||Mechanisms and functional roles of nuclear-cytoplasmic shuttling of protein phosphatase 2A
||Institute of Basic Medical Sciences
磷酸酶 Protein phosphatase 2A (PP2A)，屬於一種絲氨酸與蘇氨酸磷酸酶，廣泛表現真核生物細胞中，而且參與了細胞內許多的調節作用；例如：細胞的訊息傳遞（Signal transduction）、細胞週期的進行（Cell cycle progression）、細胞的生長（Cell growth）以及參與細胞凋亡（Apoptosis）的進行等，而當PP2A功能失常時，則會導致正常細胞的病變。PP2A的組成為三元複合體，主要由一個結構次單元，稱之為A次單元；及一個催化次單元，稱之為C次單元；以及高多樣性且具有調節功能的B次單元所組成。目前普遍認為PP2A的受質特異性（Substrate specificity）與亞細胞分佈特性（Subcellular localization）是由B次單元所調控。而我們的研究則主要是探討B56γ3調節次單元對於PP2A次細胞分佈的調控機制與其不同分佈位置對於細胞功能的影響。我們證實B56γ3的分佈是多變的，在恆穩態與細胞週期G1期的細胞中，B56γ3大致呈現核質均勻分佈(Homogenous)；然而隨著細胞週期進入G1與S期臨界期與S期時，B56γ3則明顯集中表現於細胞核內，同時在核內的PP2A/AC dimer也隨之增加。另一方面，大量表現B56γ3於細胞核內時，亦能提升PP2A/AC dimer於細胞核內的分佈，且細胞核內PP2A活性也相對來得高。但當抑制內生性B56γ2與B56γ3的表現時，則可顯著降低PP2A於細胞核內的分佈與活性。在NIH3T3細胞中，大量表現B56γ3可降低p27在蘇氨酸187上的磷酸化，並提高p27蛋白質分子的表現來延緩細胞的生長。當抑制HeLa細胞內內生性B56γ3的表現後，則會造成p27蛋白質的降解因而加速細胞生長。接著，我們也持續研究B56γ3的核運送機制，我們發現核運送受體蛋白-Importin α與Importin β會分別直接與B56γ3上的氨基酸306到405這一段區域結合。且當B56γ3上的絲胺酸440被突變成持續磷酸化時，可顯著增加B56γ3進入細胞核的情形，並減緩細胞的生長。但當絲胺酸440被突變成持續去磷酸化時，則會降低B56γ3於細胞核內的表現與影響PP2A B56γ3對於細胞生長調控的能力。此外，絲胺酸440上的磷酸化也會隨著細胞週期的進行而有所改變，進而影響B56γ3蛋白的穩定性。結論，我們已證實PP2A的調控次單元-B56γ3其核質穿梭的機制是依細胞週期而進行的，並藉由調控細胞週期調控因子，如p27，來調控細胞週期的進行。再者，B56γ3調節次單元可能是以透過“非典型的NLS”序列來調控進入細胞核，經由Importin α-dependent與Importin α-independent的機制來被送入細胞核內。
Protein phosphatase 2A (PP2A) is one major serine/threonine protein phosphatase in eukaryotic cells that has a multitude of functions inside the cell, acting through various targets in cell signal pathways. PP2A consists of a catalytic subunit (PP2A/C) which forms a stable complex with the scaffold subunit (PP2A/A), and this heterodimer (PP2A/AC) associates with regulatory proteins, termed regulatory subunits, to form trimeric holoenzymes attributed with distinct substrate specificity and targeted to different subcellular compartments. In this study, we demonstrated that the subcellular localization of the regulatory subunit B56γ3 is regulated in a cell cycle-dependent manner. Notably, B56γ3 is most concentrated in the nucleus at the G1/S interface and S phase. The S phase-specific nuclear enrichment of B56γ3 was accompanied by an increment of nuclear PP2A/AC dimer and also by nuclear PP2A activity. Overexpression of B56γ3 promoted the nuclear accumulation of PP2A/AC, whereas silencing both B56γ2 and B56γ3 blocked the S phase-specific nuclear accumulation of PP2A/AC and S phase-specific increases in the nuclear phosphatase activity of PP2A. In NIH3T3 cells, overexpressing B56γ3 reduced the phosphorylation of p27 at Thr187, and the protein level of p27 was concomitantly elevated, leading to a delay in the G1 to S transition and to retarded cell proliferation. Consistently, when endogenous B56γ3 expression was knocked down in HeLa cells, p27 protein expression was reduced, alone with increased cell proliferation. Further, to determine the nuclear transport, B56γ3 was shown to directly interact with nuclear transport receptors Importin α and β via a tentative nuclear targeting sequence (amino acid 306-405). B56γ3 was phosphorylated at residue Ser440, and a phosphorylation mimetic mutant (S440D) showed significantly increased nuclear localization of B56γ3 and inhibition of cell proliferation. Conversely, the dephosphorylation defective mutant of Ser440 (S440A) reduced the nuclear accumulation of B56γ3 and impaired the inhibitory effect on cell proliferation and the retardation of cell growth. In addition, the B56γ3 protein levels varied during the course of cell cycle progression, and its phosphorylation of Ser440 played a critical role in stabilizing B56γ3. In conclusion, our data demonstrates that the B56γ3-regulatory subunit of PP2A shuttles between the nucleus and cytoplasm in a cell cycle-dependent manner to regulate cell cycle progression with the participation of p27, a cell-cycle controller. Furthermore, these results suggest that the B56γ3 regulatory subunit may enter nucleus through a non-classical NLS-mediated and both Importin α-dependent and Importin α-independent mechanism.
