©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stimulation of Human Insulin Receptor Gene Expression by Retinoblastoma Gene Product (*)

(Received for publication, May 19, 1995; and in revised form, July 6, 1995)

Wen-jun Shen Haeyoung S. Kim Sophia Y. Tsai (§)

From the Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Multiple cis-acting elements have been defined to be important for the transcriptional regulation of the human insulin receptor (hIR) gene expression. We report here that one of these elements also mediated the stimulation of hIR promoter activity by the retinoblastoma gene product (Rb). The cis-element responsible for Rb stimulation was localized to the GA and GC boxes situated between -643 to -607 of the hIR gene. We have previously demonstrated that these GA and GC boxes bind Sp1 with high affinity and are responsible for E1a activation of hIR promoter activity. Mutation of these sequences completely abolished Rb-dependent enhancement of hIR promoter activity. In addition, we localized three regions in the N-terminal domain of Rb to be involved in stimulation of hIR promoter activity. Our results represent one of the first studies to demonstrate a functional importance assigned to the multiple phosphorylation sites in the N terminus of Rb. Finally, the mechanism by which Rb activates the hIR promoter are presented.


INTRODUCTION

Insulin plays an important role in the regulation of cellular metabolism in addition to stimulating the growth and proliferation of cells. It binds to the insulin receptor with high affinity and exerts its effects by stimulating glucose, ion, and amino acid influx, and by modifying rate-limiting enzymes involved in glucose, lipid, and protein metabolism (Straus, 1984). Upon binding insulin, the insulin receptor phosphorylates multiple tyrosine residues of insulin receptor substrate-1 (IRS-1), (^1)which then binds to various SH2 domain-containing signal transduction proteins. The tyrosine kinase activity of the insulin receptor is tightly associated with its biological activity (for reviews, see Czech(1985), Kahn (1985), and Rosen(1987)).

The human insulin receptor (hIR) gene has been isolated and characterized by different groups (Seino et al., 1989; Lee et al., 1992; Araki et al., 1987; McKeon et al., 1990; Tewari et al., 1989), and its promoter is located within 700 bp upstream of the ATG codon. Multiple transcription initiation sites were mapped within the GC-rich region of the hIR promoter (Seino et al., 1989; Tewari et al., 1989). Like other housekeeping genes, the hIR promoter lacks the TATA and CAAT boxes. It is extremely GC-rich and contains multiple GC boxes that are binding sites for the mammalian transcription factor Sp1 (Lee et al., 1992; Araki et al., 1991; McKeon et al., 1990; Kadonaga et al., 1986). There are two GA and four overlapping GC boxes at -643 to -594, and these have been shown to be important for the expression of the hIR gene (Lee et al., 1992). Mutations of the GC and GA boxes markedly reduced the transcription of the hIR gene. Gel-shift analyses showed that Sp1 binds to the cluster of four GC boxes located from -593 to -618 (Lee et al., 1992; Araki et al., 1991; Cameron et al., 1992). In addition to the GC boxes, binding sites for two novel factors, insulin receptor nuclear factors; I and II (IRNF-I and IRNF-II), have also been demonstrated to bind to the -530 to -550 and the -500 to -520 regions of the hIR promoter, respectively. Mutations that abolished IRNF-I and IRNF-II binding greatly reduced the expression of the hIR gene, indicating that both factors are important for the hIR gene expression (Lee et al., 1992). Recently, our group has demonstrated that the E1a adenoviral oncoprotein can also transactivate hIR, and this is mediated through the Sp1-binding sites (Kim et al., 1994). Loss of binding activity of Sp1 to the GA and GC boxes located between -643 to -607 results in reduction of the basal activity and the loss of E1a inducibility of the hIR gene.

Sp1 is a GC box-specific binding protein, which activates the transcription of many viral and cellular genes, including the SV40, epidermal growth factor receptor, insulin-like growth factor binding protein-2, and transforming growth factor-beta1 gene as well as the hIR gene (Lee et al., 1992; Dynan and Tjian, 1983; Xu et al., 1993; Boisclair et al., 1993; Kim et al., 1991). The structure and function of Sp1 have been extensively characterized. The DNA-binding domain of Sp1 is located in the C terminus and consists of three C(2)H(2) type zinc-finger motifs which bind to the GC boxes (Hoey et al., 1993; Kadonaga et al., 1986). There are two glutamine rich activation domains, A and B, which have transcriptional activity (Courey et al., 1989). These activation domains interact with the TFIID complex which consists of the TATA-binding protein (TBP) and the multiple TBP-associated factors (TAFIIs) (Gill and Tjian, 1992). The gene encoding TAFII-110 has been cloned and shown to interact with the A and B domains of Sp1 to mediate Sp1-dependent transcription (Hoey et al., 1993).

