Regulation of the Raf–MEK–ERK pathway by protein phosphatase 5

Abstract

The Raf-MEK-ERK pathway couples growth factor, mitogenic and extracellular matrix signals to cell fate decisions such as growth, proliferation, migration, differentiation and survival. Raf-1 is a direct effector of the Ras GTPase and is the initiating kinase in this signalling cascade. Although Raf-1 activation is well studied, little is known about how Raf-1 is inactivated. Here, we used a proteomic approach to identify molecules that may inactivate Raf-1 signalling. Protein phosphatase 5 (PP5) was identified as an inactivator that associates with Raf-1 on growth factor stimulation and selectively dephosphorylates an essential activating site, Ser 338. The PP5-mediated dephosphorylation of Ser 338 inhibited Raf-1 activity and downstream signalling to MEK, an effect that was prevented by phosphomimetic substitution of Ser 338, or by ablation of PP5 catalytic function. Furthermore, depletion of endogenous PP5 increased cellular phospho-Ser 338 levels. Our results suggest that PP5 is a physiological regulator of Raf-1 signalling pathways.

A 19S proteasomal subunit cooperates with an ERK MAPK-regulated degron to regulate accumulation of Fra-1 in tumour cells

“…Eluted proteins were separated on 4-12% NuPAGE gels (Invitrogen) and stained with Colloidal Coomassie (BioRad, Gladesville, NSW, Australia). Gel lanes of interest were sliced into multiple pieces and processed for analysis by mass spectrometry as described previously (von Kriegsheim et al, 2006).…”

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

“…Previous studies have shown that PP5 harboring a tetratricopeptide (TPR) domain selectively dephosphorylates and inhibits Raf-1 activity and downstream signaling of the MEK-ERK pathway (22). The crystal structure of PP5 revealed a mechanism of autoinhibition by its TPR domain inhibiting the C-terminal phosphatase domain, and activation by Hsp90 (44).…”

“…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). PP5 was also shown to physically interact with the heat shock protein (Hsp) 90, which is complexed with the transcription factor heat shock factor 1 (HSF1), a key regulatory protein responsible in Hsp synthesis (23).…”

High Yield Expression of Serine/Threonine Protein Phosphatase Type 5, and a Fluorescent Assay Suitable for Use in the Detection of Catalytic Inhibitors

“…Notably, PP5 has been shown to associate with several proteins that affect signal transduction networks [1][2][3][4][5][6][14][15][16][17][18]. When PP5 is displaced from these biological partners, phosphatase activity is greatly decreased.…”

“…However, the advent of siRNA and antisense oligonucleotides capable of specifically suppressing individual phosphatases has greatly aided our understanding of the biological roles of ser/thr protein phosphatases. From such studies serine/threonine protein phosphatase type 5 (PP5) has emerged as an important regulator of hormone/stress-induced signaling networks that propagate cellular responses controlling growth and stress-induced apoptosis [1][2][3][4][5][6][7]. Nonetheless, the development of small molecule inhibitors of PP5 has been hampered because human PP5 expressed in bacteria has very low catalytic activity, and the [ 32 P]based phosphatase assays [8][9][10] commonly used to measure activity are not easily adaptable to high throughput screening assays.…”