Regulatory B Subunits of Protein Phosphatase 2A Are Involved in Site-specific Regulation of Tau Protein Phosphorylation

Overexpression of amyloid precursor protein with the Swedish mutation causes abnormal hyperphosphorylation of the microtubule-associated protein tau. Hyperphosphorylated isoforms of tau are major components of neurofibrillary tangles, which are histopathological hallmarks of Alzheimer's disease. Protein phosphatase 2A (PP2A), a major tau protein phosphatase, consists of a structural A subunit, catalytic C subunit, and a variety of regulatory B subunits. The B subunits have been reported to modulate function of the PP2A holoenzyme by regulating substrate binding, enzyme activity, and subcellular localization. In the current study, we characterized regulatory B subunit-specific regulation of tau protein phosphorylation. We showed that the PP2A B subunit PPP2R2A mediated dephosphorylation of tau protein at Ser-199, Ser-202/Thr-205, Thr-231, Ser-262, and Ser-422. Down-regulation of PPP2R5D expression decreased tau phosphorylation at Ser-202/Thr-205, Thr-231, and Ser-422, which indicates activation of the tau kinase glycogen synthase kinase 3 beta (GSK3beta) by PP2A with PPP2R5D subunit. The level of activating phosphorylation of the GSK3beta kinase Akt at Thr-308 and Ser-473 were both increased by PPP2R5D knockdown. We also characterized B subunit-specific phosphorylation sites in tau using mass spectrometric analysis. Liquid chromatography-mass spectrometry revealed that the phosphorylation status of the tau protein may be affected by PP2A, depending on the specific B subunits. These studies further our understanding of the function of various B subunits in mediating site-specific regulation of tau protein phosphorylation.

Effect on Cell Cycle Progression by N-Myc Knockdown in SK-N-BE(2) Neuroblastoma Cell Line and Cytotoxicity with STI-571 Compound / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Neuroblastoma is a common tumor in childhood, and generally exhibits heterogeneity and a malignant progression. MYCN expression and amplification profiles frequently correlate with therapeutic prognosis. Although it has been reported that MYCN silencing causes differentiation and apoptosis in human neuroblastoma cells, MYCN expression influences the cytotoxic potential of chemotherapeutic drugs via the deregulation of the cell cycle. STI-571 may constitute a promising therapeutic agent against neuroblastoma, particularly in cases in which c-Kit is expressed preferentially in MYCN-amplified neuroblastoma. MATERIALS AND METHODS: To determine whether STI-571 exerts a synergistic effect on cytotoxicity with MYCN expression, we assessed apoptotic cell death and cell cycle distribution after 72 h of exposure to STI-571 with or with out treatment of SK-N-BE(2) neuroblastoma cells with MYCN siRNA. RESULTS: MYCN siRNA-treated SK-N-BE(2) cells did not affect apoptosis and cells were arrested in G0/G1 phase after STI-571 treatment. CONCLUSIONS: siRNA therapy targeted to MYCN may not be effective when administered in combination with STI-571 treatment in cases of neuroblastoma. Therefore, chemotherapeutic drugs that target S or G2-M phase may prove ineffective when applied to cells arrested in the G0/1 phase as the result of MYCN knockdown and STI-571 treatment.