Shc is a member of a highly conserved family of adaptor proteins that act downstream of receptors for a variety of extracellular messengers, including several oncogenically activated growth factor receptors. Shc lacks a recognizable catalytic domain but is very well equipped for protein-protein interactions. It contains both an amino-terminal phosphotyrosine-binding (PTB) domain and a carboxy-terminal Src-homology 2 (SH2) domain. These domains recognize phosphorylated tyrosine residues present on other proteins in a sequence specific manner. Shc is itself phosphorylated on at least three tyrosine residues that are thought to function as binding sites for other proteins. In the current model for Shc function downstream of growth factor receptor protein-tyrosine kinases, Shc uses its PTB domain to associate with activated receptors. Upon association with these receptors, Shc becomes tyrosine phosphorylated, creating a docking site for another cytoplasmic adaptor molecule, Grb-2. In this model, the sole function of Shc is to link Grb-2 to activated growth factor receptors.

To investigate whether Shc's sole function is to mediate the association of Grb-2 with activated growth factor receptors, we have created growth factor receptor mutants where a traditional Shc binding site has been destroyed and replaced with a Grb-2 binding site. If Shcs sole function is to link Grb-2 to activated receptors, then a receptor that bypasses the need for Shc and binds directly to Grb-2 should retain its signaling capabilities. We found that the receptor mutant that no longer binds to Shc is deficient in activating downstream signaling events. Replacing the Shc binding site with a Grb-2 binding site does not rescue receptor signaling. Our results suggest that Shc's function is more complicated than solely linking Grb-2 to activated growth factor receptors. We have also identified a novel protein that binds to Shcs SH2 domain in growth factor-stimulated cells and are currently purifying this protein in hopes to identify it by mass spectrometry.

In collaboration with the lab of Partho Ghosh, I also investigated Internal B (InlB), a protein that is expressed on the surface of Lysteria monocytogenes, mediates entry of L. monocytogenes into its host cell by binding to the HGF receptor. I have compared signaling by HGF and InlB and found that while InlB and HGF are equipotent in their ability to turn on the HGF receptor, InlB is a much better activator of the Ras-MAP kinase pathway. I went on to analyze a number of InlB deletion mutants and found that in contrast to what has been published previously, multiple InlB domains are involved in the activation of cellular signaling pathways. In addition, my results provide evidence that InlB affects the Ras-MAP kinase pathway through the HGF receptor and through a second, HGF receptor-independent, pathway.

PUBLICATIONS (resulting from this training)

Copp J, Marino M, Banerjee M, Ghosh P, van der Geer P. (2003) Multiple regions of internalin B contribute to its ability to turn on the Ras-mitogen-activated protein kinase pathway. J Biol Chem. 278:7783-9.

Marino M, Banerjee M, Copp J, Dramsi S, Chapman T, van der Geer P, Cossart P, Ghosh P. (2004) Characterization of the calcium-binding sites of Listeria monocytogenes InlB. Biochem Biophys Res Commun. 316:379-86.

Banerjee M, Copp J, Vuga D, Marino M, Chapman T, van der Geer P, Ghosh P. (2004) GW domains of the Listeria monocytogenes invasion protein InlB are required for potentiation of Met activation. Mol Microbiol. 52:257-71.

Wilhelmsen K, Copp J, Glenn G, Hoffman RC, Tucker P, van der Geer P. (2004) Purification and identification of protein-tyrosine kinase-binding proteins using synthetic phosphopeptides as affinity reagents. Mol Cell Proteomics. 3:887-95.

Copp J, Wiley S, Ward MW, van der Geer P. (2005) Hypertonic shock inhibits growth factor receptor signaling, induces caspase-3 activation, and causes reversible fragmentation of the mitochondrial network. Am J Physiol Cell Physiol. 288:C403-15.