Rb has been shown to regulate cell cycle progression and repress cell growth and differentiation (Bookstein et al., 1990; Horowitz et al., 1990). It has been demonstrated that Rb supresses growth related gene expression by directly inactivate transcription factors, including E2F, ATF-2, and Elf-1 (Bagchi et al., 1991; Chellappan et al., 1991; Wang et al., 1993; Kim et al., 1992a, 1992b). Hypophosphorylated forms of Rb have been shown to interact with E2F and its related family members through the conserved Rb pocket (Buchkovich et al., 1989; Chen et al., 1989; Pietenpol et al., 1990), and Rb-E2F interaction inhibits the transactivation function of E2F. Hyperphosphorylation of Rb by cyclin-cdk complexes, however, cause Rb to release E2F which is then free to transactivate its target gene (Bagchi et al., 1991; Chellappan et al., 1991; Dynlacht et al., 1994). Alternatively, Rb regulates cellular proliferation and cell cycle control by activating cellular genes, including c-fos, TGF-beta1, and c-jun through a conserved cis-activating element, termed the retinoblastoma control element (RCE). RCE are GC-rich sequences, present in the promoter of the above genes. Sp1 binds to and stimulates transcription through the RCE motif (Kim et al. 1991, 1992a, 1992b; Pietenpol et al., 1991; Robbins et al., 1990; Yu et al., 1992; Chen et al., 1994). A protease sensitive Sp1 negative regulator (Sp1-I), which inhibits Sp1 binding to the Sp1 binding site of the c-jun promoter, has recently been identified (Chen et al., 1994). The inhibition of Sp1 binding by Sp1-I was reversed by the addition of bacterially expressed recombinant Rb, suggesting that Rb may sequester Sp1-I and release Sp1 from its inhibitor. The functional domain of Rb which interacts with the putative Sp1-I has not yet been defined.

Insulin interacts with the insulin receptor to exert its role in long term cell proliferation and growth. Since Sp1 has been shown to be important for the regulation of transcription of the hIR gene and Rb has been shown to be able to regulate cell growth through either Sp1 binding to the RCE elements or via the E2F pathway, we investigated whether Rb can regulate hIR gene expression in a human hepatoma cell line, HepG2. Our results demonstrate that Rb stimulates the expression of the hIR gene through Sp1 binding sites in the promoter. In addition overexpression of Sp1 can also augment hIR promoter activity, suggesting Sp1 can substitute for Rb to stimulate the hIR gene expression. Our results are consistent with the hypothesis that Rb can sequester Sp1-I, releasing Sp1 from the inhibitory factor Sp1-I, and allowing Sp1 to activate the hIR promoter activity. Finally and most importantly, we have defined the N-terminal domain of the Rb molecule responsible for the stimulation of the hIR gene promoter activity. This represents one of the first demonstrations of a functional role for these N-terminal regions of Rb.


MATERIALS AND METHODS

Plasmids

The wild type and 5`-deletion plasmids phIRCAT-1819, -873, -772, -643, -607, -574, and the linker-scanning mutants, phIRCAT-873 (LSGA, LSGC, and LSGAGC) have been described previously (Lee et al., 1992; Kim et al., 1994). The pCMV-Sp1 and pCMV-Sp1(N539) were constructed by inserting a pair of oligonucleotides 5`-AGCTTATGC-3` and 5`-TCGAGCATA-3` into the XhoI fragment of pPACSp1 and pPACSp1(N539) (Gift from Dr. R. Tjian), and subcloned into the HindIII site of pCMV4 (Andersson et al., 1989). The pCMV-Rb expression vector was described previously (Qin et al., 1992).

Transfections and CAT Assays

The human hepatoma cell line, HepG2, was maintained in 5% CO(2) as a monolayer culture in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 µg/ml penicillin, and 100 µg/ml streptomycin. Transient transfections were carried out by using 5 µg of the reporter and 2 µg of expression plasmids by the standard calcium phosphate precipitation method (Walker et al., 1983). The total amount of DNA used for transfections were kept constant by the addition of plasmid backbone as control. Cells were harvested 44-48 h. after transfection and lysed by three freeze and thaw cycles. Protein concentrations of cell extracts were quantified by the standard Bradford assay (Bradford, 1976), and 10-50 µg of protein were analyzed for CAT activity by TLC (Lee et al., 1992) or phase extraction methods (Seed et al., 1988). In the case of the TLC assays, the plates were counted by blot analyzer (Betascope 603, betaetagen), and CAT activity was calculated as percent conversion of the substrate. Three independent transfection assays were carried out for each experiment, and the fold of stimulation represents the inducibility of hIR promoter activity.


