DNA Synthesis In Quiescent Cells Research Articles

Who gets a license: DNA synthesis in quiescent cells re-entering the cell cycle.

The precise regulation of the entry into S phase is critical for preventing genome instability. The first step in the initiation of eukaryotic DNA synthesis occurs in G1 phase cells and involves the loading of the conserved MCM helicase onto multiple origins of replication in a process known as origin licensing. In proliferating metazoan cells, an origin-licensing checkpoint delays initiation until high levels of MCM loading occur, with excess origins being licensed. One function of this checkpoint is to ensure that S phase can be completed in the face of replication stress by activation of dormant MCM bound origins. However, when both metazoan and yeast cells enter S phase from quiescence or G0 phase, a non-growing but reversible cell cycle state, origins are significantly under-licensed. In metazoan cells, under-licensing is the result of a compromised origin-licensing checkpoint. In budding yeast, our study has revealed that under-licensing can be attributed to the chromatin structure at a class of origins that is inhibitory to the binding of MCM. Thus, defects in multiple pathways may contribute to the failure to fully license origins in quiescent cells re-entering the cell cycle, thereby promoting a higher risk of genome instability.

Inactivation of Retinoblastoma Protein Does Not Overcome the Requirement for Human Cytomegalovirus UL97 in Lamina Disruption and Nuclear Egress

Human cytomegalovirus (HCMV) encodes one conventional protein kinase, UL97. During infection, UL97 phosphorylates the retinoblastoma tumor suppressor protein (pRb) on sites ordinarily phosphorylated by cyclin-dependent kinases (CDK), inactivating the ability of pRb to repress host genes required for cell cycle progression to S phase. UL97 is important for viral DNA synthesis in quiescent cells, but this function can be replaced by human papillomavirus type 16 E7, which targets pRb for degradation. However, viruses in which E7 replaces UL97 are still defective for virus production. UL97 is also required for efficient nuclear egress of viral nucleocapsids, which is associated with disruption of the nuclear lamina during infection, and phosphorylation of lamin A/C on serine 22, which antagonizes lamin polymerization. We investigated whether inactivation of pRb might overcome the requirement of UL97 for these roles, as pRb inactivation induces CDK1, and CDK1 phosphorylates lamin A/C on serine 22. We found that lamin A/C serine 22 phosphorylation during HCMV infection correlated with expression of UL97 and was considerably delayed in UL97-null mutants, even when E7 was expressed. E7 failed to restore gaps in the nuclear lamina seen in wild-type but not UL97-null virus infections. In electron microscopy analyses, a UL97-null virus expressing E7 was as impaired as a UL97-null mutant in cytoplasmic accumulation of viral nucleocapsids. Our results demonstrate that pRb inactivation is insufficient to restore efficient viral nuclear egress of HCMV in the absence of UL97 and instead argue further for a direct role of UL97 in this stage of the infectious cycle.

ChemInform Abstract: Microbial Secondary Metabolites Inhibiting Oncogene Functions

Streptomyces and other microorganisms produce antibiotics, anticancer agents and enzyme inhibitors as secondary metabolites. Microorganisms are a treasury of organic compounds which have various structures and biological activities. Therefore, isolation of the specific bioactive products in culture broths is dependent upon the testing method. Since oncogene theory has been extensively developed, we have screened oncogene function inhibitors as a new group of microbial secondary metabolites. Oncogene functions include acting as a modified growth factor receptor (erb B, erb B 2 fms), acting as a growth factor itself (sis, hst), the tyrosine kinase activity (src oncogene group), and acting as a G protein to activate phosphatidylinositol turnover (ras).Erbstatin is an inhibitor of erb B, erb B 2 and src products-associated tyrosine kinase. In cell culture it inhibited EGF receptor internalization, delayed the onset of EGF-induced DNA synthesis in quiescent cells, and induced normal phenotypes in src oncogene expressing cells. It inhibited the growth of human breast and esophageal carcinomas in nude mice. Lavendustin A was isolated from Streptomyces as a potent inhibitor of tyrosine kinase. It is a novel compound and about 50 times stronger than erbstatin in in vitro inhibition of tyrosine kinase. Many oncogenes including ras, src, sis, fms, and fes are considered to activate cellular phosphatidylinositol turnover. We have isolated psi-tectorigenin, inostamycin and piericidin B1 N-oxide as inhibitors of inositol incorporation into phospholipids. Thus, various inhibitors of oncogene functions including novel structures have been discovered in microbial secondary metabolites. They are useful for the dynamic study of oncogenes.

