scholarly journals Complementation of Defective Colony-Stimulating Factor 1 Receptor Signaling and Mitogenesis by Raf and v-Src

1999 ◽  
Vol 19 (2) ◽  
pp. 1101-1115 ◽  
Author(s):  
Natasha Aziz ◽  
Holly Cherwinski ◽  
Martin McMahon
Keyword(s):  
Map Kinase ◽  
Cyclin D1 ◽  
Colony Stimulating Factor ◽  
3T3 Cells ◽  
Mitogenic Response ◽  
Induced Expression ◽  
Map Kinase Pathway ◽  
Nih 3T3 ◽  
Kinase Pathway ◽  
Stimulating Factor

ABSTRACT Ras-activated signal transduction pathways are implicated in the control of cell proliferation, differentiation, apoptosis, and tumorigenesis, but the molecular mechanisms mediating these diverse functions have yet to be fully elucidated. Conditionally active forms of Raf, v-Src, and MEK1 were used to identify changes in gene expression that participate in oncogenic transformation, as well as in normal growth control. Activation of Raf, v-Src, and MEK1 led to induced expression of c-Myc and cyclin D1. Induction of c-Myc mRNA by Raf was an immediate-early response, whereas the induction of cyclin D1 mRNA was delayed and inhibited by cycloheximide. Raf activation also resulted in the induction of an established c-Myc target gene, ornithine decarboxylase (ODC). ODC induction by Raf was mediated, in part, by tandem E-boxes contained in the first intron of the gene. Activation of the human colony-stimulating factor 1 (CSF-1) receptor in NIH 3T3 cells leads to activation of the mitogen-activated protein (MAP) kinase pathway and induced expression of c-Fos, c-Myc, and cyclin D1, leading to a potent mitogenic response. By contrast, a mutated form of this receptor fails to activate the MAP kinases or induce c-Myc and cyclin D1 expression and fails to elicit a mitogenic response. The biological significance of c-Myc and cyclin D1 induction by Raf and v-Src was confirmed by the demonstration that both of these protein kinases complemented the signaling and mitogenic defects of cells expressing this mutated form of the human CSF-1 receptor. Furthermore, the induction of c-Myc and cyclin D1 by oncogenes and growth factors was inhibited by PD098059, a specific MAP kinase kinase (MEK) inhibitor. These data suggest that the Raf/MEK/MAP kinase pathway plays an important role in the regulation of c-Myc and cyclin D1 expression in NIH 3T3 cells. The ability of oncogenes such as Raf and v-Src to regulate the expression of these proteins reveals new lines of communication between cytosolic signal transducers and the cell cycle machinery.

Reconstituted human granulocyte-macrophage colony-stimulating factor receptor transduces growth-promoting signals in mouse NIH 3T3 cells: comparison with signalling in BA/F3 pro-B cells

1993 ◽  
Vol 13 (3) ◽  
pp. 1440-1448
Author(s):  
S Watanabe ◽  
A L Mui ◽  
A Muto ◽  
J X Chen ◽  
K Hayashida ◽  
...  
Keyword(s):  
Colony Stimulating Factor ◽  
3T3 Cells ◽  
Gm Csf ◽  
High Affinity ◽  
Macrophage Colony Stimulating Factor ◽  
Growth Promoting ◽  
Macrophage Colony ◽  
Nih 3T3 ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a critical role in growth and differentiation of myeloid cells. We previously reconstituted high-affinity human GM-CSF receptor (hGM-CSFR) in a pro-B cell line, BA/F3, by cotransfecting alpha- and beta-chain cDNA clones and showed that the reconstituted receptor could transduce growth-promoting signals. The high-affinity hGM-CSFR was also reconstituted in mouse NIH 3T3 cells, but its ability to transduce signals in fibroblasts remained undetermined. In the present study, we further characterized signal transduction by the reconstituted hGM-CSFR in both NIH 3T3 cells and BA/F3 cells. We found that the reconstituted hGM-CSFR transduces signals in NIH 3T3 fibroblasts and BA/F3 cells in response to hGM-CSF to activate transcription of the c-fos, c-jun, and c-myc proto-oncogenes. hGM-CSF also induces protein tyrosine phosphorylation and DNA synthesis in both cell types. These results indicated that hGM-CSFR is functional in fibroblasts, that signal transduction via hGM-CSFR in fibroblasts involves tyrosine kinase(s), and that association of hGM-CSFR with a factor(s) specific to hematopoietic cell lineage is not essential to transduce growth-promoting signals.

Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes

1987 ◽  
Vol 7 (7) ◽  
pp. 2378-2387 ◽  
Author(s):  
C W Rettenmier ◽  
M F Roussel ◽  
R A Ashmun ◽  
P Ralph ◽  
K Price ◽  
...  
Keyword(s):  
Growth Factor ◽  
Disulfide Bonds ◽  
Mononuclear Phagocyte ◽  
Transformed Cells ◽  
Colony Stimulating Factor ◽  
3T3 Cells ◽  
Membrane Bound ◽  
Nih 3T3 ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

NIH 3T3 cells cotransfected with the human c-fms proto-oncogene together with a 1.6-kilobase cDNA clone encoding a 256-amino-acid precursor of the human mononuclear phagocyte colony-stimulating factor CSF-1 (M-CSF) undergo transformation by an autocrine mechanism. The number of CSF-1 receptors on the surface of transformed cells was regulated by ligand-induced receptor degradation and was inversely proportional to the quantity of CSF-1 produced. A tyrosine-to-phenylalanine mutation at position 969 near the receptor carboxyl terminus potentiated its transforming efficiency in cells cotransfected by the CSF-1 gene but did not affect receptor downmodulation. CSF-1 was synthesized as an integral transmembrane glycoprotein that was rapidly dimerized through disulfide bonds. The homodimer was externalized at the cell surface, where it underwent proteolysis to yield the soluble growth factor. Trypsin treatment of viable cells cleaved the plasma membrane form of CSF-1 to molecules of a size indistinguishable from that of the extracellular growth factor, suggesting that trypsinlike proteases regulate the rate of CSF-1 release from transformed cells. The data raise the possibility that this form of membrane-bound CSF-1 might stimulate receptors on adjacent cells through direct cell-cell interactions.

Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor

1990 ◽  
Vol 10 (11) ◽  
pp. 5601-5608
Author(s):  
M Reedijk ◽  
X Q Liu ◽  
T Pawson
Keyword(s):  
Cell Transformation ◽  
Cellular Transformation ◽  
Colony Stimulating Factor ◽  
Gtpase Activating Protein ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Fibroblast Transformation ◽  
Insert Region ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

The interactions of the macrophage colony-stimulating factor 1 (CSF-1) receptor with potential targets were investigated after ligand stimulation either of mouse macrophages or of fibroblasts that ectopically express mouse CSF-1 receptors. In Rat-2 cells expressing the mouse CSF-1 receptor, full activation of the receptor and cellular transformation require exogenous CSF-1, whereas NIH 3T3 cells expressing mouse c-fms are transformed by autocrine stimulation. Activated CSF-1 receptors physically associate with a phosphatidylinositol (PI) 3'-kinase. A mutant CSF-1 receptor with a deletion of the kinase insert region was deficient in its ability to bind functional PI 3'-kinase and to induce PI 3'-kinase activity precipitable with antiphosphotyrosine antibodies. In fibroblasts, CSF-1 stimulation also induced the phosphorylation of the GTPase-activating protein (GAP)-associated protein p62 on tyrosine, although GAP itself was a relatively poor substrate. In contrast to PI 3'-kinase association, phosphorylation of p62 and GAP was not markedly affected by deletion of the kinase insert region. These results indicate that the kinase insert region selectively enhances the CSF-1-dependent association of the CSF-1 receptor with active PI 3'-kinase. The insert deletion mutant retains considerable transforming activity in NIH 3T3 cells (G. Taylor, M. Reedijk, V. Rothwell, L. Rohrschneider, and T. Pawson, EMBO J. 8:2029-2037, 1989). This mutant was more seriously impaired in Rat-2 cell transformation, although mutant-expressing Rat-2 cells still formed small colonies in soft agar in the presence of CSF-1. Therefore, phosphorylation of GAP and p62 through activation of the CSF-1 receptor does not result in full fibroblast transformation. The interaction between the CSF-1 receptor and PI 3'-kinase may contribute to c-fms fibroblast transformation and play a role in CSF-1-stimulated macrophages.