RESULTS

Rb Stimulates the Expression of the hIR Gene

Rb is believed to regulate cell growth through two different pathways: via direct binding to transcription factors, such as E2F or through GC-rich DNA sequences. Sequence analysis of the hIR promoter indicated the existence of E2F as well as Sp1 binding sites. To investigate whether Rb regulates hIR gene expression, a construct containing the hIR promoter fused to a bacterial CAT reporter (hIR-1819) was co-transfected into HepG2 cells in the presence or absence of Rb. As indicated in Fig. 1, the promoter activity of the hIR-linked reporter is increased about 5.5-fold in the presence of Rb (lane 1 versus lane 2). As a control, the HepG2 cells were transfected with the pSV2CAT plasmid and the CAT activity is unaltered in the presence or absence of Rb (lanes 3 and 4). These results indicate that Rb enhances hIR gene expression.


Figure 1: Stimulation of hIR promoter by Rb. HepG2 cells were transfected with 5 µg of reporter constructs, phIRCAT-1819 or pSV2CAT, with or without expression vector pCMV-Rb (2 µg). Cells were harvested at 44 h. after transfection, and CAT activity assayed by TLC.



Localization of the Rb Response Elements in the hIR Promoter

To map the regions conferring Rb activation on the hIR gene, various 5`-deletion mutants of the hIR promoter fused to the CAT reporter were transfected in the presence or absence of the Rb expression vector. As summarized in Fig. 2, the construct containing a deletion to -873 has a similar fold of induction of CAT activity as that of the wild type. Further deletion to -772 only had a moderate effect on the inducibility by Rb. The deletion construct -643 showed decreased Rb-dependent stimulation of hIR gene expression, and further deletion to -607 had a dramatic effect on the inducibility by Rb. The deletion constructs -607 and -574, which still contain the putative E2F binding sites, showed no stimulation of the hIR gene expression in the presence of Rb. These results clearly indicate that deletion of the sequences upstream of -643 has no significant effect on Rb inducibility of the hIR promoter, however, deletion to -607 completely abolishes Rb-dependent stimulation of the hIR promoter. This suggests that the sequences between -643 to -607, which harbor two GA and three GC boxes, are important for the Rb-dependent regulation of the hIR gene.


Figure 2: Localization of the Rb-response elements in the hIR promoter. The indicated 5`-deletion constructs of hIR promoter were co-transfected with or without expression vector, pCMV-Rb, into HepG2 cells. Cells were harvested 44 h. after transfection, and assayed for CAT activity. Data shown here is a summary of three independent transfection experiments. Fold of stimulation represents Rb-dependent hIR-reporter induction.



Use of Linker-scanning Mutants to Further Define the GA, GC Boxes as the Rb Response Element

To further define the Rb responsive element between -643 and -607, linker-scanning mutants which have mutations in the GA boxes (LSGA), the GC boxes (LSGC), or both the GA and GC boxes (LSGAGC), were used in the transfection of HepG2 cells in the presence or the absence of Rb. As shown in Fig. 3, although the mutation in the GA boxes (LSGA) reduces the stimulation of the hIR gene expression by Rb, the mutation in the GC boxes (LSGC) has a much greater decrease in the Rb-dependent stimulation of hIR gene expression. Similar mutation with both GA and GC boxes mutated (LSGAGC) also abolishes Rb inducibility. These results indicate that the GC boxes between -643 and -607 are essential for the stimulation of the hIR promoter by Rb.


Figure 3: Effect of the GA and GC box mutation on Rb stimulation. The wild type phIR CAT-873 or the linker scanning constructs with mutations in GA and GC boxes were co-transfected with or without expression vector pCMV-Rb into HepG2 cells. Cells were harvested 44 h after transfection and assayed for CAT activity. Data shown here is a summary of three independent transfection experiments. Fold of stimulation represents Rb-dependent hIR-reporter induction.



