Inducible Protein Complex Research Articles

β-amyloid, microglia, and the inflammasome in Alzheimer's disease.

There is extensive evidence that accumulation of mononuclear phagocytes including microglial cells, monocytes, and macrophages at sites of β-amyloid (Aβ) deposition in the brain is an important pathological feature of Alzheimer's disease (AD) and related animal models, and the concentration of these cells clustered around Aβ deposits is several folds higher than in neighboring areas of the brain [1-5]. Microglial cells phagocytose and clear debris, pathogens, and toxins, but they can also be activated to produce inflammatory cytokines, chemokines, and neurotoxins [6]. Over the past decade, the roles of microglial cells in AD have begun to be clarified, and we proposed that these cells play a dichotomous role in the pathogenesis of AD [4, 6-11]. Microglial cells are able to clear soluble and fibrillar Aβ, but continued interactions of these cells with Aβ can lead to an inflammatory response resulting in neurotoxicity. Inflammasomes are inducible high molecular weight protein complexes that are involved in many inflammatory pathological processes. Recently, Aβ was found to activate the NLRP3 inflammasome in microglial cells in vitro and in vivo thereby defining a novel pathway that could lead to progression of AD [12-14]. In this manuscript, we review possible steps leading to Aβ-induced inflammasome activation and discuss how this could contribute to the pathogenesis of AD.

The NLRP4-DTX4 axis: a key suppressor of TBK1 and innate antiviral signaling

The NLRP4-DTX4 axis: a key suppressor of TBK1 and innate antiviral signaling

TSLP Signaling Network Revealed by SILAC-Based Phosphoproteomics

Thymic stromal lymphopoietin (TSLP) is a cytokine that plays diverse roles in the regulation of immune responses. TSLP requires a heterodimeric receptor complex consisting of IL-7 receptor α subunit and its unique TSLP receptor (gene symbol CRLF2) to transmit signals in cells. Abnormal TSLP signaling (e.g. overexpression of TSLP or its unique receptor TSLPR) contributes to the development of a number of diseases including asthma and leukemia. However, a detailed understanding of the signaling pathways activated by TSLP remains elusive. In this study, we performed a global quantitative phosphoproteomic analysis of the TSLP signaling network using stable isotope labeling by amino acids in cell culture. By employing titanium dioxide in addition to antiphosphotyrosine antibodies as enrichment methods, we identified 4164 phosphopeptides on 1670 phosphoproteins. Using stable isotope labeling by amino acids in cell culture-based quantitation, we determined that the phosphorylation status of 226 proteins was modulated by TSLP stimulation. Our analysis identified activation of several members of the Src and Tec families of kinases including Btk, Lyn, and Tec by TSLP for the first time. In addition, we report TSLP-induced phosphorylation of protein phosphatases such as Ptpn6 (SHP-1) and Ptpn11 (Shp2), which has also not been reported previously. Co-immunoprecipitation assays showed that Shp2 binds to the adapter protein Gab2 in a TSLP-dependent manner. This is the first demonstration of an inducible protein complex in TSLP signaling. A kinase inhibitor screen revealed that pharmacological inhibition of PI-3 kinase, Jak family kinases, Src family kinases or Btk suppressed TSLP-dependent cellular proliferation making them candidate therapeutic targets in diseases resulting from aberrant TSLP signaling. Our study is the first phosphoproteomic analysis of the TSLP signaling pathway that greatly expands our understanding of TSLP signaling and provides novel therapeutic targets for TSLP/TSLPR-associated diseases in humans.

Adenomatous Polyposis Coli Protein Associates with C/EBP β and Increases Bacillus anthracis Edema Toxin-stimulated Gene Expression in Macrophages

