The TIS21 immediate-early gene and leukemia-associated BTG1 gene encode proteins with similar sequences. Two-hybrid analysis identified a protein that interacts with TIS21 and BTG1. Sequence motifs associated with S-adenosyl-L-methionine binding suggested this protein might have methyltransferase activity. A glutathione S-transferase (GST) fusion of the putative methyltransferase modifies arginine residues, in appropriate protein substrates, to form NG-monomethyl and NG,NG-dimethylarginine (asymmetric). We term the protein- arginine N-methyltransferase (EC) gene “PRMT1,” for rotein-aginine ethylransferase 1. GST-TIS21 and GST-BTG1 fusion proteins qualitatively and quantitatively modulate endogenous PRMT1 activity, using control and hypomethylated RAT1 cell extracts as methyl-accepting substrates. PRMT1 message appears ubiquitous, and is constitutive in mitogen-stimulated cells. Modulation of PRMT1 activity by transiently expressed regulatory subunits may be an additional mode of signal transduction following ligand stimulation. The TIS21 immediate-early gene and leukemia-associated BTG1 gene encode proteins with similar sequences. Two-hybrid analysis identified a protein that interacts with TIS21 and BTG1. Sequence motifs associated with S-adenosyl-L-methionine binding suggested this protein might have methyltransferase activity. A glutathione S-transferase (GST) fusion of the putative methyltransferase modifies arginine residues, in appropriate protein substrates, to form NG-monomethyl and NG,NG-dimethylarginine (asymmetric). We term the protein- arginine N-methyltransferase (EC) gene “PRMT1,” for rotein-aginine ethylransferase 1. GST-TIS21 and GST-BTG1 fusion proteins qualitatively and quantitatively modulate endogenous PRMT1 activity, using control and hypomethylated RAT1 cell extracts as methyl-accepting substrates. PRMT1 message appears ubiquitous, and is constitutive in mitogen-stimulated cells. Modulation of PRMT1 activity by transiently expressed regulatory subunits may be an additional mode of signal transduction following ligand stimulation. INTRODUCTIONThe protein products of the immediate-early/primary response genes are thought to act as “third messengers,” mediating phenotypic alterations in cells in response to ligands such as growth factors, hormones, neurotransmitters, cytokines, and neurotrophins. Many immediate-early genes encode transcription factors (e.g. Fos, Jun, Egr-1) that initiate transcriptional cascades required for proliferation or differentiation (Herschman, 25Herschman H.R. Annu. Rev. Biochem. 1991; 60: 281-319Google Scholar). Other ligand-induced immediate-early genes encode paracrine mediators of cellular communication whose products (e.g. prostaglandin synthase-2, inducible nitric- oxide synthase, and cytokines such as MCP-1) modulate the behavior of neighboring cells (Smith and Herschman, 60Smith J.B. Herschman H.R. J. Biol. Chem. 1995; 270: 16756-16765Google Scholar).Because immediate-early/primary response genes have been cloned on the basis of their induction characteristics, rather than the functions of their protein products, a number of these genes encode proteins whose biological roles have not yet been determined. One such immediate-early gene is TIS21. The TIS21 cDNA was cloned by differential screening, both from a cDNA library prepared from mitogen-treated, quiescent murine Swiss 3T3 cells (Fletcher et al., 18Fletcher B.S. Lim R.W. Varnum B.C. Kujubu D.A. Koski R.K. Herschman H.R. J. Biol. Chem. 1991; 266: 14511-14518Google Scholar) and from a cDNA library prepared from nerve growth factor-treated rat PC12 pheochromocytoma cells (Bradbury et al., 5Bradbury A. Possenti R. Shooter E.M. Tirone F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3353-3357Google Scholar). The predicted rat and mouse TIS21 proteins differ at only four out of 158 amino acid residues. We demonstrated, by metabolic labeling followed by immunoprecipitation, that maximal TIS21 protein synthesis occurs within the first hour after exposure to ligand, both in mitogen-stimulated Swiss 3T3 cells and in nerve growth factor-stimulated PC12 cells (Varnum et al., 65Varnum B.C. Reddy S.T. Koski R.A. Herschman H.R. J. Cell. Physiol. 