Michael Gilman

Controlled Dimerization of ErbB Receptors Provides Evidence for Differential Signaling by Homo- and Heterodimers

ABSTRACT The four members of the ErbB family of receptor tyrosine kinases are involved in a complex array of combinatorial interactions involving homo- and heterodimers. Since most cell types express more than one member of the ErbB family, it is difficult to distinguish the biological activities of different homo- and heterodimers. Here we describe a method for inducing homo- or heterodimerization of ErbB receptors by using synthetic ligands without interference from the endogenous receptors. ErbB receptor chimeras containing synthetic ligand binding domains (FK506-binding protein [FKBP] or FKBP-rapamycin-binding domain [FRB]) were homodimerized with the bivalent FKBP ligand AP1510 and heterodimerized with the bifunctional FKBP-FRB ligand rapamycin. AP1510 treatment induced tyrosine phosphorylation of ErbB1 and ErbB2 homodimers and recruitment of Src homology 2 domain-containing proteins (Shc and Grb2). In addition, ErbB1 and ErbB2 homodimers activated downstream signaling pathways leading to Erk2 and Akt phosphorylation. However, only ErbB1 homodimers were internalized upon AP1510 stimulation, and only ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to form foci; however, cells expressing ErbB2 homodimers displayed a five- to sevenfold higher focus-forming ability. Using rapamycin-inducible heterodimerization we show that c-Cbl is unable to associate with ErbB1 in a ErbB1-ErbB2 heterodimer most likely because ErbB2 is unable to phosphorylate the c-Cbl binding site on ErbB1. Thus, we demonstrate that ErbB1 and ErbB2 homodimers differ in their abilities to transform fibroblasts and provide evidence for differential signaling by ErbB homodimers and heterodimers. These observations also validate the use of synthetic ligands to study the signaling and biological specificity of selected ErbB dimers in any cell type.

Human and Drosophila Homeodomain Proteins That Enhance the DNA-Binding Activity of Serum Response Factor

Cells with distinct developmental histories can respond differentially to identical signals, suggesting that signals are interpreted in a fashion that reflects a cells identity. How this might occur is suggested by the observation that proteins of the homeodomain family, including a newly identified human protein, enhance the DNA-binding activity of serum response factor, a protein required for the induction of genes by growth and differentiation factors. Interaction with proteins of the serum response factor family may allow homeodomain proteins to specify the transcriptional response to inductive signals. Moreover, because the ability to enhance the binding of serum response factor to DNA resides within the homeodomain but is independent of homeodomain DNA-binding activity, this additional activity of the homeodomain may account for some of the specificity of action of homeodomain proteins in development.

Multiple protein-binding sites in the 5'-flanking region regulate c-fos expression.

We tested sequences flanking the mouse c-fos gene for the ability to form specific DNA-protein complexes with factors present in crude nuclear extracts prepared from mammalian cells. Three such complexes were detected. One complex formed in a region necessary for the induction of c-fos expression by serum growth factors. Two additional complexes formed at sequences that contribute to basal c-fos promoter activity in vivo. These complexes represent three novel sequence-specific DNA-binding activities which appear to participate in the regulation of c-fos transcription.

Cell-free activation of a DNA-binding protein by epidermal growth factor

GROWTH factors such as platelet-derived growth factor and epidermal growth factor (EGF) bind to and activate cell-surface receptors with intrinsic tyrosine kinase activities1. Receptor activation elicits multiple physiological changes in target cells, including alterations in gene expression2–4. Receptor tyrosine kinase signalling involves recruitment of proteins into a signalling complex through interactions between receptor autophosphorylation sites and the src-homology region-2 (SH2) domains on these signalling proteins5–9. Diverse signals can subsequently be generated, depending on the specific receptor and cell type2,10. How such signals are transmitted to the nucleus is poorly understood, but because the transcriptional activation of many genes by growth factors occurs in the absence of new protein synthesis4, one or more signals emanating from growth factor receptors must directly affect transcription factors. We report here the activation by EGF of a DNA-binding protein in a cell-free system where activation of DNA binding requires ligand, receptor, ATP and phosphotyrosine–SH2 interactions.

Platelet-derived growth factor induces phosphorylation of multiple JAK family kinases and STAT proteins.

Receptors for interferons and other cytokines signal through the action of associated protein tyrosine kinases of the JAK family and latent cytoplasmic transcription factors of the STAT family. Genetic and biochemical analysis of interferon signaling indicates that activation of STATs by interferons requires two distinct JAK family kinases. Loss of either of the required JAKs prevents activation of the other JAK and extinguishes STAT activation. These observations suggest that JAKs provide interferon receptors with a critical catalytic signaling function and that at least two JAKs must be incorporated into an active receptor complex. JAK and STAT proteins are also activated by ligands such as platelet-derived growth factor (PDGF), which act through receptors that possess intrinsic protein tyrosine kinase activity, raising questions about the role of JAKs in signal transduction by this class of receptors. Here, we show that all three of the ubiquitously expressed JAKs--JAK1, JAK2, and Tyk2--become phosphorylated on tyrosine in both mouse BALB/c 3T3 cells and human fibroblasts engineered to express the PDGF-beta receptor. All three proteins are also associated with the activated receptor. Through the use of cell lines each lacking an individual JAK, we find that in contrast to interferon signaling, PDGF-induced JAK phosphorylation and activation of STAT1 and STAT3 is independent of the presence of any other single JAK but does require receptor tyrosine kinase activity. These results suggests that the mechanism of JAK activation and JAK function in signaling differs between receptor tyrosine kinases and interferon receptors.

