蛋白質去磷酸酶2A型(PP2A)是一種由三種單體組成的酵素，它包含了一個催化單元(C)、一個結構單元(A)、以及一個可變的調節性單元(B)。這個可變的B單元(B, B',與B''家族)支配著PP2A的受質特異性及其在細胞內的表現位置，PP2A在細胞許多方面的功能都扮演了重要的角色。ppp2r2b是負責轉錄Bbeta可變單元的基因，Bbeta1與Bbeta2則是選擇性拼接下所轉譯出的兩種產物，它們具有不同的N端。第十二型脊髓小腦萎縮症 (SCA12)與ppp2r2b的5'-UTR產生過度擴增CAG三核苷酸有關。本研究目的是將胞器專一性的細胞質Bbeta1與粒線體Bbeta2等次單元，分別轉殖入神經纖維母細胞SK-N-SH，次將細胞經篩選之後，建立起永久表現的細胞株，維持其細胞生長，並使用西方墨漬法、共軛焦顯微鏡、PP2A酵素活性分析等方式深入探討這些轉殖細胞。轉殖細胞株皆分別過度表現Bbeta1與Bbeta2，其中Bbeta2的PP2A活性有顯著提昇，此外轉殖的Bbeta2共存於粒線體，Bbeta1則存在細胞質。Bbeta1與Bbeta2過度表現會造成tau過度表現與磷酸化，此現象與PI3-kinase/Akt/GSK-3beta pathway的活化相關，Bbeta1與Bbeta2過度表現也會破壞F-actin的完整性，影響adherens junction的分佈，而且 autophagosome生成也會增加。此外Bbeta2過度表現會促進神經分化，並且提昇Bcl-2表現。本研究證實所建立的神經退化性細胞模式，可做神經退化性疾病的發生及治療的參考。

Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme consisting of a catalytic subunit (C), a structural subunit (A), and a variable regulatory subunit (B). Variable B subunits (B, B', and B'' families) dictate PP2A substrate specificity and subcellular localization. PP2A plays an essential role in many aspects of cellular functions. ppp2r2b encodes Bbeta regulatory subunit, and Bbeta1 and Bbeta2 are two novel splice variant of the neuronal Bbeta regulatory subunit with divergent N-terminal tails. The CAG trinucleotide expansion in ppp2r2b 5'-UTR is involved in Spinocerebellar ataxias 12(SCA 12).We transferred neuron-specific Bbeta1 and Bbeta2 subunits into neuroblastoma cells SK-N-SH. The selected cells were established as permanent cell lines and maintained in media. Western blotting, confocal microscope, and PP2A activity assay are used to investigate the stably transfected cells.
The PP2A activities were elevated in Bbeta2 cones. More experiments demonstrated that Bbeta1 and are Bbeta2 is colocalized in cytopasma and mitochondria, respectively. In addition, Bbeta1 and Bbeta2 overexpression in cones caused tau overexpression and phosphorylation that are associated with PI3-kinase/Akt pathway activation. Moreover, Bbeta1 and Bbeta2 overexpression abolishes the structure of F-actin, affects adherens junction distribution and increases the expression of Bcl-2 and autophagosome formation. Bbeta2 accelerates neuron differentiation. The work provided a cell model for studying the onset of neuron disease pathogenesis and possible therapy.

Alessi DR, Caudwell FB, Andjelkovic M, Hemmings BA, Cohen P. Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase. FEBS Lett. 1996;399:333-8.

Andjelković M, Jakubowicz T, Cron P, Ming XF, Han JW, Hemmings BA. Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors. Proc Natl Acad Sci USA. 1996;93:5699-704

Bellacosa A, Franke TF, Gonzalez-Portal ME, Datta K, Taguchi T, Gardner J, Cheng JQ, Testa JR, Tsichlis PN. Structure, expression and chromosomal mapping of c-akt: relationship to v-akt and its implications. Oncogene. 1993;8:745-54.

Bijur GN, De Sarno P, Jope RS. Glycogen synthase kinase-3beta facilitates staurosporine- and heat shock-induced apoptosis. Protection by lithium. J Biol Chem. 2000 17;275:7583-90.

Bijur GN, Jope RS. Rapid accumulation of Akt in mitochondria following phosphatidylinositol 3-kinase activation. J Neurochem. 2003;87:1427-35.

Borgatti P, Martelli AM, Bellacosa A, Casto R, Massari L, Capitani S, Neri LM. Translocation of Akt/PKB to the nucleus of osteoblast-like MC3T3-E1 cells exposed to proliferative growth factors. FEBS Lett. 2000;477:27-32.

