Activation of the Rap1 Guanine Nucleotide Exchange Gene,CalDAG-GEF I, in BXH-2 Murine Myeloid Leukemia

Abstract

Here we report the recurrent proviral activation of the Rap1-specific guanine nucleotide exchange factorCalDAG-GEF I (Kawasaki, H., Springett, G. M., Toki, S., Canales, J. J., Harlan, P., Blumenstiel, J. P., Chen, E. J., Bany, I. A., Mochizuki, N., Ashbacher, A., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998)Proc. Natl. Acad. Sci. U. S. A. 95, 13278–13283; Correction (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 318) gene in BXH-2 acute myeloid leukemia. We also show thatCalDAG-GEF I encodes two protein isoforms, a full-length isoform (CalDAG-GEF Ia) and a C-terminally truncated isoform (CalDAG-GEF Ib). Expression of the full-length CalDAG-GEF Ia isoform in Rat2 fibroblasts enhances growth in low serum, whereas expression in Swiss 3T3 cells causes morphological transformation and increased saturation density. In FDCP1 myeloid cells, CalDAG-GEF Ia expression increases growth and saturation density in the presence of the diacylglycerol analogs phorbol 12-myristate 13-acetate (PMA), which activates CalDAG-GEF Ia exchange activity. Likewise, in 32Dcl3 myeloblast cells, CalDAG-GEF Ia expression increases cell adherence to fibronectin in response to PMA and calcium ionophore and allows higher saturation densities and prolonged growth on fibronectin-coated plates. These effects were correlated with increased Rap1, but not Ras, protein activation following PMA and calcium ionophore treatment. Our results suggest that Rap1-GTP delivers signals that favor progression through the cell cycle and morphological transformation. The identification ofCalDAG-GEF I as a proto-oncogene in BXH-2 acute myeloid leukemia is the first evidence implicating Rap1 signaling in myeloid leukemia. Here we report the recurrent proviral activation of the Rap1-specific guanine nucleotide exchange factorCalDAG-GEF I (Kawasaki, H., Springett, G. M., Toki, S., Canales, J. J., Harlan, P., Blumenstiel, J. P., Chen, E. J., Bany, I. A., Mochizuki, N., Ashbacher, A., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998)Proc. Natl. Acad. Sci. U. S. A. 95, 13278–13283; Correction (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 318) gene in BXH-2 acute myeloid leukemia. We also show thatCalDAG-GEF I encodes two protein isoforms, a full-length isoform (CalDAG-GEF Ia) and a C-terminally truncated isoform (CalDAG-GEF Ib). Expression of the full-length CalDAG-GEF Ia isoform in Rat2 fibroblasts enhances growth in low serum, whereas expression in Swiss 3T3 cells causes morphological transformation and increased saturation density. In FDCP1 myeloid cells, CalDAG-GEF Ia expression increases growth and saturation density in the presence of the diacylglycerol analogs phorbol 12-myristate 13-acetate (PMA), which activates CalDAG-GEF Ia exchange activity. Likewise, in 32Dcl3 myeloblast cells, CalDAG-GEF Ia expression increases cell adherence to fibronectin in response to PMA and calcium ionophore and allows higher saturation densities and prolonged growth on fibronectin-coated plates. These effects were correlated with increased Rap1, but not Ras, protein activation following PMA and calcium ionophore treatment. Our results suggest that Rap1-GTP delivers signals that favor progression through the cell cycle and morphological transformation. The identification