scholarly journals The Carboxyl Terminus of v-Abl Protein Can Augment SH2 Domain Function

2000 ◽  
Vol 74 (10) ◽  
pp. 4495-4504 ◽  
Author(s):  
David Warren ◽  
Andrew J. Heilpern ◽  
Kent Berg ◽  
Naomi Rosenberg
Keyword(s):  
Map Kinase ◽  
Protein Interactions ◽  
Leukemia Virus ◽  
Sh2 Domain ◽  
Carboxyl Terminus ◽  
Abl Tyrosine Kinase ◽  
Mitogen Activated Protein ◽  
Map Kinase Pathway ◽  
Nih 3T3 ◽  
Kinase Pathway

ABSTRACT Abelson murine leukemia virus (Ab-MLV) transforms NIH 3T3 and pre-B cells via expression of the v-Abl tyrosine kinase. Although the enzymatic activity of this molecule is absolutely required for transformation, other regions of the protein are also important for this response. Among these are the SH2 domain, involved in phosphotyrosine-dependent protein-protein interactions, and the long carboxyl terminus, which plays an important role in transformation of hematopoietic cells. Important signals are sent from each of these regions, and transformation is most likely orchestrated by the concerted action of these different parts of the protein. To explore this idea, we compared the ability of the v-Src SH2 domain to substitute for that of v-Abl in the full-length P120 v-Abl protein and in P70 v-Abl, a protein that lacks the carboxyl terminus characteristic of Abl family members. Ab-MLV strains expressing P70/S2 failed to transform NIH 3T3 cells and demonstrated a greatly reduced capacity to mediate signaling events associated with the Ras-dependent mitogen-activated protein (MAP) kinase pathway. In contrast, Ab-MLV strains expressing P120/S2 were indistinguishable from P120 with respect to these features. Analyses of additional mutants demonstrated that the last 162 amino acids of the carboxyl terminus were sufficient to restore transformation. These data demonstrate that an SH2 domain with v-Abl substrate specificity is required for NIH 3T3 transformation in the absence of the carboxyl terminus and suggest that cooperativity between the extreme carboxyl terminus and the SH2 domain facilitates the transmission of transforming signals via the MAP kinase pathway.

scholarly journals Inhibition of colony-stimulating factor-1 activity by monoclonal antibodies to the human CSF-1 receptor

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1786-1793
Author(s):  
CJ Sherr ◽  
RA Ashmun ◽  
JR Downing ◽  
M Ohtsuka ◽  
SG Quan ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Mononuclear Phagocytes ◽  
Colony Stimulating Factor ◽  
3T3 Cells ◽  
Specific Receptor ◽  
Inhibitory Antibodies ◽  
Nih 3T3 ◽  
Stimulating Factor ◽  
Macrophage Precursors ◽  
Nih 3T3 Cells

Four of 12 monoclonal antibodies (MoAbs) directed to different epitopes in the extracellular domain of the human colony-stimulating factor-1 receptor (CSF-1R, the c-fms proto-oncogene product) specifically inhibit CSF-1 binding to receptor-bearing cells. All four antibodies abrogated CSF-1-dependent colony formation by human bone marrow-derived macrophage precursors and by mouse NIH-3T3 cells expressing a transduced human c-fms gene. In addition, one of these antibodies (designated MoAb 2–4A5) interfered with the ligand-independent proliferation of NIH-3T3 cells transformed by an oncogenic, mutant c- fms allele. Unlike CSF-1 itself, neither MoAb 2–4A5 nor the other three inhibitory antibodies (MoAbs 12–2D6, 12–3A1, and 12–3A3) induced CSF-1R internalization or degradation. These antibodies should prove useful not only for identifying and quantitating CSF-1R on receptor-bearing cells but for abrogating specific receptor signals that govern the proliferation and survival of human mononuclear phagocytes.