The production of cAMP from Bacillus anthracis edema toxin (ET) activates gene expression in macrophages through a complex array of signaling pathways, most of which remain poorly defined. In this study, the tumor suppressor protein adenomatous polyposis coli (APC) was found to be important for the up-regulation of previously defined ET-stimulated genes (Vegfa, Ptgs2, Arg2, Cxcl2, Sdc1, and Cebpb). A reduction in the expression of these genes after ET exposure was observed when APC was disrupted in macrophages using siRNA or in bone marrow-derived macrophages obtained from C57BL/6J-Apc(Min) mice, which are heterozygous for a truncated form of APC. In line with this observation, ET increased the expression of APC at the transcriptional level, leading to increased amounts of APC in the nucleus. The mechanism utilized by APC to increase ET-induced gene expression was determined to depend on the ability of APC to interact with C/EBP β, which is a transcription factor activated by cAMP. Coimmunoprecipitation experiments found that APC associated with C/EBP β and that levels of this complex increase after ET exposure. A further connection was uncovered when silencing APC was determined to reduce the ET-induced phosphorylation of C/EBP β at Thr-188. This ET-mediated phosphorylation of C/EBP β was blocked by glycogen synthase kinase 3 (GSK-3) inhibitors, suggesting that GSK-3 is involved in the activation of C/EBP β and supporting the idea of APC helping direct interactions between GSK-3 and C/EBP β. These results indicate that ET stimulates gene expression by promoting the formation of an inducible protein complex consisting of APC and C/EBP β.

SOS response dynamics in Escherichia coli bacterial cells upon ultraviolet irradiation

A mathematical model of induced mutations in Escherichia coli bacterial cells upon ultraviolet irradiation is developed. The concentration dynamics of inducible protein complexes synthesized in the course of the SOS response of E. coli is described. The mutation induction at translesion synthesis is studied. The solutions to the model are based on experimental data concerning the expression of the main genes of the SOS system of E. coli bacteria.

Purification and characterization of zinc α2‐glycoprotein‐Prolactin inducible protein complex from human seminal plasma

Zinc alpha2-glycoprotein (ZAG) is present at high concentration in human seminal plasma, and considered as soluble homologue of MHC-I. ZAG is a well-known biomarker for prostate and breast carcinomas. We have purified a naturally occurring complex of ZAG with Prolactin inducible protein (PIP), which is also a well-known biomarker for the same. The ZAG-PIP complex has been isolated and purified by simple chromatographic techniques in a reproducible two-step process, using ion exchange and gel-permeation chromatography and subsequently identified by MS. The complex between ZAG and PIP is formed by non-covalent interactions. The purity and molecular mass was determined by SDS-PAGE, which shows the bands corresponding to 40 kDa and 14 kDa, which were also confirmed by MALDI-TOF. Dynamic light scattering (DLS) experiments also showed hydrodynamic radii corresponding to 54, 40 and 14 kDa for ZAG-PIP complex, ZAG and PIP respectively. The concentration dependent aggregation of this complex has also been observed. Fluorescence analysis reveals that complexes have similar binding affinities as native ZAG, for their proposed ligands like arachidonic acid, polyethylene glycol and synthetic peptide. This is the first report on purification and characterization of a naturally occurring complex of ZAG-PIP in human seminal fluid.

Quantitative proteomics using stable isotope labeling with amino acids in cell culture

Stable isotope labeling with amino acids in cell culture (SILAC) is a simple in vivo labeling strategy for mass spectrometry-based quantitative proteomics. It relies on the metabolic incorporation of nonradioactive heavy isotopic forms of amino acids into cellular proteins, which can be readily distinguished in a mass spectrometer. As the samples are mixed before processing in the SILAC methodology, the sample handling errors are also minimized. Here we present protocols for using SILAC in the following types of experiments: (i) studying inducible protein complexes, (ii) identification of Tyr kinase substrates, (iii) differential membrane proteomics and (iv) studying temporal dynamics using SILAC 5-plexing. Although the overall time is largely dependent on the rate of cell growth and various sample processing steps employed, a typical SILAC experiment from start to finish, including data analysis, should take anywhere between 20 and 25 d.

Identification of inducible protein complexes in the phenol degrader Pseudomonas sp. strain phDV1 by blue native gel electrophoresis and mass spectrometry

Pseudomonas sp. strain phDV1, being a phenol degrading bacterium, has been found to utilize phenol as sole carbon source via the meta pathway. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes involved in the aromatic degradation pathway. In particular, the Pseudomonas sp. strain phDV1 proteome was monitored under two different growth substrate conditions, using glucose or phenol as sole carbon source. The protein complexes map was compared by BN-PAGE after fractionation by sucrose density centrifugation of the cell extracts. Multiple differences were detected. Further, analysis and identification of the subunit composition of these complexes was carried out using MALDI-TOF MS, allowing the identification of 49 proteins. Additionally, functional information regarding protein-protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. Application of this functional proteomics method resulted in an higher number of the identified proteins.