Human papillomavirus 16 E7 inactivator of retinoblastoma family proteins complements human cytomegalovirus lacking UL97 protein kinase

Several different families of DNA viruses encode proteins that inactivate the cellular retinoblastoma tumor suppressor protein (pRb), which normally functions to bind E2F transcription factors and restrict expression of genes necessary for cellular processes including DNA replication. Human cytomegalovirus (HCMV) UL97, a protein kinase functionally orthologous to cellular cyclin-dependent kinases, phosphorylates pRb on inactivating residues during HCMV infection. To assess if such phosphorylation is biologically relevant, we tested whether the human papillomavirus type 16 E7 protein, which inactivates pRb family proteins by direct binding and destabilization, could substitute for UL97 during HCMV infection. In the absence of UL97, expression of wild-type E7 protein, but not a mutant E7 unable to bind pRb family proteins, restored E2F-responsive cellular gene expression, late viral gene expression, and viral DNA synthesis to levels normally observed during wild-type virus infection of quiescent cells. UL97-null mutants exhibited more pronounced defects in virus production and DNA synthesis in quiescent cells as compared to serum-fed, cycling cells. E7 expression substantially enhanced infectious virus production in quiescent cells, but did not complement the defects observed during UL97-null virus infection of cycling cells. Thus, a primary role of UL97 is to inactivate pRb family proteins during infection of quiescent cells, and this inactivation likely abets virus replication by induction of cellular E2F-responsive genes. Our findings have implications for human cytomegalovirus disease and for drugs that target UL97.

Human cytomegalovirus pp71: a new viral tool to probe the mechanisms of cell cycle progression and oncogenesis controlled by the retinoblastoma family of tumor suppressors.

The DNA tumor virus oncogenes (adenovirus E1A, simian virus 40 (SV40) large T antigen, and papillomavirus E7) have been instrumental in illuminating the molecules and mechanisms of cell cycle progression and carcinogenesis. However, since these multifunctional proteins target so many important cellular regulators, it is sometimes difficult to establish the functional importance of any individual interaction. Perhaps a herpesvirus protein, newly defined as a cell cycle regulator, can help address these issues. Like the DNA tumor virus proteins, the human cytomegalovirus (HCMV) pp71 protein contains a retinoblastoma protein (Rb) binding motif (LxCxD), and stimulates DNA synthesis in quiescent cells. Unlike E1A, T antigen, and E7, pp71 expression does not induce apoptosis, nor does it cooperate to transform primary cells. Determining how pp71 induces cell cycle progression without invoking apoptosis or leading to cellular transformation may help in defining the signals that ultimately lead to these processes.

The human cytomegalovirus UL82 gene product (pp71) accelerates progression through the G1 phase of the cell cycle.

As viruses are reliant upon their host cell to serve as proper environments for their replication, many have evolved mechanisms to alter intracellular conditions to suit their own needs. For example, human cytomegalovirus induces quiescent cells to enter the cell cycle and then arrests them in late G(1), before they enter the S phase, a cell cycle compartment that is presumably favorable for viral replication. Here we show that the protein product of the human cytomegalovirus UL82 gene, pp71, can accelerate the movement of cells through the G(1) phase of the cell cycle. This activity would help infected cells reach the late G(1) arrest point sooner and thus may stimulate the infectious cycle. pp71 also induces DNA synthesis in quiescent cells, but a pp71 mutant protein that is unable to induce quiescent cells to enter the cell cycle still retains the ability to accelerate the G(1) phase. Thus, the mechanism through which pp71 accelerates G(1) cell cycle progression appears to be distinct from the one that it employs to induce quiescent cells to exit G(0) and subsequently enter the S phase.

Human cytomegalovirus pp71 stimulates cell cycle progression by inducing the proteasome-dependent degradation of the retinoblastoma family of tumor suppressors.

