Identification of the receptor component of the IkappaBalpha-ubiquitin ligase.

NF-kappaB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IkappaBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin-proteasome system to eliminate inhibitory regulators. Here we isolate the pIkappaBalpha-ubiquitin ligase (pIkappaBalpha-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-kappaB inhibitor pIkappaBalpha. Taking advantage of its high affinity to pIkappaBalpha, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIkappaBalpha degradation motif as an F-box/WD-domain protein belonging to a recently distinguished family of beta-TrCP/Slimb proteins. This component, which we denote E3RSIkappaB (pIkappaBalpha-E3 receptor subunit), binds specifically to pIkappaBalpha and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(IkappaB), which tightly binds pIkappaBalpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIkappaBalpha and consequently NF-kappaB activation. E3RS(IkappaB) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.

Inhibition of NF-kappa-B cellular function via specific targeting of the I-kappa-B-ubiquitin ligase.

Activation of the transcription factor NF-kappa B is a paradigm for signal transduction through the ubiquitin-proteasome pathway: ubiquitin-dependent degradation of the transcriptional inhibitor I kappa B in response to cell stimulation. A major issue in this context is the nature of the recognition signal and the targeting enzyme involved in the proteolytic process. Here we show that following a stimulus-dependent phosphorylation, and while associated with NF-kappa B, I kappa B is targeted by a specific ubiquitin-ligase via direct recognition of the signal-dependent phosphorylation site; phosphopeptides corresponding to this site specifically inhibit ubiquitin conjugation of I kappa B and its subsequent degradation. The ligase recognition signal is functionally conserved between I kappa B alpha and I kappa B beta, and does not involve the nearby ubiquitination site. Microinjection of the inhibitory peptides into stimulated cells abolished NF-kappa B activation in response to TNF alpha and the consequent expression of E-selectin, an NF-kappa B-dependent cell-adhesion molecule. Inhibition of NF-kappa B function by specific blocking of ubiquitin ligase activity provides a novel approach for intervening in cellular processes via regulation of unique proteolytic events.

Stimulation-dependent I kappa B alpha phosphorylation marks the NF-kappa B inhibitor for degradation via the ubiquitin-proteasome pathway.

The nuclear translocation of NF-kappa B follows the degradation of its inhibitor, I kappa B alpha, an event coupled with stimulation-dependent inhibitor phosphorylation. Prevention of the stimulation-dependent phosphorylation of I kappa B alpha, either by treating cells with various reagents or by mutagenesis of certain putative I kappa B alpha phosphorylation sites, abolishes the inducible degradation of I kappa B alpha. Yet, the mechanism coupling the stimulation-induced phosphorylation with the degradation has not been resolved. Recent reports suggest a role for the proteasome in I kappa B alpha degradation, but the mode of substrate recognition and the involvement of ubiquitin conjugation as a targeting signal have not been addressed. We show that of the two forms of I kappa B alpha recovered from stimulated cells in a complex with RelA and p50, only the newly phosphorylated form, pI kappa B alpha, is a substrate for an in vitro reconstituted ubiquitin-proteasome system. Proteolysis requires ATP, ubiquitin, a specific ubiquitin-conjugating enzyme, and other ubiquitin-proteasome components. In vivo, inducible I kappa B alpha degradation requires a functional ubiquitin-activating enzyme and is associated with the appearance of high molecular weight adducts of I kappa B alpha. Ubiquitin-mediated protein degradation may, therefore, constitute an integral step of a signal transduction process.

Costimulation requirement for AP-1 and NF-kappa B transcription factor activation in T cells.

The transcriptional activity of the IL-2 promoter requires T-cell costimulation delivered by the TCR and the auxiliary receptor CD28. Several transcription factors participate in IL-2 promoter activation, among which are AP-1-like factors and NF-kappa B. Protein phosphorylation has an important role in the regulation of these two factors: (1) it induces the transactivating capacity of the AP-1 protein c-Jun; and (2) it is involved in the release of the cytoplasmic inhibitor, I kappa B, from NF-kappa B, allowing translocation of the latter into the nucleus. We have recently shown that both phosphorylation processes require T-cell costimulation. Furthermore, in activated T cells, the kinetics of the two phosphorylation events are essentially similar. According to our results, however, the kinases responsible for the two processes are distinct entities. Whereas TPCK inhibits phosphorylation of I kappa B and, consequently, activation of NF-kappa B, it markedly enhances the activity of JNK, the MAP kinase-related kinase that phosphorylates the transactivation domain of c-Jun. We, therefore, propose the activation scheme presented in FIGURE 3 for T-cell costimulation. Costimulation results in the activation of a signaling pathway that leads to the simultaneous induction of the two transcription factors, AP-1 and NF-kappa B. Integration of the signals generated by TCR and CD28 engagement occurs along this pathway, which then bifurcates to induce I kappa B phosphorylation and NF-kappa B activation on the one hand, and JNK activation and c-Jun phosphorylation on the other. We are currently engaged in defining where the two signals integrate along the AP-1/NF-kappa B pathway.