N-terminal Region of Rb Is Important for the Stimulation of hIR Promoter Activity

Having identified the sequences within hIR promoter that are important for the Rb stimulation, we carried out experiments to identify the region within Rb that are important for this stimulation. For this purpose, we carried out cotransfection experiments using a series of mutants which have deletions within various parts of the Rb sequence (Qian et al., 1992). As shown in Fig. 4, deletion of 52 amino acids at the N-terminal end of Rb (between AA 37 and 89) completely abolishes the stimulation of hIR promoter activity by Rb. Deletion of another adjacent 51 amino acids (between amino acids 89 and 140), also significantly reduces the stimulation of hIR promoter activity by Rb. Similarly, deletion of the 46 amino acids immediately N-terminal to the A pocket (between amino acids 343 and 389), reduces the ability of Rb to stimulate the hIR promoter. In addition, deletion of the sequences between the A and B pockets (between amino acids 580 and 614), results in decreased stimulation of hIR promoter activity by Rb. In contrast, mutations in the region within the A or B pocket domain (between amino acids 389-580 and 614-775), or the C-terminal region of Rb (between amino acids 775 and 892), do not decrease the ability of Rb to stimulate the hIR promoter activity. These results indicate that the N-terminal region of Rb are important for its stimulation of the hIR promoter. In contrast, the A, B pocket domains and the C-terminal region are not essential for this stimulation. Since the A and B domains and C-terminal region of Rb are important for its interaction with E2F (Qian et al., 1992), these results are consistent with our previous observation that stimulation of hIR promoter activity by Rb is not mediated through E2F.


Figure 4: Stimulation of hIR promoter by Rb mutants. Reporter construct phIRCAT-1819 were cotransfected with or without different Rb deletion mutants into HepG2 cells. These mutants are gifts of Dr. D. J. Templeton (Qian et al., 1992). The deletion end points are shown in the Fig. Cells were harvested 44 h after transfection and assayed for CAT activity. A and B represent the pocket domains of Rb which are important for T-antigen and E1a viral protein binding. Fold of stimulation represents Rb dependent increase of hIR-reporter activity. Three separate experiments were carried out with duplicates, and the results are expressed as means ± S.D. Statistical significance were determined by performing analysis of variance using Fisher's post-hoc test. Fold of stimulation obtained with d1, d2, d7, and d10 are significantly lower than the fold of stimulation obtained with the wild type Rb (p = 0.005).



Overexpression of Sp1 Enhances hIR Gene Expression

We and others have previously shown that Sp1 binds to the GA and GC boxes between -643 and -607 of the hIR promoter (Lee et al., 1992; Araki et al. 1991). In addition, we have also demonstrated that these GA and GC boxes are required for the efficient expression of the hIR gene. Since these Sp1 binding sites were required for Rb-dependent stimulation, we asked whether Sp1 can substitute for Rb stimulation. To investigate this, a Sp1 expression vector, pCMV-Sp1, was co-transfected together with the hIR promoter-linked CAT construct phIRCAT-873 or the linker-scanning constructs with mutations in the GA and GC boxes into HepG2 cells. As shown in Fig. 5, Sp1 also stimulates the expression of the wild type hIR promoter (Fig. 5, lane 1 compared to lane 2), and the level of stimulation is similar to that seen with Rb (lane 3 compared to lane 2). Analogous to the stimulation exerted by Rb, mutation of the GA boxes (LSGA) does not significantly reduce the stimulation of the hIR gene expression by Sp1 (lanes 4, 5, and 6), but mutation of the GC boxes (LSGC) greatly decreases the stimulation of hIR gene expression by Sp1 (lanes 7, 8, and 9). Mutation of both the GA and GC boxes (LSGAGC) also completely eliminates the stimulation by Sp1 (lanes 10, 11, and 12). These results indicate that the GC box binding sites of Sp1 are essential for both the Sp1- and Rb-dependent activation of the hIR promoter.


Figure 5: Stimulation of hIR promoter by Sp1 and Rb. 2 µg of either Rb or Sp1 expression vector, pCMV-Rb or pCMV-Sp1, were co-transfected with 5 µg of the wild type phIR CAT-873 or the linker scanning constructs with mutations in GA and GC boxes into HepG2 cells. PCMV control plasmid have been used in the control co-transfection with hIR constructs.



Sp1 and Rb Stimulate hIR Gene Expression through a Similar Pathway

To investigate whether Rb and Sp1 stimulate the expression of the hIR gene through the same or different pathways, we titrated the Sp1-enhancement of the hIR promoter activity. For this purpose, increasing amounts of the Sp1 expression vector, pCMV-Sp1 were co-transfected into HepG2 cells with a constant amount of the hIR reporter construct. Fig. 6a (bullet) shows that the CAT reporter activities increase proportionally to the added amount of Sp1 expression vector until it reaches a saturating level at 2 µg. A similar experiment was carried out with increasing amounts of the Rb expression vector, and as shown in Fig. 6b (bullet), the CAT reporter activities also increase according to the amount of Rb expression vector added until a saturating level of Rb was used. We then asked whether co-expression of either Sp1 or Rb in the presence of saturating amounts of Rb or Sp1 respectively will further increase the hIR promoter activity. As illustrated in Fig. 6a (), the addition of Sp1 at saturating amounts of Rb showed no further increase in the hIR expression. It is the same with co-expression of Rb at saturating level of Sp1 (6b ), indicating that the enhancement of hIR promoter activity by Rb and Sp1 is likely mediated through the same pathway.


