Immunoglobulin heavy chain binding protein (BiP/GRP78) is a resident endoplasmic reticulum protein that binds tightly to a number of incompletely assembled or aberrant proteins. BiP also binds ATP and can be purified by ATP affinity chromatography. Here we show that an ATPase activity co‐purifies with BiP prepared from canine pancreas. The BiP‐associated ATPase has a high affinity for ATP but a low turnover number, suggesting a regulatory, rather than an enzymatic role. We also show that submicromolar levels of ATP or ADP decrease the rate of adsorption of [125I]BiP to nitrocellulose filters coated with protein or non‐ionic detergents. In contrast, micromolar levels of AMP increase the rate of adsorption. Furthermore, ATP and ADP decrease the susceptibility of BiP to proteolytic degradation, whereas AMP was found to enhance degradation slightly. Adenine nucleotides may therefore induce or stabilize different conformations of BiP even when ATP hydrolysis does not occur.

c-Cbl down-regulates receptor tyrosine kinases by conjugating ubiquitin to them, leading to receptor internalization and degradation. The ubiquitin protein ligase activity of c-Cbl (abbreviated as E3 activity) is mediated by its RING finger domain. We show here that the E3 activity of c-Cbl is negatively regulated by other domains present in the amino-terminal half of the protein (the TKB and linker helix domains) and that this negative regulation is removed when the protein is phosphorylated on tyrosine residues. Protease digestion studies indicate that tyrosine phosphorylation alters the conformation of c-Cbl. We also show that mutation of certain conserved tyrosine residues to glutamate can constitutively activate the E3 activity of c-Cbl. In particular, a Y371E mutant shows constitutive E3 activity while retaining the ability to bind epidermal growth factor receptor (EGFR). The Y371E mutant also has altered protease sensitivity from wild type, instead resembling the proteolytic pattern seen with tyrosine-phosphorylated c-Cbl. Mutation of the homologous tyrosine residue in Cbl-b to glutamate also leads to E3 activation while retaining EGFR-binding ability. These studies argue that Tyr-371 plays a key role in activating the E3 activity of c-Cbl and that the Y371E mutant may partially mimic phosphorylation at that site. However, Tyr-371 point mutants of c-Cbl are still able to undergo phosphorylation-induced E3 activation, and we show that Tyr-368 can also be phosphorylated in addition to Tyr-371, and contributes to activation.The c-Cbl proto-oncogene was first discovered as the cellular homologue of v-Cbl, a viral transforming gene from the Cas NS-1 murine retrovirus, which causes pre-B cell lymphomas and myelogenous leukemias in mice (1). The transforming gene v-Cbl is a truncation mutant of c-Cbl, which itself does not transform cells (2). Human c-Cbl encodes a widely expressed cytosolic protein of 906 amino acids, which is a prominent substrate of a variety of tyrosine kinases and undergoes binding interactions with a large number of intracellular signaling molecules (3, reviewed in Refs. 4 -8). Two other related genes exist in mammals, Cbl-b (9) and Cbl-3 (10), and Cbl homologues have been identified in Caenorhabditis elegans (11) and Drosophila (12).The amino terminus of c-Cbl contains a conserved functional domain that binds phosphotyrosine, composed of a four-helix bundle, a Ca 2ϩ -binding EF hand domain, and a variant SH2 domain (13). These structural components together comprise a functional unit that has been called the TKB domain (for tyrosine-kinase binding). A short helical linker region connects the TKB domain to a RING finger domain that contains two bound zinc ions. The TKB, linker helix, and RING domains are well conserved among all Cbl family members.Important clues regarding Cbl function have come from genetic studies in C. elegans and Drosophila, in which it was found that Cbl family members act to negatively regulate receptor tyrosine kinases (11,12). Subsequent studies in mammalian cells ...

Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro–imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

Signal transduction by the EGF 1 (1) receptor requires activation of the tyrosine kinase of the receptor to activate downstream signaling molecules. Downstream signaling pathways activated by the EGF receptor include the classic Ras/Raf/ mitogen-activated protein kinase pathways, phospholipase C, and PI3-kinase. Although some signaling molecules are activated by direct binding to the activated EGF receptor, mediated by the direct binding of their SH2 domains to phosphorylated tyrosine residues, other signaling molecules do not appear to bind directly to the receptor itself, yet they are activated in a receptor-dependent manner. With regard to the EGF receptor, examples of the former include phospholipase C, whereas an example of the latter type of interaction is PI3-kinase, which does not appear to bind to the EGF receptor. In recent years it has become clear that large scaffolding molecules such as insulin-regulated substrate-1, Gab1, Gab2, and perhaps c-Cbl, may function in cooperation with growth factor receptors to regulate activation of downstream signaling molecules that do not bind directly to growth factor receptors (1-4). These molecules are all relatively large in size (M r 95,000 -130,000) and contain numerous tyrosine residues that become phosphorylated following receptor engagement. In our studies we have focused upon c-Cbl, a 120,000-dalton protein with 22 tyrosine residues.c-Cbl is the first member of the Cbl family of scaffolding molecules that include c-Cbl, Cbl-b, Cbl-3, D-Cbl from Drosophila and the Caenorhabditis elegans homologue sli-1 (5, 6). Engagement of numerous receptors results in the phosphorylation of c-Cbl; these receptors include the EGF receptor (7-9), the interleukin-3 receptor (10, 11), the erythropoietin receptor (8, 10), the prolactin receptor (12), integrins (13,14), the T-cell receptor (15,16), and the B-cell receptor (17, 18). c-Cbl has been shown to be associated with numerous signaling molecules (Src, Fyn, Lyn, Syk, ZAP70, and PI3-kinase) (8,11,16,17,19) as well as several adapter molecules (Shc, Crk, and Grb2) (9, 16, 17, 20 -22). The association of c-Cbl with PI3-kinase suggests that c-Cbl could function as a scaffolding molecule that regulates activation of downstream signaling molecules. c-Cbl can be phosphorylated by both Src-like kinases as well as members of the Syk/ZAP70 family of tyrosine kinases (23-29); however, it is not clear which specific tyrosine residues are phosphorylated by these kinases and whether different receptors utilize different tyrosine kinases to phosphorylate different regions of c-Cbl.Complicating the view of c-Cbl as merely a molecular scaffold is the observation that the C. elegans homologue of c-Cbl, sli-1, functions as a negative regulator of the C. elegans EGF receptor homologue let-63 (5). Consistent with this observation, overexpression of c-Cbl in fibroblasts results in the down-regulation of the EGF receptor (30,31). A mechanism explaining this observation was revealed when it was realized that the RING finger motif of c-Cbl could ...

Immunoglobulin heavy-chain binding protein (BiP, GRP-78) associates tightly in the endoplasmic reticulum (ER) with newly synthesized proteins that are incompletely assembled, have mutant structures, or are incorrectly glycosylated. The function of BiP has been suggested to be to prevent secretion of incorrectly folded or incompletely assembled protein, to promote folding or assembly of proteins, or to solubilize protein aggregates within the ER lumen. Here we examine the interaction of BiP with newly synthesized polypeptides in an in vitro protein translation-translocation system. We find that BiP forms tight complexes with nonglycosylated yeast invertase and incorrectly disulphide-bonded prolactin, but does not associate detectably with either glycosylated invertase or correctly disulphide-bonded prolactin. Moreover, BiP associates detectably only with completed chains of prolactin, not with chains undergoing synthesis. We conclude that BiP recognizes and binds with high affinity in vitro to aberrantly folded or aberrantly glycosylated polypeptides, but not to all nascent chains as they are folding.