ABSTRACT
Tnk1/Kos1 is a non-receptor protein tyrosine kinase implicated in negative regulation of cell growth by a mechanism involving inhibition of Ras activation and requiring Tnk1/Kos1's intrinsic catalytic activity. Tnk1/Kos1 null mice were created by homologous recombination by deleting the catalytic domain. Upon aging, both Tnk1+/- and Tnk1-/- mice develop spontaneous tumors, including lymphomas and carcinomas at high rates (i.e. 27%, and 43%, respectively), indicating that Tnk1/Kos1 is a tumor suppressor. Tissues from Tnk1/Kos1-null mice exhibit proportionally higher levels of basal and growth factor-stimulated Ras activation. Mechanistically, Tnk1/Kos1 requires either or both Y277 and Y287 sites to be intact for enzymatic activity and phosphorylation of its substrate, growth factor receptor binding protein 2 (Grb2). Data indicate that following tyrosine phosphorylation of Grb2 by Tnk1/Kos1, the Grb2-Sos1 guanine exchange factor (GEF) complex that mediates growth factor stimulated Ras activation becomes disrupted, resulting in the reversal of Ras activation. Conversely, the loss of Tnk1/Kos1 activity results in constitutive activation of Ras due to prolonged stabilization/activation of the Grb2-Sos1 GEF activity. Tnk1/Kos1 is the first tyrosine kinase discovered to have tumor suppressor activity, and the mechanism of spontaneous tumor formation involves constitutive, indirect activation of Ras. Thus, Ras may display "oncogenic activity" without undergoing "oncogenic" mutation. We now find that a cohort of patients with diffuse large B-cell lymphoma (DLBCL) display downregulation of Tnk1/Kos1 that may account for tumorigenesis in humans.
Subject(s)
Fetal Proteins/physiology , Protein-Tyrosine Kinases/physiology , Tumor Suppressor Proteins/physiology , Animals , DNA-Binding Proteins/metabolism , Fetal Proteins/genetics , GRB2 Adaptor Protein/metabolism , Heterozygote , Homozygote , Humans , Lymphoma, B-Cell/genetics , Mice , Mice, Knockout , Neoplasms, Experimental/genetics , Phosphorylation , Protein-Tyrosine Kinases/deficiency , Protein-Tyrosine Kinases/genetics , SOS1 Protein/metabolism , Signal Transduction , Transcription Factors/metabolism , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/genetics , ras Proteins/metabolismABSTRACT
Tnk1/Kos1 is a non-receptor protein tyrosine kinase found to be a tumor suppressor. It negatively regulates cell growth by indirectly suppressing Ras activity. We identified and characterized the critical cis-elements required for Tnk1/Kos1's promoter activity. Results indicate that the murine Tnk1 promoter lacks a conventional TATA, CAAT or initiator element (Inr) but contains multiple transcription start sites. Transcription is initiated by a TATA-like element composed of an AT rich sequence at -30 (30 bp upstream) from the major transcription start site and an Inr-like element that overlaps the multiple start sites. Deletion analysis of the m-Tnk1 promoter reveals the presence of both positive (-25 to -151) and negative (-151 to -1201) regulatory regions. The three GC boxes which bind Sp1 and Sp3 with high affinity, an AP2 site (that overlaps with an AML1 site) and a MED1 site comprise the necessary cis-elements of the proximal promoter required for both constitutive and inducible Tnk1/Kos1 expression. Importantly, results reveal that cellular stress reverses the repression of Tnk1/Kos1 and induces its expression through increased high affinity interactions between nuclear proteins Sp1, Sp3, AP2 and MED1 for the m-Tnk1 promoter. These findings provide a mechanism by which the m-Tnk1 promoter can be dynamically regulated during normal growth.
