Mechanism of transmembrane signal transduction

Candice Klingbeil

Appointment Period: 1993-1996, Grant Years: [08,09,10,11]

Part of my project examines the interactions between the epidermal growth factor (EGF) receptor and its peptide substrates. The crystal structure of the insulin receptor has provided insight into how protein tyrosine kinases may bind to their substrates. It was shown that several basic residues created a positively charged surface.

The epidermal growth factor receptor contains five autophosphorylation sites in its C-terminal region. Synthetic peptides based on the major autophosphorylation site at Tyr 1173 were tested as substrates of the intracellular domain of the epidermal growth factor receptor. A peptide containing acidic residues N-terminal to the substrate Tyr as well as the Tyr-Met-Xaa-Met motif of the insulin receptor substrate 1 had a Km value of 15 microM, the lowest value for a synthetic peptide reported to date. Another important residue contributing to substrate binding is the Tyr itself, or more specifically, the hydroxyl group of the Tyr. Substituting Phe for Tyr results in a peptide that is ineffective as an inhibitor of kinase phosphorylation. However, substitution of a Ser residue does not restore a functional substrate, indicating specificity for the Tyr hydroxyl. Secondary structure algorithms predicted that the peptide substrate based on the native sequence at Tyr 1173 would have a propensity to adopt a helical conformation in solution. Circular dichroism spectroscopy confirmed this prediction. The secondary structure of the peptide substrate is significant in its consistency with the idea that secondary structure is an important determinant in substrate recognition by protein tyrosine kinases.

Another part of my project involves a mutant EGF receptor, common in breast, lung, and brain cancer, in which a part of the extracellular ligand binding domain is deleted. These receptors do not bind EGF; however, they have a low level constitutive activity. They are also not downregulated and this failure to attenuate signalling is believed to be the major mechanism which enhances tumorigenicity.

PUBLICATIONS (resulting from this training, and some recent ones)

Klingbeil CK, Gill GN, Cadena DL. (1995) Analysis of substrate recognition determinants in a synthetic peptide containing the Tyr 1173 autophosphorylation site of the epidermal growth factor receptor. Arch Biochem Biophys. 316:745-50.

Huang HS, Nagane M, Klingbeil CK, Lin H, Nishikawa R, Ji XD, Huang CM, Gill GN, Wiley HS, Cavenee WK. (1997) The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers is mediated by threshold levels of constitutive tyrosine phosphorylation and unattenuated signaling. J Biol Chem. 272:2927-35.

Klingbeil CK, Gill GN. (1999) A basic residue, Lys 782, composes part of the ATP-binding site on the epidermal growth factor receptor tyrosine kinase. Arch Biochem Biophys. 363:27-32.

Sieg DJ, Hauck CR, Ilic D, Klingbeil CK, Schaefer E, Damsky CH, Schlaepfer DD. (2000) FAK integrates growth-factor and integrin signals to promote cell migration. Nat Cell Biol. 2:249-56.

Hauck CR, Klingbeil CK, Schlaepfer DD. (2000) Focal adhesion kinase functions as a receptor-proximal signaling component required for directed cell migration. Immunol Res. 21:293-303.

Klingbeil CK, Hauck CR, Hsia DA, Jones KC, Reider SR, Schlaepfer DD. (2001) Targeting Pyk2 to beta 1-integrin-containing focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null cells. J Cell Biol. 152:97-110.

Shi JD, Bullock C, Hall WC, Wescott V, Wang H, Levitt DJ, Klingbeil CK. (2002) In vivo pharmacodynamic effects of Hu1D10 (remitogen), a humanized antibody reactive against a polymorphic determinant of HLA-DR expressed on B cells. Leuk Lymphoma. 43:1303-12.