The putative protein tyrosine kinase domain (TKD) of the ErbB3 (HER3) receptor protein was generated as a histidinetagged recombinant protein (hisTKD-B3) and characterized enzymologically. CD spectroscopy indicated that the hisTKD-B3 protein assumed a native conformation with a secondary structure similar to that of the epidermal growth factor (EGF) receptor TKD. However, when compared with the EGF receptorderived protein, hisTKD-B3 exhibited negligible intrinsic protein tyrosine kinase activity. Immune complex kinase assays of full-

We have isolated cDNA clones from a human placental library that code for a low molecular weight GTPbinding protein originally designated Gp (also called G25K).This identification is based on comparisons with the available peptide sequences for the purified human Gp protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins (-50% identical), and the rac proteins ('=70% identical). The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell-division-cycle protein CDC42. The human placental gene, which we designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein.GTP-binding proteins play key roles in a number of biological processes, including the hormonal regulation of adenylyl cyclase and phospholipases, visual transduction, protein synthesis, protein trafficking, secretion, and cell growth. The members of the growing class of low molecular weight GTP-binding proteins, with the ras oncogene products as prototypes, consist of only a single 21-to 28-kDa polypeptide (1). These proteins include the yeast YPT1 and SEC4 proteins, which have been implicated in transport through the Golgi and exocytosis (2, 3); the mammalian rho proteins, which are substrates for ADP-ribosylation by botulinum toxin (4, 5); the mammalian rap (6-8), rab (9), ral (10), and rac (10, 11) proteins; the bovine and yeast ARF proteins (12), which promote the cholera toxin-induced ADP-ribosylation of GU in the Golgi compartment; and the human bovine Gp (G25K) proteins (13,14).The well-known transforming properties of the ras oncogenes (15) and the recent observation that the raplA protein can counteract ras transformation (8) suggest that members of this protein family play key roles in cell growth pathways.Recently the Gp protein was shown to be an excellent substrate for the tyrosine kinase activity of the purified human placental epidermal growth factor (EGF) receptor (16), which suggests the possible involvement of this protein in the actions of the EGF receptor. We have now cloned a full-length cDNA for Gp from a human placental library. (14) were labeled using [y-32P]ATP and T4 kinase. A third oligonucleotide probe (P1') was prepared from a cDNA clone that was isolated from the placental cDNA library (see Results). Hybridization was carried out under low stringency at 420C in 6x SSC (1x is 0.15 M NaCJ/0.015 M sodium citrate, pH 7)/5x Denhardt's solution (1x is 0.02% Ficoll/0.02% polyvinylpyrrolidone/0.02% bovine serum albumin)/1% SDS/50 mM Tris'HCI, pH 7.5, containing denatured salmon sperm DNA at 50 tug/ml. Filters were washed in 2 x SSC at 200C and then in 0.2x SSC/0.1% SDS at 500C. Hybridizing phages were plaque-purified and confirmed positive by South...

The ErbB2/ErbB3 heregulin co-receptor has been shown to couple to phosphoinositide (PI) 3-kinase in a heregulin-dependent manner. The recruitment and activation of PI 3-kinase by this co-receptor is presumed to occur via its interaction with phosphorylated TyrXaa-Xaa-Met (YXXM) motifs occurring in the ErbB3 C terminus. In this study, mutant ErbB3 receptor proteins expressed in COS7 cells were used to investigate PI 3-kinase-dependent signaling pathways activated by the ErbB2/ErbB3 co-receptor. We observed that a mutant ErbB3 protein with each of its six YXXM motifs containing a Tyr 3 Phe substitution was unable to bind either the p85 regulatory or p110 catalytic subunit of PI 3-kinase. However, restoration of a single YXXM motif was sufficient to mediate association with the PI 3-kinase holoenzyme, although at a lower level than wild-type ErbB3. When ErbB3 YXXM motifs were restored in pairs, evidence for cooperativity between two, those incorporating Tyr-1273 and Tyr-1286, was observed. Interestingly, we have shown that an apparent association of PI 3-kinase activity with ErbB2/Neu was due to the residual presence of ErbB3 in ErbB2 immunoprecipitates. The necessity of ErbB3 association with PI 3-kinase for downstream signaling to the effector kinase Akt was also investigated. Here, the heregulin-dependent translocation of Akt to the plasma membrane and its subsequent activation was observed in intact NIH-3T3 fibroblasts. Recruitment of PI 3-kinase to ErbB3 was required for both activities, and it appeared that ErbB2 activation alone was not sufficient to activate PI 3-kinase signaling in these cells.The type I subfamily of receptor protein-tyrosine kinases is composed of four members: the prototypical epidermal growth factor receptor (ErbB1/HER1), ErbB2 (HER2/Neu), ErbB3 (HER3), and ErbB4 (HER4). Interestingly, this family of receptors can generate a wide variety of cellular signals by mixing and matching to form various co-receptor signaling complexes (1-4). Of the various heterodimers and homodimers formed, the ErbB2/ErbB3 dimer constitutes a high affinity co-receptor for heregulin (5), which is capable of potent mitogenic signaling.In particular, ErbB3 has been characterized as a major mediator of heregulin-dependent activation of the phosphoinositide (PI) 1 3-kinase pathway (6 -14). One general mechanism of recruitment and activation of PI 3-kinase involves the binding of the tandem Src homology 2 (SH2) domains of its p85 regulatory subunit to phosphorylated YXXM motifs found in signaling proteins (15, 16). ErbB3 is particularly well adapted to mediate PI 3-kinase signaling, because it contains in its Cterminal phosphorylation domain six such consensus p85 binding motifs (17). Two previous studies have investigated the role of these YXXM motifs in ErbB3 signaling. One study showed that phosphopeptides containing the various ErbB3 YXXM motifs could inhibit p85 association with ErbB3 (9). Additionally, we have shown via the yeast two-hybrid system that these phosphorylated motifs can directly associate with th...

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Coulometric and spectroscopic analyses were performed on the three cytochrome components (cytochrome d, cytochrome b558, and the cytochrome previously described as cytochrome a1) of the purified cytochrome d complex, a terminal oxidase of the Escherichia coli aerobic respiratory chain. On the basis of heme extraction, spectroscopic, and coulometric data, the "cytochrome a1" component was identified as a b-type cytochrome: cytochrome b595. The pyridine hemochromogen technique revealed the presence of two molecules of protoheme IX per cytochrome d complex. This quantity of protoheme IX fully accounted for the sum of the cytochrome b558 and cytochrome b595 components as determined coulometrically. The renaming of cytochrome a1 as cytochrome b595 was further indicated by the lack of any heme a in the complex and by its resolved reduced-minus-oxidized spectrum. The latter was found to be similar to that of cytochrome c peroxidase, which contains protoheme IX. Coulometric titrations and carbon monoxide binding titrations revealed that there are two molecules of cytochrome d per complex. A convenient measurement of the amount of cytochrome b558 was found to be the beta-band at 531 nm since cytochrome b558 was observed to be the only component of the cytochrome d complex with a peak at this wavelength. By use of this method and the extinction coefficient for the purified cytochrome b558, it was estimated that there is one molecule of cytochrome b595 and one of cytochrome b558 per cytochrome complex.