蛋白質磷酸酶2A型 (以下簡稱PP2A)是由三種次單元所組成的磷酸酶，分別是提供PP2A酵素活性36-kDa的C次單元、做為骨架功能65-kDa的A次單元、以及最為多變的調節次單元B。到現在至少已經發現了四種B次單元家族，分別為B, B’, B’’, 以及B’’’ ，而次單元B被認為能決定PP2A的受質專一性以及PP2A在細胞中的位置。根據研究指出，B’家族中其中的一種異構型B56γ3具有抑制細胞轉型以及轉化成腫瘤的能力。與之前的研究相符，我們觀察到當短暫與穩定表現B56γ3於NIH3T3及HeLa細胞中時，B56γ3在細胞核與質中均有分布，然而決定B56γ3在細胞內位置的詳細機制仍然不清楚。我們以軟體分析B56γ3的胺基酸序列並找到了四個可能的核定位訊號(nuclear localization signal, 以下簡稱NLS)以及三段可能的核輸出訊號(nuclear export signal, 以下簡稱NES)。我們依序將四段可能的NLS從B56γ3的C端開始切除，並探討這些截切掉可能的NLS後的B56γ3序列在細胞內的分布情形。令人訝異的是，在短暫表現或是穩定表現這些截切的B56γ3突變型於細胞中的實驗裡，沒有任何一段可能的NLS序列在去除後會造成B56γ3失去入核的能力; 然而我們發現，在去除掉可能的NLS4, NLS3, 以及NLS2之後在細胞核內表現的B56γ3反而大量增加，但是在NIH3T3細胞中可能的NLS1以及NLS2之間銜接的序列被去除之後，前面所述增加核內表現的現象隨即消失，但在HeLa細胞中這個現象則沒有改變。接著，我們短暫表現接合黃色螢光蛋白(yellow fluorescence protein)與完整或切除掉可能NLS後的B56γ3序列於細胞中，再利用螢光顯微鏡觀察後，我們再度發現，所有完整及突變型仍然具有入核的能力。此外，為了探討這些截切的突變型是否仍具有與AC核心酶(core enzyme)結合的能力以形成一個具有完整功能性的PP2A完全酶，我們利用了免疫沉澱分析法分析並比較完整及截切的B56γ3突變型，發現當失去了可能的NLS1以及NLS2之間橋接的序列後，B56γ3會顯著地失去與AC核心酶相接的能力。我們的研究揭露了非標準型的NLS可能參與調控B56γ3進入細胞核的過程，以及一段對於B56γ3與AC核心酶產生結合能力相當重要的序列。

Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme which consists of a 36-kDa catalytic C subunit, a 65-kDa structural A subunit, and a variable regulatory B subunit. There are at least four subfamilies of the B subunit, named B, B’, B’’, and B’’’. The B subunit has been thought to determine the substrate specificity and subcellular localization of the enzyme. B56γ3, one of the B’ isoform, has been shown to have roles in suppressing cell transformation and tumorigenesis. Consistent with previous findings, we have observed B56γ3 in NIH3T3 and HeLa cells existing in both nucleus and cytoplasm in cells transiently and stably expressing exogenous B56γ3. The mechanisms underlying the subcellular localization of B56γ3 are not clear. We have found four putative nuclear localization signals (NLSs) and three putative nuclear export signals (NESs) within B56γ3 by sequence analyses using software. We serially truncated the four putative NLSs from the C terminus of B56γ3 and investigated the subcellular localization of these NLS truncation mutants of B56γ3. Surprisingly, none of the serially NLS-truncated B56γ3 mutants showed impaired nuclear localization in both transient and stable expression experiments in both NIH3T3 and HeLa cells. Nevertheless, we found a significant increase of nuclear localization of B56γ3 after truncations of putative NLS 4, NLS 3, and NLS 2. However, the phenotype was diminished in NIH3T3 cells but not in HeLa cells when the linker segment between putative NLS 1 and NLS 2 was deleted. In addition, fluorescence microscopy analysis of transiently expressed yellow fluorescent protein (YFP)-fused full-length and NLS truncated mutants showed that all forms of B56γ3 were capable of locating to the nucleus. Furthermore, to examine whether these truncation mutants of B56γ3 can associate with the AC core enzyme to form a functional trimeric holoenzyme, we performed co-immunoprecipitation analysis and found that the truncated B56γ3 mutants without the linker segment between NLS 1 and 2 greatly lost the ability to associate with the AC core enzyme. Our findings reveal that a noncanonical NLS may be involved in regulating B56γ3 nuclear localization and demonstrate that a motif near the C terminus is critical for B56γ3 association with the AC core enzyme.

