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 oncogene-induced 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 PR55γ and PR55δ as inhibitors of c-Jun NH₂-terminal kinase (JNK) activation by UV irradiation. We show that PR55γ 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 PR55γ 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, PR55γ. Protein Phosphatase type 2A (PP2A) represent a family of holoenzyme complexes involved in wide range of activities such as growth, differentiation, and cell death. The PP2A holoenzyme complex is made up of a catalytic, a structural, and one of various “B” subunits. These “B” subunits are thought to provide the substrate specificity required for PP2A activity. Previous work on PP2A has mostly been derived by inhibiting the catalytic subunit through chemical inhibition, as such inhibiting all of the pathways associated with PP2A. To identify individual “B” subunits involved in specific cellular processes we have generated a “B” subunit gene knockdown library, which allows us to inhibit each of the known “B” subunits individually. One of the many pathways regulated by PP2A is the c-Jun NH₂-terminal kinase (JNK) kinase pathway, which, depending on stimulus, can affect either cell survival or cell proliferation. Here we report that the “B” subunit PR55γ acts as a negative regulator of JNK activity and cell death. We show that PR55γ influences JNK activity by inhibiting one of its upstream regulators, the proto-oncogene c-SRC, through dephosphorylation at one of the key residues on c-SRC, a site we show to be critical for c-SRC activation following cell stress. Overall our work describes the novel function of a specific PP2A subunit involved in cell survival and identifies a novel mechanism of c-SRC regulation.