Figure Contents Ⅷ
Appendix Contents Ⅹ
Abbreviations list Ⅺ
Protein Phosphatase 2A (PP2A) 2
The multimeric structure of PP2A 3
The regulatory B subunits 4
The biological roles of PP2A 6
The subcellular localization of PP2A holoenzyme 9
The B56γ family 11
The functions of PP2A as a tumor suppressor 12
The regulatory subunit B56γ3 13
The regulation of nuclear-cytoplasmic shuttling mechanism 14
Nuclear protein import mechanism 15
The dynamic localization of PP2A 16
The post translation modification of PP2A 17
Research Motivation 18
Materials and Methods 21
Antibodies and Reagents 22
Cell Culture, Cell Line and Transfection 22
Selection of Cells stably Expressing HA-tagged B56γ3, B56γ3 shRNA or Vector only 23
Cell Cycle Synchronize 23
Cell Cycle Analysis 24
Western Blotting and Co-immunoprecipitation 24
Subcellular Fractionation 25
Immunofluorescence and Microscopy 25
Measurement of the PP2A Activity 26
Recombination Protein Preparation 26
In Vitro Pulldown Analysis 27
OptiPrep Density Gradient Centrifugation 27
The subcellular distribution of the B56γ3 subunit is regulated in a cell cycle-dependent manner 30
B56γ3 overexpression increases nuclear localization of the PP2A/A and PP2A/C subunits of PP2A 32
Biochemical fraction reveal S phase-specific increase in nuclear distribution of B56γ3, PP2A/A and PP2A/C subunits 34
The B56γ subunits regulate nuclear PP2A activity that is increased in an S phase-specific manner 37
B56γ3 regulates the G1 to S transition of the cell cycle and regulates p27KIP1 levels 38
Lack the potential NLS sequence does not affect the nuclear accumulation of B56γ3 42
Ser440-phosphorylation within the linker segment of B56γ3 is critical for S-phase nuclear accumulation 43
The total protein expression and phosphorylated level (S440) of B56γ3 are changed with cell cycle progression 44
The phosphorylation at Ser440 whether or not will affect the protein stability of B56γ3 45
Identify the Importin protein interactive sequence of B56γ3 46
Identification of segments within a.a. 306-405 which to affect Importin α or Importin β binds to B56γ3 47
Phosphorylation defective mutant at Ser440 of B56γ3 results in the reduction of interaction Importin α and β 48
Conclusion & Discussion 50
The B56γ3 subunit shuttles between the nucleus and cytoplasm in a cell-cycle dependent manner 51
B56γ3 overexpression increases the nuclear localization of A and C subunits 52
The nuclear PP2A activity is increased by B56γ3 in an S phase-dependent manner 53
PP2A AB56γ3C monitors the cell proliferation through regulating the cell cycle progression 54
The B56γ3-containing PP2A holoenzyme regulates the G1 to S transition, by modulating p27 protein level 55
Regulation of nuclear localization of B56γ3 57
Phosphorylation of B56γ3 at Ser440 regulates the protein stability 59
Curriculum Vitae 122
1. Janssens V, Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J. 2001;353:417-439.
2. Sontag E. Protein phosphatase 2A: the Trojan Horse of cellular signaling. Cell Signal. 2001;13:7-16.
3. Lechward K, Awotunde OS, Swiatek W, Muszynska G. Protein phosphatase 2A: variety of forms and diversity of functions. Acta Biochim Pol. 2001;48:921-933.
4. Eichhorn PJ, Creyghton MP, Bernards R. Protein phosphatase 2A regulatory subunits and cancer. Biochim Biophys Acta. 2009;1795:1-15.
5. 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;277:20750-20755.
6. Xing Y, Xu Y, Chen Y, et al. Structure of protein phosphatase 2A core enzyme bound to tumor-inducing toxins. Cell. 2006;127:341-353.
7. Shi Y. Assembly and structure of protein phosphatase 2A. Sci China C Life Sci. 2009;52:135-146.
8. Kong M, Ditsworth D, Lindsten T, Thompson CB. Alpha4 is an essential regulator of PP2A phosphatase activity. Mol Cell. 2009;36:51-60.
9. Arroyo JD, Hahn WC. Involvement of PP2A in viral and cellular transformation. Oncogene. 2005;24:7746-7755.
10. Mumby M. The 3D structure of protein phosphatase 2A: new insights into a ubiquitous regulator of cell signaling. ACS Chem Biol. 2007;2:99-103.
11. Yang J, Phiel C. Functions of B56-containing PP2As in major developmental and cancer signaling pathways. Life Sci. 2010;87:659-666.