Boya P, González-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, Métivier D, Meley D, Souquere S, Yoshimori T, Pierron G, Codogno P, Kroemer G. Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol. 2005;25:1025-40.

Brami-Cherrier K, Valjent E, Garcia M, Pagès C, Hipskind RA, Caboche J. Dopamine induces a PI3-kinase-independent activation of Akt in striatal neurons: a new route to cAMP response element-binding protein phosphorylation. J Neurosci. 2002;22:8911-21.

Brazil DP, Hemmings BA. Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem Sci. 2001;26:657-64.

Brunk UT, Terman A. The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis. Eur J Biochem. 2002 ;269:1996-2002.

Coffer PJ, Woodgett JR. Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families. Eur J Biochem. 1991 ;201:475-81.

Cohen P and Frame S. The renaissance of GSK3. Nat Rev Mol Cell Biol. 2001;2:769-76. Review.

Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature. 1995;378:785-9.

Cross DA, Culbert AA, Chalmers KA, Facci L, Skaper SD, Reith AD. Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J Neurochem. 2001;77:94-102.

Crowder RJ, Freeman RS. Glycogen synthase kinase-3 beta activity is critical for neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but not for death caused by nerve growth factor withdrawal. J Biol Chem. 2000;275:34266-71.

Cuervo AM.Autophagy: many paths to the same end. Mol Cell Biochem. 2004;263:55-72.

Dagda RK, Zaucha JA, Wadzinski BE, Strack S. A developmentally regulated, neuron-specific splice variant of the variable subunit Bbeta targets protein phosphatase 2A to mitochondria and modulates apoptosis. J Biol Chem. 2003;278:24976-85.

Dale TC. Signal transduction by the Wnt family of ligands. Biochem J. 1998;329:209-23.

Ebneth A, Godemann R, Stamer K, Illenberger S, Trinczek B, Mandelkow E. Overexpression of tau protein inhibits kinesin-dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum: implications for Alzheimer's disease. J Cell Biol. 1998 Nov 2;143(3):777-94.

Embi N, Rylatt DB, Cohen P. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur J Biochem. 1980;107:519-27.

Farr GH 3rd, Ferkey DM, Yost C, Pierce SB, Weaver C, Kimelman D. Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification. J Cell Biol. 2000;148:691-702.

Frame S and Cohen P. GSK3 takes centre stage more than 20 years after its discovery. Biochem J. 2001;359:1-16.

Gates J, Peifer M. Can 1000 reviews be wrong? Actin, alpha-Catenin, and adherens junctions. Cell. 2005;123:769-72.

Goedert M. Neurofibrillary pathology of Alzheimer's disease and other tauopathies. Prog Brain Res. 1998;117:287-306. Review. No abstract available.

Hetman M, Cavanaugh JE, Kimelman D, Xia Z. Role of glycogen synthase kinase-3beta in neuronal apoptosis induced by trophic withdrawal. J Neurosci. 2000;20:2567-74.

Hoffmann R, Lee VM, Leight S, Varga I, Otvos L Jr. Unique Alzheimer's disease paired helical filament specific epitopes involve double phosphorylation at specific sites. Biochemistry. 1997;36:8114-24.

Holmes SE, O'Hearn EE, McInnis MG, Gorelick-Feldman DA, Kleiderlein JJ, Callahan C, Kwak NG, Ingersoll-Ashworth RG, Sherr M, Sumner AJ, Sharp AH, Ananth U, Seltzer WK, Boss MA, Vieria-Saecker AM, Epplen JT, Riess O, Ross CA, Margolis RL.
Expansion of a novel CAG trinucleotide repeat in the 5' region of PPP2R2B is associated with SCA12. Nat Genet. 1999;23:391-2.

Hughes K, Nikolakaki E, Plyte SE, Totty NF, Woodgett JR. Modulation of the glycogen synthase kinase-3 family by tyrosine phosphorylation. EMBO J. 1993;12:803-8.

Janssens V and Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J. 2001;353:417-39.

Jicha GA, Weaver C, Lane E, Vianna C, Kress Y, Rockwood J, Davies P. cAMP-dependent protein kinase phosphorylations on tau in Alzheimer's disease. J Neurosci. 1999;19:7486-94.

Kazantsev AG. Cellular pathways leading to neuronal dysfunction and degeneration. Drug News Perspect. 2007;20:501-9.

Kim L, Liu J, Kimmel AR. The novel tyrosine kinase ZAK1 activates GSK3 to direct cell fate specification. Cell. 1999;99:399-408.

Lee G, Newman ST, Gard DL, Band H, Panchamoorthy G. Tau interacts with src-family non-receptor tyrosine kinases. J Cell Sci. 1998;111:3167-77.

Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell. 2004;6:463-77.

Liao Y, Hung MC. A new role of protein phosphatase 2a in adenoviral E1A protein-mediated sensitization to anticancer drug-induced apoptosis in human breast cancer cells. Cancer Res. 2004;64:5938-42.

Lilienfeld DE, Perl DP. Projected neurodegenerative disease mortality in the United States, 1990-2040. Neuroepidemiology. 1993;12:219-28.

Lovestone S, Reynolds CH. The phosphorylation of tau: a critical stage in neurodevelopment and neurodegenerative processes.Neuroscience. 1997;78(2):309-24.

Matsuo ES, Shin RW, Billingsley ML, Van deVoorde A, O'Connor M, Trojanowski JQ, Lee VM. Biopsy-derived adult human brain tau is phosphorylated at many of the same sites as Alzheimer's disease paired helical filament tau. Neuron. 1994;13(4):989-1002.

Mayer RE, Hendrix P, Cron P, Matthies R, Stone SR, Goris J, Merlevede W, Hofsteenge J, Hemmings BA. Structure of the 55-kDa regulatory subunit of protein phosphatase 2A: evidence for a neuronal-specific isoform. Biochemistry. 1991;30:3589-97.

Meier R, Alessi DR, Cron P, Andjelković M, Hemmings BA. Mitogenic activation, phosphorylation, and nuclear translocation of protein kinase Bbeta. J Biol Chem. 1997;272:30491-7.

Nixon RA, Wegiel J, Kumar A, Yu WH, Peterhoff C, Cataldo A, Cuervo AM. Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study. J Neuropathol Exp Neurol. 2005;64(2):113-22.

Pap M, Cooper GM. Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-Kinase/Akt cell survival pathway. J Biol Chem. 1998;273:19929-32.

Price NE, Mumby MC. Brain protein serine/threonine phosphatases. Curr Opin Neurobiol. 1999;9:336-42.

Resjö S, Göransson O, Härndahl L, Zolnierowicz S, Manganiello V, Degerman E. Protein phosphatase 2A is the main phosphatase involved in the regulation of protein kinase B in rat adipocytes. Cell Signal. 2002;14:231-8.

Schild A, Schmidt K, Lim YA, Ke Y, Ittner LM, Hemmings BA, Götz J. Altered levels of PP2A regulatory B/PR55 isoforms indicate role in neuronal differentiation. Int J Dev Neurosci. 2006;24:437-43.

Schonthal AH. Role of serine/threonine protein phosphatase 2A in cancer. Cancer Lett. 2001;170:1-13.

Scott CW, Spreen RC, Herman JL, Chow FP, Davison MD, Young J, Caputo CB. Phosphorylation of recombinant tau by cAMP-dependent protein kinase. Identification of phosphorylation sites and effect on microtubule assembly. J Biol Chem. 1993;268(2):1166-73.

Shaw M, Cohen P, Alessi DR. Further evidence that the inhibition of glycogen synthase kinase-3beta by IGF-1 is mediated by PDK1/PKB-induced phosphorylation of Ser-9 and not by dephosphorylation of Tyr-216. FEBS Lett. 1997;416:307-11.

Shintani T, Klionsky DJ. Autophagy in health and disease: a double-edged sword. Science. 2004;306:990-5.

Sohal RS, Mockett RJ, Orr WC. Mechanisms of aging: an appraisal of the oxidative stress hypothesis. Free Radic Biol Med. 2002;33:575-86.

Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C, Sasaki T, Ruland J, Penninger JM, Siderovski DP, Mak TW. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell. 1998;95:29-39.

Strack S, Ruediger R, Walter G, Dagda RK, Barwacz CA, Cribbs JT. Protein phosphatase 2A holoenzyme assembly: identification of contacts between B-family regulatory and scaffolding A subunits. J Biol Chem. 2002;277:20750-5.

Strack S, Zaucha JA, Ebner FF, Colbran RJ, Wadzinski BE. Brain protein phosphatase 2A: developmental regulation and distinct cellular and subcellular localization by B subunits. J Comp Neurol. 1998;392:515-27.

Woodgett JR. Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J. 1990;9:2431-8.

Woodgett JR. Judging a protein by more than its name: GSK-3.
Sci STKE. 2001; 2001:RE12.

Zheng-Fischhöfer Q, Biernat J, Mandelkow EM, Illenberger S, Godemann R, Mandelkow E. Sequential phosphorylation of Tau by glycogen synthase kinase-3beta and protein kinase A at Thr212 and Ser214 generates the Alzheimer-specific epitope of antibody AT100 and requires a paired-helical-filament-like conformation. Eur J Biochem. 1998;252:542-52.