1. Moorhead, G.B., Trinkle-Mulcahy, L. & Ulke-Lemee, A. Emerging roles of nuclear protein phosphatases. Nat Rev Mol Cell Biol 8, 234-44 (2007).
2. Hemmings, B.A. et al. alpha- and beta-forms of the 65-kDa subunit of protein phosphatase 2A have a similar 39 amino acid repeating structure. Biochemistry 29, 3166-73 (1990).
3. Wang, S.S. et al. Alterations of the PPP2R1B gene in human lung and colon cancer. Science 282, 284-7 (1998).
4. Wera, S. et al. Deregulation of translational control of the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A leads to multinucleated cells. J Biol Chem 270, 21374-81 (1995).
5. Groves, M.R., Hanlon, N., Turowski, P., Hemmings, B.A. & Barford, D. The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs. Cell 96, 99-110 (1999).
6. Stone, S.R., Hofsteenge, J. & Hemmings, B.A. Molecular cloning of cDNAs encoding two isoforms of the catalytic subunit of protein phosphatase 2A. Biochemistry 26, 7215-20 (1987).
7. Khew-Goodall, Y. & Hemmings, B.A. Tissue-specific expression of mRNAs encoding alpha- and beta-catalytic subunits of protein phosphatase 2A. FEBS Lett 238, 265-8 (1988).
8. Orgad, S. et al. The structure of protein phosphatase 2A is as highly conserved as that of protein phosphatase 1. FEBS Lett 275, 44-8 (1990).
9. Arino, J. et al. Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana. Plant Mol Biol 21, 475-85 (1993).
10. Janssens, V. & Goris, J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 353, 417-39 (2001).
11. Sontag, E., Nunbhakdi-Craig, V., Bloom, G.S. & Mumby, M.C. A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle. J Cell Biol 128, 1131-44 (1995).
12. Tehrani, M.A., Mumby, M.C. & Kamibayashi, C. Identification of a novel protein phosphatase 2A regulatory subunit highly expressed in muscle. J Biol Chem 271, 5164-70 (1996).
13. Mumby, M.C., Russell, K.L., Garrard, L.J. & Green, D.D. Cardiac contractile protein phosphatases. Purification of two enzyme forms and their characterization with subunit-specific antibodies. J Biol Chem 262, 6257-65 (1987).
14. Usui, H. et al. Three distinct forms of type 2A protein phosphatase in human erythrocyte cytosol. J Biol Chem 263, 3752-61 (1988).
15. Kuo, Y.C. et al. 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 283, 1882-92 (2008).
16. Silverstein, A.M., Barrow, C.A., Davis, A.J. & Mumby, M.C. Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits. Proc Natl Acad Sci U S A 99, 4221-6 (2002).
17. Griswold-Prenner, I., Kamibayashi, C., Maruoka, E.M., Mumby, M.C. & Derynck, R. Physical and functional interactions between type I transforming growth factor beta receptors and Balpha, a WD-40 repeat subunit of phosphatase 2A. Mol Cell Biol 18, 6595-604 (1998).
18. Mayer-Jaekel, R.E. et al. The 55 kd regulatory subunit of Drosophila protein phosphatase 2A is required for anaphase. Cell 72, 621-33 (1993).
