Ser81 Survivin induced PKA-PI3K pathway (Sandra F, et al.
Indones Biomed J.
: 157-62
DOI: 10.
18585/inabj.
RESEARCH ARTICLE
Ser81 Survivin Induced Protein Kinase A (PKA)-dependent Phosphatidylinositol 3-kinase (PI3K) Activity Ferry Sandra1,2,3,4,E.
Roya Khosravi-Far4 Department of Biochemistry and Molecular Biology.
Faculty of Dentistry.
Trisakti University.
Jl.
Kyai Tapa No.
Jakarta.
Indonesia BioCORE Laboratory.
Faculty of Dentistry.
Trisakti University.
Jl.
Kyai Tapa No.
Jakarta.
Indonesia Prodia Clinical Laboratory.
Prodia Tower.
Jl.
Kramat Raya No.
Jakarta.
Indonesia Department of Pathology.
Harvard Medical School.
BIDMC.
Research North, 99 Brookline Ave.
Boston.
MA 02215.
USA
Corresponding author.
E-mail: ferrysandra@gmail.
Abstract Abstrak ACKGROUND: Our previous report showed that phosphorylated-survivin at Ser81 induces survivin back loop to activate protein kinase A (PKA) in the cytoprotection mechanism.
Activated PKA could possibly induce the cytoprotection via Phosphatydilinositol 3-kinase (PI3K).
Therefore our current study was conducted to investigate the possibility of survivin-PKA-PI3K signaling ATAR BELAKANG: Penelitian kami sebelumnya memperlihatkan bahwa survivin yang terfosforilasi pada Ser81, dapat menginduksi survivin back loop sehingga mengaktifkan protein kinase A (PKA) dalam mekanisme pertahanan sel.
PKA yang teraktivasi dapat menginduksi pertahanan sel lewat Phosphatydilinositol 3-kinase (PI3K).
Oleh karena itu, kami melakukan penelitian ini untuk mengetahui kemungkinan adanya jalur pensinyalan survivin-PKA-PI3K.
METHODS: Viral productions by BOSC23 cells of Survivin.
Antisense Survivin (Survivin-AS) and Ser81Ala mutant (Survivin-S81A) in pMSCV-IRES-GFP vector with cytomegalovirus (CMV) promoter were conducted.
L929 cells were pretreated with/without PKI 6-22 amide and infected with viral particle of Survivin.
Survivin-AS.
Survivin-S81A or vector only.
Cells were harvested, lysed and immunoprecipitated with anti-PI3K .
antibody and immunoblotted to detect PI3K .
and phospho-(Ty.
p85 PI3K.
To conirm PI3K activation.
PI3K Activity Assay was conducted by using phosphoinositide fraction containing PtdIns.
P2 and .
P]ATP.
RESULTS: Immunoblot and PI3K activity results showed similar results.
Upon infection of virus with survivin, a markedly increased level of tyrosine phosphorylation of p85 PI3K or PI3K activity in L929 cells was seen.
Low levels of tyrosine phosphorylation of p85 PI3K or PI3K activity were observed for Survivin-AS and Survivin-S81Aviral-infected L929 cells.
With higher concentrations of Survivin-viral-infection, levels of tyrosine phosphorylation of p85 PI3K or PI3K activity in L929 cells were gradually METODE: Dilakukan produksi virus Survivin.
Antisense Survivin (Survivin-AS) dan Ser81Ala mutant (SurvivinS81A) pada plasmid pMSCV-IRES-GFP dengan promoter cytomegalovirus (CMV) oleh sel BOSC23.
Sel L929 diberikan perlakuan dengan/tanpa PKI 6-22 amide sebelum diinfeksikan dengan virus Survivin.
Survivin-AS.
SurvivinS81A atau vektor saja.
Sel kemudian dikumpulkan, dilisis, dan di-imunopresipitasi dengan antibodi anti-PI3K .
dan di-imunoblot untuk mendeteksi PI3K .
and phospho(Ty.
p85 PI3K.
Untuk mengkonirmasi adanya aktivasi PI3K, tes aktivitas PI3K dilakukan dengan menggunakan fraksi fosfoinositida dan PtdIns.
