D-type cyclins and their cyclin-dependent kinases: G1 phase integrators of the mitogenic response.
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Animals↗
Biomedical subjects
M F Roussel
Publications and source records attributed to M F Roussel.
Signal transduction by the macrophage-colony-stimulating factor receptor (CSF-1R).
The macrophage-specific colony-stimulating factor 1 (CSF-1 or M-CSF) is required throughout the G1 phase of the cell cycle to regulate both immediate and delayed early responses necessary for cell proliferation. These are triggered by the binding of the growth factor to the colony-stimulating factor 1 receptor and the activation of its intrinsic tyrosine-specific protein kinase. Phosphorylation of the colony-stimulating factor 1 receptor on specific tyrosine residues enables it to bind directly to cytoplasmic effector proteins, which in turn relay receptor-induced signals through multiple-signal transduction pathways. The activity of p21ras as well as transcription factors of the ets gene family appears to be required for colony-stimulating factor 1 to induce the c-myc gene, and the latter response is essential to ensure cell proliferation. Genes within the fos/jun or activator protein 1 family are targeted via a parallel and independently regulated signal transduction pathway. The continuous requirement for colony-stimulating factor 1 after the immediate early response is initiated indicates that expression of additional delayed early response genes, although contingent on previously induced gene products, might also depend on colony-stimulating factor 1-induced signals. Among the growth factor-regulated delayed early response genes are D-type G1 cyclins, which play an important role in cell-cycle progression.
Animals↗
Dual control of myc expression through a single DNA binding site targeted by ets family proteins and E2F-1.
NIH3T3 cells expressing a mutant colony-stimulating factor-1 receptor (CSF-1R) containing a phenylalanine for tyrosine substitution in the tyrosine kinase domain at codon 809 exhibit defective myc regulation and do not enter S phase when stimulated by CSF-1. Enforced expression of either ets-1 or ets-2 in these cells restores their mitogenic response, albeit less efficiently than myc itself, suggesting that ets proteins may regulate c-myc expression. Ets-1 transactivates reporter genes driven by the human and mouse c-myc promoters through the binding site for the transcription factor E2F, the latter being required for E1A- and serum-induced c-myc expression. Analysis of E2F-1 sequences identified a minimal DNA binding domain that is related to those of ets proteins. Although E2F and ets proteins interact with similar consensus DNA binding sites, in vitro binding assays revealed that E2F can bind DNA as a homodimer, whereas ets proteins bind these sites as monomers. E2F and ets proteins do not form heterodimers in vitro and do not transactivate c-myc synergistically. Thus, E2F-1 and ets family members may independently regulate c-myc transcription through the same binding site at different times following growth factor stimulation.
Amino Acid Sequence↗
Activation of Src family kinases by colony stimulating factor-1, and their association with its receptor.
The receptor for the macrophage colony stimulating factor-1 (CSF-1R) is a transmembrane glycoprotein with intrinsic tyrosine kinase activity. CSF-1 stimulation promotes the growth of cells of the macrophage lineage and of fibroblasts engineered to express CSF-1R. We show that CSF-1 stimulation resulted in activation of three Src family kinases, Src, Fyn and Yes. Concomitant with their activation, all three Src family kinases were found to associate with the ligand-activated CSF-1 receptor. These interactions were also demonstrated in SF9 insect cells co-infected with viruses encoding the CSF-1 receptor and Fyn, and the isolated SH2 domain of Fyn was capable of binding the CSF-1R in vitro. Analysis of mutant CSF-1Rs revealed that the 'kinase insert' (KI) domain of CSF-1R was not required for interactions with Src family kinases, but that mutation of one of the receptor autophosphorylation sites, Tyr809, reduced both their binding and enzymatic activation. Because fibroblasts expressing this receptor mutant are unable to form colonies in semi-solid medium or to grow in chemically defined medium in the presence of CSF-1, the Src family kinases may play a physiological role in the mitogenic response to CSF-1.
3T3 Cells↗
Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts.
Mammalian D-type cyclins are growth factor-regulated, delayed early response genes that are presumed to control progression through the G1 phase of the cell cycle by governing the activity of cyclin-dependent kinases (cdks). Overexpression of mouse cyclin D1 in serum-stimulated mouse NIH-3T3 and rat-2 fibroblasts increased their rates of G0 to S- and G1- to S-phase transit by several hours, leading to an equivalent contraction of their mean cell generation times. Although such cells remained contact inhibited and anchorage dependent, they manifested a reduced serum requirement for growth and were smaller in size than their normal counterparts. Ectopic expression of cyclin D2 in rodent fibroblasts, either alone or together with exogenous cdk4, shortened their G0- to S-phase interval and reduced their serum dependency, but cyclin D2 alone did not alter cell size significantly. When cells were microinjected during the G1 interval with a monoclonal antibody specifically reactive to cyclin D1, parental rodent fibroblasts and derivatives overexpressing this cyclin were inhibited from entering S phase, but cells injected near the G1/S phase transition were refractory to antibody-induced growth suppression. Thus, cyclin D1, and most likely D2, are rate limiting for G1 progression.