Proteasome‐mediated degradation of transcriptional activators correlates with activation domain potency in vivo

We show that the intracellular concentration of transcriptional activator proteins is regulated by the proteasome‐mediated protein degradation pathway. The rate of degradation of activators by proteasomes correlates with activation domain potency in vivo. Mutations either in the activation domain residues involved in target protein interaction or in the DNA‐binding domain residues essential for DNA binding abolish the transcriptional activation function in vivo and render the activator resistant to degradation by proteasomes. Finally, using a rapamycin‐regulated gene expression system, we show that recruiting activation domains to DNA‐bound receptor proteins greatly enhanced the rate of degradation of reconstituted activators. These observations suggest that in mammalian cells efficient recruitment of activator–target protein complexes to the promoter means that they are subjected to rapid degradation by proteasomes. We propose that proteasome‐mediated control of the intracellular levels of transcriptional activators could play an important role in the regulation of gene expression.

Ribonuclease Protection Assay

Sequence‐specific hybridization probes of high specific activity are prepared by cloning the probe sequence downstream of a bacteriophage promoter. The plasmid is cleaved with a restriction enzyme, and the plasmid DNA is transcribed with bacteriophage RNA polymerase, which efficiently transcribes the cloned sequence into a discrete RNA species of known specific activity and high abundance. The RNA is purified by removal of the DNA template, protein, and the unincorporated label. Alternatively, the probe is purified by gel electrophoresis, as described in a support protocol. The probe RNA is hybridized to sample RNAs and the hybridization reactions are treated with ribonuclease to remove free probe, leaving intact fragments of probe annealed to homologous sequences in the sample RNA. These fragments are analyzed by electrophoresis on a sequencing gel and the presence of the target mRNA is revealed by the appearance of an appropriately sized fragment of the probe.

Multiple sequence elements of a single functional class are required for cyclic AMP responsiveness of the mouse c-fos promoter

Agents that elevate the intracellular concentration of cyclic AMP (cAMP) rapidly and transiently induce expression of the c-fos proto-oncogene in BALB/c 3T3 cells. We show that the mouse c-fos promoter-enhancer region contains multiple elements that contribute to cAMP responsiveness of the promoter in transient expression assays. The most potent element was found to correspond to a previously mapped basal promoter element and protein-binding site located 65 base pairs upstream of the transcriptional initiation site. This element and two less potent sites contained a match to the cAMP response element (CRE) core sequence defined in several mammalian genes. The relative potencies of these elements corresponded with their relative affinities for cellular factors that bound to the CRE in vitro. Mutation of all three elements failed to abolish completely cAMP responsiveness of the c-fos promoter in the transient expression assay. However, we present evidence that this residual responsiveness may have been due to sequences present in vector DNA. Finally, we show, by using a new microinjection competition assay, that a double-stranded oligonucleotide carrying the major c-fos CRE is sufficient to block induction of the endogenous c-fos gene by cAMP. Therefore, induction of the endogenous gene requires positively acting cellular factors that interact with a single functional class of regulatory sites in the c-fos gene. Unrelated regulatory elements, such as the serum response element and putative AP-2 sites, are not by themselves sufficient to mediate the cAMP response.

YY1 facilitates the association of serum response factor with the c-fos serum response element.

YY1 is a multifunctional transcription factor that acts as an activator or repressor in different contexts. YY1 binds to multiple sites in the mouse c-fos promoter, inducing at each site a sharp DNA bend. Binding of YY1 to a site situated between the cyclic AMP response element (CRE) and the TATA box bends the DNA in a way that interferes with the interaction of proteins bound at the CRE and TATA elements, resulting in repression of transcription. Here, we show that binding of YY1 to a different site in the c-fos promoter has a different result. Binding of YY1 to the c-fos serum response element (SRE) enhances the binding of serum response factor (SRF). This enhancement requires the binding of YY1 to SRE DNA. YY1 and SRF can cooccupy the SRE at least transiently. In the region of overlapping contact, YY1 contacts DNA in the major groove, while SRF contacts DNA in the minor groove. YY1 also enhances the association of SRF with the SRE in transfected insect cells. Thus, although YY1 induces similar structural changes in DNA at different binding sites, it can have distinct local effects on protein-DNA and protein-protein interactions. These data support a general role for YY1 in the building of highly organized promoter complexes.

Expression cloning of a novel zinc finger protein that binds to the c-fos serum response element

Induction of c-fos transcription by serum growth factors requires the serum response element (SRE). The SRE is a multifunctional element which responds to several positively and negatively acting signals. To identify cellular proteins that might mediate functions of the SRE, we screened a human cDNA expression library with an SRE probe. We report the isolation and characterization of SRE-ZBP, a previously unidentified SRE-binding protein. SRE-ZBP is a member of the C2H2 zinc finger family of proteins exemplified by TFIIIA and the Drosophila Krüppel protein. The seven tandemly repeated zinc finger motifs in SRE-ZBP are sufficient for high-affinity binding to the SRE. We show that SRE-ZBP is a nuclear protein and identify a candidate cellular protein encoded by the SRE-ZBP gene. Because we cannot detect any DNA-binding activity attributable to the endogenous protein, we propose that SRE-ZBP activity may be subject to posttranslational regulation. Like c-fos mRNA, SRE-ZBP mRNA is serum inducible in HeLa cells, but with slower kinetics. The role of SRE-ZBP in the regulation of c-fos transcription remains unestablished, but this protein binds to a region of the SRE where mutations lead to derepression.