P2 dan .
P]ATP.
HASIL: Hasil imunoblot dan aktivitas PI3K memperlihatkan hasil yang serupa.
Setelah infeksi dengan virus Survivin, terlihat peningkatan level p85 PI3K yang terfosforilasi pada tirosin.
Peningkatan aktivitas PI3K juga Didapatkan level yang rendah pada p85 PI3K yang terfosforilasi pada tirosin dan aktivitas PI3K pada infeksi virus Survivin-AS dan Survivin-S81A.
Dengan konsentrasi virus Survivin yang semakin tinggi, maka level p85 PI3K The Indonesian Biomedical Journal.
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No.
December 2014, p.
However, when L929 cells were pretreated with PKI 6-22 amide, prior to Survivin-viral-infection, level of tyrosine phosphorylation level of p85 PI3K or PI3K activity was detected much lower.
CONCLUSION: Our result suggest that Ser81 Survivin play role in inducing PI3K activation and the Survivin-PI3K signaling pathway was PKA-dependent.
Print ISSN: 2085-3297.
Online ISSN: 2355-9179 yang terfosforilasi pada tirosin dan aktivitas PI3K semakin meningkat pula.
Akan tetapi, ketika sel L929 diberikan perlakuan dengan PKI 6-22 amide, sebelum infeksi dengan Survivin, level p85 PI3K yang terfosforilasi pada tirosin dan aktivitas PI3K akan rendah sekali.
KESIMPULAN: Ser81 Survivin sangat penting dalam mengiduksi aktivasi PI3K dan jalur pensinyalan SurvivinPI3K merupakan jalur yang bergantung pada PKA.
KEYWORDS: Ser81.
Survivin.
PKA.
PI3K.
L929 KATA KUNCI: Ser81.
Survivin.
PKA.
PI3K.
L929 Indones Biomed J.
: 157-62
Introduction Among Inhibitor of mammalian Inhibitor of Apoptosis Proteins (IAP.
family, survivin is the smallest member, containing a single Baculovirus IAP Repeat (BIR), which is the hallmark of these molecules.
Transcription of the survivin gene is strictly cell cycle-regulated, and peaks at mitosis.
Survivin plays several roles in homeostatic networks, including the control of mitosis, the regulation of apoptosis and the cellular stress response.
Therefore, the presence of survivin in cancer has been linked to resistance to apoptosis, metastasis, cell cycle checkpoints bypass and resistance to therapy.
In addition, in some cancers, survivin have identiied survivin as a Aorisk factorAo for poor prognosis and disease recurrence.
In response to cell death stimulation, survivin physically complexes with X-linked IAP (XIAP), then in turn promotes XIAP stability and synergistic inhibition of caspase-9 activation.
Besides that.
Phosphatydilinositol 3-kinase (PI3K).
Akt, -catenin.
T-cell factor (Tc.
Lymphoid enhancer-binding factor (Le.
and many others implicated in the expression of survivin.
Our previous report showed that Survivin dominant negative enhanced Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)Aos activity in inducing apoptosis.
In accordance, a recent report showed a posititve feedback loop connecting survivin expression in tumor cells to PI3K/ Akt, followed by secretion of Vascular Endothelial Growth Factor (VEGF) and angiogenesis.
Amino acid residue Thr34 that lies within surAvivin BIR domain, was shown as an important amino acid in regulation of apoptosis.
Phosphorylated-survivin at Thr34 bound to caspase-9 in HeLa cells.
In addition, initiated from Scansite result.
Ser81 was shown as another potential target to inhibit survivin-modulated cytoprotection as well as to sensitize eficacy of TRAIL or other related apoptotic inducers.
Phosphorylated-survivin at Ser81 was later known mediating survivin back loop to activate protein kinase A (PKA) in the cytoprotection mechanism.
As being reported, phosphodiesterase 4 (PDE.
control intracellular cyclic adenosine monophosphate .
AMP) levels by catalyzing their hydrolysis and inactivating second .
PDE4 inhibition promotes PKA-dependent resolution of established neutrophilic inlammation by inhibition of the PI3K/Akt/myeloid cell leukemia 1 (Mcl-.
