scholarly journals Leukemia-Related Transcription Factor TEL Is Negatively Regulated through Extracellular Signal-Regulated Kinase-Induced Phosphorylation

2004 ◽  
Vol 24 (8) ◽  
pp. 3227-3237 ◽  
Author(s):  
Kazuhiro Maki ◽  
Honoka Arai ◽  
Kazuo Waga ◽  
Ko Sasaki ◽  
Fumihiko Nakamura ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Effect ◽  
Phosphorylation Sites ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT TEL is an ETS family transcription factor that possesses multiple putative mitogen-activated protein kinase phosphorylation sites. We here describe the functional regulation of TEL via ERK pathways. Overexpressed TEL becomes phosphorylated in vivo by activated ERK. TEL is also directly phosphorylated in vitro by ERK. The inducible phosphorylation sites are Ser213 and Ser257. TEL binds to a common docking domain in ERK. In vivo ERK-dependent phosphorylation reduces trans-repressional and DNA-binding abilities of TEL for ETS-binding sites. A mutant carrying substituted glutamates on both Ser213 and Ser257 functionally mimics hyperphosphorylated TEL and also shows a dominant-negative effect on TEL-induced transcriptional suppression. Losing DNA-binding affinity through phosphorylation but heterodimerizing with unmodified TEL could be an underlying mechanism. Moreover, the glutamate mutant dominantly interferes with TEL-induced erythroid differentiation in MEL cells and growth suppression in NIH 3T3 cells. Finally, endogenous TEL is dephosphorylated in parallel with ERK inactivation in differentiating MEL cells and is phosphorylated through ERK activation in Ras-transformed NIH 3T3 cells. These data indicate that TEL is a constituent downstream of ERK in signal transduction systems and is physiologically regulated by ERK in molecular and biological features.

scholarly journals Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4

1998 ◽  
Vol 18 (11) ◽  
pp. 6605-6615 ◽  
Author(s):  
Mohamed H. Ladha ◽  
Kwang Y. Lee ◽  
Todd M. Upton ◽  
Michael F. Reed ◽  
Mark E. Ewen
Keyword(s):  
Cyclin D1 ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Cyclin Dependent Kinase ◽  
3T3 Cells ◽  
Rate Limiting Step ◽  
Mitogen Activated Protein ◽  
Nih 3T3 ◽  
Rate Limiting ◽  
Nih 3T3 Cells

ABSTRACT The synthesis of cyclin D1 and its assembly with cyclin-dependent kinase 4 (CDK4) to form an active complex is a rate-limiting step in progression through the G1 phase of the cell cycle. Using an activated allele of mitogen-activated protein kinase kinase 1 (MEK1), we show that this kinase plays a significant role in positively regulating the expression of cyclin D1. This was found both in quiescent serum-starved cells and in cells expressing dominant-negative Ras. Despite the observation that cyclin D1 is a target of MEK1, in cycling cells, activated MEK1, but not cyclin D1, is capable of overcoming a G1 arrest induced by Ras inactivation. Either wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in reversing the G1 arrest induced by inhibition of Ras activity. In quiescent NIH 3T3 cells expressing either ectopic cyclin D1 or activated MEK1, cyclin D1 is able to efficiently associate with CDK4; however, the complex is inactive. A significant percentage of the cyclin D1-CDK4 complexes are associated with p27 in serum-starved activated MEK1 or cyclin D1 cell lines. Reduction of p27 levels by expression of antisense p27 allows for S-phase entry from quiescence in NIH 3T3 cells expressing ectopic cyclin D1, but not in parental cells.

scholarly journals Mutations in the SAM Domain of the ETV6-NTRK3 Chimeric Tyrosine Kinase Block Polymerization and Transformation Activity