Figure 6: a, dose-dependent activation of hIR by Sp1. Reporter construct phIRCAT-1819 was co-transfected with increasing amount of expression vector pCMV-Sp1 into HepG2 cells in the presence () or absence (bullet) of pCMV-Rb. b, dose-dependent activation of hIR by Rb. Reporter construct phIRCAT-1819 was co-transfected with increasing amount of expression vector pCMV-Rb into HepG2 cells in the presence () or absence (bullet) of pCMV-Sp1.




DISCUSSION

Rb is a 110-kDa nuclear protein that is modified by cell cycle-regulated hyperphosphorylations (Bandara et al., 1991; Buchkovich et al., 1989; Chen et al., 1989). Mutations in the RB-1 gene have been observed in a wide variety of tumors including retinoblastoma, osteosarcomas, bladder carcinomas, small-cell lung carcinomas, prostate carcinomas, and cervical carcinomas. The wide variety of tumors carrying mutated RB-1 genes suggest that Rb has an important role in the regulation of normal cell proliferation (for review, see Lees et al.(1991), Weinberg (1992), and Horowitz et al.(1990)). It has also been reported that functional replacement of the RB-1 gene can suppress tumorigenic phenotypes and inhibit growth in proliferating cells (Huang et al., 1991; Bookstein et al., 1990; Sumegi et al., 1990; Takahashi et al., 1991; Templeton et al., 1991). Rb has been shown to be able to complex with the oncoproteins encoded by several DNA tumor viruses, including adenovirus E1a, simian virus 40 T antigen, and human papillomavirus 7 (DeCaprio et al., 1989; Dyson et al., 1989; Whyte et al., 1988). Recently, Rb has also been shown to bind to the cellular E2F transcription factor, and repress E2F-mediated gene expression thereby suppressing cellular proliferation. The cell cycle regulated hyper-phosphorylated form of Rb, however, can no longer associate with E2F, and thereby loses the ability to repress E2F-regulated transactivation (Bagchi et al., 1991; Dynlacht et al., 1994; Buchkovich et al., 1989; Chen et al., 1989; Pietenpol et al., 1990). These results suggested that Rb has a very important role in regulating cell cycle progression and in repressing cell growth and differentiation.

Another target of Rb is the Sp1 family of transcription factors which binds to the RCE and activate RCE containing genes. Robbins et al.(1990) demonstrated that in the c-fos promoter, Rb can repress the transcription of c-fos through the RCE and reduce the AP-1 stimulatory activity. Similarly, Kim et al. (1992a) showed that RCE-like sequences are important for positive regulation of insulin-like growth factor II (IGF-II) gene by human Rb. Furthermore, they have shown that the TGF-beta1 and c-fos promoters can both be positively and negatively regulated by Rb through the same RCE element in different cell types (Kim et al., 1992b). Subsequently, they demonstrated that Rb regulates the expression of IGF-II through Sp1 (Kim et al., 1992b), and that Sp1 binding to DNA is not required, since a GAL4-Sp1 fusion protein can also confer the transcriptional regulation by Rb.

We have shown here that Rb can stimulate the expression of the hIR gene by 5-6-fold. Rb-dependent stimulation is mediated through sequences between -643 and -607, which contains two GA and three GC boxes through which Sp1 binds. By either deleting the GC-rich sequences between -643 and -607 or mutating the GC boxes for Sp1 binding in this region, we have demonstrated that the Rb-dependent stimulation is eliminated. Our results indicate that the Rb-dependent stimulation of the hIR promoter is mediated through Sp1-binding sites. These results are similar to those observed with the c-jun and TGF-beta1 promoters (Kim et al., 1991; 1992a; Chen et al., 1994).

