Ser81 Survivin Induced Protein Kinase a (PKA)-dependent Phosphatidylinositol 3-kinase (PI3K) Activity

BACKGROUND: 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 pathway.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 (p85) antibody and immunoblotted to detect PI3K (p85) and phospho-(Tyr) p85 PI3K. To confirm PI3K activation, PI3K Activity Assay was conducted by using phosphoinositide fraction containing PtdIns(4,5)P2 and [32P]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-S81A-viral-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 increased. 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.


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 is the hallmark of these molecules.(1,2) Transcription of the survivin gene is strictly cell cycle-regulated, and peaks at mitosis.(1,3) Survivin plays several roles in homeostatic networks, including the control of mitosis, the regulation of apoptosis and the cellular stress response.(1) 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 'risk factor' for poor prognosis and disease recurrence. (1,4) 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. (1,5) Besides that, Phosphatydilinositol 3-kinase (PI3K), Akt, β-catenin, T-cell factor (Tcf), Lymphoid enhancer-binding factor (Lef) and many others implicated in the expression of survivin. (6)(7)(8) Our previous report showed that Survivin dominant negative enhanced Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)'s activity in inducing apoptosis.(7) 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. (6) Amino acid residue Thr34 that lies within sur¬vivin BIR domain, was shown as an important amino acid in regulation of apoptosis. (9) Phosphorylated-survivin at Thr34 bound to caspase-9 in HeLa cells. (9) 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. (7) Phosphorylated-survivin at Ser81 was later known mediating survivin back loop to activate protein kinase A (PKA) in the cytoprotection mechanism.
(10) As being reported, phosphodiesterase 4 (PDE4) control intracellular cyclic adenosine monophosphate (cAMP) levels by catalyzing their hydrolysis and inactivating second messengers.(11) PDE4 inhibition promotes PKA-dependent resolution of established neutrophilic inlammation by inhibition of the PI3K/Akt/myeloid cell leukemia 1 (Mcl-1) pathway. (11) Due to our previous study in PKA-activation upon phosphorylation of survivin at Ser81 (10), 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 pathway.

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 (7,10). Similar protocols were conducted. Briely, Survivin, Survivin-AS, Ser81Ala mutants (Survivin-S81A) cDNAs were inserted in pMSCV-IRES-GFP vector with cytomegalovirus (CMV) promoter. Each vector was transformed in DH5α Escherichia coli, puriied and conirmed. Each cDNA was transfected in BOSC23 cells by calcium phosphate method with additon of pCl3-EcotR for 10 hours at 37°C. Viruses were harvested at 72 hours after transfection and tittered using NIH3T3 cells. L929 cells were cultured in α-DMEM containing 10% horse serum. Infection was carried out using viral product of BOSC23 cells (Survivin, Survivin-AS, Survivin-S81A or vector only) for 48 hours.

Survivin induced tyrosine phosphorylation of p85 PI3K
Similar levels of p85 PI3K were observed for all lanes (Figure 1)

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 (   tyrosine phosphorylation of p85 PI3K in L929 cells were gradually increased (lane 2-4 from left). A clear band of tyrosine phosphorylation of p85 PI3K was detected for 75x107 viral particle/ml Survivin-viral-infected L929 cells (lane 4 from left). However, when L929 cells were pretreated with 4 μM 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 (lane 5 from left) compared to the one without PKI 6-22 amide pretreatment (lane 4 from left). Evidence for the existence of a positive feedback loop connecting survivin expression in tumor cells to PI3K/Akt was reported.(6) This signal enhanced β-catenin-Tcf/Lefdependent transcription followed by secretion of VEGF and angiogenesis. (6) 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 activity. Based on our previous report, phosphorylation of Ser81 was crucial for activation of PKA.(10) 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 TRAIL's activity in inducing apoptosis. (7) PKA was reported to have potential in regulating insulin receptor substrate-1 (IRS-1) and growth factor receptor bound protein 2-associated binding protein 2 (GAB2), adaptors that normally integrate receptor/ nonreceptor tyrosine kinase signaling into PI3K/Akt. (13)  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. (14)(15)(16)(17) 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. (14)(15)(16)(17)(18)(19) Increase in PIP3 recruits Akt to the membrane where is activated by other kinases also dependent on PIP3. (14) Another positive feedback mechanism that decrease of E-cadherin, lowers PTEN and thereby increases PIP3, further activating Akt. (20) Many components of this pathway have been described as causal factors in cancer. (14,15,17,18,20) Activation of PI3K and AKT are reported to occur in breast, ovarian, blood, pancreatic, esophageal, gallbladder and other cancers. (14,17) 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 cytoprotection. However, further investigation to disclose downstream of Survivin-PKA-PI3K signaling pathway is necessary.
We thanks to Aristi Papaioannou from Department of Pathology, Harvard Medical School, for lab assistance.