Subject(s)
Promoter Regions, Genetic , Protein-Tyrosine Kinases/genetics , Transcription Initiation Site , 3T3 Cells , Animals , Base Sequence , Mediator Complex Subunit 1 , Mice , Molecular Sequence Data , Protein-Tyrosine Kinases/biosynthesis , Regulatory Sequences, Nucleic Acid , Sp1 Transcription Factor/genetics , Sp1 Transcription Factor/metabolism , Sp3 Transcription Factor/genetics , Sp3 Transcription Factor/metabolism , Transcription Factor AP-2/genetics , Transcription Factor AP-2/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional ActivationABSTRACT
Tnk1/Kos1 is a non-receptor protein tyrosine kinase implicated in negatively regulating cell growth in a mechanism requiring its intrinsic catalytic activity. Tnk1/Kos1 null mice were created by homologous recombination by deleting the catalytic domain. Both Tnk1(+/-) and Tnk1(-/-) mice develop spontaneous tumors, including lymphomas and carcinomas, at high rates [27% (14 of 52) and 43% (12 of 28), respectively]. Tnk1/Kos1 expression is silenced in tumors that develop in Tnk1(+/-) mice but not in adjacent uninvolved tissue, and silencing occurs in association with Tnk1 promoter hypermethylation. Tissues and murine embryonic fibroblasts derived from Tnk1/Kos1-null mice exhibit proportionally higher levels of basal and epidermal growth factor-stimulated Ras activation that results from increased Ras-guanine exchange factor (GEF) activity. Mechanistically, Tnk1/Kos1 can directly tyrosine phosphorylate growth factor receptor binding protein 2 (Grb2), which promotes disruption of the Grb2-Sos1 complex that mediates growth factor-induced Ras activation, providing dynamic regulation of Ras GEF activity with suppression of Ras. Thus, Tnk1/Kos1 is a tumor suppressor that functions to down-regulate Ras activity.
Subject(s)
Neoplasms, Experimental/genetics , Protein-Tyrosine Kinases/physiology , Alleles , Animals , Base Sequence , Blotting, Western , Cells, Cultured , DNA Primers , Epigenesis, Genetic , Gene Silencing , Gene Targeting , Immunoprecipitation , Mice , Mice, Knockout , Protein-Tyrosine Kinases/geneticsABSTRACT
Relaxin, a hormone in the insulin superfamily, is synthesized by the corpus luteum of the rat ovary. Expression of relaxin precursor mRNA in rats is sharply induced after day 10 of pregnancy and plateaus on days 15 to 20 (parturition occurs on day 23). In an effort to understand this induction, we cloned the gene and carried out promoter analyses by transient transfection and chromatin immunoprecipitation methods. The single gene is 2.9 kilobases and is composed of two exons and one intron. There are alternative splice acceptor sites, 3 base pairs apart, which account for the inclusion of an extra codon in about 10% of the transcripts. The induction of transcription by day 15 was observed by the binding of polymerase II and histone H3 acetylation at the promoter region. There is a functional STAT binding site, about 3.8 kb upstream from the transcriptional start site, that is occupied by STAT3 on day 6 of pregnancy, when relaxin expression is minimal; on day 15, when expression is maximal, STAT3 is replaced by STAT5a. These data are consistent with STAT5 playing a role in the induction of relaxin expression.
Subject(s)
Genes/genetics , Relaxin/genetics , Animals , Base Sequence , Cells, Cultured , Chromatin/genetics , Chromatin/metabolism , Cloning, Molecular , DNA/chemistry , DNA/genetics , Exons , Female , Gene Expression , Gene Expression Regulation, Developmental , Granulosa Cells/cytology , Granulosa Cells/metabolism , Humans , Introns , Luciferases/genetics , Luciferases/metabolism , Male , Molecular Sequence Data , Precipitin Tests/methods , Pregnancy , Protein Precursors/genetics , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Relaxin/metabolism , Sequence Analysis, DNA , Transcription Initiation Site , TransfectionABSTRACT
Kinase of embryonic stem cells (Kos1), a nonreceptor protein tyrosine kinase (NRPTK), was identified and cloned from differentiating murine embryonic stem cells. Kos1 is localized on mouse chromosome 11 that corresponds to human chromosome 17p13.1 and is homologous to Tnk1, Ack1 and Ack2, making it a new member of the Ack family of NRPTKs. Kos1 is a ubiquitously expressed 47-kDa protein with autotyrosine kinase activity that is developmentally regulated during embryogenesis. Kos1 is also upregulated following IL3 withdrawal from factor-dependent murine NSF/N1.H7 cells that undergo apoptosis, suggesting a role in growth inhibition. Stable overexpression of Kos1 inhibits growth of NIH 3T3 cells, while the kinase-dead Kos1(CN) promotes cell growth in both liquid culture and soft agar. In addition, forced expression of Kos1 inhibits Ras activity in an indirect mechanism that results in the downregulation of the Ras-Raf1-MAPK growth pathway. Furthermore, overexpression of Kos1 in NCI-H82 lung cancer cells that express oncogenic Ha-Ras(G12V) inhibits cell growth under reduced serum (0.5%) conditions in close association with the upregulation of the Ras inhibitor, Rap1A. Collectively, these data support a negative regulatory role for Kos1 in regulating the Ras-Raf1-MAPK growth pathway by a mechanism that requires its autotyrosine kinase activity.
