PME-1 Protects Extracellular Signal-Regulated Kinase Pathway Activity from Protein Phosphatase 2A–Mediated Inactivation in Human Malignant Glioma

Abstract

Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway activity is regulated by the antagonist function of activating kinases and inactivating protein phosphatases. Sustained ERK pathway activity is commonly observed in human malignancies; however, the mechanisms by which the pathway is protected from phosphatasemediated inactivation in the tumor tissue remain obscure. Here, we show that methylesterase PME-1-mediated inhibition of the protein phosphatase 2A promotes basal ERK pathway activity and is required for efficient growth factor response. Mechanistically, PME-1 is shown to support ERK pathway signaling upstream of Raf, but downstream of growth factor receptors and protein kinase C. In malignant gliomas, PME-1 expression levels correlate with both ERK activity and cell proliferation in vivo. Moreover, PME-1 expression significantly correlates with disease progression in human astrocytic gliomas (n = 222). Together, these observations identify PME-1 expression as one mechanism by which ERK pathway activity is maintained in cancer cells and suggest an important functional role for PME-1 in the disease progression of human astrocytic gliomas.

Relevance Rank Platform (RRP) for Functional Filtering of High Content Protein–Protein Interaction Data*

“…48 h, transfection cells were harvested, resuspended in 2 ϫ 1 ml membrane lysis buffer (11), and mechanically disrupted. Lysates were cleared by centrifugation, and strep purification from subsequent supernatants was performed using 0.2 ml Strep-Tactin Superflow columns (IBA) following the manufacturer's instructions.…”

“…To this end, we set up to purify a de novo protein complex by one-step Strep-tag method (15). PME-1 (PPME1) is a methylesterase that promotes ERK pathway activity in human glioblastoma by inhibition of protein phosphatase 2A (PP2A) (11). For single-step affinity purification coupled with mass spectrometry (AP-MS) identification of PME-1 interactome, we established a cell line stably expressing either Strep-tag alone (Strep) or Strep-PME-1 fusion protein.…”

“…In addition, MAPK3 (ERK1) and AKT1 were included in the RRP library as internal controls representing known PME-1 effector proteins (11,23). The relevance of the PC-3 cell model for assessing PME-1 interactome function was validated by robust inhibition of colony growth by PME-1 depletion ( Supplemental Fig.…”

“…5F), further indicating its potential involvement in cancer biology. were included to RRP analysis as previously validated PME-1 effector genes (11,23). FunCoup similarity index (min.…”

“…As a relevant read-out for the proof-of-principle test, we chose oncogenic function of established oncoproteins Pin1 (9, 10) and PME-1 (11). The resulting platform, designed as relevance rank platform (RRP) 1 , consists of several subsequent filtering steps, each of which produces a ranking, and whose combination then determines the final RRP rank between the interactome proteins.…”