2004 ◽  
Vol 24 (11) ◽  
pp. 4636-4650 ◽  
Author(s):  
Cristina E. Tognon ◽  
Cameron D. Mackereth ◽  
Aruna M. Somasiri ◽  
Lawrence P. McIntosh ◽  
Poul H. B. Sorensen
Keyword(s):  
Tyrosine Kinases ◽  
Fusion Proteins ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
3T3 Cells ◽  
Sam Domain ◽  
Binding Interface ◽  
Transformation Activity ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT The 12p13 ETV6 (TEL) gene is frequently targeted by chromosomal translocations in human malignancies, resulting in the formation of oncogenic ETV6 gene fusions. Many of the known partner genes encode protein tyrosine kinases (PTKs), generating fusion proteins that function as chimeric PTKs. ETV6-NTRK3 (EN), comprised of the ETV6 SAM domain fused to the NTRK3 PTK, is unique among ETV6 chimeric oncoproteins, as it is expressed in cancers of multiple lineages. We initially hypothesized that, similar to other ETV6-PTK chimeras, SAM-mediated dimerization of EN leads to constitutive activation of the PTK and downstream signaling cascades. However, when the EN SAM domain was replaced with an inducible FK506 binding protein (FKBP) dimerization system, resulting FKBP-NTRK3 chimeras failed to transform NIH 3T3 cells even though PTK activation was preserved. It was recently shown that the ETV6 SAM domain has two potential interacting surfaces, raising the possibility that this domain can mediate protein polymerization. We therefore mutated each EN SAM binding interface in a manner shown previously to abolish self-association of wild-type ETV6. Each mutation completely blocked the ability of EN to polymerize, to activate its PTK, and to transform NIH 3T3 cells. Furthermore, EN itself formed large polymeric structures within cells while mutant EN proteins were present only as monomers. Finally, we observed a dominant negative effect on the transformation of isolated SAM domains coexpressed in EN-transformed cells. Taken together, our results suggest that higher-order polymerization may be a critical requirement for the transformation activity of EN and possibly other ETV6-PTK fusion proteins.

Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A

1989 ◽  
Vol 9 (6) ◽  
pp. 2453-2463
Author(s):  
P Yaciuk ◽  
J K Choi ◽  
D Shalloway
Keyword(s):  
Protein Kinase ◽  
3T3 Cells ◽  
Triple Mutant ◽  
Dependent Protein Kinase ◽  
Mutant Proteins ◽  
Tetradecanoyl Phorbol Acetate ◽  
Dependent Protein ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.

scholarly journals The Transcription Factor GATA4 Is Activated by Extracellular Signal-Regulated Kinase 1- and 2-Mediated Phosphorylation of Serine 105 in Cardiomyocytes

2001 ◽  
Vol 21 (21) ◽  
pp. 7460-7469 ◽  
Author(s):  
Qiangrong Liang ◽  
Russell J. Wiese ◽  
Orlando F. Bueno ◽  
Yan-Shan Dai ◽  
Bruce E. Markham ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Phosphorylation Site ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Mitogen Activated Protein Kinase ◽  
Cardiomyocyte Hypertrophy ◽  
Hypertrophic Growth ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

ABSTRACT The zinc finger-containing transcription factor GATA4 has been implicated as a critical regulator of multiple cardiac-expressed genes as well as a regulator of inducible gene expression in response to hypertrophic stimulation. Here we demonstrate that GATA4 is itself regulated by the mitogen-activated protein kinase signaling cascade through direct phosphorylation. Site-directed mutagenesis and phospho-specific GATA4 antiserum revealed serine 105 as the primary site involved in agonist-induced phosphorylation of GATA4. Infection of cultured cardiomyocytes with an activated MEK1-expressing adenovirus induced robust phosphorylation of serine 105 in GATA4, while a dominant-negative MEK1-expressing adenovirus blocked agonist-induced phosphorylation of serine 105, implicating extracellular signal-regulated kinase (ERK) as a GATA4 kinase. Indeed, bacterially purified ERK2 protein directly phosphorylated purified GATA4 at serine 105 in vitro. Phosphorylation of serine 105 enhanced the transcriptional potency of GATA4, which was sensitive to U0126 (MEK1 inhibitor) but not SB202190 (p38 inhibitor). Phosphorylation of serine 105 also modestly enhanced the DNA binding activity of bacterially purified GATA4. Finally, induction of cardiomyocyte hypertrophy with an activated MEK1-expressing adenovirus was blocked with a dominant-negative GATA4-engrailed-expressing adenovirus. These results suggest a molecular pathway whereby MEK1-ERK1/2 signaling regulates cardiomyocyte hypertrophic growth through the transcription factor GATA4 by direct phosphorylation of serine 105, which enhances DNA binding and transcriptional activation.

scholarly journals Cell transformation mediated by homodimeric E2A-HLF transcription factors.