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Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D type G1 cyclins.
Murine D type cyclins associate with a catalytic subunit (p34PSK-J3) with properties distinct from known cyclin-dependent kinases (cdks). Mouse p34PSK-J3 shows less than 50% amino acid identity to p34cdc2, p33cdk2, and p36cdk3, lacks a PSTAIRE motif, and does not bind to p13suc1. Cyclin D1-p34PSK-J3 complexes accumulate in macrophages during G1 and decline in S phase, whereas complexes involving cyclins D2 and D3 form in proliferating T cells. Although histone H1 kinase activity is not detected in cyclin D or PSK-J3 immunoprecipitates, cyclin D-p34PSK-J3 complexes assembled in vitro stably bind and phosphorylate the retinoblastoma gene product (pRb) and an Rb-like protein (p107) but do not interact with pRb mutants that are functionally inactive. Thus, p34PSK-J3 is a cyclin D-regulated catalytic subunit that acts as an Rb (but not H1) kinase.
Amino Acid Sequence↗
Regulation of CYL/cyclin D genes by colony-stimulating factor 1.
The proliferative effects of colony-stimulating factor 1 (CSF-1) on macrophages are exerted only throughout the G1 phase of the cell cycle. Genetic targets of the delayed early response to CSF-1 include novel G1 cyclin (CYL or cyclin D) genes. In macrophages, cyclin D1 is induced early in G1 and is expressed throughout the cell cycle as long as CSF-1 is present. The cyclin D1 protein turns over rapidly in CSF-1-stimulated cells and its level declines precipitously upon CSF-1 withdrawal. Cyclin D2 is induced later in G1 and its expression is periodic, whereas cyclin D3 is not expressed in macrophages but is regulated by growth factors in other cell types. The cyclin D1 protein associates during G1 with a polypeptide antigenically related to p34cdc2 and binds in vitro to a histone H1 kinase present in lysates of CSF-1-starved macrophages. The instability of the cyclin D1 protein and its ability to rescue a cyclin-dependent kinase activity from growth factor-deprived macrophages together suggest that the cyclin D protein is the dynamic partner in the complex. The timing of expression of cyclin D genes suggests that they act to link growth factor signals with cell cycle transitions during G1.
Amino Acid Sequence↗
Random mutagenesis of CSF-1 receptor (FMS) reveals multiple sites for activating mutations within the extracellular domain.
Retroviral vectors containing human FMS protooncogene cDNA were reconfigured to allow single-step excision and reinsertion of restriction fragments encoding short segments of the extracellular domain of the colony-stimulating factor 1 receptor (CSF-1R). Fragments ligated into M13 bacteriophages were subjected to random chemical mutagenesis on both strands and recloned into the parental vector to create libraries of FMS genes containing mutations restricted to predefined target cassettes. Transfection of retroviral vector libraries into NIH/3T3 cells gave rise to transformed foci from which cellular DNA was amplified by the polymerase chain reaction (PCR), using primers flanking the mutagenized target sequences. Amplified fragments from individual primary transformants were recloned into intact FMS vector plasmids, and those with transforming activity were subjected to nucleotide sequence analysis. Alternatively, retroviruses rescued from transformed cells by superinfection with helper virus were used to generate secondary transformants containing unique copies of proviral DNA, whose sequences were determined after PCR amplification. Novel activating mutations were identified within sequences separating the third and fourth immunoglobulin-like loops, as well as within non-covalently stabilized loop 4 of the CSF-1R extracellular domain. Thus, FMS mutations able to convert human CSF-1R to an active oncoprotein are not restricted to those previously identified at codon 301. This approach should be generally applicable for defining activating mutations in related growth factor receptors, including those for platelet-derived growth factor and Steel factor (KIT ligand), in which ligand-independent oncoprotein variants have not been identified.
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Genomic organization, chromosomal localization, and independent expression of human cyclin D genes.
