Extracellular signal-regulated kinase (ERK) signalling plays a central role in various biological processes, including cell migration, but it remains unknown what factors directly regulate the strength and duration of ERK activation. We found that, among the B56 family of protein phosphatase 2A (PP2A) regulatory subunits, B56γ1 suppressed EGF-induced cell migration on collagen, bound to phosphorylated-ERK, and dephosphorylated ERK, whereas B56α1 and B56β1 did not. B56γ1 was immunolocalized in nuclei. The IER3 protein was immediately highly expressed in response to costimulation of cells with EGF and collagen. Knockdown of IER3 inhibited cell migration and enhanced dephosphorylation of ERK. Analysis of the time course of PP2A-B56γ1 activity following the costimulation showed an immediate loss of phosphatase activity, followed by a rapid increase in activity, and this activity then remained at a stable level that was lower than the original level. Our results indicate that the strength and duration of the nuclear ERK activation signal that is initially induced by ERK kinase (MEK) are determined at least in part by modulation of the phosphatase activity of PP2A-B56γ1 through two independent pathways.
References
[1]
Anderson NG, Maller JL, Tonks NK, Sturgill TW (1990) Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature 343: 651–653.
[2]
Gomez N, Cohen P (1991) Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases. Nature 353: 170–173.
[3]
Crews CM, Alessandrini A, Erikson RL (1992) The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science 258: 478–480.
[4]
Wortzel I, Seger R (2011) The ERK Cascade: Distinct functions within various subcellular organelles. Genes Cancer 2: 195–209.
[5]
Murphy LO, Blenis J (2006) MAPK signal specificity: the right place at the right time. Trends Biochem Sci 31: 268–275.
[6]
Sacks DB (2006) The role of scaffold proteins in MEK/ERK signalling. Biochem Soc Trans 34: 833–836.
[7]
Brandman O, Meyer T (2008) Feedback loops shape cellular signals in space and time. Science 322: 390–395.
[8]
Fowler T, Sen R, Roy AL (2011) Regulation of primary response genes. Mol Cell 44: 48–60.
[9]
Traverse S, Gomez N, Paterson H, Marshall C, Cohen P (1992) Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells Comparison of the effects of nerve growth factor and epidermal growth factor. Biochem J 288: 351–355.
[10]
Marshall CJ (1995) Specificity of receptor tyrosine kinase signalling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80: 179–185.
[11]
Weber JD, Raben DM, Phillips PJ, Baldassare JJ (1997) Sustained activation of extracellular-signal-regulated kinase 1 (ERK1) is required for the continued expression of cyclin D1 in G1 phase. Biochem J 326: 61–68.
[12]
Morino N, Mimura T, Hamasaki K, Tobe K, Ueki K, et al. (1995) Matrix/integrin interaction activates the mitogen-activated protein kinase, p44erk-1 and p42erk-2. J Biol Chem 270: 269–273.
[13]
Kahan C, Seuwen K, Meloche S, Pouyssegur J (1992) Coordinate, biphasic activation of p44 mitogen-activated protein kinase and S6 kinase by growth factors in hamster fibroblasts Evidence for thrombin-induced signals different from phosphoinositide turnover and adenylylcyclase inhibition. J Biol Chem 267: 13369–13375.
[14]
Miyamoto S, Teramoto H, Gutkind JS, Yamada KM (1996) Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors. J Cell Biol 135: 1633–1642.
[15]
Eliceiri BP, Klemke R, Stromblad S, Cheresh DA (1998) Integrin αvβ3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis. J Cell Biol 140: 1255–1263.
[16]
Kawahara E, Nakada N, Hikichi T, Kobayashi J, Nakanishi I (2002) EGF and β1 integrin convergently regulate migration of A431 carcinoma cell through MAP kinase activation. Exp Cell Res 272: 84–91.
[17]
Howe AK, Aplin AE, Juliano RL (2002) Anchorage-dependent ERK signaling-mechanisms and consequences. Curr Opin Genet Dev 12: 30–35.
[18]
Tian YC, Chen YC, Chang CT, Hung CC, Wu MS, et al. (2007) Epidermal growth factor and transforming growth factor-β1 enhance HK-2 cell migration through a synergistic increase of matrix met alloproteinase and sustained activation of ERK signaling pathway. Exp Cell Res 313: 2367–2377.
