The Src SH3 domain is required for DNA synthesis induced by platelet-derived growth factor and epidermal growth factor.

The Src family of protein tyrosine kinases has been implicated in the response of cells to platelet-derived growth factor (PDGF) or epidermal growth factor (EGF). We recently described a microinjection approach that we used to demonstrate that kinase activity of Src family members is required for PDGF- and EGF-induced S-phase entry of fibroblasts. We have now used this approach to ask whether a functional SH3 domain of Src is required to transduce the mitogenic signal upon PDGF or EGF stimulation. Microinjection of plasmids encoding Src mutants lacking the SH3 domain (SrcDeltaSH3) or point-mutated within the ligand binding surface of the SH3 domain, but with intact kinase domains, inhibited the mitogenic effect of PDGF and EGF in fibroblasts. SrcDeltaSH3 could still associate with the PDGF receptor, suggesting that the inhibitory effect of the Src SH3 mutants was brought about by a failure of the PDGF receptor.SrcDeltaSH3 complex to relay the mitogenic signal further downstream. Chimeric molecules in which the Src SH3 domain was replaced with that of spectrin or Lck also blocked PDGF-induced DNA synthesis, whereas a chimera containing the Fyn SH3 domain did not. These data suggest that the Src or Fyn SH3 domain is required either for correct substrate selection or to recruit other proteins to the PDGF receptor.

Src family protein tyrosine kinases and cellular signal transduction pathways.

Members of the Src family of protein tyrosine kinases are thought to be involved in signal transduction pathways that control growth and cellular architecture. In recent years it has been shown that they interact with receptor tyrosine kinases (such as the platelet-derived growth factor receptor) and with receptors that themselves lack intrinsic tyrosine kinase activity (such as the interleukin-2 receptor). In some cases they are required for mitogenic signalling by these receptors. They are also activated in response to stress and during mitosis.

Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions.

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its C-terminal tail. The repressed state is achieved through intramolecular interactions involving the phosphorylated tail, the Src homology 2 (SH2) domain and the SH3 domain. Both the SH2 and SH3 domains have also been shown to mediate the intermolecular interaction of Src with several proteins. To test which amino acids of the Src SH3 domain are important for these interactions, and whether the intra- and intermolecular associations involve the same residues, we carried out a detailed mutational analysis of the presumptive interaction surface. All mutations of conserved hydrophobic residues had an effect on both inter- and intramolecular interactions of the Src SH3 domain, although not all amino acids were equally important. Chimeric molecules in which the Src SH3 domain was replaced with those of spectrin or Lck showed derepressed kinase activity, whereas a chimera containing the Fyn SH3 domain was fully regulated. Since spectrin and Lck SH3 domains share the conserved hydrophobic residues characteristic of SH3 domains, other amino acids must be important for specificity. Mutational analysis of non- or semi-conserved residues in the RT and n-Src loops showed that some of these were also involved in inter- and intramolecular interactions. Stable transfection of selected SH3 domain mutants into NIH-3T3 cells showed that despite elevated levels of phosphotyrosine, the cells were morphologically normal, indicating that the SH3 domain was required for efficient transformation of NIH-3T3 cells by Src.

Macromolecular chelation as an improved mechanism of protease inhibition: structure of the ecotin-trypsin complex.

The 2.4 A crystal structure (R = 0.180) of the serine protease inhibitor ecotin was determined in a complex with trypsin. Ecotin's dimer structure provides a second discrete and distal binding site for trypsin and, as shown by modelling experiments, other serine proteases. The second site is approximately 45 A from the reactive/active site of the complex and features 13 hydrogen bonds, including six that involve carbonyl oxygen atoms and four bridged by water molecules. Contacts ecotin makes with trypsin's active site are similar to, though more extensive than, those found between trypsin and basic pancreatic trypsin inhibitor. The side chain of ecotin Met84 is found in the substrate binding pocket of trypsin where it makes few contacts, but also does not disrupt the solvent structure or cause misalignment of the scissile bond. This first case of protein dimerization being used to augment binding energy and allow chelation of a target protein provides a new model for protein-protein interactions and for protease inhibition.

Expression of the protease inhibitor ecotin and its co-crystallization with trypsin.

We have expressed the serine protease inhibitor ecotin to high levels (greater than 400 mg/l of cell culture) in its natural mileau, the Escherichia coli periplasm, using the endogenous signal peptide and the heterologous tac promoter. After induction, functional, soluble ecotin comprises 15% of total cellular protein. This expression system has facilitated initiation of a crystallographic study to determine the structural basis for inhibition of the pancreatic serine proteases by ecotin. Ecotin was co-crystallized with rat trypsin mutant D102N. Preliminary crystallographic analysis of co-crystals showed that they diffract to at least 2.7 A, and indicate that they belong to the monoclinic space group, P21. The cell constants are a = 52.0 A, b = 93.3 A, c = 160.7 A, and beta = 96 degrees. Four molecules each of trypsin and ecotin are found in the asymmetric unit.