A Novel Caspase-2 Complex Containing TRAF2 and RIP1

The enzymatic activity of caspases is implicated in the execution of apoptosis and inflammation. Here we demonstrate a novel nonenzymatic function for caspase-2 other than its reported proteolytic role in apoptosis. Caspase-2, unlike caspase-3, -6, -7, -9, -11, -12, and -14, is a potent inducer of NF-kappaB and p38 MAPK activation in a TRAF2-mediated way. Caspase-2 interacts with TRAF1, TRAF2, and RIP1. Furthermore, we demonstrate that endogenous caspase-2 is recruited into a large and inducible protein complex, together with TRAF2 and RIP1. Structure-function analysis shows that NF-kappaB activation occurs independent of enzymatic activity of the protease and that the caspase recruitment domain of caspase-2 is sufficient for the activation of NF-kappaB and p38 MAPK. These results demonstrate the inducible assembly of a novel protein complex consisting of caspase-2, TRAF2, and RIP1 that activates NF-kappaB and p38 MAPK through the caspase recruitment domain of caspase-2 independently of its proteolytic activity.

The pkr kinase promoter binds both Sp1 and Sp3, but only Sp3 functions as part of the interferon-inducible complex with ISGF-3 proteins

The protein kinase regulated by RNA (PKR) is an important mediator of the antiviral and antiproliferative actions of interferon (IFN). The promoter of the PKR gene contains a novel 15-bp element designated KCS that is required for both basal and IFN-inducible transcription, with KCS function dependent upon both position and orientation relative to the ISRE element. Novel inducible protein complexes (iKIBP1, iKIBP2) that require both the KCS and the ISRE element sequences for their formation have been identified and characterized. Transcription factors Sp1 and Sp3 were found to be KCS-binding proteins by electrophoretic mobility shift analyses (EMSA) and Sepharose bead-KCS oligonucleotide pull-down assays. However, only Sp3 but not Sp1 was a constituent of the inducible iKIBP complexes. EMSA also identified STAT1, STAT2, and IRF-9 as components of the iKIBP complexes, indicating that ISGF-3 participates in iKIBP complex formation. Proteins bound at the KCS element in the absence of ISRE were able to recruit both STAT1 and STAT2 to the KCS element; recruitment was dependent upon IFN-α treatment. Chromatin immunoprecipitation assays revealed that the binding of Sp3, similar to STAT1 and STAT2, at the PKR promoter in vivo was IFN-dependent, but that Sp1 binding was not dependent upon IFN treatment. These results, taken together, strongly suggest a role for Sp1 in basal and Sp3 in inducible transcription of PKR and that a potential function of the KCS element is to facilitate the recruitment of ISGF-3 complex components to the PKR promoter to stimulate transcription.

Cutting edge: chromatin remodeling at the IL-4/IL-13 intergenic regulatory region for Th2-specific cytokine gene cluster.

During the differentiation of naive Th cells into Th2 effector cells, the entire IL-4/IL-13 locus is remodeled into an accessible chromatin conformation. Here we show that ectopic expression and activation of Stat6 or GATA-3 in Th cells developing under Th1-polarizing conditions lead to the induction of chromatin remodeling not only at the flanking regions of the IL-4 and IL-13 genes but also at the IL-4/IL-13 intergenic regulatory region for the IL-4/IL-13/IL-5 gene cluster. Furthermore, we demonstrate that GATA-3 and another Th2-specific, inducible protein complex interact with the IL-4/IL-13 intergenic DNase I hypersensitive region specifically in Th2 cells.

Activation of intercellular adhesion molecule 1 expression by Helicobacter pylori is regulated by NF-kappaB in gastric epithelial cancer cells.

