Characterization of Signal Transducing Enzymes

Investigators

Abstract

The class of mammalian biologically active polypeptides called growth factors influence the proliferation, differentiation, motility, maintenance and apoptosis of target cells. The ErbB family of receptors which include the epidermal growth factor receptor (EGFR), ErbB2, ErbB3 and ErbB4 mediate the biological actions of a family of growth factors which are structurally related to EGF. The EGFR, ErbB2 and ErbB4 are protein tyrosine kinases, whereas ErbB3 has no known intrinsic enzymatic activity. Engagement of growth factor by the extracellular domain of an ErbB initiates a process of hetero- and homo-oligomerization of ErbBs, transphosphorylation of the ErbBs in the complex and subsequent signaling. It is important to note that no known extracellular growth factor ligand for ErbB2 has been identified. Relatively little is known regarding the molecular mechanism of ErbB kinase activation and how specificity of signaling is conferred. To generate insight into ErbB signaling our laboratory is studying the enzymology of the ErbBs. To this end, we have generated epitope-tagged versions of each of the ErbB receptors to facilitate an in vitro determination of the enzymatic parameters towards specific substrates. Synthetic peptide substrates corresponding to specific phosphorylation sites within the COOH-terminal regions of the ErbBs have been prepared for this analysis. In addition, mutant ErbBs are being generated in which tyrosine to phenylalanine mutations have been made at known transphosphorylation sites. These mutants will be used in intact cells to test various hypotheses which arise from the in vitro enzymatic studies. An additional area of interest involves the role of the EGFR kinase activity in EGF-dependent signaling. Gene targeting and inactivation of the murine EGFR results in strain-dependent phenotypes that range from death in utero to postnatal abnormalities in skin, kidney, lung, gastrointestinal tract, and brain. However, recent work from our lab has demonstrated that some EGF-dependent signals, such as activation of MAP kinase and the pro-survival kinase Akt, do not require the EGFR kinase activity in cells which also express ErbB2. Since EGFR and ErbB2 are almost always coexpressed in fibroblasts and epithelial cells, certain aspects of EGF signalling in vivo may be EGFR kinase-independent. To more fully understand the in vivo function of the EGFR kinase activity we are in the process of generating a knock-in mouse which is homozygous for the kinase-inactive form of the EGFR. In the event that the phenotype of this knock-in mouse is indistinguishable from that of the EGFR knockout mouse (i.e. early lethality), we plan to cross the F1 heterozygotic knock-in mouse with mice carrying the loxP-modified alleles of the wild type EGFR (conditional knockout). The role of the protein-tyrosine phosphatase SHP-2 as a positive mediator of ErbB receptor signalling. The class of mammalian biologically active polypeptides called growth factors influence the proliferation, differentiation, motility, maintenance and apoptosis of target cells. On-going studies in my laboratory are directed towards understanding the mechanism of action and biological role of growth factors which signal through the ErbB family of receptors . To address these issues, we utilize techniques common to protein biochemistry, cell biology and molecular biology. The ErbB family of receptors which include the epidermal growth factor receptor (EGFR), ErbB2, ErbB3 and ErbB4 mediate the biological actions of a family of growth factors which are structurally related to EGF. Members of this family of mitogens such as EGF and amphiregulin require the presence of the EGFR on cells for signaling, whereas signal transduction by the heregulins (HRGs; neu differentiation factor, neuregulin, acetylcholine receptor-inducing activity, glial growth factor) is initiated via an interaction with either ErbB3 or ErbB4. Previous work from our laboratory has demonstrated that the Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase SHP-2 is an essential positive mediator of ligand-induced signalling by all of the ErbB receptors (Deb et al., 1998). We found that there was a common requirement for the catalytic activity and both SH2 domains in mitogen-activated protein (MAP) kinase activation by EGF and the heregulins. However, at the present time the mechanism of action and substrate target responsible for this SHP-2 function is unknown. To better understand this function we are generating mutant forms of SHP-2 which are constitutively-activated in vitro and evaluating the ability of these mutants to signal in cells in the absence of ErbB activation or extracellular stimuli. A mechanistic evaluation of these data will provide information regarding downstream targets of SHP-2 and position SHP-2 function within the well characterized MAP kinase cascade.