12. Healy AM, Zolnierowicz S, Stapleton AE, Goebl M, DePaoli-Roach AA, Pringle JR. CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization, and homology to the B subunit of mammalian type 2A protein phosphatase. Mol Cell Biol. 1991;11:5767-5780.
13. Mayer-Jaekel RE, Ohkura H, Gomes R, et al. The 55 kd regulatory subunit of Drosophila protein phosphatase 2A is required for anaphase. Cell. 1993;72:621-633.
14. Kuo YC, Huang KY, Yang CH, Yang YS, Lee WY, Chiang CW. Regulation of phosphorylation of Thr-308 of Akt, cell proliferation, and survival by the B55alpha regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt. J Biol Chem. 2008;283:1882-1892.
15. Turowski P, Myles T, Hemmings BA, Fernandez A, Lamb NJ. Vimentin dephosphorylation by protein phosphatase 2A is modulated by the targeting subunit B55. Mol Biol Cell. 1999;10:1997-2015.
16. McCright B, Rivers AM, Audlin S, Virshup DM. The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm. J Biol Chem. 1996;271:22081-22089.
17. Okamoto K, Kamibayashi C, Serrano M, Prives C, Mumby MC, Beach D. p53-dependent association between cyclin G and the B' subunit of protein phosphatase 2A. Mol Cell Biol. 1996;16:6593-6602.
18. Ito A, Kataoka TR, Watanabe M, et al. A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation. EMBO J. 2000;19:562-571.
19. Shtrichman R, Sharf R, Kleinberger T. Adenovirus E4orf4 protein interacts with both Balpha and B' subunits of protein phosphatase 2A, but E4orf4-induced apoptosis is mediated only by the interaction with Balpha. Oncogene. 2000;19:3757-3765.
20. Kleinberger T, Shenk T. Adenovirus E4orf4 protein binds to protein phosphatase 2A, and the complex down regulates E1A-enhanced junB transcription. J Virol. 1993;67:7556-7560.
21. Bondesson M, Ohman K, Manervik M, Fan S, Akusjarvi G. Adenovirus E4 open reading frame 4 protein autoregulates E4 transcription by inhibiting E1A transactivation of the E4 promoter. J Virol. 1996;70:3844-3851.
22. Kanopka A, Muhlemann O, Petersen-Mahrt S, Estmer C, Ohrmalm C, Akusjarvi G. Regulation of adenovirus alternative RNA splicing by dephosphorylation of SR proteins. Nature. 1998;393:185-187.
23. Van Hoof C, Goris J. Phosphatases in apoptosis: to be or not to be, PP2A is in the heart of the question. Biochim Biophys Acta. 2003;1640:97-104.
24. Kitajima TS, Sakuno T, Ishiguro K, et al. Shugoshin collaborates with protein phosphatase 2A to protect cohesin. Nature. 2006;441:46-52.
25. Xu Y, Xing Y, Chen Y, et al. Structure of the protein phosphatase 2A holoenzyme. Cell. 2006;127:1239-1251.
26. Yan Z, Fedorov SA, Mumby MC, Williams RS. PR48, a novel regulatory subunit of protein phosphatase 2A, interacts with Cdc6 and modulates DNA replication in human cells. Mol Cell Biol. 2000;20:1021-1029.
27. Voorhoeve PM, Hijmans EM, Bernards R. Functional interaction between a novel protein phosphatase 2A regulatory subunit, PR59, and the retinoblastoma-related p107 protein. Oncogene. 1999;18:515-524.
28. Moreno CS, Park S, Nelson K, et al. WD40 repeat proteins striatin and S/G(2) nuclear autoantigen are members of a novel family of calmodulin-binding proteins that associate with protein phosphatase 2A. J Biol Chem. 2000;275:5257-5263.
29. Dunphy WG. The decision to enter mitosis. Trends Cell Biol. 1994;4:202-207.
30. Lee TH, Solomon MJ, Mumby MC, Kirschner MW. INH, a negative regulator of MPF, is a form of protein phosphatase 2A. Cell. 1991;64:415-423.
31. Lee TH, Turck C, Kirschner MW. Inhibition of cdc2 activation by INH/PP2A. Mol Biol Cell. 1994;5:323-338.
32. Borgne A, Meijer L. Sequential dephosphorylation of p34(cdc2) on Thr-14 and Tyr-15 at the prophase/metaphase transition. J Biol Chem. 1996;271:27847-27854.
33. Clarke PR, Hoffmann I, Draetta G, Karsenti E. Dephosphorylation of cdc25-C by a type-2A protein phosphatase: specific regulation during the cell cycle in Xenopus egg extracts. Mol Biol Cell. 1993;4:397-411.
34. Felix MA, Cohen P, Karsenti E. Cdc2 H1 kinase is negatively regulated by a type 2A phosphatase in the Xenopus early embryonic cell cycle: evidence from the effects of okadaic acid. EMBO J. 1990;9:675-683.
35. Yamashita K, Yasuda H, Pines J, et al. Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitosis-like state in BHK21 cells. EMBO J. 1990;9:4331-4338.
36. Chou DM, Petersen P, Walter JC, Walter G. Protein phosphatase 2A regulates binding of Cdc45 to the prereplication complex. J Biol Chem. 2002;277:40520-40527.