Interactions between leukocytes and epithelial cells may play a key role in Helicobacter pylori-associated gastric mucosal inflammation. This process is mediated by various cell adhesion molecules. The present study examined the molecular mechanisms leading to H. pylori-induced epithelial cell intercellular adhesion molecule-1 (ICAM-1; also called CD54) expression. Coculture of epithelial cells with cytotoxin-associated gene pathogenicity island-positive (cag PAI(+)) H. pylori strains, but not with a cag PAI(-) strain or H. pylori culture supernatants, resulted in upregulation of steady-state mRNA levels and cell surface expression of ICAM-1. Coculture with H. pylori induced an increase in luciferase activity in cells which were transfected with a luciferase reporter gene linked to the 5'-flanking region of the ICAM-1 gene. H. pylori activated the ICAM-1 promoter via the NF-kappaB binding site. An inducible nuclear protein complex bound to the ICAM-1 NF-kappaB site and was identified as the NF-kappaB p50-p65 heterodimer. H. pylori induced the degradation of IkappaB-alpha, a major cytoplasmic inhibitor of NF-kappaB, and stimulated the expression of IkappaB-alpha mRNA. Pretreatment of epithelial cells with pyrrolidine dithiocarbamate, which blocks NF-kappaB activation, inhibited H. pylori-induced ICAM-1 expression. THP-1 macrophagic cells, peripheral blood mononuclear cells, and purified neutrophils adhered to H. pylori-infected epithelial cells to a greater extent than to uninfected cells. These results show that H. pylori directly induces expression of ICAM-1 on gastric epithelial cells in an NF-kappaB-dependent manner that may support leukocyte attachment during inflammation.

Grb2 Forms an Inducible Protein Complex with CD28 through a Src Homology 3 Domain-Proline Interaction

CD28 provides a costimulatory signal that results in optimal activation of T cells. The signal transduction pathways necessary for CD28-mediated costimulation are presently unknown. Engagement of CD28 leads to its tyrosine phosphorylation and subsequent binding to Src homology 2 (SH2)-containing proteins including the p85 subunit of phosphatidylinositol 3'-kinase (PI3K); however, the contribution of PI3K to CD28-dependent costimulation remains controversial. Here we show that CD28 is capable of binding the Src homology 3 (SH3) domains of several proteins, including Grb2. The interaction between Grb2 and CD28 is mediated by the binding of Grb2-SH3 domains to the C-terminal diproline motif present in the cytoplasmic domain of CD28. While the affinity of the C-terminal SH3 domain of Grb2 for CD28 is greater than that of the N-terminal SH3 domain, optimal binding requires both SH3 domains. Ligation of CD28, but not tyrosine-phosphorylation, is required for the SH3-mediated binding of Grb2 to CD28. We propose a model whereby the association of Grb2 with CD28 occurs via an inducible SH3-mediated interaction and leads to the recruitment of tyrosine-phosphorylated proteins such as p52(shc) bound to the SH2 domain of Grb2. The inducible interaction of Grb2 to the C-terminal region of CD28 may form the basis for PI3K-independent signaling through CD28.

A18 kDa cadmium inducible protein Complex: its isolation and characterisation from rice ( Oryza sativa L.) seedlings

Summary The present study shows the pattern of cadmium absorption and distribution in growing seedlings of two Indian rice ( Oryza sativa L.) cvs. Ratna and Jaya. Cadmium was readily absorbed by roots of growing seedlings and its localisation was greater in roots than shoots. Low translocation of cadmium from roots to shoots was observed. Seedlings exposed for 20 days under 500 umol/L Cd 2+ in the medium showed accumulation of Cd 2+ to a level far beyond the ambient. Under 500 umol/L Cd 2+ in the growth medium about 2.2 to 2.8 umols Cd 2+ g −1 dry wt. equivalent to 245 to 311 ppm Cd 2+ was observed in roots and 0.5 to 1.2 μmol Cd 2+ g −1 dry wt. equivalent to 56 to 134 ppm Cd 2+ was noted in shoots in 20-day-grown seedlings. One Cd 2+ binding protein complex with an apparent molecular weight of 18 kDa was purified by DEAE-cellulose chromatography, Sephadex G-200 gel filteration and fast protein liquid chromatography (FPLC). This complex had 4 sulfhydryl groups per protein molecule. The specific Cd 2+ content of the complex was 3.7μmol mg −1 peptide. The results suggest that Cd 2+ binds possibly with the help of 4-SH groups to the peptide in mercaptide bonds and that this complex, which has a comparatively higher molecular weight than conventional phytochelatins, helps in sequestration of excess Cd 2+ ions in rice plants.

