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Nonphosphorylatable substrate analogs selectively block autophosphorylation and activation of the insulin receptor,
S E Shoelson1, M F White, C R Kahn
1Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02215.
Abstract:
The receptors for insulin and epidermal growth factor undergo tyrosine autophosphorylation in response to ligand stimulation, while pp60v-src is an unregulated tyrosine kinase. In this report we show that each of the kinases phosphorylates an exogenous peptide that corresponds to the insulin proreceptor sequence 1142-1153. When the kinases were pre-phosphorylated, saturable Michaelis-Menten kinetics were observed. However, when the kinases had not been pre-phosphorylated biphasic kinetics were observed; at progressively higher substrate concentrations (greater than Km) less substrate phosphorylation was seen. Furthermore, when the kinases had not been pre-phosphorylated kinase autophosphorylation was inhibited at high substrate concentrations. On this basis we postulated that the substrate inhibition of substrate phosphorylation resulted directly from substrate inhibition of kinase autophosphorylation. To test this we designed additional peptides to function specifically as inhibitors of the kinases. Each of the 3 tyrosine residues within the substrate sequence were replaced either by 4-methoxyphenylalanine or phenylalanine, residues structurally similar to tyrosine but unable to accept phosphoryl transfer. Both analogs inhibited insulin and epidermal growth factor receptor autophosphorylation, whereas only the Phe-substituted analog inhibited pp60v-src phosphorylation. These data suggest that autophosphorylation of tyrosine residues near the kinase active site is a generalized mechanism for tyrosine kinase activation and that activation can be selectively blocked by substrates and nonphosphorylatable analogs.
Insights
Tyrosine kinases, including insulin and epidermal growth factor receptors, activate through autophosphorylation. This process can be selectively blocked by non-phosphorylatable analogs, revealing a generalized activation mechanism for these crucial enzymes.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Insulin and epidermal growth factor receptors are tyrosine kinases that activate via autophosphorylation upon ligand binding.
- pp60v-src is an unregulated tyrosine kinase involved in cellular signaling.
Purpose of the Study:
- To investigate the kinetics of tyrosine kinase phosphorylation using an exogenous peptide substrate.
- To determine the role of autophosphorylation in tyrosine kinase activation and substrate inhibition.
- To explore the potential for selective inhibition of tyrosine kinase activity.
Main Methods:
- Kinetic analysis of tyrosine kinases (insulin receptor, epidermal growth factor receptor, pp60v-src) phosphorylating an exogenous peptide.
- Design and synthesis of non-phosphorylatable peptide analogs by replacing tyrosine residues.
- Assessment of peptide analog inhibition on kinase autophosphorylation and substrate phosphorylation.
Main Results:
- Pre-phosphorylation of kinases resulted in Michaelis-Menten kinetics.
- Unphosphorylated kinases exhibited biphasic kinetics with substrate inhibition at higher concentrations.
- Substrate inhibition correlated with inhibition of kinase autophosphorylation.
- Non-phosphorylatable analogs selectively inhibited kinase activity, with differential effects on receptor tyrosine kinases versus pp60v-src.
Conclusions:
- Autophosphorylation of tyrosine residues near the kinase active site is a general mechanism for tyrosine kinase activation.
- Kinase activation can be modulated by substrate concentration and selectively blocked by non-phosphorylatable analogs.
- These findings offer insights into the regulation of tyrosine kinase signaling pathways.