Abstract
The role of Shc as a substrate of receptors for growth factors and cytokines is well established. To gain further insight into the function of Shc in signal transduction, we used an affinity method to identify potential Shc-binding proteins. Incubation of bovine brain lysates with a glutathione S-transferase (GST)-Shc fusion protein immobilized on glutathione-Sepharose beads resulted in the binding of cellular proteins of approximately 115, 110, and 100 kDa as well as those of 50 and 17 kDa. Amino acid sequencing of tryptic peptides revealed that the 100-kDa protein was almost identical to beta-adaptin and that the 110- and 115-kDa proteins were almost identical to alphaA-adaptin. Using immunoblot analysis, anti-alpha-adaptin antibody recognized several proteins of 100 approximately 115 kDa, and anti-beta-adaptin antibody recognized a 100-kDa protein, suggesting that alphaA-, alphaC-, and beta-adaptins are bound to the GST-Shc fusion protein. Immunoblot analysis with anti-alpha-adaptin antibody revealed that alpha-adaptin was coimmunoprecipitated with Shc from PC12, KB, and COS cell lysates, suggesting a specific interaction of Shc and adaptins in intact cells. A binding study using mutant GST-Shc fusion proteins revealed that the collagen homologous region (amino acids 233-377) of Shc was required for adaptin binding. Conversely, the collagen homologous region of Shc inhibited the binding of adaptins to GST-Shc. In addition, adaptin was able to bind mutant fusion proteins containing amino acids 233-369, 233-355, 346-369, and 346-355 of Shc, but failed to bind a mutant containing amino acids 233-345, suggesting that amino acids 346-355 (RDLFDMKPFE) in the collagen homologous region of Shc are required for adaptin binding. Thus, this study indicates the specific interaction of Shc with alpha- and beta-adaptin components of plasma membrane adaptor proteins that are thought to be involved in receptor endocytosis.
Highlights
Many receptor tyrosine kinases such as those for epidermal growth factor (EGF),1 platelet-derived growth factor, and insulin transmit intracellular signals for cell proliferation and differentiation [1]
Amino acid sequencing of the fragments of the 110-kDa protein yielded the sequences KSEFRQNLGRMYLFYGNK, KTSVQFQNFSPTVVHPGDL, and KYLQVGHLLREPNAQAQMYRLTLRTK, and that of the 115-kDa protein yielded KTSVQFQNFSPTVVHPGDL. These amino acid sequences were almost identical to the mouse ␣A-adaptin, but not ␣C-adaptin, of the adaptor complex (Fig. 1B) [24]. These results indicate that ␣A- and -adaptins bind to Shc expressed as fusion protein with glutathione S-transferase (GST)
Amino acid sequencing revealed that the 100-kDa protein is -adaptin, whereas immunoblot analysis with antibodies to ␣- and -adaptins disclosed that the 100-kDa protein consists of ␣and -adaptins
Summary
Many receptor tyrosine kinases such as those for epidermal growth factor (EGF), platelet-derived growth factor, and insulin transmit intracellular signals for cell proliferation and differentiation [1]. A number of proteins that contain SH2 domains have been identified, including phospholipase C-␥1, Ras GTPase-activating protein, the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase, Grb, and Shc [5]. Many of these proteins contain one or more SH3 domains, which interact with proline-rich sequences, serving as linker molecules to bring other proteins into the signaling complex [5]. Tyrosine 317 within the collagen homologous region of Shc is suggested to be the binding site of Grb2 [6], and Shc interacts with tyrosine-phosphorylated growth factor receptors such as EGF receptors, TrkA, and ErbB-2 (14 –16). To gain further insight into the function of Shc in signal transduction, we tried to identify Shc-binding proteins using glutathione S-transferase (GST)-Shc fusion proteins
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