ofCalDAG-GEF I as a proto-oncogene in BXH-2 acute myeloid leukemia is the first evidence implicating Rap1 signaling in myeloid leukemia. Retroviral insertional mutagenesis in BXH-2 recombinant inbred mice induces a high incidence of acute myeloid leukemia (AML),1 and the proviral integration sites in the leukemias provide powerful genetic tags for disease gene identification (2Jonkers J. Berns A. Biochim. Biophys. Acta.. 1996; 1287: 29-57Google Scholar, 3Wolff L. Biochim. Biophys. Acta.. 1997; 1332: F67-F104Google Scholar). During the past several years, a number of disease genes have been identified in BXH-2 AMLs by proviral tagging. They include a tumor suppressor gene, Neurofibromatosis type 1 (Nf1); a gene with homology to the lymphoid-restricted type II membrane protein Jaw1, Mrv integration site 1 (Mrvi1); a gene encoding a hematopoietic cell growth and differentiation factor, myeloblastosis oncogene (Myb); three homeobox genes, homeobox A7 (Hoxa7), homeobox A9 (Hoxa9), and myeloid ecotropic viral integration site 1 (Meis1); a zinc finger protein gene, ecotropic viral integration site 9 (Evi9); and a novel cytokine receptor (Evi27) (4Buchberg A.M. Bedigian H.G. Jenkins N.A. Copeland N.G. Mol. Cell. Biol... 1990; 10: 4658-4666Google Scholar, 5Largaespada D.A. Shaughnessy Jr., J.D. Jenkins N.A. Copeland N.G. J. Virol.. 1995; 69: 5095-5102Google Scholar, 6Li J. Shen H. Himmel K.L. Dupuy A.J. Largaespada D.A. Nakamura T. Shaughnessy Jr., J.D. Jenkins N.A. Copeland N.G. Nat. Genet.. 1999; 23: 33-348Google Scholar, 7Moskow J.J. Bullrich F. Huebner K. Daar I.O. Buchberg A.M. Mol. Cell. 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Importantly, two of these genes,NF1 and HOXA9, are known human myeloid leukemia disease genes, validating the usefulness of this approach for human disease gene identification (9Nakamura T. Largaespada D.A. Lee M.P. Johnson L.A. Ohyashiki K. Toyama K. Chen S.J. Willman C.L. Chen I.M. Feinberg A.P. Jenkins N.A. Copeland N.G. Shaughnessy Jr., J.D. Nat. Genet... 1996; 12: 154-158Google Scholar, 13Gutmann D.H. Collins F.S. Neuron.. 1993; 10: 335-343Google Scholar). Mutations in the human NF1 gene are responsible for the cancer predisposition syndrome neurofibromatosis type 1 (13Gutmann D.H. Collins F.S. Neuron.. 1993; 10: 335-343Google Scholar).NF1 encodes neurofibromin, a GTPase-activating protein (GAP) that is active on the four true Ras proteins (Ha-Ras, K-RasA, K-RasB, and N-Ras) as well as R-Ras (14DeClue J.E. Papageorge A.G. Fletcher J.A. Diehl S.R. Ratner N. Vass W.C. Lowy D.R. Cell.. 1992; 69: 265-273Google Scholar, 15Martin G.A. Viskochil D. Bollag G. McCabe P.C. Crosier W.J. Haubruck H. Conroy L. Clark R. O'Connell P. Cawthon R.M. Innis M.A. McCormick F. Cell.. 1990; 63: 843-849Google Scholar, 16Ohba Y. Mochizuki N. Yamashita S. Chan A.M. Schrader J.W. Hattori S. Nagashima K. Matsuda M. J. Biol. Chem... 2000; 275: 20020-20026Google Scholar). In BXH2 AMLs, about 15% of the AMLs have proviral insertions in the Nf1 gene (4Buchberg A.M. Bedigian H.G. Jenkins N.A. Copeland N.G. Mol. Cell. Biol... 1990; 10: 4658-4666Google Scholar, 5Largaespada D.A. Shaughnessy Jr., J.D. Jenkins N.A. Copeland N.G. J. Virol.. 1995; 69: 5095-5102Google Scholar). These proviral insertions inhibit neurofibromin expression, and no wild type neurofibromin is expressed in these AMLs (5Largaespada D.A. Shaughnessy Jr., J.D. Jenkins N.A. Copeland N.G. J. Virol.. 