Bonni A, Brunet A, West AE, Datta SR, Takasu MA, et al. (1999) Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 286: 1358–1362.
[21]
Murphy LO, MacKeigan JP, Blenis J (2004) A network of immediate early gene products propagates subtle differences in mitogen-activated protein kinase signal amplitude and duration. Mol Cel Biol 24: 144–153.
[22]
Joslin EJ, Opresko LK, Wells A, Wiley HS, Lauffenburger DA (2007) EGF-receptor-mediated mammary epithelial cell migration is driven by sustained ERK signaling from autocrine stimulation. J Cell Sci 120: 3688–3699.
[23]
Murphy LO, Smith S, Chen RH, Fingar DC, Blenis J (2002) Molecular interpretation of ERK signal duration by immediate early gene products. Nat Cell Biol 4: 556–564.
[24]
Sun H, Charles CH, Lau LF, Tonks NK (1993) MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell 75: 487–493.
[25]
Alessi DR, Gomez N, Moorhead G, Lewis T, Keyse SM, et al. (1995) Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines. Curr Biol 5: 283–295.
[26]
Chajry N, Martin PM, Cochet C, Berthois Y (1996) Regulation of p42 mitogen-activated-protein kinase activity by protein phosphatase 2A under conditions of growth inhibition by epidermal growth factor in A431 cells. Eur J Biochem 235: 97–102.
Gronda M, Arab S, Iafrate B, Suzuki H, Zanke BW (2001) Hematopoietic protein tyrosine phosphatase suppresses extracellular stimulus-regulated kinase activation. Mol Cell Biol 21: 6851–6858.
[29]
Noordman YE, Jansen PA, Hendriks WJ (2006) Tyrosine-specific MAPK phosphatases and the control of ERK signaling in PC12 cells. J Mol Signal 1: 4.
[30]
Hashigasako A, Machide M, Nakamura T, Matsumoto K, Nakamura T (2004) Bi-directional regulation of Ser-985 phosphorylation of c-met via protein kinase C and protein phosphatase 2A involves c-Met activation and cellular responsiveness to hepatocyte growth factor. J Biol Chem 279: 26445–26452.
[31]
van Kanegan MJ, Strack S (2009) The protein phosphatase 2A regulatory subunits B′β and B′δ mediate sustained TrkA neurotrophin receptor autophosphorylation and neuronal differentiation. Mol Cell Biol 29: 662–674.
[32]
Ugi S, Imamura T, Ricketts W, Olefsky JM (2002) Protein phosphatase 2A forms a molecular complex with Shc and regulates Shc tyrosine phosphorylation and downstream mitogenic signaling. Mol Cell Biol 22: 2375–2387.
[33]
Abraham D, Podar K, Pacher M, Kubicek M, Welzel N, et al. (2000) Raf-1-associated protein phosphatase 2A as a positive regulator of kinase activation. J Biol Chem 275: 22300–22304.
[34]
Ory S, Zhou M, Conrads TP, Veenstra TD, Morrison DK (2003) Protein phosphatase 2A positively regulates Ras signaling by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 binding sites. Curr Biol 13: 1356–13564.
[35]
Adams DG, Coffee RL, Zhang H, Pelech S, Strack S, et al. (2005) Positive regulation of Raf1-MEK1/2-ERK1/2 signalling by protein serine/threonine phosphatase 2A holoenzymes. J Biol Chem 280: 42644–42654.
[36]
Mao L, Yang L, Arora A, Choe ES, Zhang G, et al. (2005) Role of protein phosphatase 2A in mGluR5-regulated MEK/ERK phosphorylation in neurons. J Biol Chem 280: 12602–12610.
[37]
Bae D, Ceryak S (2009) Raf-independent, PP2A-dependent MEK activation in response to ERK silencing. Biochem Biophys Res Commun 385: 523–527.
[38]
Zhou B, Wang ZX, Zhao Y, Brautigan DL, Zhang ZY (2002) The specificity of extracellular signal-regulated kinase 2 dephosphorylation by protein phosphatases. J Biol Chem 277: 31818–31825.
