Rapamycin inhibition of baculovirus recombinant (BVr) ribosomal protein S6 kinase (S6K1) is mediated by an event other than phosphorylation

Abstract

Background: Ribosomal protein S6 kinase 1(S6K1) is an evolutionary conserved kinase that is activated in response to growth factors and viral stimuli to influence cellular growth and proliferation. This downstream effector of target of rapamycin (TOR) signaling cascade is known to be directly activated by TOR- kinase mediated hydrophobic motif (HM) phosphorylation at Threonine 412 (T412). Selective loss of this phosphorylation by inactivation of TOR kinase or activation/recruitment of a phosphatase has accordingly been implicated in mediating inhibition by rapamycin.

Findings: We present evidence that baculovirus driven expression of S6K1 in insect cells (Sf9) fails to activate the enzyme and instead renders it modestly active representing 4-6 folds less activity than its fully active mammalian counterpart. Contrary to the contention that viral infection activates TOR signaling pathway, we report that BVr enzyme fails to exhibit putative TOR dependent phosphorylation at the HM and the resultant phosphorylation at the activation loop (AL) of the enzyme, correlating with the level of activity observed. Surprisingly, the BVr enzyme continued to exhibit sensitivity to rapamycin that remained unaffected by mutations compromised for TOR phosphorylation (T412A) or deletions compromised for TOR binding (ΔNH 2-46/ΔCT104).

Conclusions: These data together with the ability of the BVr enzyme to resist inactivation by phosphatases indicate that inhibition by rapamycin is not mediated by any phosphorylation event in general and TOR dependent phosphorylation in particular.

Figure 1

Comparative activity and rapamycin response of BVr and HEK S6K. Sf9 cells infected with HA-S6K1α1 virus at an M.O.I of ≤ 1 in TNM-FH media supplemented with 5% FBS and HEK-293 cells transiently transfected with HA-S6K1 in DMEM containing 5% FBS were grown for 24 and 48 hours respectively. Cells were exposed to rapamycin or ethanol for 15 min at indicated concentrations before harvest. Lysates were clarified and used for inmunoprecipitation with α HA antibody. Immune complexes recovered were subjected to kinase assays in presence of ³²pATP using GST-S6 or S6 peptide as a substrate. Samples were separated on a 12% SDS-PAGE gel transferred on to a PVDF membrane for autoradiography or immunoblotting. Peptide kinase assays were performed by standard procedure and the activity in Cpm (Counts per minute) normalized to 100% for comparison. The data is a representative expression of four independent experiments.

Figure 2

Status of activating phosphorylations in HEK and BVrS6K1. HA-S6Kα1 immuno-precipitated from HEK 293 cells or Sf9 cells treated with or without rapamycin (50nM), were subjected to immune-complex kinase assay using GST-S6 as substrate and separated on 12% SDS-PAGE gel for autoradiography. Alternatively immune complexes were transferred on to a PVDF membrane for probing with indicated antibodies. The results represent typical of three independent observations.

Figure 3

BVr-S6K1 is resistant to phosphatase inactivation. S6Kα1-WT immuno-precipitated with anti-HA antibodies from infected Sf9, HEK 293, CHO or NIH-3T3 cells as above. The immunoprecipitate was split into aliquots and incubated with indicated concentrations of acid phosphatase (3a, 3b) or protein phosphatase 2A (b) for 30 min and washed with buffer containing phosphatase inhibitors or okadaic acid (5nM) respectively, followed by excessive washing with kinase buffer and processed for S6 phosphorylation assay as above.

Figure 4

Tor signaling input is not required for rapamycin inhibition of BVr enzyme. Sf9 cells were infected with S6Kα1WT, S6Kα1T412A and T252A (a) or S6Kα1WT & S6K ΔNH_2-46/ΔCT₁₀₄ (b) recombinant viruses at an M.O.I ≤ 1. Cells were exposed to rapamycin (50 nM) or vehicle, 24 h post infection for 15 min and harvested, lysed for IPs and subjected to S6 kinase as above. The figure is representative of three independent experiments.