The oncoproteins of the DNA tumor viruses, adenovirus E1A, simian virus 40 T antigen, and papillomavirus E7, each interact with the retinoblastoma family of tumor suppressors, leading to cell cycle stimulation, apoptosis induction, and cellular transformation. These proteins utilize a conserved LXCXE motif, which is also found in cellular proteins, to target the retinoblastoma family. Here, we describe a herpesvirus protein that shares a subset of the properties of the DNA tumor virus oncoproteins but maintains important differences as well. The human cytomegalovirus pp71 protein employs an LXCXD motif to attack the retinoblastoma family members and induce DNA synthesis in quiescent cells. pp71 binds to and induces the degradation of the hypophosphorylated forms of the retinoblastoma protein and its family members p107 and p130 in a proteasome-dependent manner. However, pp71 does not induce apoptosis and fails to transform cells. Thus, the similarities and differences in comparison to E1A, T antigen, and E7 make pp71 an interesting new tool with which to further dissect the role of the retinoblastoma/E2F pathway in cellular growth control and carcinogenesis.

Antisense-mediated depletion of p300 in human cells leads to premature G1 exit and up-regulation of c-MYC.

The cAMP-response element-binding protein (CREB)-binding protein and p300 are two highly conserved transcriptional coactivators and histone acetyltransferases that integrate signals from diverse signal transduction pathways in the nucleus and also link chromatin remodeling with transcription. In this report, we have examined the role of p300 in the control of the G(1) phase of the cell cycle in nontransformed immortalized human breast epithelial cells (MCF10A) and fibroblasts (MSU) by using adenovirus vectors expressing p300-specific antisense sequences. Quiescent MCF10A and MSU cells expressing p300-specific antisense sequences synthesized p300 at much reduced levels and exited G(1) phase without serum stimulation. These cells also showed an increase in cyclin A and cyclin A- and E-associated kinase activities characteristic of S phase induction. Further analysis of the p300-depleted quiescent MCF10A cells revealed a 5-fold induction of c-MYC and a 2-fold induction of c-JUN. A direct target of c-MYC, CAD, which is required for DNA synthesis, was also found to be up-regulated, indicating that up-regulation of c-MYC functionally contributed to DNA synthesis. Furthermore, S phase induction in p300-depleted cells was reversed when antisense c-MYC was expressed in these cells, indicating that up-regulation of c-MYC may directly contribute to S phase induction. Adenovirus E1A also induced DNA synthesis and increased the levels of c-MYC and c-JUN in serum-starved MCF10A cells in a p300-dependent manner. Our results suggest an important role of p300 in cell cycle regulation at G(1) and raise the possibility that p300 may negatively regulate early response genes, including c-MYC and c-JUN, thereby preventing DNA synthesis in quiescent cells.

A novel putative transcription factor protein MYT2 that preferentially binds supercoiled DNA and induces DNA synthesis in quiescent cells

Myelin transcription factor 2 (MYT2), a putative transcription factor found in the human central nervous system, was cloned from an expression cDNA library from human T-cells. MYT2 shares weak similarity to bacterial type I topoisomerases and shares 63% sequence identity to a replicase from Leuconostoc mesenteroides. MYT2 preferentially binds supercoiled DNA (scDNA). Incubation of MYT2 and scDNA at or above equal molar ratios generated topoisomer-like patterns that were abolished by deproteination. Thus, MYT2 appears to relax scDNA via a non-enzymatic mechanism. The banding pattern of MYT2–scDNA complexes was shown to be quantisized, saturable and sequence-independent. Microinjection of MYT2 mRNA induced G o growth-arrested NIH 3T3 cells to enter the S phase of the cell cycle.

Epidermal growth factor signaling and mitogenesis in Plcg1 null mouse embryonic fibroblasts.

Gene targeting techniques and early mouse embryos have been used to produce immortalized fibroblasts genetically deficient in phospholipase C (PLC)-gamma1, a ubiquitous tyrosine kinase substrate. Plcg1(-/-) embryos die at embryonic day 9; however, cells derived from these embryos proliferate as well as cells from Plcg1(+/+) embryos. The null cells do grow to a higher saturation density in serum-containing media, as their capacity to spread out is decreased compared with that of wild-type cells. In terms of epidermal growth factor receptor activation and internalization, or growth factor induction of mitogen-activated protein kinase, c-fos, or DNA synthesis in quiescent cells, PLcg1(-/-) cells respond equivalently to PLcg1(+/+) cells. Also, null cells are able to migrate effectively in a wounded monolayer. Therefore, immortalized fibroblasts do not require PLC-gamma1 for many responses to growth factors.

