c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an as yet undefined mechanism. We demonstrate here, using the yeast two-hybrid system and an in vitro binding assay, that the c-Cbl RING finger domain interacts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacted with the wild-type c-Cbl RING finger domain but not with a RING finger domain that lacks the amino acids that are deleted in 70Z-Cbl, an oncogenic mutant of c-Cbl. The in vitro interaction was enhanced by sequences on both the N-and C-terminal sides of the RING finger. In vivo and in vitro experiments revealed that c-Cbl and UbcH7 synergistically promote the ligand-induced ubiquitination of the epidermal growth factor receptor (EGFR). In contrast, 70Z-Cbl markedly reduced the ligand-induced, UbcH7-mediated ubiquitination of the EGFR. MG132, a proteasome inhibitor, significantly prolonged the ligand-induced phosphorylation of both the EGFR and c-Cbl. Thus, cCbl plays an essential role in the ligand-induced ubiquitination of the EGFR by a mechanism that involves an interaction of the RING finger domain with UbcH7. This mechanism participates in the down-regulation of tyrosine kinase receptors and loss of this function, as occurs in the naturally occurring 70Z-Cbl isoform, probably contributes to oncogenic transformation.

An RNA molecule, 340 nucleotides in length and designated H1 RNA, copurifies with RNase P activity from extracts of HeLa cells or isolated HeLa cell nuclei. When the genomic DNA of various organisms is probed with H1 cDNA in Southern hybridization assays, only mammalian DNA gives a positive signal. The gene coding for H1 RNA in human cells is present in one to three copies per cell. The nucleotide sequence of HI RNA, which shows little homology to the known sequences of its analogs from prokaryotes and yeast, can be drawn as a two-dimensional, hydrogen-bonded structure that resembles similar structures proposed for the RNA subunit of RNase P from these other sources. Part of the hypothetical structure is virtually identical to structures that can be drawn for analogous RNAs from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and S. octosporus.[Key Words: HeLa cells; ribonucleoprotein; RNase P; tRNA processing] Received November 1, 1988; revised version accepted January 31, 1989.RNase P, an endoribonuclease responsible for the biosynthesis of the 5' termini of mature tRNA molecules from their precursors ), was first identified in extracts of Escherichia coli (Altman and Smith 1971). Since then, similar enzymatic activities have been found in extracts of both prokaryotic and eukaryotic cells. In every case examined in detail, both RNases and proteases have been shown to inactivate the enzymatic activity [although some conflicting data have been reported in the cases of Xenopus laevis and spinach chloroplasts by Castano et al. (1986) and Wang et al. (1988), respectively]. These observations indicate that RNase P has both essential RNA and protein subunits. However, a unique RNA species with a demonstrable role in the catalytic activity of RNase P has been characterized from only a few prokaryotic organisms. The RNA subunit of RNase P from E. coli, Salmonella typhimurium, and Bacillus subtilis is responsible for the catalytic activity of RNase P from those organisms. No such demonstration of catalytic activity or ability to reconstitute the RNase P holoenzyme in vitro has been reported for the homogeneous species of RNA that have been purified from RNase P from Schizosaccharomyces pombe (Krupp et al. 1986)

Sera from certain patients with systemic lupus erythematosus (SLE) and related rheumatic diseases contain antibodies that selectively deplete extracts of HeLa cells of RNase P activity. Most of the sera that recognize RNase P, an endoribonuclease with an essential RNA subunit, also contain antibodies against another small ribonucleoprotein known as the Th antigen. A species of RNA about 400 nucleotides in length is the only RNA species found in common in all immunoprecipitates prepared with anti-RNase P antibodies.The discovery of antibodies against RNase P defines a major class of antibodies produced by patients with autoimmune disease.Sera from patients with systemic lupus erythematosus (SLE) and related rheumatic diseases contain antibodies to various small nuclear and cytoplasmic ribonucleoprotein complexes designated snRNPs and scRNPs, respectively (1). These antibodies have proved to be powerful tools for defining the structure and function of a variety of macromolecules. For example, anti-Ul RNP, anti-U2 RNP, and anti-Sm snRNP antibodies were crucial to the elucidation of the role played by the U snRNPs in the processing of pre-mRNA (2).RNase P, a ribonucleoprotein, is an enzyme essential for the biosynthesis of the 5' termini of tRNAs (3).Under certain conditions in vitro the RNA component of the enzyme from Escherichia coli and other prokaryotes exhibits catalytic activity (4). Studies of RNase P from eukaryotic cells have proceeded slowly because the enzymatic activity is labile (5, 6) and the enzyme is much less abundant than other RNPs. We previously showed that RNase P from HeLa cells has essential RNA and protein components, but we were unable to identify unequivocally any single species of RNA associated with the enzymatic activity. We now report that a subset ofpatients with SLE and related rheumatic diseases produces antibodies against RNase P. These antibodies occur in nearly 25% of patients and thus constitute a major autoantibody class. By using these anti-RNase P antibodies, we have identified a major species of RNA that copurifies with RNase P and that is selectively immunoprecipitated with the enzymatic activity from RNase P preparations.MATERIALS AND METHODS Sera. Sera were obtained from patients followed in the rheumatology clinic at the Yale University School of Medicine. Sixty sera were selected for study because of their high titers of anti-nuclear antibodies detected by immunofluorescence.Preparation of RNase P. RNase P was prepared from HeLa cells (purchased from the Massachusetts Institute of Technology Cell Culture Center and grown and prepared under sterile conditions) by an extension of methods previously described (7). All buffers were supplemented with 1 mM dithiothreitol/0.2 mM phenylmethylsulfonyl fluoride. An S20 extract was prepared from whole cells and loaded onto a column ofDEAE-Sepharose, and the fractions that contained the peak of enzymatic activity were concentrated by using Amicon Centriflo concentrating filters. This material was then loaded onto a glycer...

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Interleukin-6 (IL-6) is an important mediator of parathyroid hormone (PTH)-induced bone resorption. Serum levels of IL-6 and its soluble receptor (IL-6sR) are regulated in part by PTH. The PTH/PTH-related protein type 1 receptor is highly expressed in the liver, and in the current study we investigated whether the liver produces IL-6 or IL-6sR in response to PTH. Perfusion of the isolated rat liver with PTH-(1-84) stimulated rapid, dose-dependent production of bioactive IL-6 and the IL-6sR. These effects were observed at near physiological concentrations of the hormone such that 1 pM PTH induced hepatic IL-6 production at a rate of approximately 0.6 ng/min. In vitro, hepatocytes, hepatic endothelial cells, and Kupffer cells, but not hepatic stellate cells, were each found to produce both IL-6 and IL-6sR in response to higher (10 nM) concentrations of PTH. Our data suggest that hepatic-derived IL-6 and IL-6sR contribute to the increase in circulating levels of these cytokines induced by PTH in vivo and raise the possibility that PTH-induced, liver-derived IL-6 may exert endocrine effects on tissues such as bone.