1997 ◽  
Vol 17 (3) ◽  
pp. 1417-1424 ◽  
Author(s):  
T Inukai ◽  
T Inaba ◽  
T Yoshihara ◽  
A T Look
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Fusion Gene ◽  
Dominant Negative ◽  
Fusion Product ◽  
3T3 Cells ◽  
Par Proteins ◽  
Downstream Target ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The E2A-HLF fusion gene, created by the t(17;19)(q22;p13) chromosomal translocation in pro-B lymphocytes, encodes an oncogenic protein in which the E2A trans-activation domain is linked to the DNA-binding and protein dimerization domain of hepatic leukemia factor (HLF), a member of the proline- and acidic amino acid-rich (PAR) subfamily of bZIP transcription factors. This fusion product binds to its DNA recognition site not only as a homodimer but also as a heterodimer with HLF and two other members of the PAR bZIP subfamily, thyrotroph embryonic factor (TEF) and albumin promoter D-box binding protein (DBP). Thus, E2A-HLF could transform cells by direct regulation of downstream target genes, acting through homodimeric or heterodimeric complexes, or by sequestering normal PAR proteins into nonfunctional heterocomplexes (dominant-negative interference). To distinguish among these models, we constructed mutant E2A-HLF proteins in which the leucine zipper domain of HLF was extended by one helical turn or altered in critical charged amino acids, enabling the chimera to bind to DNA as a homodimer but not as a heterodimer with HLF or other PAR proteins. When introduced into NIH 3T3 cells in a zinc-inducible vector, each of these mutants induced anchorage-independent growth as efficiently as unaltered E2A-HLF, indicating that the chimeric oncoprotein can transform cells in its homodimeric form. Transformation also depended on an intact E2A activator region, providing further support for a gain-of-function contribution to oncogenesis rather than one based on a dominant-interfering or dominant-negative mechanism. Thus, the tumorigenic effects of E2A-HLF and its mutant forms in NIH 3T3 cells favor a straightforward model in which E2A-HLF homodimers bind directly to promoter/enhancer elements of downstream target genes and alter their patterns of expression in early B-cell progenitors.

scholarly journals Prostaglandin F2αStimulates Formation of p21ras-GTP Complex and Mitogen-activated Protein Kinase in NIH-3T3 Cells via Gq-protein-coupled Pathway

1995 ◽  
Vol 270 (15) ◽  
pp. 8984-8990 ◽  
Author(s):  
Tsuyoshi Watanabe ◽  
Iwao Waga ◽  
Zen-ichiro Honda ◽  
Kiyoshi Kurokawa ◽  
Takao Shimizu
Keyword(s):  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
3T3 Cells ◽  
Mitogen Activated Protein ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

scholarly journals Transformation of NIH 3T3 cells by cotransfection with c-src and nuclear oncogenes.

1987 ◽  
Vol 7 (10) ◽  
pp. 3582-3590 ◽  
Author(s):  
D Shalloway ◽  
P J Johnson ◽  
E O Freed ◽  
D Coulter ◽  
W A Flood
Keyword(s):  
3T3 Cells ◽  
Focus Formation ◽  
Adenovirus E1a ◽  
Rous Sarcoma ◽  
Cellular Homolog ◽  
Sarcoma Virus ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

pp60c-src, the cellular homolog of the Rous sarcoma virus transforming protein, does not completely transform cells even when present at high levels, but has been shown to be involved in polyomavirus-induced transformation when activated by polyomavirus middle T (pmt)-antigen binding. Here we show that cotransfection, but not solo transfection, of expression plasmids for c-src and either adenovirus E1A, v-myc, c-myc, or the 5' half of polyomavirus large T (pltN) antigen into NIH 3T3 cells induces anchorage-independent growth, enhanced focus formation, and, for pltN cotransfection, tumorigenicity in adult NFS mice. Enhancement of transformation was not observed with polyomavirus small t (pst) antigen. Cotransfection of c-src with pltN induced modification of pp60c-src that altered its electrophoretic mobility and in vivo phosphorylation state and stimulated its in vitro kinase activity. Similar alterations were not seen after c-src-E1A cotransfection, suggesting that at least two different mechanisms of enhancement are involved.

Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro

Science ◽  
1992 ◽  
Vol 257 (5075) ◽  
pp. 1404-1407 ◽  
Author(s):  
P Dent ◽  
W Haser ◽  
T. Haystead ◽  
L. Vincent ◽  
T. Roberts ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
3T3 Cells ◽  
Mitogen Activated Protein ◽  
Nih 3T3 ◽  
Protein Kinase Kinase ◽  
Nih 3T3 Cells
Sign in / Sign up

Export Citation Format

Share Document