19. Yan, Z., Fedorov, S.A., Mumby, M.C. & Williams, R.S. PR48, a novel regulatory subunit of protein phosphatase 2A, interacts with Cdc6 and modulates DNA replication in human cells. Mol Cell Biol 20, 1021-9 (2000).
20. Moreno, C.S. 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 275, 5257-63 (2000).
21. Muneer, S. et al. Genomic organization and mapping of the gene encoding the PP2A B56gamma regulatory subunit. Genomics 79, 344-8 (2002).
22. Chen, W. et al. Identification of specific PP2A complexes involved in human cell transformation. Cancer Cell 5, 127-36 (2004).
23. Francia, G. et al. Identification by differential display of a protein phosphatase-2A regulatory subunit preferentially expressed in malignant melanoma cells. Int J Cancer 82, 709-13 (1999).
24. Ito, A. et al. A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation. EMBO J 19, 562-71 (2000).
25. Ikeda, S., Kishida, M., Matsuura, Y., Usui, H. & Kikuchi, A. GSK-3beta-dependent phosphorylation of adenomatous polyposis coli gene product can be modulated by beta-catenin and protein phosphatase 2A complexed with Axin. Oncogene 19, 537-45 (2000).
26. Seeling, J.M. et al. Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A. Science 283, 2089-91 (1999).
27. Yamamoto, H. et al. Inhibition of the Wnt signaling pathway by the PR61 subunit of protein phosphatase 2A. J Biol Chem 276, 26875-82 (2001).
28. Turowski, P., Fernandez, A., Favre, B., Lamb, N.J. & Hemmings, B.A. Differential methylation and altered conformation of cytoplasmic and nuclear forms of protein phosphatase 2A during cell cycle progression. J Cell Biol 129, 397-410 (1995).
29. Strack, S., Zaucha, J.A., Ebner, F.F., Colbran, R.J. & Wadzinski, B.E. Brain protein phosphatase 2A: developmental regulation and distinct cellular and subcellular localization by B subunits. J Comp Neurol 392, 515-27 (1998).
30. McCright, B., Rivers, A.M., Audlin, S. & Virshup, D.M. 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 271, 22081-9 (1996).
31. Strack, S., Chang, D., Zaucha, J.A., Colbran, R.J. & Wadzinski, B.E. Cloning and characterization of B delta, a novel regulatory subunit of protein phosphatase 2A. FEBS Lett 460, 462-6 (1999).
32. Voorhoeve, P.M., Hijmans, E.M. & Bernards, R. Functional interaction between a novel protein phosphatase 2A regulatory subunit, PR59, and the retinoblastoma-related p107 protein. Oncogene 18, 515-24 (1999).
33. Fiscella, M. et al. Wip1, a novel human protein phosphatase that is induced in response to ionizing radiation in a p53-dependent manner. Proc Natl Acad Sci U S A 94, 6048-53 (1997).
34. Bollen, M. & Beullens, M. Signaling by protein phosphatases in the nucleus. Trends Cell Biol 12, 138-45 (2002).
35. Murata, K., Wu, J. & Brautigan, D.L. 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 94, 10624-9 (1997).
36. Andjelkovic, N., Zolnierowicz, S., Van Hoof, C., Goris, J. & Hemmings, B.A. The catalytic subunit of protein phosphatase 2A associates with the translation termination factor eRF1. EMBO J 15, 7156-67 (1996).
37. Okamoto, K. et al. p53-dependent association between cyclin G and the B' subunit of protein phosphatase 2A. Mol Cell Biol 16, 6593-602 (1996).
38. Chowdhury, D. et al. gamma-H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double-strand break repair. Mol Cell 20, 801-9 (2005).
39. Yan, Y. & Mumby, M.C. Distinct roles for PP1 and PP2A in phosphorylation of the retinoblastoma protein. PP2a regulates the activities of G(1) cyclin-dependent kinases. J Biol Chem 274, 31917-24 (1999).