Recently, Chen et al.(1994) showed that Rb can stimulate the c-jun promoter activity. A 20-kDa protein has been implicated to have the ability to prevent the binding of Sp1 to DNA. This Sp1 inhibitor (Sp1-I) is a heat-labile and proteinase K-sensitive protein, which presumably interacts with both Sp1 and Rb. When it binds to Sp1, it prevents Sp1 from binding to DNA, hence inhibiting the ability of Sp1 to transactivate target genes. In its presence, Rb competes with Sp1 for the Sp1-I binding, thus releasing Sp1 and allowing it to bind to the GC boxes and transactivate target genes. For the hIR, we have not been able to detect the direct association of Rb with Sp1 by immunoprecipitation and supershifts in gel mobility assays, suggesting that Rb stimulation of the hIR promoter is not through direct interaction with Sp1.^2 Therefore, it is likely that Sp1-I, similar to the c-jun system (Chen et al., 1994), interacts with Rb and releases Sp1 for the hIR gene stimulation. If this is the case, overexpression of Sp1 should be able to replace Rb in the hIR promoter stimulation. Indeed as shown in Fig. 5, overexpression of Sp1 could substitute for Rb as it enhances the hIR expression to a similar extent as the Rb-dependent activation. This is consistent with the notion that Rb sequesters Sp1-I and releases Sp1 from its inhibition, leading to the activation of the hIR promoter activity. This conclusion is further supported by the failure of Rb to further stimulate the hIR promoter activity at a saturating amount of Sp1 (Fig. 6). Thus, the enhancement of hIR promoter activity by Rb and Sp1 is likely mediated through the same pathway; and the role of Rb is to sequester Sp1-I, removing it from inhibition of the activity of Sp1.

Rb contains multiple functional domains, including two discontiguous regions, the A and B pockets, and a N- and a C-terminal domains. The A and B pockets, located at amino acids 389-580 and 614-775, are required for interactions with DNA tumor virus antigens, E1a, and transcription factor E2F (Qian et al. 1992; Hu et al., 1990; Kaelin, et al., 1990). The N-terminal domain consists of three regions which contain kinase recognition sites (Qian et al., 1992; Qin et al., 1992) and has been proposed to be important for hyperphosphorylation of Rb (Qian et al., 1992). Although this domain has been implicated to be involved in growth suppression, a biological function for this region has not been well defined (Karantza et al., 1993; Hogg et al. 1993; Dryja et al., 1993). We have shown here that the N-terminal regions (amino acids 37-89, 89-140, and 343-389) are important for stimulation of hIR promoter activity. In addition, the sequences between the A and B pockets (amino acids 580-614) which contains putative phosphorylation sites are also important for the stimulation. In contrast, the A and B pockets, which are important for interaction with E2F are not essential for activation of hIR promoter activity. These results are consistent with the hypothesis that the Rb stimulation of hIR is mediated through sequestering Sp1-I rather than binding to E2F. Most importantly, we have defined the N-terminal region of Rb to be involved in activation of hIR promoter activity. This represents one of the first assigned biological function for this region.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK-44988 (to S. Y. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798- 6251; Fax: 713-798-8227.

(^1)
The abbreviations used are: IRS-1, insulin receptor substrate-1; CAT, chloramphenicol acetyltransferase; SH2, src-homology 2; kb, kilobase(s); bp, base pair(s); hIR, human insulin receptor; Rb, retinoblastoma gene product; TBP, TATA-binding protein; RCE, retinoblastoma control element; IGF-II, insulin-like growth factor II.

(^2)
W.-J. Shen and S. Y. Tsai, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. R. Tjian (Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California at Berkeley) for the gift of constructs pPacSp1 and pPacSp1(N539), Dr. K. Helin and Dr. Ed Harlow (Laboratory of Molecular Oncology, Massachusetts General Hospital, Harvard Medical School) for the gift of pRB, Dr. D. Templeton (Institute of Pathology, Case Western Reserve University School of Medicine, University Hospital of Cleveland) for the Rb deletion mutants. We also thank members of the Dr. Tsai's laboratory and Dr. A. Cooney for critical comments.


