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SH2 domain proteins as high-affinity receptor tyrosine kinase substrates
1Department of Pharmacology, The University of Iowa, Iowa City 52242-1109.
Abstract:
Activation of a growth factor receptor tyrosine kinase (RTK) is accompanied by a rapid autophosphorylation of the receptor on tyrosine residues. Receptor activation has been shown to promote the association of signal-transducing proteins containing SH2 domains (second domain of src homology). These receptor-associated proteins can, in turn, be phosphorylated by the RTK, an event which presumably regulates their activities. It has been suggested that SH2 domains in signal-transducing proteins target these proteins as substrates of the activated RTK. To test this hypothesis, recombinant proteins were generated that contained tyrosine phosphorylation sites of the erbB3 receptor and/or the SH2 domain of c-src. Incorporation of the SH2 domain led to a decrease in KM and an increase in Vmax for the substrate. The KM determined for one chimeric SH2/erbB3 substrate was among the lowest reported for epidermal growth factor RTK substrates. Experiments with a truncated kinase lacking C-terminal autophosphorylation sites indicated that the reduction in KM for these substrates was mediated by interactions between the substrate SH2 domain and phosphotyrosine residues of the RTK. These interactions could also inhibit RTK activity. These results demonstrate that the SH2 domain can effectively target substrates to a RTK and that SH2 domain proteins can regulate RTK activity.
Insights
The SH2 domain targets proteins to receptor tyrosine kinases (RTKs), enhancing their phosphorylation. This interaction also regulates RTK activity, revealing a novel mechanism in cell signaling.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Biochemistry
Background:
- Receptor tyrosine kinases (RTKs) activate upon growth factor binding, leading to autophosphorylation.
- SH2 domains mediate protein-protein interactions, particularly with phosphotyrosine residues.
- SH2 domain proteins are hypothesized to be substrates of activated RTKs.
Purpose of the Study:
- To investigate the role of SH2 domains in targeting substrates to RTKs.
- To determine if SH2 domains influence the enzymatic activity of RTKs towards their substrates.
- To elucidate the mechanism by which SH2 domains interact with RTKs.
Main Methods:
- Generation of recombinant proteins incorporating SH2 domains and RTK phosphorylation sites.
- Enzymatic assays to measure substrate phosphorylation kinetics (KM and Vmax).
- Experiments using truncated RTKs to analyze the role of autophosphorylation sites.
Main Results:
- Incorporation of the SH2 domain decreased the KM and increased the Vmax for substrates.
- A chimeric SH2/erbB3 substrate exhibited one of the lowest KMs reported for epidermal growth factor RTK substrates.
- Interaction between the substrate SH2 domain and RTK phosphotyrosine residues mediated substrate targeting and could inhibit RTK activity.
Conclusions:
- SH2 domains effectively target substrates to RTKs.
- SH2 domain-containing proteins can regulate RTK activity through interactions with phosphotyrosine residues.
- This study provides mechanistic insights into RTK-substrate recognition and regulation.