Identification of Transcription Factor Binding Sites Important in the Regulation of the Human Interleukin-5 Gene

This study identifies three regions of the human interleukin (IL)-5 promoter involved in binding nuclear factors from activated T cells. DNase I footprinting and mobility shift assays with nuclear proteins from the human T cell clone, SP-B21, demonstrated protein interactions with each of these response elements (REs), located between positions -79 and -45 (RE-I), -123 and -92 (RE-II), and -170 and -130 (RE-III). Two of these regions, RE-II and RE-III, have not previously been described to regulate IL-5 expression in T cells. The RE-II site was shown to be critical for inducible IL-5 promoter activity in transient transfection assays in D10.G4.1 T cells, while the RE-III site functions as a negative regulatory element. The activity of the RE-II site was specifically inhibited by cyclosporin A, and transfection assays with IL-5 constructs containing mutations in the RE-II site showed greatly reduced reporter gene activity. We have defined the sequence involved in stimulation-dependent transcription and have identified constitutive as well as inducible DNA-binding protein complexes that bind to RE-II. Antibodies against at least two members of the nuclear factor of activated T cells (NFAT) family of transcription factors are capable of binding to the IL-5 RE-II complexes, although they can be distinguished from previously identified NFAT-specific complexes by several characteristics.

Complex role of protein phosphorylation in gene activation by hypoxia

Complex role of protein phosphorylation in gene activation by hypoxia. Mammalian cells are able to sense decreased oxygen tension in their environment and turn-on the expression of specific hypoxia responsive genes. The best studied of these hypoxia regulated genes is the one that encodes for erythropoietin, the glycoprotein hormone that regulates red cell production [1]. The response of the erythropoietin gene to hypoxia is mediated by an enhancer sequence located at the 3′ flanking region of the gene [2–4]. A hypoxia inducible DNA-binding protein complex, termed HIF-1, regulates the transcriptional function of the erythropoietin enhancer [5]. Most interestingly, identical HIF-1 complexes also control the responses of other hypoxia regulated genes, in what appears to be a general mechanism of oxygen sensing and response [6, 7]. These other hypoxia regulated genes include vascular endothelial growth factor, glycolytic enzymes such as pyruvate kinase and aldolase A, glucose transporter 1 and endothelin, among others. All these genes are transcriptionally activated by hypoxia and also by transition metals such as cobalt (Co) and by iron chelators such as desferrioxamine (Dfx) [reviewed in 8].

Regulation of the Human P-selectin Promoter by Bcl-3 and Specific Homodimeric Members of the NF-κB/Rel Family

P-selectin, an adhesion receptor for leukocytes, is constitutively expressed by megakaryocytes and endothelial cells. Synthesis of P-selectin is also increased by some inflammatory mediators. We characterized a previously identified kappa B site (-218GGGGGTGACCCC-207) in the promoter of the human P-selectin gene. The kappa B site was unique in that it bound constitutive nuclear protein complexes containing p50 or p52, but not inducible nuclear protein complexes containing p65. Furthermore, the element bound recombinant p50 or p52 homodimers, but not p65 homodimers. Methylation interference analysis indicated that p50 or p52 homodimers contacted the guanines at positions -218 to -214 on the coding strand and at -210 to -207 on the noncoding strand. Changes in the three central residues at -213 to -211 altered binding specificity for members of the NF-kappa B/Rel family. Mutations that eliminated binding to NF-kappa B/Rel proteins reduced by approximately 40% the expression of a reporter gene driven by the P-selectin promoter in transfected bovine aortic endothelial cells. Overexpression of p52 enhanced P-selectin promoter activity, and co-overexpression of Bcl-3 further induced promoter activity in a kappa B site-dependent manner. In contrast, overexpression of p50 repressed promoter activity; this repression was prevented by co-overexpression of Bcl-3. Similar phenomena were observed with reporter gene constructs driven by two tandem P-selectin kappa B sequences linked to the SV40 minimal promoter. These data suggest that Bcl-3 differentially regulates the effects of p50 and p52 homodimers bound to the kappa B site of the P-selectin promoter. This site may be a prototype for kappa B elements in other genes that bind specifically to p50 and/or p52 homodimers.

Multiple signals are required for function of the human granulocyte-macrophage colony-stimulating factor gene promoter in T cells.

