host of the virus , with a recombinant retrovirus expressing non tagged , wild-type HPV16 E7 and measuring the phosphorylation state of ERK1 / 2 , JNK and p38 MAP kinase as described in Material and Methods .

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As seen in Figures 1A ( NIH3T3 ) and 1B ( HFK ) , E7 expression reduced the phosphorylation state of p38 MAP kinase ( p38 ) by approximately 90 % while having no significant effect on the phosphorylation level of either the ERK or JNK MAP kinases .

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The same results were observed in NIH3T3 cells infected with HA-tagged wild-type E7 ( data not shown ) .

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Further , two different protocols were used to determine whether it is simply E7 expression or E7-dependent transformation that leads to an inhibition of p38 .

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First , we used human primary keratinocytes ( HPKIA cells ) immortalized by transfection of the entire HPV16 genome and which become spontaneously transformed at high passage number and are tumorigenic in nude mice [ 48 ] , [ 50 ] .

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As shown in Figure 1C , p38 was much less phosphorylated in the transformed ( late passage ) keratinocytes than in the immortalized ( early passage ) cells while , also here , having no significant effect on the phosphorylation levels of either the ERK1 / 2 or JNK MAP kinases .

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To further confirm this dependance of p38 dephosphorylation upon HPV-induced transformation , we then extended our study to NIH3T3 cells infected with recombinant retroviruses expressing either wild-type HPV16 E7 or transformation-deficient mutants [ 53 ] , [ 54 ] .

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As seen in Fig. 1D , the wild-type E7 reduced p38 phosphorylation as above while the transformation negative mutants had no effect on p38 phosphorylation .

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Altogether , these results demonstrate that HPV16-induced transformation reduces the p38 activity through E7 without affecting either the ERK or the JNK MAP kinases and are consistent with the reported specificity of p38 in oncogenic processes .

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HPV16 E7-dependent down-regulation of p38 is an early event in transformation .

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The above experimental systems do not permit the determination of the dynamic processes occurring during transformation .

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To determine whether inhibition of p38 is an early event in transformation , a cell model was constructed in which the transformation of normal cells can be rapidly induced and early events subsequently monitored .

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NIH3T3 cells were infected with a recombinant retrovirus in which HPV16 E7 gene expression is under the control of a promoter that is negatively regulated by tetracycline , clone 2BN11 [ 48 ] .

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The temporal sequence of the increase in E7 message ( Fig. 2A ) and the change in p38 phosphorylation state ( Fig. 2B ) and in activity , measured as the phosphorylation of its substrate , ATF2 , ( Fig. 2C ) was monitored after induction of E7 expression .

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These experiments showed that the increase in E7 mRNA and reduction of both p38 phosphorylation and activity levels started approximately 2 – 3 hrs after tet removal , which is in agreement with the previously observed time course for the appearance of E7 mRNA and protein [ 48 ] .

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HPV16 E7 expression activates the cAMP pathway .

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As the cAMP / PKA system has been demonstrated to be involved in both transformation / tumor progression [ 12 ] , [ 35 ] , [ 55 ] , [ 56 ] and regulation of p38 [ 57 ] , we next measured the effect of E7-driven transformation on the levels of cellular cAMP at both early ( 3 hrs ) and late ( 24 hrs ) time points after tet removal .

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We started with a biochemical assay in which the bioluminescence intensity level inversely correlates with cAMP intracellular mobilization ( see Materials and Methods ) .

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Cells were first treated with 100 µ M of the phosphodiesterase inhibitor , IBMX , in order to observe the activity level of endogenous adenylate cyclase and then were treated with IBMX together with 20 µ M of the pharmacologic adenylate cyclase activator , forskolin , to determine the dynamic range of increasing activity of the remaining non E7-stimulated enzyme .

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The left panel of Figure 3A shows a much greater decrease of bioluminescence after inhibition of intracellular phosphodiesterases by IBMX in the E7-transformed cells ( − tet ) while the subsequent decrease of bioluminescence after addition of the adenylate cyclase stimulator , forskolin , was similar in control and transformed cells .