37. Davis AJ, Yan Z, Martinez B, Mumby MC. Protein phosphatase 2A is targeted to cell division control protein 6 by a calcium-binding regulatory subunit. J Biol Chem. 2008;283:16104-16114.
38. Ruediger R, Van Wart Hood JE, Mumby M, Walter G. Constant expression and activity of protein phosphatase 2A in synchronized cells. Mol Cell Biol. 1991;11:4282-4285.
39. Sontag E, Nunbhakdi-Craig V, Bloom GS, Mumby MC. A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle. J Cell Biol. 1995;128:1131-1144.
40. Turowski P, Fernandez A, Favre B, Lamb NJ, Hemmings BA. Differential methylation and altered conformation of cytoplasmic and nuclear forms of protein phosphatase 2A during cell cycle progression. J Cell Biol. 1995;129:397-410.
41. Chen J, Peterson RT, Schreiber SL. Alpha 4 associates with protein phosphatases 2A, 4, and 6. Biochem Biophys Res Commun. 1998;247:827-832.
42. Murata K, Wu J, Brautigan DL. B cell receptor-associated protein alpha4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A. Proc Natl Acad Sci U S A. 1997;94:10624-10629.
43. Inui S, Sanjo H, Maeda K, Yamamoto H, Miyamoto E, Sakaguchi N. Ig receptor binding protein 1 (alpha4) is associated with a rapamycin-sensitive signal transduction in lymphocytes through direct binding to the catalytic subunit of protein phosphatase 2A. Blood. 1998;92:539-546.
44. Santoro MF, Annand RR, Robertson MM, et al. Regulation of protein phosphatase 2A activity by caspase-3 during apoptosis. J Biol Chem. 1998;273:13119-13128.
45. Ruvolo PP, Deng X, Ito T, Carr BK, May WS. Ceramide induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A. J Biol Chem. 1999;274:20296-20300.
46. Deng X, Ito T, Carr B, Mumby M, May WS, Jr. Reversible phosphorylation of Bcl2 following interleukin 3 or bryostatin 1 is mediated by direct interaction with protein phosphatase 2A. J Biol Chem. 1998;273:34157-34163.
47. Shtrichman R, Sharf R, Barr H, Dobner T, Kleinberger T. Induction of apoptosis by adenovirus E4orf4 protein is specific to transformed cells and requires an interaction with protein phosphatase 2A. Proc Natl Acad Sci U S A. 1999;96:10080-10085.
48. Marcellus RC, Chan H, Paquette D, Thirlwell S, Boivin D, Branton PE. Induction of p53-independent apoptosis by the adenovirus E4orf4 protein requires binding to the Balpha subunit of protein phosphatase 2A. J Virol. 2000;74:7869-7877.
49. Hong Y, Sarge KD. Regulation of protein phosphatase 2A activity by heat shock transcription factor 2. J Biol Chem. 1999;274:12967-12970.
50. Cairns J, Qin S, Philp R, Tan YH, Guy GR. Dephosphorylation of the small heat shock protein Hsp27 in vivo by protein phosphatase 2A. J Biol Chem. 1994;269:9176-9183.
51. Okamoto K, Beach D. Cyclin G is a transcriptional target of the p53 tumor suppressor protein. EMBO J. 1994;13:4816-4822.
52. van Lookeren Campagne M, Okamoto K, Prives C, Gill R. Developmental expression and co-localization of cyclin G1 and the B' subunits of protein phosphatase 2a in neurons. Brain Res Mol Brain Res. 1999;64:1-10.
53. Khew-Goodall Y, Hemmings BA. Tissue-specific expression of mRNAs encoding alpha- and beta-catalytic subunits of protein phosphatase 2A. FEBS Lett. 1988;238:265-268.
54. 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;392:515-527.
55. Csortos C, Zolnierowicz S, Bako E, Durbin SD, DePaoli-Roach AA. High complexity in the expression of the B' subunit of protein phosphatase 2A0. Evidence for the existence of at least seven novel isoforms. J Biol Chem. 1996;271:2578-2588.
56. Price NE, Mumby MC. Brain protein serine/threonine phosphatases. Curr Opin Neurobiol. 1999;9:336-342.
57. Lee VM. Disruption of the cytoskeleton in Alzheimer's disease. Curr Opin Neurobiol. 1995;5:663-668.
58. Billingsley ML, Kincaid RL. Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration. Biochem J. 1997;323 ( Pt 3):577-591.
59. Price NE, Wadzinski B, Mumby MC. An anchoring factor targets protein phosphatase 2A to brain microtubules. Brain Res Mol Brain Res. 1999;73:68-77.
60. Gong CX, Lidsky T, Wegiel J, Zuck L, Grundke-Iqbal I, Iqbal K. Phosphorylation of microtubule-associated protein tau is regulated by protein phosphatase 2A in mammalian brain. Implications for neurofibrillary degeneration in Alzheimer's disease. J Biol Chem. 2000;275:5535-5544.
61. Sontag E, Nunbhakdi-Craig V, Lee G, Bloom GS, Mumby MC. Regulation of the phosphorylation state and microtubule-binding activity of Tau by protein phosphatase 2A. Neuron. 1996;17:1201-1207.