1994; 158: 205-213Google Scholar). Moreover, the half-life of both mitogen- and nerve growth factor-induced TIS21 protein is less than 15 min (Varnum et al., 65Varnum B.C. Reddy S.T. Koski R.A. Herschman H.R. J. Cell. Physiol. 1994; 158: 205-213Google Scholar). Despite substantial investigation into both the structure of the TIS21 gene and the induced expression of the TIS21 message and protein, no function has been identified for this protein.The human BTG1 gene was cloned and characterized (Rimokh et al., 49Rimokh R. Rouault J.P. Wahbi K. Gadoux M. Lafage M. Archimbaud E. Charrin C. Gentilhomme O. Germain D. Genes Chromosomes Cancer. 1991; 3: 24-36Google Scholar) because of its proximity to a chromosome translocation breakpoint (8;12) associated with a chronic lymphocytic leukemia. BTG1 overexpression in murine 3T3 cells decreases growth rate and reduces colony formation, leading to the suggestion that BTG1 is an “antiproliferative gene” whose loss of function contributes to uncontrolled growth (Rouault et al., 51Rouault J.P. Rimokh R. Tessa C. Paranhos G. Ffrench M. Duret L. Garoccio M. Germain D. Samarut J. Magaud J.P. EMBO J. 1992; 11: 1663-1670Google Scholar). The human BTG1 and rodent TIS21 open reading frames share 59% identity and 75% conserved sequence similarity at the amino acid level. Moreover, the exon/intron structures of the TIS21 and BTG1 genes are remarkably similar (Varnum et al., 1991; Rimokh et al., 49Rimokh R. Rouault J.P. Wahbi K. Gadoux M. Lafage M. Archimbaud E. Charrin C. Gentilhomme O. Germain D. Genes Chromosomes Cancer. 1991; 3: 24-36Google Scholar). However, BTG1 is not the human TIS21 orthologue; the murine BTG1 cDNA has been cloned and identified as a distinct entity with an amino acid sequence identical to human BTG1 (Rouault et al., 52Rouault J.P. Samarut C. Duret L. Tessa C. Samarut J. Magaud J.P. Gene (Amst.). 1993; 129: 303-306Google Scholar). TIS21 and BTG1 are, therefore, members of a gene family.The stringent regulation of TIS21 gene expression, the short time window for the presence of TIS21 protein following ligand stimulation, and the similarities between the TIS21 and BTG1 proteins suggest that these proteins play important roles in mediating ligand-induced biological responses. We previously speculated that, during its brief availability in mitogen-stimulated cells, TIS21 might reverse a BTG1 antiproliferative barrier by formation of a TIS21-BTG1 heterodimer (Varnum et al., 65Varnum B.C. Reddy S.T. Koski R.A. Herschman H.R. J. Cell. Physiol. 1994; 158: 205-213Google Scholar). Alternatively, TIS21 and BTG1 might serve as regulatory partners that modulate the function of a common catalytic subunit. To test these hypotheses, we used the yeast two-hybrid interaction system (Fields and Song, 16Fields S. Song O. Nature. 1989; 240: 245-246Google Scholar; Fields and Sternglanz, 17Fields S. Sternglanz R. Trends Genet. 1994; 10: 286-292Google Scholar) to determine (i) whether TIS21 and BTG1 proteins can form heterodimers and/or homodimers, and (ii) whether there exist proteins that interact with both TIS21 and BTG1. INTRODUCTIONThe protein products of the immediate-early/primary response genes are thought to act as “third messengers,” mediating phenotypic alterations in cells in response to ligands such as growth factors, hormones, neurotransmitters, cytokines, and neurotrophins. Many immediate-early genes encode transcription factors (e.g. Fos, Jun, Egr-1) that initiate transcriptional cascades required for proliferation or differentiation (Herschman, 25Herschman H.R. Annu. Rev. Biochem. 1991; 60: 281-319Google Scholar). Other ligand-induced immediate-early genes encode paracrine mediators of cellular communication whose products (e.g. prostaglandin synthase-2, inducible nitric- oxide synthase, and cytokines such as MCP-1) modulate the behavior of neighboring cells (Smith and Herschman, 60Smith J.B. Herschman H.R. J. Biol. Chem. 1995; 270: 16756-16765Google Scholar).Because immediate-early/primary response genes have been cloned on the basis of their induction characteristics, rather than the functions of their protein products, a number of these genes encode proteins whose biological roles have not yet been determined. One such