p190 RhoGAP is a 190-kDa protein that stably associates with p120 RasGAP and regulates actin dynamics through members of the Rho family of small GTPases. Previous studies have indicated a direct relationship between levels of p190 tyrosine phosphorylation, the extent and kinetics of epidermal growth factor (EGF)-induced actin rearrangements, and EGF-induced cell cycle progression, suggesting that p190 links Rasmediated mitogenic signaling with signaling through the actin cytoskeleton. Determining which tyrosine residues in p190 are phosphorylated, what factors regulate phosphorylation of these sites, and what effect tyrosine phosphorylation has on p190 function is key to understanding the role(s) that p190 may play in these processes. To begin investigating these questions, we used biochemical approaches to characterize the number and relative levels of in vivo-phosphorylated tyrosine residues on endogenous p190 from C3H10T1/2 murine fibroblasts. Only two tryptic phosphopeptides containing phosphotyrosine (p-Tyr), a major site, identified as Y1105, and a minor, unidentified site, were detected. Phosphorylation of Y1105, but not the minor site, was modulated in vivo to a greater extent by overexpression of c-Src than by the EGF receptor and was efficiently catalyzed by c-Src in vitro, indicating that Y1105 is a selective and preferential target of c-Src both in vitro and in vivo. In vitro and in vivo coprecipitation analysis using glutathione S-transferase (GST) fusion proteins containing wild-type and Y1105F variants of the p190 middle domain, variants of full-length p190 ectopically expressed in COS-7 cells, and endogenous p190 and p120 in C3H10T1/2 cells revealed that p190 could bind to p120 in the presence and absence of p190 tyrosine phosphorylation. p-Tyr-independent complexes comprised 10 to 20% of the complexes formed in the presence of p-Tyr. Mutation of Y1105 from Tyr to Phe resulted in complete loss of p-Tyr-dependent complex formation, indicating that p-Y1105 was the sole p-Tyr residue mediating binding to p120. These studies describe a specific mechanism by which c-Src can regulate p190-p120 association and also document a significant role for p-Tyr-independent means of p190-p120 binding.

A murine B-cell lymphoma bearing the class II major histocompatibility complex molecule I-Ak was cultured with the protein antigen hen egg white lysozyme (HEL). The I-Ak molecules were purified, and their associated peptides were extracted for characterization. Five HEL peptides were identified. Four contained the 10 amino acid residues HEL 52-61 (DYGILQINSR) but were heterogeneous in length and flanking residues. This core sequence is known to confer a high binding affinity for I-Ak. ). An understanding of the mechanisms involved in determinant selection might be gained from an examination ofthe end products of antigen processing. Toward this end, the biochemical characterization of physiologically processed antigen in the form of peptides eluted from affinity-purified class I (8-10) and class II (11-14) MHC molecules has been initiated. The eluted peptides were found to be of low molecular weight and to have limited heterogeneity. This small size was in agreement with that of synthetic peptides, which substitute for processed antigen (15, 16). The naturally processed peptides isolated from class I molecules were 8 or 9 amino acids in length with fixed amino-and carboxylterminal ends and were derived from proteins synthesized in the cell cytosol. In contrast, peptides extracted from class II molecules were from 13 to 17 amino acids in length with variable carboxyl-terminal ends, and most were derived from cellular transmembrane glycoproteins. One peptide was recovered from bovine serum albumin, a protein found in the culture medium used to grow the cells (13).Studies in our laboratory have used hen egg white lysozyme (HEL) as a model antigen for processing and presentation by APC. HEL, a soluble nonglycosylated protein 129 amino acids in length, contains four intrachain disulfide bonds and requires intracellular processing for T-cell recognition. Analysis of the T-cell response to HEL in several mouse strains revealed that some regions were favored over others (17,18). We previously showed that the major component of the CD4 T-cell response to HEL in H-2k mice was directed toward the 10 amino acid residues 52-61 (19, 20). These residues lie partially buried inside the native molecule in a (3-sheet conformation near the active site of the enzyme. Peptides containing residues 52-61 of HEL bind with high affinity to I-Ak (5). The HEL peptide 52-61 contains amino acid residues that contact the I-Ak molecule interspersed with amino acid residues that contact the T-cell receptor (20).We are now using this well-characterized model antigen to investigate determinant selection and here report the sequences of physiologically presented HEL peptides eluted from I-Ak after processing by an APC line. MATERIALS AND METHODSIsolation of I-Ak-Associated Peptides. Peptide-loaded MHC class II molecules were purified from the I-Ak-expressing B-lymphoma line M12.C3.F6. This line was produced by DNA-mediated cotransfection of genomic clones for the Ak a and (3 chains into M12.C3, an Ia-negative B-lymphoma line (21). M12.C3.F...