62. Sontag E, Nunbhakdi-Craig V, Lee G, et al. Molecular interactions among protein phosphatase 2A, tau, and microtubules. Implications for the regulation of tau phosphorylation and the development of tauopathies. J Biol Chem. 1999;274:25490-25498.
63. Xu Y, Chen Y, Zhang P, Jeffrey PD, Shi Y. Structure of a protein phosphatase 2A holoenzyme: insights into B55-mediated Tau dephosphorylation. Mol Cell. 2008;31:873-885.
64. Shi Y. Serine/threonine phosphatases: mechanism through structure. Cell. 2009;139:468-484.
65. Lee TY, Lai TY, Lin SC, et al. The B56gamma3 regulatory subunit of protein phosphatase 2A (PP2A) regulates S phase-specific nuclear accumulation of PP2A and the G1 to S transition. J Biol Chem. 2010;285:21567-21580.
66. Zhou J, Pham HT, Walter G. The formation and activity of PP2A holoenzymes do not depend on the isoform of the catalytic subunit. J Biol Chem. 2003;278:8617-8622.
67. Strack S, Chang D, Zaucha JA, Colbran RJ, Wadzinski BE. Cloning and characterization of B delta, a novel regulatory subunit of protein phosphatase 2A. FEBS Lett. 1999;460:462-466.
68. 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;278:24976-24985.
69. Li X, Virshup DM. Two conserved domains in regulatory B subunits mediate binding to the A subunit of protein phosphatase 2A. Eur J Biochem. 2002;269:546-552.
70. Riedel CG, Katis VL, Katou Y, et al. Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I. Nature. 2006;441:53-61.
71. Flegg CP, Sharma M, Medina-Palazon C, et al. Nuclear export and centrosome targeting of the protein phosphatase 2A subunit B56alpha: role of B56alpha in nuclear export of the catalytic subunit. J Biol Chem. 2010;285:18144-18154.
72. Jin Z, Shi J, Saraf A, et al. The 48-kDa alternative translation isoform of PP2A:B56epsilon is required for Wnt signaling during midbrain-hindbrain boundary formation. J Biol Chem. 2009;284:7190-7200.
73. McCright B, Brothman AR, Virshup DM. Assignment of human protein phosphatase 2A regulatory subunit genes b56alpha, b56beta, b56gamma, b56delta, and b56epsilon (PPP2R5A-PPP2R5E), highly expressed in muscle and brain, to chromosome regions 1q41, 11q12, 3p21, 6p21.1, and 7p11.2 --> p12. Genomics. 1996;36:168-170.
74. Gigena MS, Ito A, Nojima H, Rogers TB. A B56 regulatory subunit of protein phosphatase 2A localizes to nuclear speckles in cardiomyocytes. Am J Physiol Heart Circ Physiol. 2005;289:H285-294.
75. Ito A, Koma Y, Sohda M, et al. Localization of the PP2A B56gamma regulatory subunit at the Golgi complex: possible role in vesicle transport and migration. Am J Pathol. 2003;162:479-489.
76. Hendrix P, Mayer-Jackel RE, Cron P, et al. Structure and expression of a 72-kDa regulatory subunit of protein phosphatase 2A. Evidence for different size forms produced by alternative splicing. J Biol Chem. 1993;268:15267-15276.
77. Haeberle AM, Castets F, Bombarde G, Baillat G, Bailly Y. Immunogold localization of phocein in dendritic spines. J Comp Neurol. 2006;495:336-350.
78. McCright B, Virshup DM. Identification of a new family of protein phosphatase 2A regulatory subunits. J Biol Chem. 1995;270:26123-26128.
79. Tehrani MA, Mumby MC, Kamibayashi C. Identification of a novel protein phosphatase 2A regulatory subunit highly expressed in muscle. J Biol Chem. 1996;271:5164-5170.
80. Lin SS, Bassik MC, Suh H, et al. PP2A regulates BCL-2 phosphorylation and proteasome-mediated degradation at the endoplasmic reticulum. J Biol Chem. 2006;281:23003-23012.
81. Li HH, Cai X, Shouse GP, Piluso LG, Liu X. A specific PP2A regulatory subunit, B56gamma, mediates DNA damage-induced dephosphorylation of p53 at Thr55. EMBO J. 2007;26:402-411.
82. Shouse GP, Cai X, Liu X. Serine 15 phosphorylation of p53 directs its interaction with B56gamma and the tumor suppressor activity of B56gamma-specific protein phosphatase 2A. Mol Cell Biol. 2008;28:448-456.
83. Arroyo JD, Lee GM, Hahn WC. Liprin alpha1 interacts with PP2A B56gamma. Cell Cycle. 2008;7:525-532.
84. Sablina AA, Hector M, Colpaert N, Hahn WC. Identification of PP2A complexes and pathways involved in cell transformation. Cancer Res. 2010;70:10474-10484.
85. Shouse GP, Nobumori Y, Liu X. A B56gamma mutation in lung cancer disrupts the p53-dependent tumor-suppressor function of protein phosphatase 2A. Oncogene. 2010;29:3933-3941.
