Inhibition of Mammalian Target of Rapamycin Activates Apoptosis Signal-regulating Kinase 1 Signaling by Suppressing Protein Phosphatase 5 Activity

Abstract

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces a cellular stress response characterized by rapid and sustained activation of the apoptosis signal-regulating kinase 1 (ASK1) signaling pathway and selective apoptosis of cells lacking functional p53. Here we have investigated how mTOR regulates ASK1 signaling using p53-mutant rhabdomyosarcoma cells. In Rh30 cells, ASK1 was found to physically interact with protein phosphatase 5 (PP5), previously identified as a negative regulator of ASK1. Rapamycin did not affect either protein level of PP5 or association of PP5 with ASK1. Instead, rapamycin caused rapid dissociation of the PP2A-B؆ regulatory subunit (PR72) from the PP5-ASK1 complex, which was associated with reduced phosphatase activity of PP5. This effect was dependent on expression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Down-regulation of PP5 activity by rapamycin coordinately activated ASK1, leading to elevated phosphorylation of c-Jun. Amino acid deprivation, which like rapamycin inhibits mTOR signaling, also inhibited PP5 activity, caused rapid dissociation of PR72, and activated ASK1 signaling. Overexpression of PP5, but not the PP2A catalytic subunit, blocked rapamycin-induced phosphorylation of c-Jun, and protected cells from rapamycin-induced apoptosis. The results suggest that PP5 is downstream of mTOR, and positively regulated by the mTOR pathway. The findings suggest that in the absence of serum factors, mTOR signaling suppresses apoptosis through positive regulation of PP5 activity and suppression of cellular stress.

Heat Shock Protein gp96 Interacts with Protein Phosphatase 5 and Controls Toll-like Receptor 2 (TLR2)-mediated Activation of Extracellular Signal-regulated Kinase (ERK) 1/2 in Post-hypoxic Kidney Cells

“…Rapamycin, an inhibitor of mTOR, induces a cellular stress response characterized by a rapid and sustained activation of ASK1 and selective apoptosis in p53-mutant cells. Rapamycin does not affect either protein level of PP5 or association of PP5 with ASK1, but induces rapid dissociation of PP2A-BЉ subunit (PR72) from PP5, that is associated with concomitant decreased phosphatase activity of PP5 and activating ASK1 (21). Conversely, overexpression of PP5, but not the PP2A catalytic subunit, protected cells from rapamycin-induced apoptosis (21).…”

“…Protein phosphatase PP5 is implicated in a variety of cellular processes and associates with many proteins involved in cellular signaling, such as the glucocorticoid receptor-Hsp90 complex (45,51), DNA-dependent protein kinase (52), G ␣12 /G␣ 13 subunits of the heteromeric G proteins (53), human blue-light photoreceptor cryptochrome 2 (54), Hsp90-dependent heme-regulated eukaryotic initiation factor 2␣ kinase (55), ASK1 (19,21), or A-regulatory subunit of protein phosphatase 2A (21,56). These studies have provided lines of evidence that PP5 plays important role in the regulation of the cellular responses to stress.…”

“…The protein phosphatase 5 (PP5), a member of the PPP family of serine/threonine phosphatases, ubiquitously expressed in mammalian cells (18), has been shown to act as a physiological negative regulator of the ASK1-JNK/p38 pathway that facilitates apoptosis in hypoxic and H 2 O 2 -stressed cell (19,20). Rapamycin, an inhibitor of the serine/threonine target of rapamycin (mTOR) was also shown to activate ASK1 signaling by suppressing protein phosphatase 5 activity (21). PP5 has been identified as an inactivator of the MEK-ERK pathway through its interaction with the kinase Raf-1 initiating this pathway (22).…”

Therapeutic targets: MTOR and related pathways

“…In selected models, sirolimus induces apoptosis. 33,34 Selective apoptosis was seen in cells lacking functional p53 or p27 Kip1 . 34 Based on these laboratory models, the expected clinical activity of sirolimus would be delayed tumor progression rather than tumor regression in most patients with sensitive disease; however, tumor regression could occur through sirolimus induced apoptosis.…”

The mTOR inhibitor CCI-779 induces apoptosis and inhibits growth in preclinical models of primary adult human ALL

“…For these experiments, we used a modification of the protocol of Huang et al 24 Mouse spleens were harvested and dissociated into a single-cell suspension after which erythrocytes were lysed using Tris/ammonium chloride. Ten to 20 ϫ 10 6 splenocytes were lysed in ice-cold lysis buffer, derived from mixing 1 mL of 10ϫ Lysis Buffer (Cell Signaling Technologies, Beverly, MA), 1 tablet of Complete Mini (Roche Diagnostics, Mannheim, Germany), and 100 L 100 mM phenylmethylsulfonyl fluoride (PMFS) to a final volume of 10 mL with ddH 2 O.…”