Protein Phosphatase 2A, a Negative Regulator of the ERK Signaling Pathway, Is Activated by Tyrosine Phosphorylation of Putative HLA Class II-associated Protein I (PHAPI)/pp32 in Response to the Antiproliferative Lectin, Jacalin

Abstract

Protein phosphatase 2A (PP2A) is a family of mammalian serine/threonine phosphatases that is involved in the control of many cellular functions including those mediated by extracellular signal-regulated kinase (ERK) signaling. While investigating the reversible antiproliferative effect of the dietary lectin, jacalin, which binds the Thomsen-Friedenreich antigen (galactose ␤1-3 N-acetylgalactosamine ␣-), we have found that this lectin (30 g/ml) induces rapid, transient, tyrosine phosphorylation of putative human HLA-DR-associated protein I (PHAPI, also known as the tumor suppressor pp32) in HT29 human colon cancer cells. This is accompanied by the release of PP2A from association with PHAPI, allowing increased phosphatase activity of PP2A (by 42 ؎ 10% at 10 min) and consequent complete dephosphorylation of the ERK kinase, MEK1/2, by 10 min and of ERK1/2 by 60 min. PHAPI knockdown by RNA interference abolished the effects of jacalin on PP2A activation and MEK inhibition. Thus phosphorylation of PHAPI/pp32 is a critical regulatory step in PP2A activation and ERK signaling.Protein phosphatase 2A (PP2A) 1 is a family of mammalian serine/threonine phosphatases that accounts for the major portion of serine/threonine kinase activity in most cells. PP2A is a trimeric holoenzyme complex and is composed of a catalytic C subunit, a structural A subunit, and a regulatory B-type subunit, each encoded by separate genes (1, 2). Up to 75 different trimeric ABC holoenzymes may exist. The C subunit is the enzymatically active component, A is a scaffolding component, and B acts as a targeting module that directs the enzyme to various intracellular locations and also provides substrate specificity.PP2A was initially thought to act passively and nonspecifically in antagonizing the action of kinases by removal of phosphate groups from their substrates, but it has become clear that it is tightly regulated by mechanisms that include variation in its subunit composition (3), phosphorylation, and/or methylation of its catalytic subunit and signal-regulated targeting to specific subcellular compartments (1-4). PP2A is an important negative regulator of the extracellular signal-regulated kinase (ERK) signaling pathway (5-7) and is involved in the control of many cellular functions including metabolism, transcription, translation, RNA splicing, DNA replication, cell cycle progression, transformation, and apoptosis (1-8).PHAPI, first purified by affinity with an agarose-conjugated synthetic cytoplasmic region of HLA-DR ␣ chain, is a putative HLA class II-associated cytosolic protein (9), later identified as a phosphoprotein (10). It belongs to a group of proteins containing 20 -29-residue leucine-rich repeat motifs. The principal function of these motifs seems to be the provision of a structural framework that allows protein-protein interactions (11). PHAPI is also known as pp32, a potent tumor suppressor (12-15) that is down-regulated in cancer with reciprocal increased expression of pp32r1 and pp32r2, other members of the pp32 gene family (...

Regulation of ERK Kinase by MEK1 Kinase Inhibition in the Brain

“…Direct dephosphorylation of Thr-202/Tyr-204 of ERK is conducted by its dedicated phosphatases, such as striatal enriched phosphoprotein (56) and MKP-2 (11). However, consistent with the results presented here, both PP1 and PP2A are thought to indirectly regulate ERK (57,58).…”

“…For example, PP1 inhibition with tautomycetin prevents Raf/MEK/ ERK activation (57). Conversely, the inhibition of PP2A phosphatase activity has been demonstrated to enhance the MEK/ ERK pathway (58). Therefore, evidence has been reported to suggest that these phosphatases simultaneously activate and inhibit the MEK/ERK pathway.…”

Targeting Tumor Metastasis by Regulating Nm23 Gene Expression

“…It is also notify that the over expression of Nm23-H2 inhibits cell migration and colonization (Syed et al, 2005). Nm23-H2 protein also involves in ERK signaling pathway responsible for cellular proliferation and transmission of signals through Ras/Raf/ERK cascade (Roberts and Der, 2004;Yu et al, 2004;Adams et al, 2005). Nm23-H2 over expression suppresses ERK activation and blocks the activation of Raf-1, MEK and ELK-1 regulated by ERK pathway and consecutive proliferation.…”

Enhanced Sensitivity to Cytochrome c–Induced Apoptosis Mediated by PHAPI in Breast Cancer Cells

“…PHAPI knockdown was achieved using small interfering RNA (siRNA) duplexes generated by Eurogentec. T47D cells were transfected using the Transit-TKO reagent (Mirus, Madison, WI) with either the siRNA negative control duplex or the following duplex targeting PHAPI (30) at 100 nmol/L: 5V -GGACGCCCUCUGAUGUGAATT-3V ; 5V -UUCACAUCAGAGGGCGUCCTT-3V .…”