In vivo stimulation of I kappa B phosphorylation is not sufficient to activate NF-kappa B.

NF-kappa B is a major inducible transcription factor in many immune and inflammatory reactions. Its activation involves the dissociation of the inhibitory subunit I kappa B from cytoplasmic NF-kappa B/Rel complexes, following which the Rel proteins are translocated to the nucleus, where they bind to DNA and activate transcription. Phosphorylation of I kappa B in cell-free experiments results in its inactivation and release from the Rel complex, but in vivo NF-kappa B activation is associated with I kappa B degradation. In vivo phosphorylation of I kappa B alpha was demonstrated in several recent studies, but its role is unknown. Our study shows that the T-cell activation results in rapid phosphorylation of I kappa B alpha and that this event is a physiological one, dependent on appropriate lymphocyte costimulation. Inducible I kappa B alpha phosphorylation was abolished by several distinct NF-kappa B blocking reagents, suggesting that it plays an essential role in the activation process. However, the in vivo induction of I kappa B alpha phosphorylation did not cause the inhibitory subunit to dissociate from the Rel complex. We identified several protease inhibitors which allow phosphorylation of I kappa B alpha but prevent its degradation upon cell stimulation, presumably through inhibition of the cytoplasmic proteasome. In the presence of these inhibitors, phosphorylated I kappa B alpha remained bound to the Rel complex in the cytoplasm for an extended period of time, whereas NF-kappa B activation was abolished. It appears that activation of NF-kappa B requires degradation of I kappa B alpha while it is a part of the Rel cytoplasmic complex, with inducible phosphorylation of the inhibitory subunit influencing the rate of degradation.

Antibodies in human sera against the Epstein-Barr virus encoded latent membrane protein (LMP).

Antibodies reactive with the Epstein-Barr (EBV)-encoded latent membrane protein, LMP, were detected in human sera. Membrane fractions of the EBV-carrying Raji cells and a fusion protein (LMFP), that represents the carboxy part of LMP, were used as antigens. These were assayed by the reduction of the leukocyte migration inhibition (LMI) reaction. Reactivity with LMFP was detected in 10 of 14 sera from patients with Burkitt's lymphoma (BL) and in 3 of 23 sera from healthy EBV-seropositive individuals. The antibody levels were higher in the BL sera. Since the tumor cells do not express LMP, this may be due to the high virus load in the patients.

Proteins in normal and malignant cells, cross-reacting with the latent membrane protein encoded by Epstein-Barr virus.

Human B lymphocytes transformed by infection with the Epstein-Barr virus (EBV) express a new membrane protein of 63 kDa (latent membrane protein, LMP) encoded by the virus. The function of this protein in the virus-cell interaction is not known. In this work we have identified in EBV- human and mouse cell molecules which cross-react with LMP. Two types of reagents were employed: (a) antibodies against LMP-derived synthetic peptides, affinity purified from antisera against a fusion protein containing the carboxy half of the LMP molecule and (b) antisera prepared by immunizing rabbits directly with the peptide conjugates. Cross-reactions were determined by radioimmunoblotting experiments. At least six molecules (Mr = 110, 85, 63, 53, 45 and 23 kDa), present in a variety of human cells (peripheral blood lymphocytes, B cell lines and epithelial cell lines) were found to cross-react with the LMP-derived peptides. Cross-reacting proteins were also identified in normal mouse tissues. The specificity of the cross-reacting antibodies was confirmed by inhibition experiments with the corresponding peptide. Furthermore, antibodies eluted from individual bands were shown to bind to the same band when reacted with new blots of the same extracts. Our data suggest that normal cells contain a family of highly conserved proteins cross-reacting with the LMP molecule. If, indeed, these proteins share common functions, their study may lead the way to unraveling the function of LMP.

Down-regulation of the EBV-encoded membrane protein (LMP) in Burkitt lymphomas.