86. Shouse GP, Nobumori Y, Panowicz MJ, Liu X. ATM-mediated phosphorylation activates the tumor-suppressive function of B56gamma-PP2A. Oncogene. 2011.
87. Muneer S, Ramalingam V, Wyatt R, Schultz RA, Minna JD, Kamibayashi C. Genomic organization and mapping of the gene encoding the PP2A B56gamma regulatory subunit. Genomics. 2002;79:344-348.
88. Chen W, Possemato R, Campbell KT, Plattner CA, Pallas DC, Hahn WC. Identification of specific PP2A complexes involved in human cell transformation. Cancer Cell. 2004;5:127-136.
89. Bialojan C, Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J. 1988;256:283-290.
90. Pallas DC, Shahrik LK, Martin BL, et al. Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A. Cell. 1990;60:167-176.
91. Cayla X, Ballmer-Hofer K, Merlevede W, Goris J. Phosphatase 2A associated with polyomavirus small-T or middle-T antigen is an okadaic acid-sensitive tyrosyl phosphatase. Eur J Biochem. 1993;214:281-286.
92. Kamibayashi C, Estes R, Lickteig RL, Yang SI, Craft C, Mumby MC. Comparison of heterotrimeric protein phosphatase 2A containing different B subunits. J Biol Chem. 1994;269:20139-20148.
93. Deichmann M, Polychronidis M, Wacker J, Thome M, Naher H. The protein phosphatase 2A subunit Bgamma gene is identified to be differentially expressed in malignant melanomas by subtractive suppression hybridization. Melanoma Res. 2001;11:577-585.
94. Okamoto K, Li H, Jensen MR, et al. Cyclin G recruits PP2A to dephosphorylate Mdm2. Mol Cell. 2002;9:761-771.
95. Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation of p53. Nature. 1997;387:296-299.
96. Dohoney KM, Guillerm C, Whiteford C, et al. Phosphorylation of p53 at serine 37 is important for transcriptional activity and regulation in response to DNA damage. Oncogene. 2004;23:49-57.
97. Ruediger R, Pham HT, Walter G. Alterations in protein phosphatase 2A subunit interaction in human carcinomas of the lung and colon with mutations in the A beta subunit gene. Oncogene. 2001;20:1892-1899.
98. Sablina AA, Chen W, Arroyo JD, et al. The tumor suppressor PP2A Abeta regulates the RalA GTPase. Cell. 2007;129:969-982.
99. Esplin ED, Ramos P, Martinez B, Tomlinson GE, Mumby MC, Evans GA. The glycine 90 to aspartate alteration in the Abeta subunit of PP2A (PPP2R1B) associates with breast cancer and causes a deficit in protein function. Genes Chromosomes Cancer. 2006;45:182-190.
100. Mumby M. Regulation by tumour antigens defines a role for PP2A in signal transduction. Semin Cancer Biol. 1995;6:229-237.
101. Poon IK, Jans DA. Regulation of nuclear transport: central role in development and transformation? Traffic. 2005;6:173-186.
102. Gorlich D, Mattaj IW. Nucleocytoplasmic transport. Science. 1996;271:1513-1518.
103. Weis K. Importins and exportins: how to get in and out of the nucleus. Trends Biochem Sci. 1998;23:185-189.
104. Fahrenkrog B. The nuclear pore complex, nuclear transport, and apoptosis. Can J Physiol Pharmacol. 2006;84:279-286.
105. Xu L, Massague J. Nucleocytoplasmic shuttling of signal transducers. Nat Rev Mol Cell Biol. 2004;5:209-219.
106. Vartiainen MK. Nuclear actin dynamics--from form to function. FEBS Lett. 2008;582:2033-2040.
107. Gorlich D, Kutay U. Transport between the cell nucleus and the cytoplasm. Annu Rev Cell Dev Biol. 1999;15:607-660.
108. Mattaj IW, Englmeier L. Nucleocytoplasmic transport: the soluble phase. Annu Rev Biochem. 1998;67:265-306.
109. Bogerd HP, Fridell RA, Benson RE, Hua J, Cullen BR. Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay. Mol Cell Biol. 1996;16:4207-4214.
110. Fornerod M, Ohno M, Yoshida M, Mattaj IW. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell. 1997;90:1051-1060.
111. Kohler M, Haller H, Hartmann E. Nuclear protein transport pathways. Exp Nephrol. 1999;7:290-294.
112. Goldfarb DS, Corbett AH, Mason DA, Harreman MT, Adam SA. Importin alpha: a multipurpose nuclear-transport receptor. Trends Cell Biol. 2004;14:505-514.
113. Chook YM, Blobel G. Karyopherins and nuclear import. Curr Opin Struct Biol. 2001;11:703-715.
114. Soprano KJ, Purev E, Vuocolo S, Soprano DR. Rb2/p130 and protein phosphatase 2A: key mediators of ovarian carcinoma cell growth suppression by all-trans retinoic acid. Oncogene. 2006;25:5315-5325.
115. Harreman MT, Kline TM, Milford HG, Harben MB, Hodel AE, Corbett AH. Regulation of nuclear import by phosphorylation adjacent to nuclear localization signals. J Biol Chem. 2004;279:20613-20621.
