No abstract

In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p2l"GAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src-and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with Mrs of 62,000 and 130,000 was inhibited by incubation with a Src S13 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.The Src family of protein tyrosine kinases contains two conserved domains, the Src homology 2 (SH2) and Src homology 3 (SH3) domains. SH2 and SH3 domains mediate intramolecular and intermolecular binding interactions which regulate the functional activity of these proteins in intracellular signal transduction pathways (20,37,38). The intermolecular protein-protein interactions couple Src family kinases with proteins which serve as protein substrates or which determine the subcellular localization of these enzymes (5, 20). The intramolecular binding interactions mediated by SH2 and SH3 domains regulate the catalytic activity of these kinases (20, 50, 56). SH2 and SH3 domains are also found in other protein tyrosine kinases (e.g., Abl, Fps, Syk, and Zap) as well as in cellular proteins otherwise unrelated to Src family kinases (38). These proteins include enzymes (e.g., phospholipase C--y, p21aSGAP [a Ras GTPase-activating protein], and the p85 subunit of phosphatidylinositol 3'-kinase [p85-PI-3K]), transcription factors (e.g., the p113, p91, and p84 subunits of interferon-stimulated gene factor 3), cytoskeletal proteins (e.g., tensin, cortactin, myosin 1B, and a-spectrin), and adaptor proteins that appear to serve exclusively as coupling factors that link proteins involved in signaling pathways (e.g., Grb-2/ Sem5, Nck, and Crk) (6,8,14,20,28,31,37,38 and SH3 domains are commonly found together in intracellular p...

The human homeodomain protein Phox1 interacts functionally with serum response factor (SRF) to impart serum responsive transcriptional activity to SRF-binding sites in a HeLa cell cotransfection assay. However, stable ternary complexes composed of SRF, Phox1, and DNA, which presumably mediate the transcriptional effects of Phox1 in vivo, have not been observed in vitro. Here, we report the identification, purification, and molecular cloning of a human protein that promotes the formation of stable higher-order complexes of SRF and Phox1. We show that this protein, termed SPIN, interacts with SRF and Phox1 in vitro and in vivo. Furthermore, SPIN binds specifically to multiple sequences in the c-fos promoter and interacts cooperatively with Phox1 to promote serum-inducible transcription of a reporter gene driven by the c-fos serum response element (SRE). SPIN is identical to the initiator-binding protein TFII-I. Consistent with this hypothesis, SPINexhibits modest affinity for a characterized initiator sequence in vitro. We propose that this multifunctional protein coordinates the formation of an active promoter complex at the c-fos gene, including the linkage of specific signal responsive activator complexes to the general transcription machinery.

The complete primary structure of Fel dI (International Union of Immunological Societies nomenclature), the major allergen produced by the domestic cat, Felis domesticus, was determined by protein sequence analysis and cDNA cloning. Protein sequencing of Fel dI from an immunoaffinity-purified extract of house dust revealed that the allergen is composed of two polypeptide chains. Degenerate oligonucleotides derived from the protein sequence were used in polymerase chain reaction amplification ofcat salivary gland cDNA to demonstrate that the two chains are encoded by different genes. Chain 1 of Fel dM shares amino acid homology with rabbit uteroglobin, while chain 2 is a glycoprotein with N-linked oligosaccharides.

In an effort to extend automated Edman degradation to nanomole quantities of protein, the method of sequenator analysis described by Edman and Begg (Edman, P., and Begg, G. (1967), Eur. J. Biochem. 1, 80) was modified to permit long degradations in the absence of carrier proteins. By using an aqueous 0.1 M Quadrol program with limited, combined benezene-ethyl acetate solvent extractions, as well as a change in the delivery system for heptafluorobutyric acid, it was possible to recover and identify the first 30 amino acid residues from a sequenator run on 7 nmol of myoglobin. For 3 nmol of myoglobin, 20 steps could be identified. PTH-amino acids were identified by gas-liquid chromatography and thin-layer chromatography on polyamide sheets. Without using a carrier protein the cup to prevent mechanical losses (Niall, H. D., Jacobs, J. W., Van Rietshoten, J., and Tregear, G. W. (1974), FEBS Lett. 41, 62), the repetitive yield using this program was 93-96%. The same program has been applied successfully to peptides of 14 or more residues with or without modification by Braunitzer's reagent and to a number of larger peptides and proteins including a 216 residue segment of rabbit antibody heavy chain in which a sequence of 35 steps was accomplished on 25 nmol.