A RNA interference screen identifies the Protein Phosphatase 2A subunit PR55γ as a stress-sensitive inhibitor of c-SRC

Abstract

Protein Phosphatase type 2A (PP2A) represents a family of holoenzyme complexes with diverse biological activities. Specific holoenzyme complexes are thought to be deregulated during oncogenic transformation and oncogeneinduced signaling. Since most studies on the role of this phosphatase family have relied on the use of generic PP2A inhibitors, the contribution of individual PP2A holoenzyme complexes in PP2A-controlled signaling pathways is largely unclear. To gain insight into this, we have constructed a set of shRNA vectors targeting the individual PP2A regulatory subunits for suppression by RNA interference. Here, we identify PR55c and PR55d as inhibitors of c-Jun NH 2 -terminal kinase (JNK) activation by UV irradiation. We show that PR55c binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we demonstrate to be required for JNK activation by c-SRC. We also find that the physical interaction between PR55c and c-SRC is sensitive to UV irradiation. Our data reveal a novel mechanism of c-SRC regulation whereby in response to stress c-SRC activity is regulated, at least in part, through loss of the interaction with its inhibitor, PR55c.

Structure- and Modeling-based Identification of the Adenovirus E4orf4 Binding Site in the Protein Phosphatase 2A B55α Subunit

“…However, it is intriguing that they could still partially transduce the E4orf4 toxic signal. It has been previously reported that knockdown of various B55 subunits led to stress-induced Src activation (49). Src is another major E4orf4 partner that contributes to E4orf4-induced cell death, and E4orf4 was shown to affect Src activity independently of its interaction with PP2A (19,20).…”

LB100, a small molecule inhibitor of PP2A with potent chemo- and radio-sensitizing potential

“…PP2A has been shown to dephosphorylate and therefore inhibit Src, a non-receptor tyrosine kinase that has been shown to mediate TRAIL resistance in breast cancer cells via activation of Akt pathway survival signaling. 102,103 Using the BT549 breast cancer cell line, Xu et al demonstrated that TRAIL signaling activated Src, which in turn phosphorylated caspase-8, initiating apoptosis. 104 Using immunoprecipitation, they found that TRAIL stimulation led to increased PP2A dephosphorylation of Src, abrogating Src-mediated activation of caspase-8.…”

Regulation of the Src-PP2A Interaction in Tumor Necrosis Factor (TNF)-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis

“…We chose PP2A because PP2A has been shown to target the holoenzyme to dephosphorylate Src at serine 12, a site that is important for c-Src activation upon cell stress (28). To this end, HEK293T cells co-transfected with pcDNA3-Src, PP2A/C-HA (catalytic subunit of PP2A), and caspase-8-HA were left untreated or treated with TRAIL and then harvested for Western IP.…”

“…Protein phosphatase 2A (PP2A) is the major serine-threonine phosphatase that regulates a number of cell signaling pathways (28). PP2A is a trimeric holoenzyme that consists of a catalytic subunit, a structural subunit, and a regulatory subunit (29).…”