immediate-early gene is TIS21. The TIS21 cDNA was cloned by differential screening, both from a cDNA library prepared from mitogen-treated, quiescent murine Swiss 3T3 cells (Fletcher et al., 18Fletcher B.S. Lim R.W. Varnum B.C. Kujubu D.A. Koski R.K. Herschman H.R. J. Biol. Chem. 1991; 266: 14511-14518Google Scholar) and from a cDNA library prepared from nerve growth factor-treated rat PC12 pheochromocytoma cells (Bradbury et al., 5Bradbury A. Possenti R. Shooter E.M. Tirone F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3353-3357Google Scholar). The predicted rat and mouse TIS21 proteins differ at only four out of 158 amino acid residues. We demonstrated, by metabolic labeling followed by immunoprecipitation, that maximal TIS21 protein synthesis occurs within the first hour after exposure to ligand, both in mitogen-stimulated Swiss 3T3 cells and in nerve growth factor-stimulated PC12 cells (Varnum et al., 65Varnum B.C. Reddy S.T. Koski R.A. Herschman H.R. J. Cell. Physiol. 1994; 158: 205-213Google Scholar). Moreover, the half-life of both mitogen- and nerve growth factor-induced TIS21 protein is less than 15 min (Varnum et al., 65Varnum B.C. Reddy S.T. Koski R.A. Herschman H.R. J. Cell. Physiol. 1994; 158: 205-213Google Scholar). Despite substantial investigation into both the structure of the TIS21 gene and the induced expression of the TIS21 message and protein, no function has been identified for this protein.The human BTG1 gene was cloned and characterized (Rimokh et al., 49Rimokh R. Rouault J.P. Wahbi K. Gadoux M. Lafage M. Archimbaud E. Charrin C. Gentilhomme O. Germain D. Genes Chromosomes Cancer. 1991; 3: 24-36Google Scholar) because of its proximity to a chromosome translocation breakpoint (8;12) associated with a chronic lymphocytic leukemia. BTG1 overexpression in murine 3T3 cells decreases growth rate and reduces colony formation, leading to the suggestion that BTG1 is an “antiproliferative gene” whose loss of function contributes to uncontrolled growth (Rouault et al., 51Rouault J.P. Rimokh R. Tessa C. Paranhos G. Ffrench M. Duret L. Garoccio M. Germain D. Samarut J. Magaud J.P. EMBO J. 1992; 11: 1663-1670Google Scholar). The human BTG1 and rodent TIS21 open reading frames share 59% identity and 75% conserved sequence similarity at the amino acid level. Moreover, the exon/intron structures of the TIS21 and BTG1 genes are remarkably similar (Varnum et al., 1991; Rimokh et al., 49Rimokh R. Rouault J.P. Wahbi K. Gadoux M. Lafage M. Archimbaud E. Charrin C. Gentilhomme O. Germain D. Genes Chromosomes Cancer. 1991; 3: 24-36Google Scholar). However, BTG1 is not the human TIS21 orthologue; the murine BTG1 cDNA has been cloned and identified as a distinct entity with an amino acid sequence identical to human BTG1 (Rouault et al., 52Rouault J.P. Samarut C. Duret L. Tessa C. Samarut J. Magaud J.P. Gene (Amst.). 1993; 129: 303-306Google Scholar). TIS21 and BTG1 are, therefore, members of a gene family.The stringent regulation of TIS21 gene expression, the short time window for the presence of TIS21 protein following ligand stimulation, and the similarities between the TIS21 and BTG1 proteins suggest that these proteins play important roles in mediating ligand-induced biological responses. We previously speculated that, during its brief availability in mitogen-stimulated cells, TIS21 might reverse a BTG1 antiproliferative barrier by formation of a TIS21-BTG1 heterodimer (Varnum et al., 65Varnum B.C. Reddy S.T. Koski R.A. Herschman H.R. J. Cell. Physiol. 1994; 158: 205-213Google Scholar). Alternatively, TIS21 and BTG1 might serve as regulatory partners that modulate the function of a common catalytic subunit. To test these hypotheses, we used the yeast two-hybrid interaction system (Fields and Song, 16Fields S. Song O. Nature. 1989; 240: 245-246Google Scholar; Fields and Sternglanz, 17Fields S. Sternglanz R. Trends Genet. 1994; 10: 286-292Google Scholar) to determine (i) whether TIS21 and BTG1 proteins can form heterodimers and/or homodimers, and (ii) whether there exist proteins that interact with both TIS21 and BTG1.

The Mammalian Immediate-early TIS21 Protein and the Leukemia-associated BTG1 Protein Interact with a Protein-arginine -Methyltransferase