Mitogenic signaling by colony-stimulating factor 1 and ras is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression

1992 ◽  
Vol 12 (12) ◽  
pp. 5355-5362
Author(s):  
S J Langer ◽  
D M Bortner ◽  
M F Roussel ◽  
C J Sherr ◽  
M C Ostrowski
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Colony Stimulating Factor ◽  
3T3 Cells ◽  
Semisolid Medium ◽  
Myc Gene ◽  
Nih 3T3 ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

The activity of p21ras is required for the proliferative response to colony-stimulating factor 1 (CSF-1), and signals transduced by both the CSF-1 receptor (CSF-1R) and p21ras stimulate transcription from promoter elements containing overlapping binding sites for Fos/Jun- and Ets-related proteins. A sequence encoding the DNA-binding domain and nuclear localization signal of human c-ets-2, which lacked portions of the c-ets-2 gene product necessary for trans activation, was fused to the bacterial lacZ gene and expressed from an actin promoter in NIH 3T3 cells expressing either the v-ras oncogene or human CSF-1R. Nuclear expression of the Ets-LacZ protein, confirmed by histochemical staining of beta-galactosidase, inhibited the activity of ras-responsive enhancer elements and suppressed morphologic transformation by v-ras as well as CSF-1R-dependent colony formation in semisolid medium. When CSF-1R-bearing cells expressing the Ets-LacZ protein were stimulated by CSF-1, induction of c-ets-2, c-jun, and c-fos ensued, but the c-myc response was impaired. Enforced expression of the c-myc gene overrode the suppressive effect of ets-lacZ and restored the ability of these cells to form colonies in response to CSF-1. NIH 3T3 cells engineered to express a CSF-1R (Phe-809) mutant similarly cannot form CSF-1-dependent colonies in semisolid medium and exhibit an impaired c-myc response, but expression of an exogenous myc gene resensitizes these cells to CSF-1 [M. F. Roussel, J. L. Cleveland, S. A. Shurtleff, and C. J. Sherr, Nature (London) 353:361-363, 1991]. The ability of these cells to respond to CSF-1 was also rescued by enforced expression of an endogenous c-ets-2 gene. The ets family of transcription factors therefore plays a central role in integrating both CSF-1R and ras-induced mitogenic signals and in modulating the myc response to CSF-1 stimulation.

scholarly journals Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor.

1990 ◽  
Vol 10 (11) ◽  
pp. 5601-5608 ◽  
Author(s):  
M Reedijk ◽  
X Q Liu ◽  
T Pawson
Keyword(s):  
Cell Transformation ◽  
Cellular Transformation ◽  
Colony Stimulating Factor ◽  
Gtpase Activating Protein ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Fibroblast Transformation ◽  
Insert Region ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

The interactions of the macrophage colony-stimulating factor 1 (CSF-1) receptor with potential targets were investigated after ligand stimulation either of mouse macrophages or of fibroblasts that ectopically express mouse CSF-1 receptors. In Rat-2 cells expressing the mouse CSF-1 receptor, full activation of the receptor and cellular transformation require exogenous CSF-1, whereas NIH 3T3 cells expressing mouse c-fms are transformed by autocrine stimulation. Activated CSF-1 receptors physically associate with a phosphatidylinositol (PI) 3'-kinase. A mutant CSF-1 receptor with a deletion of the kinase insert region was deficient in its ability to bind functional PI 3'-kinase and to induce PI 3'-kinase activity precipitable with antiphosphotyrosine antibodies. In fibroblasts, CSF-1 stimulation also induced the phosphorylation of the GTPase-activating protein (GAP)-associated protein p62 on tyrosine, although GAP itself was a relatively poor substrate. In contrast to PI 3'-kinase association, phosphorylation of p62 and GAP was not markedly affected by deletion of the kinase insert region. These results indicate that the kinase insert region selectively enhances the CSF-1-dependent association of the CSF-1 receptor with active PI 3'-kinase. The insert deletion mutant retains considerable transforming activity in NIH 3T3 cells (G. Taylor, M. Reedijk, V. Rothwell, L. Rohrschneider, and T. Pawson, EMBO J. 8:2029-2037, 1989). This mutant was more seriously impaired in Rat-2 cell transformation, although mutant-expressing Rat-2 cells still formed small colonies in soft agar in the presence of CSF-1. Therefore, phosphorylation of GAP and p62 through activation of the CSF-1 receptor does not result in full fibroblast transformation. The interaction between the CSF-1 receptor and PI 3'-kinase may contribute to c-fms fibroblast transformation and play a role in CSF-1-stimulated macrophages.

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