Due to our previous study in PKA-activation upon phosphorylation of survivin at Ser81 .
, we assume that the activated PKA could possibly induce cytoprotection via PI3K.
Therefore our current study was conduced to investigate the possibility of suvivin-PKA-PI3K signaling Methods Preparation of Survivin Constructs.
Viral Production and L929 Cells Infection All preparations of Antisense survivin (Survivin-AS) and Ser81Ala mutants (Survivin-S81A) were described in our previous report .
Similar protocols were conducted.
Briely.
Survivin.
Survivin-AS.
Ser81Ala mutants (Survivin-S81A) cDNAs were inserted in pMSCV-IRESGFP vector with cytomegalovirus (CMV) promoter.
Each vector was transformed in DH5 Escherichia coli, puriied and conirmed.
Each cDNA was transfected in BOSC23 cells by calcium phosphate method with additon of pCl3EcotR for 10 hours at 37AC.
Viruses were harvested at 72 hours after transfection and tittered using NIH3T3 cells.
DOI: 10.
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L929 cells were cultured in -DMEM containing 10% horse serum.
Infection was carried out using viral product of BOSC23 cells (Survivin.
Survivin-AS.
Survivin-S81A or vector onl.
for 48 hours.
Ser81 Survivin induced PKA-PI3K pathway (Sandra F, et al.
Indones Biomed J.
: 157-62
DTT, and solution C: containing 10 mM Tris-HCl buffer .
H 7.
, 0.
1 M NaCl and 1 mM DTT.
This was followed by incubation with 0.
5 mg/ml phosphoinositide fraction containing PtdIns.
P2.
Reaction buffer .
mM MgCl2, 100 mM HEPES buffer pH 7.
6, 250 M ATP containing 5 Ci of .
P]ATP) was added and incubated at 30AC for 10 Labelled-samples were then spotted on a thin layer chromatography (TLC) plate.
The assay was continued as described by Chen et al.
Cell Treatment.
Lysate Preparation and PI3K .
Immunoprecipitation L929 cells were pretreated with/without 4 M PKI 6-22 amide, a PKA inhibitor (Santa Cruz Biotechnology.
Dallas.
TX) and infected with viral particle of Survivin.
Survivin-AS.
Survivin-S81A or vector only.
Cells were then harvested and lysed in a cold lysis buffer .
mM TrisHCl buffer pH 8.
0, 500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM -glycerophosphate, 10 mM NaF, 10 mM pNPP, 0.
4 mM sodium orthovanadate, 1 mM benzamidine, 2 mM phenylmethylsulfonyl luoride (PMSF), aprotinin, 1 mg/ml leupeptin, 1 mM dithiothreitol (DTT) and 10% Nonidet P-.
The cell lysate supernatant was collected by centrifugation and incubated with monoclonal anti-PI3K .
antibody (Cell Signaling.
Beverly.
MA) immobilized with protein G Sepharose (Sigma.
St.
Louis.
MO) in immunoprecipitation buffer .
mM Tris-HCl buffer pH 0, 500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM -glycerophosphate, 10 mM NaF, 10 mM pNPP, 0.
4 mM sodium orthovanadate, 1 mM benzamidine, 2 mM PMSF, 10 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mM DTT and 1% Nonidet P-.
Survivin induced tyrosine phosphorylation of p85 PI3K Similar levels of p85 PI3K were observed for all lanes (Figure .
, showing that the same volume of proteins were electrophorated and detected.
Meanwhile, basal tyrosine phosphorylation of p85 PI3K .
ane 1 from lef.
was seen in L929 cells.
Upon infection of virus with vector only .
ane 2 from lef.
, the level of tyrosine phosphorylation of p85 PI3K in L929 cells was remained similar to the basal.
However, upon infection of virus with survivin, a markedly increased level of tyrosine phosphorylation of p85 PI3K in L929 cells was seen .
ane 3 from lef.
Low levels of tyrosine phosphorylation of p85 PI3K, even lower than the basal, were observed for Survivin-AS .
ane 4 from lef.
and Survivin-S81A-viral-infected L929 cells .
ane 5 from lef.
Phospho-(Ty.
p85 PI3K Immunoblot PI3K .