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Calculation of cAMP concentration with a standard curve showed that transformation increased cellular cAMP levels ( measured in the presence of IBMX ) , by approximately 2.5-fold at 3 hrs and 5-fold at 24 hrs without increasing total potential adenylate cyclase activity ( Figure 3A right panel ) .

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Interestingly , a similar rise in cAMP was observed in late passage compared to early HPK1A cells and , further , blockage of NHE1 activity with 2 µ M of its specific inhibitor , HOE642 , had no effect on this E7-induced increase in cAMP in either 2BN11 or HPK1A cells ( Figure S3 ) .

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To further verify this specific stimulation of cAMP mobilization in response to HPV16 E7 expression and visualize cAMP dynamics in vivo , we transfected 2BN11 cells with a PKA-based FRET construct composed of green fluorescent protein variants bound to either the PKA catalytic subunit ( Cat-YFP ) or the PKA regulatory II subunit ( RII-CFP ) and recorded FRET images with a dual-emission CCD camera .

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Increased levels of cAMP produce a decrease in FRET signal , measured as YFP-to-CFP emissions ratio , due to cAMP binding to the chimeric PKA reporter and the resultant separation of the catalytic and regulatory subunits .

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In time course experiments , when the cAMP signal was stable , cells were first treated with IBMX in order to observe the activity level of endogenous adenylate cyclase and then were perfused with IBMX together with forskolin to determine the dynamic range of increasing activity of the remaining non E7-stimulated enzyme .

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As can be seen in Figure 3B , IBMX superfusion stimulated cAMP production approximately 2-fold at 3 hrs and 4.5-fold at 24 hrs in the E7 transformed cells compared to the control cells , while the further cAMP elevation after forskolin ( FSK ) treatment was similar in both conditions .

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Altogether , these data suggest that , indeed , transformation of the cells by E7 stimulates adenylate cyclase-dependent production of cAMP above very low basal levels in the control cells .

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Figure 3C shows the relative increase in cAMP mobilization as pseudocolor changes in typical FRET experiments .

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Involvement of PKA and p38 in HPV16 E7-mediated transformation .

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To assess the role and dynamics of PKA and p38 in mediating transformation , we utilized changes in NHE1 activity as the transformation readout because we have previously demonstrated that stimulation of NHE1 occurs early in transformation and is necessary and sufficient for the further development of transformed phenotypes [ 48 ] .

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As seen in Figure 4 , the induction of cellular E7 expression and transformation by removal of tetracycline for 24 hrs stimulated NHE1 activity by 69.5 ± 6.2 % , n = 15 , P < 0.02 ( blue cross-hatched bar ) and this stimulation was blocked by 2 µ M of the specific inhibitor of the NHE1 , HOE642 ( data not shown ) .

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Inhibition of PKA by its specific inhibitor , H89 , completely abrogated the transformation-dependent increase in NHE1 activity ( green stippled bar ) while inhibition of p38 activity by either its pharmacological inhibitor , SB203580 , or by the transient expression of the dominant negative ( dn ) mutant for p38alpha ( p38AF ) further potentiated the transformation-dependent increase in NHE1 activity by approximately 2-fold ( black stripped bars ) .

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Furthermore , inhibition of p38 either with SB203580 or via transient expression of dnp38alpha blocked the abrogation of the transformation-dependent activation of NHE1 by H89 ( red stripped bars ) .

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These data strongly suggest that PKA and p38alpha are part of the same pathway in regulating the transformation-dependent stimulation of NHE1 and that PKA is up-stream of p38 .

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This conclusion is further supported by the ability of H89 to block and forskolin ( Fsk ) to potentiate the E7-dependent down-regulation of p38 phosphorylation ( Figures 4B & C ) while having no effect on the phosphorylation state of JNK and ERK ( Figure S4 ) .

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HPV16 E7-dependent transformation induces PKA-dependent phosphorylation and down-regulation of RhoA .

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However , the steps that mediate the down-regulation of p38 by PKA still need to be identified .

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RhoA is a potential candidate since it has been shown to be an upstream regulator of p38 in enhancing migration and invasion of breast cancer [ 11 ] and pancreatic carcinoma cells [ 32 ] and to be inhibited by PKA-dependent phosphorylation on serine 188 [ 11 ] , [ 58 ] .

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