A Comparative Analysis of the Interactions of the E6 Proteins from Cutaneous and Genital Papillomaviruses with p53 and E6AP in Correlation to Their Transforming Potential

A common necessity for all papillomaviruses is to induce DNA synthesis in quiescent cells. This is commonly achieved by the E7 gene product, which interferes with the function of members of the retinoblastoma family controlling transition from the G1-phase to the S-phase of the cell cycle. Uncontrolled entry into S-phase activates, however, negative growth control signals which have to be bypassed to achieve production of progeny viruses. In addition to inherent activities of the E7 protein, high risk genital types encode an E6 protein that overcomes p53-mediated G1-arrest and apoptosis in concert with the cellular factor E6AP by targeting p53 for the enhanced ubiquitin-dependent degradation. The key question, which of these functions of genital E6 and E7 proteins is responsible for the carcinogenic phenotype, is still not completely answered. In contrast to high risk genital types no immortalizing or transforming activities have been found for the E7 proteins of the high risk cutaneous HPV8 and 47. On the other hand the ability of the E6 protein to transform established rodent fibroblasts seems to be a property shared by high risk genital and cutaneous types. To examine the existence of a common E6-mediated transforming pathway for both virus groups we compared the properties of the cutaneous E6 proteins with already known functions of E6 proteins of genital viruses. For this we analyzed the E6 proteins of low risk and high risk cutaneous and genital papillomaviruses with respect to cell transformation, to their abilities to bind, degradate, and influence the activity of human p53, and to bind E6AP. The results of our study demonstrate a clear lack of interaction between the transforming E6 proteins of HPV1 and HPV8 and both cellular proteins p53 and E6AP. In contrast, we found E6AP-independent binding of HPV16 E6 and HPV6 E6 to p53, although both proteins were different in their transforming potential. Of all four proteins investigated, only HPV16 E6 was able to bind to p53 and E6AP and to induce degradation of the p53 protein in the reticulocyte system. When we investigated in frame deletion mutants of the E6 protein of HPV16 for their abilities to bind to p53 or E6AP, degradate, and inhibit the transactivation function of p53 and to transform rodent fibroblasts, no correlation between the different activities could be found. Mutants still able to bind p53 and E6AP lacked transforming ability and other mutants that were transformation-competent were deficient in p53 and E6AP binding.

The Phosphatase Inhibitor Okadaic Acid Stimulates the TSH-Induced G1–S Phase Transition in Thyroid Cells

Protein phosphorylation plays an essential role in regulating many cellular processes in eukaryotes. Signal transduction mechanisms that are reversibly controlled by protein phosphorylation require also protein phosphatases (PPs). Okadaic acid (OA), which is a potent inhibitor of protein phosphatase 2A (PP2A) and protein phosphatase 1, elicits phosphorylation of many proteins in unstimulated cells and induces different cellular responses, including transcriptional activation, shape changes, and pseudomitotic state. In this study, the effects of OA on rat thyroid cells (FRTL-5 strain) were analyzed to evaluate the role of serine/threonine phosphatases in hormone-induced thyroid cell proliferation. OA at a concentration range between 0.1 and 1 nMstimulated thyroid cell growth. Furthermore, 0.25 nMOA increased about 3.5-fold the thyrotropin (TSH)-induced DNA synthesis in quiescent cells. OA treatment also stimulated cell proliferation induced by drugs that mimic TSH effect, such as 8Br-cAMP and cholera toxin, suggesting that PP2A activity was relevant in the cAMP pathway activated by the hormone. Flow cytometry experiments showed that OA significantly increased the fraction of TSH-stimulated quiescent cells entering the S phase. In order to define the mechanisms underlying the observed stimulatory effect of OA on thyroid cell growth, expression of genes relevant in the G1–S phase transition was evaluated. A 2-fold increase in the level of cyclin D1 mRNA expression was found by Northern blot analysis in OA-treated cells. Althoughcdk2gene expression was not modulated by the same OA treatment, an increase in Cdk2 protein was revealed by immunoprecipitation experiments. Moreover, OA modifies the phosphorylation pattern of the tumor suppressor retinoblastoma protein, a key event in the G1–S phase transition. Therefore, these experiments reveal that PP2A phosphatases play an important role in thyroid cell growth and can act at multiple sites in the TSH pathways driving cells to S phase.