116. Jans DA, Xiao CY, Lam MH. Nuclear targeting signal recognition: a key control point in nuclear transport? Bioessays. 2000;22:532-544.
117. Gentry MS, Hallberg RL. Localization of Saccharomyces cerevisiae protein phosphatase 2A subunits throughout mitotic cell cycle. Mol Biol Cell. 2002;13:3477-3492.
118. Jiang W, Hallberg RL. Isolation and characterization of par1(+) and par2(+): two Schizosaccharomyces pombe genes encoding B' subunits of protein phosphatase 2A. Genetics. 2000;154:1025-1038.
119. Kinoshita K, Nemoto T, Nabeshima K, Kondoh H, Niwa H, Yanagida M. The regulatory subunits of fission yeast protein phosphatase 2A (PP2A) affect cell morphogenesis, cell wall synthesis and cytokinesis. Genes Cells. 1996;1:29-45.
120. Le Goff X, Buvelot S, Salimova E, et al. The protein phosphatase 2A B'-regulatory subunit par1p is implicated in regulation of the S. pombe septation initiation network. FEBS Lett. 2001;508:136-142.
121. Chen J, Martin BL, Brautigan DL. Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science. 1992;257:1261-1264.
122. Usui H, Inoue R, Tanabe O, et al. Activation of protein phosphatase 2A by cAMP-dependent protein kinase-catalyzed phosphorylation of the 74-kDa B' (delta) regulatory subunit in vitro and identification of the phosphorylation sites. FEBS Lett. 1998;430:312-316.
123. Xu Z, Williams BR. The B56alpha regulatory subunit of protein phosphatase 2A is a target for regulation by double-stranded RNA-dependent protein kinase PKR. Mol Cell Biol. 2000;20:5285-5299.
124. Kim KY, Baek A, Hwang JE, et al. Adiponectin-activated AMPK stimulates dephosphorylation of AKT through protein phosphatase 2A activation. Cancer Res. 2009;69:4018-4026.
125. Harper JV. Synchronization of cell populations in G1/S and G2/M phases of the cell cycle. Methods Mol Biol. 2005;296:157-166.
126. Waltregny D, De Leval L, Glenisson W, et al. Expression of histone deacetylase 8, a class I histone deacetylase, is restricted to cells showing smooth muscle differentiation in normal human tissues. Am J Pathol. 2004;165:553-564.
127. Tsuji L, Takumi T, Imamoto N, Yoneda Y. Identification of novel homologues of mouse importin alpha, the alpha subunit of the nuclear pore-targeting complex, and their tissue-specific expression. FEBS Lett. 1997;416:30-34.
128. Nourse J, Firpo E, Flanagan WM, et al. Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature. 1994;372:570-573.
129. Polyak K, Lee MH, Erdjument-Bromage H, et al. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell. 1994;78:59-66.
130. Polyak K, Kato JY, Solomon MJ, et al. p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev. 1994;8:9-22.
131. Toyoshima H, Hunter T. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell. 1994;78:67-74.
132. Coats S, Flanagan WM, Nourse J, Roberts JM. Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle. Science. 1996;272:877-880.
133. Sheaff RJ, Groudine M, Gordon M, Roberts JM, Clurman BE. Cyclin E-CDK2 is a regulator of p27Kip1. Genes Dev. 1997;11:1464-1478.
134. Vlach J, Hennecke S, Amati B. Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27. EMBO J. 1997;16:5334-5344.
135. Morisaki H, Fujimoto A, Ando A, Nagata Y, Ikeda K, Nakanishi M. Cell cycle-dependent phosphorylation of p27 cyclin-dependent kinase (Cdk) inhibitor by cyclin E/Cdk2. Biochem Biophys Res Commun. 1997;240:386-390.
136. Sutterluty H, Chatelain E, Marti A, et al. p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells. Nat Cell Biol. 1999;1:207-214.
137. Rodier G, Montagnoli A, Di Marcotullio L, et al. p27 cytoplasmic localization is regulated by phosphorylation on Ser10 and is not a prerequisite for its proteolysis. EMBO J. 2001;20:6672-6682.
138. Boehm M, Yoshimoto T, Crook MF, et al. A growth factor-dependent nuclear kinase phosphorylates p27(Kip1) and regulates cell cycle progression. EMBO J. 2002;21:3390-3401.
139. Reynisdottir I, Massague J. The subcellular locations of p15(Ink4b) and p27(Kip1) coordinate their inhibitory interactions with cdk4 and cdk2. Genes Dev. 1997;11:492-503.
140. Orend G, Hunter T, Ruoslahti E. Cytoplasmic displacement of cyclin E-cdk2 inhibitors p21Cip1 and p27Kip1 in anchorage-independent cells. Oncogene. 1998;16:2575-2583.
141. Soucek T, Yeung RS, Hengstschlager M. Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene-2. Proc Natl Acad Sci U S A. 1998;95:15653-15658.
142. Tomoda K, Kubota Y, Kato J. Degradation of the cyclin-dependent-kinase inhibitor p27Kip1 is instigated by Jab1. Nature. 1999;398:160-165.