[39]
Letourneux C, Rocher G, Porteu F (2006) B56-containing PP2A dephosphorylate ERK and their activity is controlled by the early gene IEX-1 and ERK. EMBO J 25: 727–738.
[40]
Janssens V, Goris J, van Hoof C (2005) PP2A: the expected tumor suppressor. Curr Opin Genet 15: 34–41.
[41]
Ruediger R, Pham HT, Walter G (2007) Disruption of protein phosphatase 2A subunit interaction in human cancers with mutations in the Aα subunit gene. Oncogene 20: 10–15.
[42]
Eichhorn PJ, Creyghton MP, Bernards R (2009) Protein phosphatase 2A regulatory subunits and cancer. Biochim Biophys Acta 1: 1–15.
[43]
Young MR, Lozano Y, Djorjevic A, Maier GD (1993) Protein phosphatases limit tumor motility. Int J Cancer 54: 1036–1041.
[44]
Maier GD, Wright MA, Lozano Y, Djordjevic A, Matthews JP, et al. (1995) Regulation of cytoskeletal organization in tumor cells by protein phosphatases-1 and -2A. Int J Cancer 61: 54–61.
[45]
Rocher G, Letourneux C, Lenormand P, Porteu F (2007) Inhibition of B56-containing protein phosphatase 2As by the early response gene IEX-1 leads to control of Akt activity. J Biol Chem 282: 5468–5477.
[46]
Ito A, Kataoka TR, Watanabe M, Nishiyama K, Mazaki Y, et al. (2000) A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation. EMBO J 19: 562–571.
[47]
Koma YI, Koma YI, Ito A, Watabe K, Kimura SH, et al. (2004) A truncated isoform of the PP2A B56γ regulatory subunit reduces irradiation-induced Mdm2 phosphorylation and could contribute to metastatic melanoma cell radioresistance. Histol Histopathol 19: 391–400.
[48]
Li HH, Cai X, Shouse GP, Piluso LG, Liu X (2007) A specific PP2A regulatory subunit, B56γ, mediates DNA damage-induced dephosphorylation of p53 at Thr55. EMBO J 26: 402–411.
[49]
Shouse GP, Nobumori Y, Liu X (2010) A B56γ mutation in lung cancer disrupts the p53-dependent tumor-suppressor function of protein phosphatase 2A. Oncogene 29: 3933–3941.
[50]
Ma J, Arnold HK, Lilly MB, Sears RC, Kraft AS (2007) Negative regulation of Pim-1 protein kinase levels by the B56β subunit of PP2A. Oncogene 26: 5145–5153.
[51]
Saraf A, Virshup DM, Strack S (2007) Differential expression of the B′β regulatory subunit of protein phosphatase 2A modulates tyrosine hydroxylase phosphorylation and catecholamine synthesis. J Biol Chem 282: 573–580.
[52]
Padmanabhan S, Mukhopadhyay A, Narasimhan SD, Tesz G, Czech MP, et al. (2009) A PP2A regulatory subunit regulates C. elegans insulin/IGF-1 signaling by modulating AKT-1 phosphorylation. Cell 136: 939–951.
[53]
Rodgers JT, Vogel RL, Puigserver P (2011) Clk2 and B56β mediate insulin-regulated assembly of the PP2A phosphatase holoenzyme complex on Akt. Mol Cell 41: 471–479.
[54]
Isoda M, Sako M, Suzuki K, Nishino K, Nakajo N, et al. (2011) Dynamic regulation of Emi2 by Emi2-bound Cdk1/Plk1/CK1 and PP2A-B56 in meiotic arrest of Xenopus eggs. Dev Cell 21: 506–519.
[55]
Bhasin N, Cunha SR, Cunha SR, Mudannayake M, Gigena MS, et al. (2007) Molecular basis for PP2A regulatory subunit B56α targeting in cardiomyocytes. Am J Physiol Heart Circ Physiol 293: 109–119.
[56]
Pitman MR, Barr RK, Gliddon BL, Magarey AM, Moretti PA, et al. (2011) A critical role for the protein phosphatase 2A B′α regulatory subunit in dephosphorylation of sphingosine kinase 1. Int J Biochem Cell Biol 43: 342–347.