REFERENCES

  1. Andersson, S., Davis, D. L., Dahlback, H., Jornvall, H., and Russell, D. W. (1989) J. Biol. Chem. 264,8222-8229 [Abstract/Free Full Text]
  2. Araki, E., Shimada, F., Uzawa, H., Mori, T., and Ebina, Y. (1987) J. Biol. Chem. 262,16186-16191 [Abstract/Free Full Text]
  3. Araki, E., Murakami, T., Shirotani, T., Kanai, F., Shinohara, Y., Shimada, F., Mori, M., Shichiri, M., and Ebina, Y. (1991) J. Biol. Chem. 266,3944-3948 [Abstract/Free Full Text]
  4. Bagchi, S., Weinmann, R., and Raychaudhuri, P. (1991) Cell 65,1063-1072 [Medline] [Order article via Infotrieve]
  5. Bandara, L. R., Adamczewski, J. P., Hunt, T., and La Thangue. N. B. (1991) Nature 352,249-251 [CrossRef][Medline] [Order article via Infotrieve]
  6. Boisclair, Y. R., Brown, A. L., Casola, S., and Rechler, M. M. (1993) J. Biol. Chem 268,24892-24901 [Abstract/Free Full Text]
  7. Bookstein, R., Shew, J., Chen, P., Scully, P., and Lee, W. H. (1990) Science 247,712-715 [Medline] [Order article via Infotrieve]
  8. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 [CrossRef][Medline] [Order article via Infotrieve]
  9. Buchkovich, K., Duffy, L. A., and Harlow, E. (1989) Cell 58,1097-1105 [Medline] [Order article via Infotrieve]
  10. Cameron, K. E., Resnik, J., and Webster, N. J. G. (1992) J. Biol. Chem. 267,17375-17383 [Abstract/Free Full Text]
  11. Chellappan, S. P., Hiebert, S., Mudryj, M., Horowitz, J. M., and Nevins, J. R. (1991) Cell 65,1053-1061 [Medline] [Order article via Infotrieve]
  12. Chen, L. I., Nishinaka, T., Kwan, K., Kitabayashi, I., Yokoyama, K., Fu, Y. H., Grunwald, S., and Chiu, R. (1994) Mol. Cell. Biol. 14,4380-4389 [Abstract]
  13. Chen, P. L., Scully, P., Shew, J. Y., Wang, J. Y., and Lee, W. H. (1989) Cell 58,1193-1198 [Medline] [Order article via Infotrieve]
  14. Courey, A. J., Holtzman, D. A., Jackson, S. P., and Tjian, R. (1989) Cell 59,827-836 [Medline] [Order article via Infotrieve]
  15. Cuatrecasas, P. (1972) J. Biol. Chem. 247,1980-1991 [Abstract/Free Full Text]
  16. Czech, M. P. (1985) Annu. Rev. Physiol. 47,357-381 [CrossRef][Medline] [Order article via Infotrieve]
  17. DeCaprio, J. A., Ludlow, J. W., Lynch, D., Furukawa, Y., Griffin, J., Piwnica-Worms, H., Huang, C.-H., and Livingston, D. M. (1989) Cell 58,1085-1095 [Medline] [Order article via Infotrieve]
  18. Dryja, T. P., Rapaport, J., Mcgee, T. L., Nork, T. M., and Schwartz, T. L. (1993) Am. J. Hum. Genet. 52,1122-1128 [Medline] [Order article via Infotrieve]
  19. Dynan, W. S., and Tjian, R. (1983) Cell 35,79-87 [Medline] [Order article via Infotrieve]
  20. Dynlacht, B. D., Flores, O., Lees, J. A., and Harlow, E. (1994) Genes & Dev. 8,1772-1786
  21. Dyson, N., Buchkovich, K., Whyte, P., and Harlow, E. (1989) Cell 58,249-255 [Medline] [Order article via Infotrieve]
  22. Gill, G., and Tjian, R., (1992) Curr. Opin. Cell Biol. 2,236-242
  23. Hoey, T., Weinzierl, R. O., Gill, G., Chen, J. L., Dynlacht, B. D., and Tjian, R. (1993) Cell 72,247-260 [Medline] [Order article via Infotrieve]
  24. Hogg, A., Bia, B., Onadim, Z., and Coweil, J. K. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,7351-7355 [Abstract]
  25. Horowitz, J. M., Park, S. H., Bogenmann, E., Cheng, J. C., Yandefl, D. W., Kaye, F. J., Minna, J. D., Dryja, T. P., and Weinberg, R. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,2775-2779 [Abstract]
  26. Hu, Q. J., Dyson, N., and Harlow, E. (1990) EMBO J. 9,1147-1155 [Abstract]
  27. Huang, S., Lee, W.-H., and Lee, E. Y.-H. P. (1991) Nature 350,160-162 [CrossRef][Medline] [Order article via Infotrieve]
  28. Kadonaga, J. T., Jones, K. A., and Tjian, R. (1986) Trends Biochem. Sci. 11,20-23 [CrossRef]
  29. Kaelin, W. G., Ewen, M. E., and Livingston, D. M. (1990) Mol. Cell. Biol. 10,3761-3769 [Medline] [Order article via Infotrieve]