A radioimmunoassay (RIA) has been developed and used to determine the expression of LMP-a membrane protein encoded by the LT3 region of the Epstein-Barr virus (EBV) genome-in cell lines of various origins. The RIA was highly sensitive, specific and reproducible. All EBV-negative cell lines were LMP-negative and 18 of 21 EBV-carrying cells were LMP-positive. LMP concentrations varied widely, ranging approximately from less than 4 ng up to 650 ng/mg protein. In several instances comparisons were made between lymphoblastoid (LCLs) and Burkitt lymphoma (BL) cell lines (EBV-positive or EBV-converted sublines of originally EBV-negative BL) originating from the same patient. In all such cases LMP and LMP-specific mRNA levels were higher in the LCLs. Most of the LMP was found in the cytosol fraction, yet this fraction was negative in immunoblotting tests. However, antiserum preincubated with the cytosol lost its ability to react in immunoblotting with membrane LMP, indicating that the 2 LMP forms (membrane and cytosol) are completely cross-reactive.

Levels of fibrinogen/fibrin degradation fragment E and related substances in sera and effusions of patients with malignant disease.

A radioimmunoassay (RIA) was developed and used to determine the level of fragment E [a fibrinogen/fibrin degradation product (FDP)] and of fragment-E-containing substances (FES) in sera and effusion fluids of patients with malignant diseases. Sera of patients with other diseases and sera of healthy individuals served as controls. Results were expressed as units/ml (U/ml), one unit being equivalent to 40 ng pure fragment E. Effusion fluids of both malignant and nonmalignant origin contained relatively high levels of fragment-E-containing substances, up to 7,500 U/ml. Normal sera had less than 30 U/ml, while sera of patients with a variety of neoplastic or nonneoplastic conditions contained larger amounts, reaching to hundreds and, in rare cases (some patients with rheumatoid arthritis), even thousands of U/ml. Some of the highest levels in the malignant sera were found in samples from patients with Burkitt's lymphoma and stomach cancer. About 10%-20% of the reactive material in effusions and 20%-40% in the sera consisted of fragment E. These results confirm earlier findings of high FDP levels in neoplasia. Given the higher accuracy of the radioimmunoassay and its suitability for large scale testing, it would appear worthwhile to continue such studies to explore the clinical usefulness of the RIA for fragment E.

Antigens in immune complexes from patients with breast cancer. Identification of autoantigens in immune complexes isolated from breast cancer effusions.

Sera and effusion fluids of patients with breast cancer (BC) contain immune complexes (IC). Antigens present in these complexes were isolated as follows: a pool of effusions from patients with BC was fractionated with ammonium sulfate. The proteins precipitating at 40% saturation were further fractionated by filtration through a Sephadex G-200 column. The material recovered in the first peak (molecules larger than monomeric IgG) was brought to pH 3.0 to dissociate the IC, and the mixture was filtered through a column of Sephacryl S-300 at pH 3.0. Proteins smaller than monomeric IgG were collected, radioiodinated, and used as antigens (125Ag) to search for corresponding antibodies in sera of patients with BC (BCS) and of healthy individuals (NHS). 125Ag was reacted with the sera and the immune complexes obtained were precipitated with an antiserum to human Ig and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Both NHS and BCS contained antibodies against two antigens; one of these appeared as a strong band of 17KD, the other as a doublet of approximately 25KD. It is concluded that some of the proteins in the IC from patients with BC are auto-antigens. No BC-specific antigens were identified.

The use of a monoclonal anti-idiotype antibody to study the biology of a human B cell lymphoma.

Immunoglobulin was obtained from the tumor cells of a patient with nodular lymphoma by hybridization to mouse myeloma cells. The human immunoglobulin secreted by these hybridoma cells was used as an immunogen to make murine monoclonal antibodies. Antibodies specific for idiotype, mu heavy chain and lambda light chain, were produced. One anti-idiotype antibody was used to document that idiotype-positive cells and low levels of 19S IgM idiotype were present in the patient's blood. The levels of each were found to correlate with the patient's disease activity. The monoclonal anti-idiotype was effective in eliminating idiotype-positive cells in vitro by solid phase absorption or by complement-mediated cytotoxicity. The anti-idiotype was also used to analyze the host's immunologic response to his own tumor idiotype. There was neither a detectable anti-idiotype antibody response produced in vivo nor a detectable population of T cells that expressed idiotype of could bind idiotype.