The human granulocyte-macrophage CSF (GM-CSF) gene is expressed in T cells in response to TCR activation that can be mimicked by treatment of the cells with PMA and Ca2+ ionophore. The gene contains a proximal functional promoter region (-620 to +34), as well as a powerful enhancer located 3 kb upstream, both of which are involved in the response of the gene to TCR activation. The proximal promoter contains a region termed CLEO (-54 to -31) that consists of a purine-rich element abutting an activator protein-1 (AP-1)-like site, as well as an upstream nuclear factor-kappa B (NF-kappa B) site (-85 to -76) and a CK-1 element (-101 to -92). We show in this work that mutations in either the purine-rich region of the CLEO element or the NF-kappa B site result in reduced PMA/Ca2+ activation of a 620-bp human GM-CSF promoter-luciferase reporter construct in Jurkat T cells by 65% and 50%, respectively. The major inducible protein complex that binds to the human CLEO (hCLEO) element is an AP-1-like complex that is inducible by PMA alone, but shows increased binding in response to PMA together with Ca2+ ionophore. Although the binding of this complex is not cyclosporin-sensitive, promoter induction is inhibited by cyclosporin treatment. A second weak inducible complex resembling nuclear factor of activated T cells (NF-AT) was also observed binding to the hCLEO region. By using recombinant proteins, we confirmed that AP-1, NF-ATp, and a higher order NF-ATp/AP-1 complex could all form with the hCLEO element, and we have also defined the sequence requirements for binding of each of these complexes. We found that expression of a constitutively active form of calcineurin could substitute for Ca2+ ionophore and synergize with PMA to activate the GM-CSF promoter, and conversely that mutant-activated Ras could substitute for PMA and cooperate with Ca2+ ionophore. Co-expression of Ras and calcineurin, however, did not activate the GM-CSF promoter, but required the additional expression of NF-kappa B p65. These results imply that at least three signals are required to activate the GM-CSF proximal promoter, and that the signals impinge on distinct transcription factors that bind to the hCLEO and NF-kappa B regions of the promoter.

Transcriptional Regulation of the Intercellular Adhesion Molecule-1 Gene by Inflammatory Cytokines in Human Endothelial Cells: ESSENTIAL ROLES OF A VARIANT NF-κB SITE AND p65 HOMODIMERS

Intercellular adhesion molecule-1 (ICAM-1) is greatly up-regulated on endothelial cells at sites of inflammation and is involved in leukocyte attachment and extravasation. Previously, we had shown that the ICAM-1 gene expression in human umbilical vein endothelial cells (HUVECs) was transcriptionally regulated by tumor necrosis factor-alpha (TNF-alpha) (Wertheimer, S. J., Myers, C. L., Wallace, R. W., and Parks, T. P. (1992) J. Biol. Chem. 267, 12030-12035). In the present investigation, TNF-alpha-induced transcription was found to be initiated exclusively at two sites, 40 and 41 base pairs upstream of the translation start site. Deletion analysis of the 5' regulatory region of the ICAM-1 gene revealed a 92-base pair sequence which was both necessary and sufficient to confer TNF-alpha responsiveness to a linked luciferase reporter gene in transient transfection assays. This TNF-alpha-responsive region contained a variant NF-kappa B site at -187 to -178, which when mutated, completely abolished ICAM-1 promoter activation by TNF-alpha, interleukin-1 beta, and lipopolysaccharide. Two inducible nuclear protein complexes bound to the ICAM-1 kappa B and were identified as the NF-kappa B p65 homodimer and p65/p50 heterodimer. Overexpression of p65, but not p50, transactivated the ICAM-1 promoter in a kappa B site-dependent manner in HUVECs. In addition, p65-mediated transactivation was suppressed by co-expression of p50. Our results suggest that cytokine activation of the ICAM-1 promoter in HUVECs may critically depend on p65 homodimers binding to a variant kappa B site.

Inducible binding to the c-fos serum response element during T cell activation is regulated by a phosphotyrosine-containing protein.

The proto-oncogene c-fos is an immediate-early gene, and one of the first genes transcribed after stimulation of most cells with a variety of ligands. Fos expression may be a pivotal event in converting ligand-receptor interactions at the membrane into functional modulation of cell phenotype. The serum response element (SRE) in the c-fos regulatory region participates in induction of transcription by various growth factors and by phorbol esters and subsequent squelching of transcription. We show that an inducible protein complex (Band A) binds to SRE DNA within 10 min after mitogenic stimulation of human PBL-T, and becomes nondetectable by 60 min. Band A contains the serum response factor plus additional factor(s). A protein that is phosphorylated on a tyrosine residue in resting PBL-T suppresses binding of a component of Band A to the SRE motif. Upon stimulation of the cells, this protein no longer prevents binding of DNA by Band A, and suppression of binding is restored within 30 min. The phosphorylated tyrosine residue itself is important for the protein-protein interaction.