1995; 69: 5095-5102Google Scholar). Loss of neurofibromin in myeloid cells is associated with increased and prolonged Ras activation after cytokine stimulation in chronic myeloid leukemia and in primary cells (17Largaespada D.A. Brannan C.I. Jenkins N.A. Copeland N.G. Nat. Genet.. 1996; 12: 137-143Google Scholar). These results contribute to a large body of evidence implicating aberrant regulation of Ras signaling in myeloid leukemia. The other 85% of BXH-2 AMLs do not have proviral insertions in theNf1 gene (4Buchberg A.M. Bedigian H.G. Jenkins N.A. Copeland N.G. Mol. Cell. Biol... 1990; 10: 4658-4666Google Scholar, 5Largaespada D.A. Shaughnessy Jr., J.D. Jenkins N.A. Copeland N.G. J. Virol.. 1995; 69: 5095-5102Google Scholar). This has led to the suggestion that these AMLs have insertions in or near other genes involved in Ras regulation and that these insertions activate Ras in the absence of inactivating insertions at Nf1. One class of likely targets are the guanine nucleotide exchange factors (GEF) that catalyze the exchange of GTP for GDP on Ras and consequently turn on Ras. One such GEF is RasGRP (18Ebinu J.O. Bottorff D.A. Chan E.Y.W. Stang S.L. Dunn R.J. Stone J.C. Science.. 1998; 280: 1082-1086Google Scholar, 19Tognon L.A. R.J. Mol. Cell. Biol... 1998; 18: Scholar). RasGRP is of three guanine exchange factors that have in to diacylglycerol (18Ebinu J.O. Bottorff D.A. Chan E.Y.W. Stang S.L. Dunn R.J. Stone J.C. Science.. 1998; 280: 1082-1086Google Scholar, 19Tognon L.A. R.J. Mol. Cell. Biol... 1998; 18: Scholar, D. E. I. J. Clark M. J. E. H. J. Genet.. 1997; Scholar). has been that this class of cell that through and to the Ras (18Ebinu J.O. Bottorff D.A. Chan E.Y.W. Stang S.L. Dunn R.J. Stone J.C. Science.. 1998; 280: 1082-1086Google Scholar, D. E. I. J. Clark M. J. E. H. J. Genet.. 1997; Scholar). In the we report the recurrent proviral activation of the and guanine nucleotide exchange 1 (CalDAG-GEF gene in BXH-2 CalDAG-GEF I has and have that CalDAG-GEF I is not a Ras to a Rap1 GEF H. S. J.J. P. Chen Mochizuki N. A. Matsuda M. A.M. Proc. Natl. Acad. Sci. U. S. 1998; Correction (1999) Proc. Natl. Acad. Sci. U. S. Scholar). We also show that of CalDAG-GEF I results in transformation of fibroblasts and Rap1 signaling in these This is the first evidence implicating Rap1 signaling in myeloid leukemia in human or insertions in the CalDAG-GEF were as of a large that a in J. Shen H. Himmel K.L. Dupuy A.J. Largaespada D.A. Nakamura T. Shaughnessy Jr., J.D. Jenkins N.A. Copeland N.G. Nat. Genet.. 1999; 23: 33-348Google Scholar). In of BXH-2 AMLs with the by at for and the were in of at to for The in and in of of this as in a primary in a of and and of in the with a as for of for for for of for for for with The of primary by and to 1 of the primary as in a the that the were The on a the II and to These were to of The in the primary are as and the the and The were the on and were and of BXH2 and by and as N.A. Copeland N.G. Bedigian H.G. Lee J. Scholar). or cells were in 1 at a of cells at for The were by for at to The were in and at The of protein in the the of protein and to as (5Largaespada D.A. Shaughnessy Jr., J.D. Jenkins N.A. Copeland N.G. J. Virol.. 