Murine cDNA clones for three cyclin D genes that are normally expressed during the G1 phase of the cell cycle were used to clone the cognate human genes. Bacteriophage and cosmid clones encompassing five independent genomic loci were partially sequenced and chromosomally assigned by an analysis of somatic cell hybrids containing different human chromosomes and by fluorescence in situ hybridization to metaphase spreads from normal peripheral blood lymphocytes. The human cyclin D1 gene (approved gene symbol, CCND1) was assigned to chromosome band 11q13, cyclin D2 (CCND2) to chromosome band 12p13, and cyclin D3 (CCND3) to chromosome band 6p21. Pseudogenes containing sequences related to cyclin D2 and cyclin D3 mapped to chromosome bands 11q13 and 6p21, respectively. Partial nucleotide sequence analysis of exons within each gene revealed that the authentic human cyclin D genes are more related to their mouse counterparts than to each other. These genes are ubiquitously transcribed in human tumor cell lines derived from different cell lineages, but are independently and, in many cases, redundantly expressed. The complex patterns of expression of individual cyclin D genes and their evolutionary conservation across species suggest that each family member may play a distinct role in cell cycle progression.
Amino Acid Sequence↗
Mitogenic signaling by colony-stimulating factor 1 and ras is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression.
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.
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Myc rescue of a mutant CSF-1 receptor impaired in mitogenic signalling.
The colony-stimulating factor-1 receptor (CSF-1R) mediates its pleiotropic effects through the coupling of its ligand-activated tyrosine kinase to multiple intracellular effector proteins, whose combined actions determine the magnitude and specificity of the biological response. The interaction of cytoplasmic signalling molecules with CSF-1R is mediated in part by sequence motifs flanking sites of receptor tyrosine phosphorylation. Mutation of an autophosphorylation site at tyrosine 809 in the cytoplasmic domain of human CSF-1R does not significantly reduce its ligand-stimulated tyrosine kinase activity, binding to phosphatidylinositol 3-kinase, or induction of the immediate early response genes, c-fos and junB (ref.2). Unlike cells bearing wild-type receptors, mouse NIH3T3 cells expressing mutant CSF-1R(Phe 809) were unable to grow in serum-free medium containing human recombinant CSF-1 and did not form colonies in semi-solid medium in its presence. CSF-1 induction of c-myc messenger RNA in these cells was impaired, but enforced expression of an exogenous c-myc gene restored their ability to proliferate in response to the growth factor. These studies demonstrate a receptor-mediated bifurcation of intracellular signal transduction pathways during the immediate early response and assign a central role for c-myc in CSF-1-induced mitogenesis.
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Cloning and expression of murine interleukin-1 receptor antagonist in macrophages stimulated by colony-stimulating factor 1.
Colony-stimulating factor 1 (CSF-1) can act on mature macrophages to modulate their production of inflammatory cytokines. A cDNA encoding the interleukin-1 receptor antagonist (IL-1Ra) was cloned by subtractive hybridization from a CSF-1-stimulated murine macrophage cell line, sequenced, and expressed in mammalian and bacterial cells. Mouse IL-1Ra is a 22-Kd glycoprotein that is 76% identical to its human counterpart, shows considerably less similarity to IL-1 alpha and IL-1 beta, and competes with IL-1 alpha for binding to the type I IL-1 receptor normally expressed on T cells and fibroblasts. CSF-1 treatment of mouse bone marrow-derived macrophages led to a rapid and sustained increase in IL-1Ra mRNA during the G1 phase of the cell cycle as well as to increases in mRNAs encoding IL-1 alpha and IL-1 beta. Cycloheximide inhibited CSF-1-induced IL-1 alpha mRNA synthesis, but augmented IL-1 beta mRNA production and did not affect induction of IL-1Ra mRNA. No IL-1Ra mRNA was observed in CSF-1-stimulated mouse fibroblasts engineered to express CSF-1 receptors, demonstrating that its regulation depends on cell context and can be dissociated from the proliferative response. In agreement, bacterial lipopolysaccharide, a nonmitogenic activator, also induced IL-1Ra and IL-1 mRNAs in macrophages. Unlike IL-1 alpha and beta, IL-1Ra contains a signal peptide. The kinetics of its induction and its ability to gain access to the secretory compartment imply that IL-1Ra may be secreted more efficiently than IL-1, and suggest that macrophages both positively and negatively regulate the IL-1 response.
Amino Acid Sequence↗
Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle.
Three mouse cyclin-like (CYL) genes were isolated, two of which are regulated by colony-stimulating factor 1 (CSF-1) during the G1 phase of the macrophage cell cycle. CSF-1 deprivation during G1 leads to rapid degradation of CYL proteins (p36CYL) and correlates with failure to initiate DNA synthesis. However, after entering S phase, macrophages no longer require CSF-1 and can complete cell division without expressing CYL genes. During G1, p36CYL is phosphorylated and associates with a polypeptide antigenically related to p34cdc2. The timing of p36CYL expression, its rapid turnover in the absence of CSF-1, and its phosphorylation and transient binding to a cdc2-related polypeptide suggest that CYL genes may function during S phase commitment.