The binding of immunogenic peptides to class II major histocompatibility molecules was examined at various pH values. We studied binding of peptides containing residues 52-61 from hen egg lysozyme (HEL) to I-Ak on fixed peritoneal macrophages or to solubilized affinity-purified I-Ak. Optimum binding occurred at pH 5.5-6.0 with accelerated kinetics relative to pH 7.4; equilibrium binding was also higher at pH 5.54.0 than at 7. Antigen processing involves the intracellular catabolism of proteins to produce immunogenic peptides that bind to class II major histocompatibility complex (MHC) molecules. A significant proportion of class II MHC molecules resides in endosomes (1-3), where binding of peptides appears to occur (2). Antigen processing involves late endosomes and lysosomes, since it is blocked at 180C (4). Efficient processing occurs with liposome-encapsulated antigens that are sequestered until delivery and release into lysosomes (5). These data suggest that immunogenic peptides produced in lysosomes may be recycled to endosomes to meet and bind to class II MHC molecules. Endosomes maintain an acidic luminal pH (about pH 6 for early endosomes and pH 5-5.5 in later endosomes, whereas lysosomes may attain pH 4.6-5.0). Alkalinization of the endosomal environment (e.g., by lysosomotropic amines or ionophores) disrupts endosomal/lysosomal processes (6), including antigen processing (7). In this context, class II MHC molecules would be expected to bind peptides efficiently at endosomal pH. Previously published data tend to support this hypothesis (8), but the effects of pH on peptide binding remain unclear in many aspects. Peptide dissociation has also been reported to be greater at acidic pH (pH 4.6-5.6) than at neutral pH (9), although other reports indicate that peptides remain stably bound in this pH range (10, 11).We have studied (12-14) the binding of peptides to the murine class II MHC molecule I-Ak on whole cells and as a solubilized afflinity-purified molecule (12-14 Immunogenic peptides were added in unbuffered normal saline and incubated at 37°C for 2 hr (peptides were synthesized and purified as described) (19). The cells were then washed and T-hybridoma cells were added in normal medium. T-cell responses (interleukin 2 secretion) were measured using a CTLL proliferation assay as described (18). We used the following T-cell hybridomas: 3A9, specific for the HEL peptide (21). For peptide dissociation experiments, the peptides were allowed to bind to fixed macrophages in normal medium. The cells were then washed, incubated at various pH values in the above buffers, and washed in regular medium prior to addition of T cells. For studies of the carbohydrates, fixed adherent macrophages or TA3 B-lymphoma cells were incubated with peptides in the presence of the carbohydrates, washed, and then incubated with T-hybridoma cells.

p190 RhoGAP is a multi-domain protein that is thought to regulate actin cytoskeleton dynamics. It can be phosphorylated both in vitro and in vivo at multiple sites by the Src tyrosine kinase and one or more of these sites is postulated to modulate p190 function. One of the regions which is multiply phosphorylated by Src in vitro is the N-terminal GTP binding domain. Using a partially purified, bacterially expressed recombinant protein that includes the GTP binding domain (residues 1^389), we show that GTP binds to this fragment in a specific and saturable manner that is both time-and dosedependent and that tyrosine phosphorylation of this fragment by c-Src results in a loss of GTP binding activity. These findings suggest that tyrosine phosphorylation of the p190 N-terminal domain can alter its ability to bind GTP.z 2000 Federation of European Biochemical Societies.

The binding of a lysozyme and ovalbumin peptide to purified class II major histocompatibiity molecules in detergents was increased by the addition of certain lipids. Natural lipids from B lymphoma cells enhanced the binding and so did phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and cardiolipin. Phosphatidylethanolamine, sphingomyelin, and cholesterol had no effect. There was no major difference between the effects of a phospholipid and its lyso derivative. As studied with phosphatidylcholine, the increase in peptide binding was also dependent on the fatty acid composition of the lipid. (MHC) proteins (1,2). The extent of binding of peptides to histocompatibility molecules, which can be studied in detergent solutions (1-7), usually correlates with their immunogenicity in vivo (2-4). There are, however, indications that the interactions of peptides with class II MHC may be quantitatively different on live antigen-presenting cells. For example, the binding of antigenic peptides to class II MHC molecules on live cells was reported to be much faster than that found in solution (8-10). A similar result was found for the dissociation of peptides bound to the class II MHC molecules (11).We recently found that the labeling of class II MHC molecules to peptides containing a photoreactive compound was influenced by phospholipids, both in the extent as well as in the relative degree ofthe a or p chain labeling (12). When tested on cell membranes that contained class II MHC proteins, photoreactive conjugates of the hen egg white lysozyme (HEL) peptide from residues 46 to 61 labeled exclusively the a chain of I-Ak (13). Conversely, I-Ak purified in an equimolar solution of MEGA 8 and MEGA 9 detergents was labeled to similar extent on both chains. However, the addition of certain lysophospholipids resulted in strong binding, with lysophosphatidylcholine predominantly labeling the a chain and lysophosphatidylserine labeling the ,B chain. We concluded that class II MHC molecules contained dialyzable components required for their binding integrity and that, most likely, these components were lipids.Lipid-protein interactions are of critical importance in maintaining the biological activity of a number of membrane proteins (for review, see refs. 14-16). These interactions may have different degrees of specificity and often induce conformational changes in the protein critical for their function (14,(17)(18)(19)(20)(21)(22). Lipids, by interacting directly with the MHC protein or by changing the structure ofthe detergent micelles, may induce conformational changes reflected in the distal binding site of the protein (23,24). Consistent with this hypothesis are the observations indicating that class II MHC molecules reconstituted in lipid monolayers present peptides differently, depending on the lipid composition (25). Other studies have shown that treatment of antigen-presenting cells with purified phospholipase A2 or C, or with cerulenin, an antibiotic that interferes with lipid metabolism, greatly dimi...