1995; 69: 5095-5102Google Scholar). The were with or to and with the The CalDAG-GEF Ia and CalDAG-GEF were a the to Ia are and were and the This a to the of the CalDAG-GEF Ia to full-length CalDAG-GEF are and These site and a site to the and of the were with and and the and sites of the This a to the of the CalDAG-GEF the CalDAG-GEF Ia and CalDAG-GEF expression were for expression were in to that protein for CalDAG-GEF Ia and protein for CalDAG-GEF The CalDAG-GEF Ia the expression and the cell The cells were in in and were and at were after cells and at The following cell were with Ia or and in Swiss and were the which D. of Rat2 and Swiss 3T3 were in with and in FDCP1 and were in cell in with and and were and FDCP1 and cells were in 1 Retroviral by of and or cells and Swiss in of a of high and growth with four after the cells were to were for after in Rat2 cells were for by at cells in of or in plates. or in the were and with and Rat2 cells were also in as D.A. D.A. H. E. M. R. Oncogene.. 1992; Scholar). of Swiss 3T3 were for of cells after to These cells were and at in of or and cells and were after the cells were growth and saturation FDCP1 were at cells in of growth with or phorbol 12-myristate 13-acetate and for cells were three in and at a density of 1 cells in growth the PMA also in a of at a of 1 were at the for cell by and The activation of Ras and Rap1 were as in that the of GTP and GTP GDP to Ras or Rap1 Proc. Natl. Acad. Sci. U. S. 1995; Scholar, K. Y. I. J. Biol. Chem... 1998; Scholar, Oncogene.. 2000; 19: Scholar). cells were and in Ras the cells were in a and after and the by the were in the and the other for 1 at Ras on protein to which The Ras four in and GTP and GDP to Ras were by The were in and GTP in by to and the and the by the that is to 1 The the and the of GTP with of The of GTP GDP in the other of the by GDP to GTP and and which the of GTP as Rap1 activation but are no for Rap1, the of to to GTP of the on Oncogene.. 2000; 19: Scholar). The GTP and as the of GTP and GDP to in the other of the were in the absence of with GTP to to GTP by a as of the for Rap1 cells were in the GTP and a the Rap1 were by the to for which the of by to the and the GTP and as insertions the first of the CalDAG-GEF I gene were identified as of a large to proviral insertions that are near J. Shen H. Himmel K.L. Dupuy A.J. Largaespada D.A. Nakamura T. Shaughnessy Jr., J.D. Jenkins N.A. Copeland N.G. Nat. Genet.. 1999; 23: 33-348Google Scholar). of two of the that were in the first of the of the human gene also I H. S. J.J. P. Chen Mochizuki N. A. Matsuda M. A.M. Proc. Natl. Acad. Sci. U. S. 1998; Correction (1999) Proc. Natl. Acad. Sci. U. S. Scholar). of of the leukemias that CalDAG-GEF I expression with other BXH-2 leukemias and that not viral I 1 These results show that viral integration at CalDAG-GEF I to I CalDAG-GEF were in BXH-2 leukemias This the has not been in H. S. J.J. P. Chen Mochizuki N. A. Matsuda M. A.M. Proc. Natl. Acad. Sci. U. S. 1998; Correction (1999) Proc. Natl. Acad. Sci. U. S. Scholar). were by viral integration at CalDAG-GEF I 1 This to for the CalDAG-GEF I in CalDAG-GEF are expressed in Expression in and by and Expression not in are also expressed in the with the expression and of we that the is also expressed in not The to of CalDAG-GEF I but not with not In the of the is for that after the by of with a that the and are at a in is expressed of the human CalDAG-GEF a site that after the site This site is also in the of a number of and human in which at this site at the of the