Amino Acid Sequence↗
The carboxy-terminal catalytic domain of the GTPase-activating protein inhibits nuclear signal transduction and morphological transformation mediated by the CSF-1 receptor.
To determine whether ras p21 products are necessary for signal transduction mediated by the colony stimulating factor-1 receptor (CSF-1R, the c-fms proto-oncogene product), we determined whether CSF-1R and ras activate a common nuclear target and whether the interruption of ras action affects CSF-1R signal transduction. Expression of the NVL3 retrotransposon was activated to the same extent in NIH-3T3 cells by both ras and v-fms oncogenes, and the ras-responsive element located in the long terminal repeat of NVL3 was demonstrated to be a common target for oncogene action. Human recombinant CSF-1 stimulated expression of the NVL3 element 30-fold in NIH-3T3 cells that contained human CSF-1R. Expression of the carboxy-terminal 374 amino acid residues of the human ras GTPase-activating protein (GAP) in cells containing CSF-1R was able to inhibit CSF-1 induction of NVL3 expression by 90%. Expression of the catalytic domain of GAP was also able to suppress transformation by either v-fms or ligand-activated CSF-1R. Expression of the c-jun proto-oncogene was activated by CSF-1R but was insensitive to the action of the catalytic domain of GAP. These results provide genetic evidence that in NIH-3T3 cells, ras p21 is involved in signal transduction mediated by CSF-1R.
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Novel mammalian cyclins (CYL genes) expressed during G1.
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Amino Acid Sequence↗
Regulation of cell growth and differentiation by the colony-stimulating factor 1 receptor.
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Animals↗
Signal-response coupling mediated by the transduced colony-stimulating factor-1 receptor and its oncogenic fms variants in naive cells.
Colony-stimulating factor-1 (CSF-1 or M-CSF) supports the proliferation and survival of mononuclear phagocytes by binding to a receptor (CSF-1R) encoded by the c-fms proto-oncogene. Whereas the CSF-1R kinase is normally regulated by ligand, receptors bearing 'activating mutations' act constitutively as enzymes and can transform fibroblasts and haemopoietic cells of different lineages. Introduction of human CSF-1R enables mouse NIH-3T3 cells to form colonies in agar in response to human CSF-1 and to proliferate in serum-free medium supplemented with CSF-1, albumin, transferrin and insulin. Similarly, expression of human CSF-1R in interleukin 3-dependent mouse FDC-P1 myeloid cells enables them to grow in CSF-1. High levels of CSF-1R expression in FDC-P1 cells can induce factor-independent growth which is abrogated by a 'neutralizing' monoclonal antibody to the receptor. Therefore, critical mutations in the c-fms gene or overexpression of CSF-1R in immature myeloid precursors might each contribute to leukaemia.
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Structural features of the colony-stimulating factor 1 receptor that affect its association with phosphatidylinositol 3-kinase.
The colony-stimulating factor 1 receptor (CSF-1R), immunoprecipitated with either anti-phosphotyrosine or anti-receptor antibodies from lysates of ligand-stimulated cells, is associated with a phosphatidylinositol (PtdIns) 3-kinase activity. The ligand-independent transforming efficiencies of human CSF-1R mutants containing certain amino acid substitutions at codon 301 in their extracellular domains correlated directly with their levels of associated lipid kinase activity. A tyrosine kinase defective CSF-1R mutant (CSF-1R[met616]), containing a mutated ATP binding site, lacked associated PtdIns 3-kinase activity in immune complexes recovered from CSF-1-stimulated cells. However, CSF-1R[met616] associated with PtdIns 3-kinase when phosphorylated in trans in CSF-1-stimulated cells coexpressing an enzymatically competent CSF-1R tyrosine kinase. Another CSF-1R mutant, (CSF-1R[delta KI]), lacking 67 amino acids from its intracellular 'kinase insert' domain, exhibited a partially impaired ligand-dependent mitogenic response and a significant reduction in its associated PtdIns 3-kinase activity. Ligand-stimulated CSF-1R[delta KI] molecules contained levels of phosphotyrosine almost equivalent to wild-type receptors, but were phosphorylated at different sites in vitro. Therefore, the association of CSF-1R with active PtdIns 3-kinase required the receptor tyrosine kinase activity, was triggered by receptor phosphorylation on tyrosine and, in this series of mutants, correlated with their mitogenic potential. Although the receptor KI domain strongly contributes to the association with PtdIns 3-kinase, this region is not strictly essential for the interaction.
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