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

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).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.S6K1 is a ubiquitously expressed serine/threonine protein kinase that phosphorylates 40S ribosomal protein S6, and coordinates cellular growth and proliferation. Multiple independent phosphorylations have been proposed to account for complete activation of the enzyme in response to growth factor stimulation [1]. A battery of protein kinases coordinate to accomplish the activation of the enzyme through a series of phosphorylation events that culminate in phosphorylating Threonine 412 (T412) at HM and Threonine 252 (T252) at the AL [2-7]. The dynamics of these critical phosphorylations, in particular the one at the HM therefore, dictates the activation state of the enzyme. Accordingly selective loss of this TOR kinase dependent phosphorylation has been implicated in mediating the inhibitory effects of rapamycin, through direct inactivation of TOR kinase, or through activation/recruitment of a phosphatase [8-11]. In addition to Insulin and other growth factor stimulation, S6K1 has also been reported to