9305458 Haigler Growth factors such as EGF stimulate cell replication by binding to transmembrane receptors with inducible protein-tyrosine kinase activity. The activated receptors undergo autophosphorylation, then phosphorylate tyrosine residues of other regulatory proteins. Recent studies indicate that non-catalytic src-homology 2 (SH2) domains mediate these interactions by binding to phosphotyrosine residues located within specific amino acid sequences. The focus of this research is the possible role in cellular signal transduction for Annexin I, a substrate for the EGF receptor/kinase whose biological function is unknown. The proposed function of tyrosine-phosphorylation of Annexin I is to promote binding to the SH2 domain of phospholipase C (PLC)-gamma-1 and perhaps other regulatory proteins. The model is supported by the following: 1) the amino acid sequence around the phosphorylation site of Annexin I matches the consensus sequence of the site to which the SH2 domain of PLC-gamma-1 is known to bind; and 2) preliminary experiments showed that Annexin I phosphorylated on tyr-21 (P-tyr- Annexin I) by the EGF receptor/kinase bound with high affinity to a fusion protein containing SH2 and SH3 domains from PLC-gamma-1. Hypothetical functions of P-tyr-Annexin I binding to regulatory proteins include modulation of their catalytic activity or shuttling to the plasma membrane via the calcium-dependent phospholipid binding activity of Annexin I. Planned experiments will characterize the SH2/SH3 binding properties of Annexin I in vitro. The binding of 32P-labelled P-tyr-Annexin I to fusion proteins containing SH2 and/or SH3 domains from PLC-gamma-1 and other regulatory proteins will be measured to delineate the regions which contain the binding sites, the dissociation constants of the interactions and binding specificities will be determined. In vitro assays will determine whether P-tyr-Annexin I binding to PLC- gamma-1 modulates its catalytic activity and wh ether binding to PLC-gamma-1 changes the calcium and phospholipid binding characteristics of P-tyr-Annexin I. %%% Growth factors such as epidermal growth factor (EGF) stimulate cell replication by binding to transmembrane receptors. The activated receptors then act as enzymes to add phosphate groups, first to themselves, and then to specific amino acids of other regulatory proteins. The focus of this research is the possible role in cellular signal transduction for "Annexin I", a member of a family of calcium-binding proteins which is also a target for addition of phosphate group by the EGF receptor enzyme. The calcium binding Annexins have been identified in many cell types, and it has been widely supposed that they are important cellular regulatory molecules, but until now, no one has obtained convincing evidence as to their real physiological function. The preliminary results on which this project is based provide the first solid clue as to a biological function of Annexin I. This research, thus, will help to define the function of this class of regulatory molecules and the interaction of regulatory proteins involved in the signal transduction pathway by which epidermal growth factor stimulates cell division.***