Specific activation of p85-p110 phosphatidylinositol 3'-kinase stimulates DNA synthesis by ras- and p70 S6 kinase-dependent pathways.

We have developed a polyclonal antibody that activates the heterodimeric p85-p110 phosphatidylinositol (PI) 3'-kinase in vitro and in microinjected cells. Affinity purification revealed that the activating antibody recognized the N-terminal SH2 (NSH2) domain of p85, and the antibody increased the catalytic activity of recombinant p85-p110 dimers threefold in vitro. To study the role of endogenous PI 3'-kinase in intact cells, the activating anti-NSH2 antibody was microinjected into GRC + LR73 cells, a CHO cell derivative selected for tight quiescence during serum withdrawal. Microinjection of anti-NSH2 antibodies increased bromodeoxyuridine (BrdU) incorporation fivefold in quiescent cells and enhanced the response to serum. These data reflect a specific activation of PI 3'-kinase, as the effect was blocked by coinjection of the appropriate antigen (glutathione S-transferase-NSH2 domains from p85 alpha), coinjection of inhibitory anti-p110 antibodies, or treatment of cells with wortmannin. We used the activating antibodies to study signals downstream from PI 3'-kinase. Although treatment of cells with 50 nM rapamycin only partially decreased anti-NSH2-stimulated BrdU incorporation, coinjection with an anti-p70 S6 kinase antibody effectively blocked anti-NSH2-stimulated DNA synthesis. We also found that coinjection of inhibitory anti-ras antibodies blocked both serum- and anti-NSH2-stimulated BrdU incorporation by approximately 60%, and treatment of cells with a specific inhibitor of MEK abolished antibody-stimulated BrdU incorporation. We conclude that selective activation of physiological levels of PI 3'-kinase is sufficient to stimulate DNA synthesis in quiescent cells. PI 3'-kinase-mediated DNA synthesis requires both p70 S6 kinase and the P21ras/MEK pathway.

Requirement for c-Src Catalytic Activity and the SH3 Domain in Platelet-derived Growth Factor BB and Epidermal Growth Factor Mitogenic Signaling

The Src family protein-tyrosine kinases are required for mitogenic signaling from the platelet-derived growth factor (PDGF), colony stimulating factor-1, and epidermal growth factor (EGF) receptor protein-tyrosine kinases (RPTK) (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700; Roche, S., Koegl, M., Barone, M. V., Roussel, M. F., and Courtneidge, S. A.(1995) Mol. Cell. Biol. 15, 1102-1109). In NIH3T3 fibroblasts, c-Src, Fyn, and c-Yes associate with the activated PDGF receptor, are substrates for receptor phosphorylation, and are themselves activated. Src family catalytic function is required for RPTK mitogenic signaling as evidenced by the SH2-dependent dominant negative phenotype exhibited by kinase-inactive Src and Fyn mutants (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700). Here, we have generated clonal Src- murine fibroblast cell lines overexpressing various murine c-Src mutants and studied the effect of these mutant Src proteins on PDGF- and EGF-induced mitogenesis. Two c-Src SH3 domain mutants, Y133F and Y138F, each inhibited PDGF BB- and EGF-induced DNA synthesis in quiescent cells. This demonstrates an involvement of the Src SH3 domain in PDGFbeta and EGF receptor mitogenic signaling. Since both Tyr-133 and Tyr-138 are located on the ligand binding surface of the SH3 domain, these results suggest that the c-Src SH3 domain is required for PDGF and EGF mitogenic signaling. The dominant negative effect of either single mutant on PDGF receptor signaling was reversed by a second SH2-inactivating mutation. We conclude that the c-Src SH3 domain function requires the SH2 domain in the case of the PDGF receptor, presumably because binding of c-Src to the receptor via its SH2 domain is a prerequisite for the SH3 domain function. In contrast, SH2 function is apparently not essential for the SH3 function in EGF receptor signaling.

Antiproliferative and c-myc mRNA suppressive effect of tranilast on newborn human vascular smooth muscle cells in culture.