  30. Kahn, C. R. (1985) Annu. Rev. Med. 36,429-451 [CrossRef][Medline] [Order article via Infotrieve]
  31. Karantza, V., Maroo, A., Fay, D., and Sedicy, J. M. (1993) Mol. Cell. Biol. 13,6640-6652 [Abstract]
  32. Kim, H. S., Lee, J.-K., and Tsai, S. Y. (1994) Mol. Endocrinol. 9,178-186 [Abstract]
  33. Kim, S. J., Lee, H. D., Robbins, P. D., Busatn, K., Spom, M. B., and Roberts, A. B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,3052-3056 [Abstract]
  34. Kim, S. J., Onwuta, U. S., Lee, Y. I., Li, R., Botchan, M. R., and Robbins, P. D. (1992a) Mol. Cell. Biol. 12,2455-2463 [Abstract]
  35. Kim, S. J., Wagner, S., Liu, F., O'Reilly, M. A., Robbins, P. D., and Green, M. R. (1992b) Nature 358,331-334 [CrossRef][Medline] [Order article via Infotrieve]
  36. Kolterman, O. G., Gray, R. S., Griffin, J., Burstein, P., Insel, J., Scarlett, A., and Olefsky, J. M. (1981) J. Clin. Invest. 68,957-969 [Medline] [Order article via Infotrieve]
  37. Lee, J.-K., Tam, J. W., Tsai, M.-J., and Tsai, S. Y. (1992) J. Biol. Chem. 267,4638-4645 [Abstract/Free Full Text]
  38. Lees, J. A., Buchkovich, K. J., Marshak, D. R., Anderson, C. W., and Harlow, E. (1991) EMBO J. 10,4279-4290 [Abstract]
  39. McKeon, C., and Pham, T. (1991) Biochem. Biophys. Res. Commun. 174,721-728 [Medline] [Order article via Infotrieve]
  40. McKeon, C., Moncada, V., Pham, T., Salvatore, P., Kadowaki, T., Accili, D., and Taylor, S. I. (1990) Mol. Endocrinol. 4,647-656 [Abstract]
  41. Pietenpol, J. A., Stein, R. W., Moran, E., Yaciuk, P., Schlegel, R., Lyons, R. M., Pittelkow, M. R., Munger, K., Howley, P. M., and Moses, H. L. (1990) Cell 61,777-785 [Medline] [Order article via Infotrieve]
  42. Qian, Y.-Y., Luckey, C., Horton, L., Esser, M., and Templeton, D. J. (1992) Mol. Cell. Biol. 12,5363-5372 [Abstract]
  43. Qin, X.-Q., Chittenden, T., Livingston, D., and Kaelin, W. G. (1992) Genes & Dev. 6,953-964
  44. Robbins, P. D., Horowitz, J. M., and Mulligan, R. C. (1990) Nature 346,668-671 [CrossRef][Medline] [Order article via Infotrieve]
  45. Rosen, O. M. (1987) Science 237,1452-1458 [Medline] [Order article via Infotrieve]
  46. Seed, B., and Sheen, J. Y. (1988) Gene (Amst.) 67,271-277 [CrossRef][Medline] [Order article via Infotrieve]
  47. Seino, S., Seino, M., Neshi, S., and Bell, G. I. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,114-118 [Abstract]
  48. Straus, D. S. (1984) Endocr. Rev. 5,356-369 [Medline] [Order article via Infotrieve]
  49. Sumegi, J., Uzvolgyi, E., and Klein, G. (1990) Cell Growth & Differ. 2,247-250
  50. Takahashi, R., Hashimoto, T., Xu, H.-J., Hu, S.-X., Maatsui, T., Miki, T., Bigo-Marshall, H., Saronson, S. A., and Benedict, W. F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,5257-5261 [Abstract]
  51. Templeton, D. J., Park, S. H., Lanier, L., and Weinberg, R. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,3033-3037 [Abstract]
  52. Tewari, D. S., Cook, D. M., and Taub, R. (1989) J. Biol. Chem. 264,16238-16245 [Abstract/Free Full Text]
  53. Walker, M. D., Edlund, T., Boulet, A. M., and Rutter, W. J. (1983) Nature 306,557-561 [Medline] [Order article via Infotrieve]
  54. Wang, P. N., To, H., Lee, W. H., and Lee, E. Y. H. (1993) Oncogene 8,279-288 [Medline] [Order article via Infotrieve]
  55. Weinberg, R. A. (1992) Cancer Surv. 12,43-57 [Medline] [Order article via Infotrieve]
  56. Whyte, P., Buchkovich, K. J., Horowitz, J. M., Friend, S. H., Raybuck, M., Weinberg, R. A., and Harlow, E. (1988) Nature 34,471-475
  57. Xu, J., Thompson, K. L., Shephard, L. B., Hudson, L. G., and Gir, G. N. (1993) J. Biol. Chem. 268,16065-16073 [Abstract/Free Full Text]
  58. Yu, D., Matin, A., and Hung, M.-C. (1992) J. Biol. Chem. 267,10203-10206 [Abstract/Free Full Text]

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