-immunoprecipitated samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene diluoride (PVDF) sheet.
After blocking with 5 % skim milk in Tris-buffered saline (TBS, 150 mM NaCl and 50 mM Tris-HCl, pH 7.
, the sheet was incubated with monoclonal anti-PI3K .
(Cell Signalin.
or rabbit polyclonal anti-phospho-(Ty.
p85 PI3K binding motif antibodies.
The secondary antibody was horseradish peroxidase-conjugated sheep anti-mouse (Amersham.
Piscataway.
NJ) or donkey anti-rabbit IgG antibody (Amersha.
The bound antibodies were visualized using the ECL system (Amersha.
, captured using Alliance 4.
7 (UVItech.
Ltd.
Cambridge.
UK) and quantiied using UVIband software (UVItech.
Ltd.
Figure 1.
Survivin induced PI3K phosphorylation.
L929 cells were infected with 200x107 viral particle/ml retrovirus of Survivin.
Survivin-AS.
Survivin-S81A or vector merely for 48 hours and cell sorted.
Sorted cells were then lysed, immunoprecipitated using anti-p85 PI3K antibody and immunoblotted using anti-phospho(Ty.
p85 PI3K antibody.
Detailed procedures were described in "MethodsAy.
This experiment was repeated 3 times.
PI3K Activity Assay PI3K .
-immunoprecipitated samples were washed with serial solutions as follows, solution A: PBS containing 1% Nonidet P-40 and 1 mM DTT, solution B: consisting 1 M Tris-HCl buffer .
H 7.
, 0.
5 M LiCl and 1 mM Survivin induced PI3K activity As shown in Figure 2, basal PtdIns.
,4,.
P3 (PIP.
smeary dot was seen .
ane 1 from lef.
A clear PIP3 dot was seen for Survivin-viral-infected L929 cells .
ane 3 from lef.
Meanwhile, smeary PIP3 dots were observed for Vector Results The Indonesian Biomedical Journal.
Vol.
No.
December 2014, p.
ane 2 from lef.
Survivin-AS .
ane 4 from lef.
and Survivin-S81A-viral-infected L929 cells .
ane 5 from lef.
Print ISSN: 2085-3297.
Online ISSN: 2355-9179 tyrosine phosphorylation of p85 PI3K in L929 cells were gradually increased .
ane 2-4 from lef.
A clear band of tyrosine phosphorylation of p85 PI3K was detected for 75x107 viral particle/ml Survivin-viral-infected L929 cells .
ane 4 from lef.
However, when L929 cells were pretreated with 4 M PKI 6-22 amide, a synthetic peptide that acts as PKA inhibitor, prior to 75x107 viral particle/ml Survivinviral-infection, tyrosine phosphorylation level of p85 PI3K was detected much lower .
ane 5 from lef.
compared to the one without PKI 6-22 amide pretreatment .
ane 4 from lef.
PKI 6-22 amide inhibited Survivin-induced PI3K Basal PIP3 smeary dot was seen (Figure 4, lane 1 from Upon infection of higher concentrations of virus with survivin, levels of PIP3 dots were gradually clearly seen .
ane 3-5 from lef.
A clear PIP3 dot was detected for 75x107 viral particle/ml Survivin-viral-infected L929 cells .
ane 5 from lef.
However, when L929 cells were pretreated with 4 M PKI 6-22 amide, prior to 75x107 viral particle/ml Survivin-viral-infection, smeary PIP3 dot was resulted .
ane 2 from lef.
Figure 2.
Survivin induced PI3K activity.
L929 cells were infected with 200x107 viral particle/ml retrovirus of Survivin.
Survivin-AS.
Survivin-S81A or vector merely for 48 hours and cell sorted.
Sorted cells were then lysed and subjected to PI3K activity assay.
Detailed procedures were described in "MethodsAy.
This experiment was repeated 3 times.
Figure 3.
Survivin induced PI3K phosphorylation via PKA L929 cells were pretreated with/without 4 M PKI 6-22 amide for 2 hours prior to infection with different concentrations of Survivin retrovirus for 48 hours as indicated in the panel.