1. Newborn human vascular smooth muscle cells (VSMCs) proliferated faster and were more sensitive to platelet-derived growth factor-BB (PDGF-BB) than those from adults. In this study, we investigated mechanism of the inhibitory effect of tranilast on PDGF-BB-induced proliferation of VSMCs from newborns. 2. Tranilast (30-300 microM) concentration-dependently inhibited the VSMC proliferation in randomly growing cultures stimulated with PDGF-BB. 3. Tranilast (30-300 microM) concentration-dependently inhibited the [3H]-thymidine incorporation into DNA in VSMCs that had been synchronized by 48 h serum depletion and then stimulated by addition of PDGF-BB. However, tranilast had little influence on unscheduled DNA synthesis in quiescent cells or on RNA and protein synthesis, unlike aphidicolin, actimomycin D, and cycloheximide. 4. In synchronized VSMC cultures, tranilast still inhibited the PDGF-BB-induced DNA synthesis even when added 18 h after stimulation of the quiescent cells. The mode of the antiproliferative action of tranilast was different from that of NiCl2, genistein, or staurosporin. In addition, flow cytometry of synchronized VSMCs treated with tranilast revealed a blockade of PDGF-inducible cell-cycle progression at the G1/S checkpoint. 5. Northern blotting showed that tranilast (30-300 microM) concentration-dependently suppressed constitutive c-myc mRNA expression even when added 18 h after PDGF-BB-stimulation of quiescent VSMCs. Tranilast still had an inhibitory effect on the induction of c-myc mRNA when de novo protein synthesis was inhibited by cycloheximide and did not shorten the degradation of c-myc mRNA at the post-transcriptional level, demonstrating that tranilast directly inhibited c-myc mRNA expression at the transcriptional level. 6. These results suggest that the inhibitory effect of tranilast on PDGF-BB-induced proliferation is due to S-phase blockade and may be, at least in part, involved in the direct suppression of c-myc gene expression. Tranilast did not cause cell toxicity and may therefore hold promising potential for the prevention of vascular proliferative diseases.

SV40 Large T Antigen Transactivates the Human cdc2 Promoter by Inducing a CCAAT Box Binding Factor

Cyclin-dependent protein kinases (Cdks) play a key role in the cell division cycle of eukaryotic cells. Cdc2, the first mammalian Cdk that was discovered, is expressed in S phase and functions in the G2 to M phase transition. By transfecting segments of the human cdc2 promoter linked to a reporter gene into monkey kidney (CV-1) cells, we identified the region containing the Sp1, E2F, and two CCAAT box binding sites as essential and sufficient for basal transcription. SV40 large T antigen (SV40-LT) is a viral oncoprotein that transactivates viral and cellular promoters and induces DNA synthesis in quiescent cells. SV40-LT transactivated wild-type cdc2 promoter/reporter constructs in a dose-dependent manner, coinciding with an increase in endogenous cdc2 mRNA. A mutant promoter from which the two CCAAT box motifs were deleted was 8-fold less sensitive to SV40-LT. Activation by SV40-LT did not require its ability to bind the retinoblastoma or p53 tumor suppressor proteins. SV40-LT induced a specific CCAAT box-binding factor (CBF) in CV-1 and COS-7 cells, as judged by gel shift and Southwestern analyses. Similar results were obtained in human fibroblasts expressing a conditional SV40-LT. The SV40-LT-inducible CBF appears to be novel and differs from the CBF that activates heat shock protein 70 gene expression.

All ErbB Receptors Other Than the Epidermal Growth Factor Receptor Are Endocytosis Impaired

Four transmembrane tyrosine kinases constitute the ErbB receptor family: the epidermal growth factor (EGF) receptor, ErbB-2, ErbB-3, and ErbB-4. We have measured the endocytic capacities of all four members of the EGF receptor family, including ErbB-3 and ErbB-4, which have not been described previously. EGF-responsive chimeric receptors containing the EGF receptor extracellular domain and different ErbB cytoplasmic domains (EGFR/ErbB) have been employed. The capacity of these growth factor-receptor complexes to mediate 125I-EGF internalization, receptor down-regulation, receptor degradation, and receptor co-immunoprecipitation with AP-2 was assayed. In contrast to the EGF receptor, all EGFR/ErbB receptors show impaired ligand-induced rapid internalization, down-regulation, degradation, and AP-2 association. Also, we have analyzed the heregulin-responsive wild-type ErbB-4 receptor, which does not mediate the rapid internalization of 125I-heregulin, demonstrates no heregulin-regulated receptor degradation, and fails to form association complexes with AP-2. Despite the substantial differences in ligand-induced receptor trafficking between the EGF and ErbB-4 receptors, EGF and heregulin have equivalent capacities to stimulate DNA synthesis in quiescent cells. These results show that the ligand-dependent down-regulation mechanism of the EGF receptor, surprisingly, is not a property of any other known ErbB receptor family member. Since endocytosis is thought to be an attenuation mechanism for growth factor-receptor complexes, these data imply that substantial differences in attenuation mechanisms exist within one family of structurally related receptors.