CalDAG-GEF I These I which the and are after a at this site a of The CalDAG-GEF is to a truncated CalDAG-GEF include the Ras the and the to that the and or by the human and CalDAG-GEF I in the the site in the and the In other These that the CalDAG-GEF I two through which a full-length CalDAG-GEF and a truncated CalDAG-GEF CalDAG-GEF I were expression through of were for to the to expression and to to the of This in with a and a CalDAG-GEF with a The to the expression as we cells and expression to the we were to CalDAG-GEF protein expression by This with in we were to protein not We have also to CalDAG-GEF These results suggest that the CalDAG-GEF protein is or at low for effects of the CalDAG-GEF Ia expression for CalDAG-GEF Ia Ia) and for to CalDAG-GEF Ia protein in and Ia cells expressed protein The Rat2 cell with CalDAG-GEF or and in Rat2 were and of of were for growth in and growth in or with or a diacylglycerol were cells of large in not The number of in in higher for Ia for the with and PMA higher to a number of of CalDAG-GEF Ia in Rat2 and Swiss 3T3 at low in Rat2 The number of after cells of cells or Rat2 cells is in CalDAG-GEF Swiss 3T3 of Swiss 3T3 cells, Swiss 3T3 cells and of four of Swiss 3T3 cells primary and and saturation density in Swiss The number of Swiss 3T3 cells at is for cells and for Swiss 3T3 cells The in or In the number of of Swiss or Swiss 3T3 cells is are the number that after and growth to are the number that after and growth to In to Rat2 cells, Swiss 3T3 cells with Ia after These as of cells, at high density and in The of these increased with in were in with the Ia These were in in Swiss 3T3 cells also a higher saturation density The Ia and were also to cells the myeloid cell and FDCP1 to the effects of CalDAG-GEF Ia on myeloid The growth and of and FDCP1 cells are on the presence of in the is the these cells by have that Rap1 a in cell to fibronectin K. Hattori M. N. S. K. T. Mol. Cell. Biol... 2000; 20: Scholar, E. R.M. Y. M. J. Biol... 2000; Scholar, N. Hattori M. H. N. J. Biol. Chem... 1999; and fibronectin in of these The of PMA in the absence of a in in but not cells, which in fibronectin-coated not PMA and calcium cells a in adherence to fibronectin with FDCP1 cells CalDAG-GEF Ia at high not but a in growth and saturation density with the in growth and saturation density in the presence of or PMA activates the guanine nucleotide exchange of CalDAG-GEF I H. S. J.J. P. Chen Mochizuki N. A. Matsuda M. A.M. Proc. Natl. Acad. Sci. U. S. 1998; Correction (1999) Proc. Natl. Acad. Sci. U. S. Scholar). expression ofCalDAG-GEF Ia in cells in increased activation in myeloid cells, we and in or Ia of These cells were of and for and with calcium ionophore and PMA to activate CalDAG-GEF Ia for CalDAG-GEF cells in at with cells The of not CalDAG-GEF Ia and cells this This is with other that CalDAG-GEF I not in cells H. S. J.J. P. Chen Mochizuki N. A. Matsuda M. A.M. Proc. Natl. Acad. Sci. U. S. 1998; Correction (1999) Proc. Natl. Acad. Sci. U. S. Scholar). Ia expression at high the of Rap1 that in response to has been in cells Oncogene.. 2000; 19: Rap1 protein in cells a high of activation with Ras This has been to to the presence of a at in Rap1, which Oncogene.. 