40. Takenaka, I., Morin, F., Seizinger, B.R. & Kley, N. Regulation of the sequence-specific DNA binding function of p53 by protein kinase C and protein phosphatases. J Biol Chem 270, 5405-11 (1995).
41. Kumar, M. & Spandau, D.F. The effect of phosphorylation on the antigenic reactivity of p53 in cultured human keratinocytes. Biochem Biophys Res Commun 214, 744-53 (1995).
42. Leung-Pineda, V., Ryan, C.E. & Piwnica-Worms, H. Phosphorylation of Chk1 by ATR is antagonized by a Chk1-regulated protein phosphatase 2A circuit. Mol Cell Biol 26, 7529-38 (2006).
43. Kitajima, T.S. et al. Shugoshin collaborates with protein phosphatase 2A to protect cohesin. Nature 441, 46-52 (2006).
44. Pemberton, L.F. & Paschal, B.M. Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic 6, 187-98 (2005).
45. Kalderon, D., Roberts, B.L., Richardson, W.D. & Smith, A.E. A short amino acid sequence able to specify nuclear location. Cell 39, 499-509 (1984).
46. Robbins, J., Dilworth, S.M., Laskey, R.A. & Dingwall, C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 64, 615-23 (1991).
47. Izaurralde, E. et al. A role for the M9 transport signal of hnRNP A1 in mRNA nuclear export. J Cell Biol 137, 27-35 (1997).
48. Fischer, U., Huber, J., Boelens, W.C., Mattaj, I.W. & Luhrmann, R. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82, 475-83 (1995).
49. Cho, U.S. & Xu, W. Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme. Nature 445, 53-7 (2007).
50. Xu, Y. et al. Structure of the protein phosphatase 2A holoenzyme. Cell 127, 1239-51 (2006).
51. Nakai, K. & Horton, P. PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24, 34-6 (1999).
52. Mulder, N.J., Kersey, P., Pruess, M. & Apweiler, R. In silico characterization of proteins: UniProt, InterPro and Integr8. Mol Biotechnol 38, 165-77 (2008).
53. Li, X. et al. SENP1 mediates TNF-induced desumoylation and cytoplasmic translocation of HIPK1 to enhance ASK1-dependent apoptosis. Cell Death Differ 15, 739-50 (2008).
54. Harreman, M.T. et al. Regulation of nuclear import by phosphorylation adjacent to nuclear localization signals. J Biol Chem 279, 20613-21 (2004).
55. Itoh, M. et al. Nuclear export of glucocorticoid receptor is enhanced by c-Jun N-terminal kinase-mediated phosphorylation. Mol Endocrinol 16, 2382-92 (2002).
56. Xu, Z. & Williams, B.R. The B56alpha regulatory subunit of protein phosphatase 2A is a target for regulation by double-stranded RNA-dependent protein kinase PKR. Mol Cell Biol 20, 5285-99 (2000).
57. Usui, H. 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 430, 312-6 (1998).
58. Sherman, M.P., de Noronha, C.M., Heusch, M.I., Greene, S. & Greene, W.C. Nucleocytoplasmic shuttling by human immunodeficiency virus type 1 Vpr. J Virol 75, 1522-32 (2001).
59. Li, X. & Virshup, D.M. Two conserved domains in regulatory B subunits mediate binding to the A subunit of protein phosphatase 2A. Eur J Biochem 269, 546-52 (2002).
60. Strack, S. et al. Protein phosphatase 2A holoenzyme assembly: identification of contacts between B-family regulatory and scaffolding A subunits. J Biol Chem 277, 20750-5 (2002).
61. Ruediger, R., Fields, K. & Walter, G. Binding specificity of protein phosphatase 2A core enzyme for regulatory B subunits and T antigens. J Virol 73, 839-42 (1999).