Fibroblast Growth Factor (FGF) 3 from Xenopus laevis (XFGF3) Binds with High Affinity to FGF Receptor 2

We demonstrate that purified fibroblast growth factor (FGF) 3 from Xenopus laevis (XFGF3) activates the mitogen-activated protein kinase pathway and induces DNA synthesis in quiescent cells. To characterize the high affinity cell surface receptors that mediate these responses, the ligand binding domains of different FGF receptors (FGFR) were expressed on COS-1 cells, and their affinity for XFGF3 was determined. Unlabeled XFGF3 efficiently competed with 125I-FGF1 for binding to the IIIb and IIIc isoforms of FGFR2, giving 50% displacement (ID50) at 0.3-0.8 nM. Higher XFGF3 concentrations were needed to displace 125I-FGF1 from FGFR3 and FGFR1 (ID50 approximately 4 and 21 nM, respectively), indicating that XFGF3 has a lower affinity for these receptors. No association of XFGF3 with FGFR4 was found using this assay. FGFR2 isoforms isolated from both mouse and Xenopus showed similar high affinity binding of XFGF3 as determined by direct binding assays (Kd values in the range of 0.2-0.6 nM). These results indicate that the binding specificity of XFGF3 is different from that of other FGFs, and identifies FGFR2 as its high affinity receptor.

Phospholipase C-gamma 1 can induce DNA synthesis by a mechanism independent of its lipase activity.

Inositol phospholipid-specific phospholipase C (PLC) is involved in several signaling pathways leading to cellular growth and differentiation. Our previous studies reported the induction of DNA synthesis in quiescent NIH 3T3 cells after microinjection of PLC and the inhibition of serum- or Ras-stimulated DNA synthesis by a mixture of monoclonal antibodies to PLC-gamma 1. In the course of our investigation of anti-PLC-gamma 1 monoclonal antibodies, we found that each antibody exerts different inhibitory effects on the phosphatidylinositol-hydrolyzing activity of PLC-gamma 1 and that the inhibition of enzymatic activity does not correlate with the inhibition of DNA synthesis observed in the microinjection assay. PLC-gamma 1 with defective enzymatic activity was synthesized by substituting phenylalanine for histidine within the PLC-gamma 1 catalytic domain at amino acids 335 and 380, and mutant enzymes were expressed using a vaccinia expression system. The mutant enzymes were purified and microinjected into quiescent NIH 3T3 cells to evaluate their mitogenic activity. A moderate induction of DNA synthesis occurred after injection of mutant PLC-gamma 1. This mitogenic activity was inhibited by an antibody (alpha E 8-4) that does not significantly inhibit PLC-gamma 1 enzyme activity, which indicates that something else has to be inhibited. Furthermore, the partial induction of DNA synthesis observed with mutant PLC-gamma 1 was increased to levels seen with wild-type PLC-gamma 1 by coinjection of mutant PLC-gamma 1 with two second messengers, diacylglycerol and inositol trisphosphate. These results suggest that the mitogenic activity of PLC-gamma 1 does not exclusively result from the enzymatic activity of the lipase and that another activity inherent to the PLC-gamma 1 molecule can also induce DNA synthesis in quiescent cells.

An Amino-Terminal Fragment of SV40 T Antigen Induces Cellular DNA Synthesis in Quiescent Rat Cells

SV40 large T antigen can stimulate cellular DNA synthesis in quiescent cells. Here we report that an SV40 mutant, which expresses only the amino-terminal 147 amino acids of this protein and which does not make small t antigen, stimulates cellular DNA synthesis in quiescent rat cells at levels similar to those achieved with serum stimulation. Thus, the amino-terminal 147 amino acids of large T antigen define a domain of this protein that is sufficient for efficient stimulation of cellular DNA synthesis in the absence of small t antigen.