2000; 19: Scholar). of leukemia integration sites in BXH-2 has identified a GEF gene, CalDAG-GEF that is a for leukemia integration in BXH2 integration I to increased CalDAG-GEF that CalDAG-GEF I is a I is the GEF to as a myeloid leukemia disease gene in or The gene is in by to the active GEF in human myeloid the of which in activation M. S. A. 1999; Scholar). CalDAG-GEF I is to RasGRP H. S. J.J. P. Chen Mochizuki N. A. Matsuda M. A.M. Proc. Natl. Acad. Sci. U. S. 1998; Correction (1999) Proc. Natl. Acad. Sci. U. S. J.O. Bottorff D.A. Chan E.Y.W. Stang S.L. Dunn R.J. Stone J.C. Science.. 1998; 280: 1082-1086Google Scholar, 19Tognon L.A. R.J. Mol. Cell. Biol... 1998; 18: Scholar, D. E. I. J. Clark M. J. E. H. J. Genet.. 1997; Scholar). RasGRP has been to have GEF for but for other Ras such as or H. S. J.J. P. Chen Mochizuki N. A. Matsuda M. A.M. Proc. Natl. Acad. Sci. U. S. 1998; Correction (1999) Proc. Natl. Acad. Sci. U. S. Scholar, J.O. Bottorff D.A. Chan E.Y.W. Stang S.L. Dunn R.J. Stone J.C. Science.. 1998; 280: 1082-1086Google Scholar, 19Tognon L.A. R.J. Mol. Cell. Biol... 1998; 18: Scholar). In CalDAG-GEF I GEF for but high for Rap1 H. S. J.J. P. Chen Mochizuki N. A. Matsuda M. A.M. Proc. Natl. Acad. Sci. U. S. 1998; Correction (1999) Proc. Natl. Acad. Sci. U. S. Scholar). CalDAG-GEF and a identified CalDAG-GEF have also been to have exchange for R-Ras and Y. Mochizuki N. Yamashita S. Chan A.M. Schrader J.W. Hattori S. Nagashima K. Matsuda M. J. Biol. Chem... 2000; 275: 20020-20026Google Scholar, S. Mochizuki N. Y. M. Y. H. Nagashima K. Matsuda M. J. Biol. Chem... 2000; 275: Scholar). J. G. M. S. M. A. N. J. Biol. 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M. 1995; Scholar, Chen J. McCormick F. M. Proc. Natl. Acad. Sci. U. S. 1995; Scholar, P. S. P. Biol... 1997; Scholar). CalDAG-GEF the truncated of CalDAG-GEF is in that no other identified have a truncated The to the of CalDAG-GEF is that as a of CalDAG-GEF to activity. The identified CalDAG-GEF is the is to the of GEF the of the is not is for the effects of RasGRP in fibroblasts L.A. R.J. Mol. Cell. Biol... 1998; 18: Scholar). The of catalyze the exchange of GDP for GTP on Ras but the of the increases the of this P. I. L. E. A. Biophys. 1995; Scholar). The and of protein with a of the protein the of the D. J. 1998; Scholar). The of three The first two of the and a of the D. J. 1998; Scholar). The and the of the protein the and the with the This of with other The of the the and is not and CalDAG-GEF I. The of a of CalDAG-GEF CalDAG-GEF in and human that the and of the the and the that this of the protein have and CalDAG-GEF CalDAG-GEF Ia by with and a that also to and CalDAG-GEF Ia and other CalDAG-GEF to human D. E. I. J. Clark M. J. E. H. J. Genet.. 1997; Scholar). The has been in a of cell and cell G.A. G. Scholar, F. F. Nat. Genet... 1997; 15: Scholar). and gene have been in these is that activation of the gene or growth genes, such as and are the targets for in this F. F. Nat. Genet... 1997; 15: Scholar). identification ofCalDAG-GEF I as a proto-oncogene in BXH-2 that activation also in of these these that is to effects on has been in in the and of cells in K. Hattori M. N. S. K. T. Mol. Cell. Biol... 2000; 20: Scholar, E. R.M. Y. M. J. Biol... 2000; Scholar, N. Hattori M. H. N. J. Biol. Chem... 1999; Scholar). that CalDAG-GEF I gene activation a in the of these acute myeloid leukemia diacylglycerol phorbol 12-myristate 13-acetate GTPase-activating protein guanine nucleotide exchange factors and guanine nucleotide exchange 1 Ras