[57]
Dozier C, Bonyadi M, Baricault L, Tonasso L, Darbon JM (2004) Regulation of Chk2 phosphorylation by interaction with protein phosphatase 2A via its B′ regulatory subunit. Biol Cell 96: 509–517.
[58]
Mukhopadhyay NK, Gilchrist D, Gordon GJ, Chen CJ, Bueno R, et al. (2004) Integrin-dependent protein tyrosine phosphorylation is a key regulatory event in collagen-IV-mediated adhesion and proliferation of human lung tumor cell line, Calu-1. Ann Thorac Surg 78: 450–457.
[59]
Kawahara E, Yasunori O, Nakanishi I, Kazushi I, Kojima S, et al. (1993) The expression of invasive behaviour of differentiated squamous cell carcinoma cell line evaluated by an in vitro invasion model. Jpn J Cancer Res 84: 409–418.
[60]
Kawahara E, Tokuda R, Nakanishi I (1999) Migratory phenotype of HSC-3 squamous carcinoma cell line induced by EGF and PMA: Relevance to migration of loosening of adhesion and vinculin-associated focal contacts with prominent filopodia. Cell Biol Int 23: 163–174.
[61]
Yebra M, Filard EJ, Bayna EM, Kawahara E, Becker JC, et al. (1995) Induction of carcinoma cell migration on vitronectin by NF-κB-dependent gene expression. Mol Biol Cell 6: 841–850.
[62]
Seeling JM, Miller JR, Gil R, Moon RT, White R, et al. (1999) Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A. Science 283: 2089–2091.
[63]
Ito A, Koma Y, Sohda M, Watanabe K, Nagano T, et al. (2003) Localization of the PP2A B56ΖΖΖ regulatory subunit at the Golgi complex Possible role in vesicle transport and migration. Am J Pathol 162: 479–489.
[64]
Kawahara E, Saito A, Kobayashi J, Maenaka S, Minamoto T, et al. (2005) Adhesiveness of β5 integrin variant lacking FNK767–769 is similar to that of the prototype containing FNKFNK764–769. Cell Biol Int 29: 521–528.
[65]
Nakada N, Kuroda K, Kawahara E (2005) Protein phosphatase 2A regulatory subunit Bβ promotes MAP Kinase-mediated migration of A431 cells. Cell Physiol Biochem 15: 19–28.
[66]
Garcia J, Ye Y, Arranz V, Letourneux C, Pezeron G, et al. (2002) IEX-1: a new ERK substrate involved in both ERK survival activity and ERK activation. EMBO J 21: 5151–5163.
[67]
Charles CH, Yoon JK, Simske JS, Lau LF (1993) Genomic structure, cDNA sequence, and expression of gly96, a growth factor-inducible immediate-early gene encoding a short-lived glycosylated protein. Oncogene 8: 797–801.
[68]
McCright B, Rivers AM, Audlin S, Virshup DM (1996) 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: 22081–22089.
[69]
Chen J, Martin BL, Brautigan DL (1992) Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science 257: 1261–1264.
[70]
Chen J, Parsons S, Brautigan DL (1994) Tyrosine phosphorylation of protein phosphatase 2A in response to growth stimulation and v-src transformation of fibroblasts. J Biol Chem 269: 7957–7962.
[71]
Schlaepfer DD, Broome MA, Hunter T (1997) Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins. Mol Cell Biol 17: 1702–1713.
[72]
Ortega-Lazaro JC, del Mazo J (2003) Expression of the B56δ subunit of protein phosphatase 2A and Mea1 in mouse spermatogenesis. Identification of a new B56γ subunit (B56γ4) specifically expressed in testis. Cytogenet Genome Res 103: 345–351.
[73]
Muneer S, Ramalingam V, Wyatt R, Schultz RA, Minna JD, et al. (2002) Genomic organization and mapping of the gene encoding the PP2A B56γ regulatory subunit. Genomics 79: 344–348.
[74]
Shouse GP, Cai X, Liu X (2008) Serine 15 phosphorylation of p53 directs its interaction with B56γ and the tumor suppressor activity of B56γ-specific protein phosphatase 2A. Mol Cell Biol 28: 448–456.