Cell were sorted, lysed, immunoprecipitated using anti-p85 PI3K antibody and immunoblotted using anti-phospho-(Ty.
p85 PI3K Detailed procedures were described in "MethodsAy.
This experiment was repeated 3 times.
PKI 6-22 amide inhibited Survivin-induced tyrosine phosphorylation of p85 PI3K To conirm same proteins were loaded, levels of p85 PI3K were observed.
Yet, we found similar levels of p85 PI3K (Figure .
Basal tyrosine phosphorylation of p85 PI3K .
ane 1 from lef.
was seen in L929 cells.
Upon infection of higher concentrations of virus with survivin, levels of Discussion Evidence for the existence of a positive feedback loop connecting survivin expression in tumor cells to PI3K/Akt was reported.
This signal enhanced -catenin-Tcf/Lefdependent transcription followed by secretion of VEGF and angiogenesis.
In accordance, our current results had shown that Survivin could induce tyrosine phosphorylation of p85 PI3K and PI3K activation speciically.
While Survivin induced tyrosine phosphorylation of p85 PI3K and PI3K activity.
Vector merely and Survivin-AS did not induce neither tyrosine phosphorylation of p85 PI3K nor PI3K Based on our previous report, phosphorylation of Ser81 was crucial for activation of PKA.
In our current results, we found phosphorylation of Ser81 was also crucial in inducing tyrosine phosphorylation of p85 PI3K and activating PI3K.
Survivin-PKA-PI3K signal pathway possibly play important role in anti-apoptosis/cytoprotection.
In the opposite.
Survivin-S81A-viral-infection was reported to enhance TRAILAos activity in inducing apoptosis.
PKA was reported to have potential in regulating insulin receptor substrate-1 (IRS-.
and growth factor receptor bound protein 2-associated binding protein 2 (GAB.
, adaptors that normally integrate receptor/ nonreceptor tyrosine kinase signaling into PI3K/Akt.
Ser81 Survivin induced PKA-PI3K pathway (Sandra F, et al.
Indones Biomed J.
: 157-62
DOI: 10.
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as causal factors in cancer.
,15,17,18, .
Activation of PI3K and AKT are reported to occur in breast, ovarian, blood, pancreatic, esophageal, gallbladder and other .
Taken together, our current results suggest that Ser81 Survivin play an important role in inducing PI3K activation and the Survivin-PI3K signaling pathway was PKA-dependent.
This provides important information to understand the mechanism induced by Survivin in mediating However, further investigation to disclose downstream of Survivin-PKA-PI3K signaling pathway is Acknowledgement We thanks to Aristi Papaioannou from Department of Pathology.
Harvard Medical School, for lab assistance.
Figure 4.
Survivin induced PI3K activity via PKA.
L929
cells were pretreated with/without 4 M PKI 6-22 amide for 2 hours prior to infection with different concentrations of Survivin retrovirus for 48 hours as indicated in the panel.
TNF- was Cell were sorted, lysed and subjected to PI3K activity assay.
Detailed procedures were described in "MethodsAy.
This experiment was repeated 3 times.
This reveals a new route for PKA to activate a pathway that promotes proliferation, inhibits apoptosis and enhances .
This report is in accordance with our previous report, showing that phosphorylated-Survivin at Ser81 induced PKA back loop.
In addition, our current results emphasized that Survivin-induced PI3K activation was mediated by PKA, since PKI 6-22 amide inhibited Survivin-induced pathway, as shown by lower tyrosine phosphorylation of p85 PI3K and PI3K activity upon pretreatment of PKI 6-22 amide prior to Survivin-viral Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/PI3K/Akt constitute an important pathway regulating the signaling of multiple biological processes such as apoptosis, metabolism, cell proliferation and cell growth.
PTEN is a non-redundant phosphatase, counteracting one of the most critical cancerpromoting pathways: the PI3K/Akt signaling pathway and its main substrate PIP3 is the product of PI3K.
Increase in PIP3 recruits Akt to the membrane where is activated by other kinases also dependent on PIP3.
Another positive feedback mechanism that decrease of E-cadherin, lowers PTEN and thereby increases PIP3, further activating Akt.
Many components of this pathway have been described References