143. Connor MK, Kotchetkov R, Cariou S, et al. CRM1/Ran-mediated nuclear export of p27(Kip1) involves a nuclear export signal and links p27 export and proteolysis. Mol Biol Cell. 2003;14:201-213.
144. Kalderon D, Roberts BL, Richardson WD, Smith AE. A short amino acid sequence able to specify nuclear location. Cell. 1984;39:499-509.
145. Nakai K, Horton P. PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci. 1999;24:34-36.
146. Mulder NJ, Kersey P, Pruess M, Apweiler R. In silico characterization of proteins: UniProt, InterPro and Integr8. Mol Biotechnol. 2008;38:165-177.
147. Hubner S, Xiao CY, Jans DA. The protein kinase CK2 site (Ser111/112) enhances recognition of the simian virus 40 large T-antigen nuclear localization sequence by importin. J Biol Chem. 1997;272:17191-17195.
148. Xiao CY, Hubner S, Jans DA. SV40 large tumor antigen nuclear import is regulated by the double-stranded DNA-dependent protein kinase site (serine 120) flanking the nuclear localization sequence. J Biol Chem. 1997;272:22191-22198.
149. Briggs LJ, Stein D, Goltz J, et al. The cAMP-dependent protein kinase site (Ser312) enhances dorsal nuclear import through facilitating nuclear localization sequence/importin interaction. J Biol Chem. 1998;273:22745-22752.
150. Yamashita M, Dimayuga P, Kaul S, et al. Phosphatase activity in the arterial wall after balloon injury: effect of somatostatin analog octreotide. Lab Invest. 1999;79:935-944.
151. Jeon KI, Jono H, Miller CL, et al. Ca2+/calmodulin-stimulated PDE1 regulates the beta-catenin/TCF signaling through PP2A B56 gamma subunit in proliferating vascular smooth muscle cells. FEBS J. 2010;277:5026-5039.
152. Slingerland J, Pagano M. Regulation of the cdk inhibitor p27 and its deregulation in cancer. J Cell Physiol. 2000;183:10-17.
153. Montagnoli A, Fiore F, Eytan E, et al. Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation. Genes Dev. 1999;13:1181-1189.
154. Carrano AC, Eytan E, Hershko A, Pagano M. SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat Cell Biol. 1999;1:193-199.
155. Tsvetkov LM, Yeh KH, Lee SJ, Sun H, Zhang H. p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27. Curr Biol. 1999;9:661-664.
156. Morishita D, Katayama R, Sekimizu K, Tsuruo T, Fujita N. Pim kinases promote cell cycle progression by phosphorylating and down-regulating p27Kip1 at the transcriptional and posttranscriptional levels. Cancer Res. 2008;68:5076-5085.
157. Hong F, Larrea MD, Doughty C, Kwiatkowski DJ, Squillace R, Slingerland JM. mTOR-raptor binds and activates SGK1 to regulate p27 phosphorylation. Mol Cell. 2008;30:701-711.
158. Shin I, Rotty J, Wu FY, Arteaga CL. Phosphorylation of p27Kip1 at Thr-157 interferes with its association with importin alpha during G1 and prevents nuclear re-entry. J Biol Chem. 2005;280:6055-6063.
159. McAllister SS, Becker-Hapak M, Pintucci G, Pagano M, Dowdy SF. Novel p27(kip1) C-terminal scatter domain mediates Rac-dependent cell migration independent of cell cycle arrest functions. Mol Cell Biol. 2003;23:216-228.
160. Pemberton LF, Paschal BM. Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic. 2005;6:187-198.
161. Nardozzi JD, Lott K, Cingolani G. Phosphorylation meets nuclear import: a review. Cell Commun Signal. 2010;8:32.
162. Efthymiadis A, Briggs LJ, Jans DA. The HIV-1 Tat nuclear localization sequence confers novel nuclear import properties. J Biol Chem. 1998;273:1623-1628.
163. Fagotto F, Gluck U, Gumbiner BM. Nuclear localization signal-independent and importin/karyopherin-independent nuclear import of beta-catenin. Curr Biol. 1998;8:181-190.
164. Peifer M, Berg S, Reynolds AB. A repeating amino acid motif shared by proteins with diverse cellular roles. Cell. 1994;76:789-791.
165. Yano R, Oakes ML, Tabb MM, Nomura M. Yeast Srp1p has homology to armadillo/plakoglobin/beta-catenin and participates in apparently multiple nuclear functions including the maintenance of the nucleolar structure. Proc Natl Acad Sci U S A. 1994;91:6880-6884.
166. Glaser ND, Lukyanenko YO, Wang Y, Wilson GM, Rogers TB. JNK activation decreases PP2A regulatory subunit B56alpha expression and mRNA stability and increases AUF1 expression in cardiomyocytes. Am J Physiol Heart Circ Physiol. 2006;291:H1183-1192.
167. LeNoue-Newton M, Watkins GR, Zou P, et al. The E3 ubiquitin ligase- and protein phosphatase 2A (PP2A)-binding domains of the Alpha4 protein are both required for Alpha4 to inhibit PP2A degradation. J Biol Chem. 2011;286:17665-17671.