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Microinjection of a protein-tyrosine-phosphatase inhibits insulin action in Xenopus oocytes
M F Cicirelli1, N K Tonks, C D Diltz
1Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle 98195.
Abstract:
A protein-tyrosine-phosphatase (PTPase 1B; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), specific for phosphotyrosyl residues, was microinjected into Xenopus oocytes. This resulted in a 3- to 5-fold increase in PTPase activity over endogenous levels. The PTPase blocked the insulin-stimulated phosphorylation of tyrosyl residues on endogenous proteins, including a protein having a molecular mass in the same range as the beta subunit of the insulin or insulin-like growth factor I receptor. PTPase 1B also blocked the activation of an S6 peptide kinase--i.e., an enzyme recognizing a peptide having the sequence RRLSSLRA found in a segment of ribosomal protein S6 and known to be activated early in response to insulin. On the other hand, the insulin stimulation of an S6 kinase, detected by using 40S ribosomes as substrate, was unaffected even though PTPase 1B partially prevented the phosphorylation of ribosomal protein S6 in vivo. Mono Q chromatography of insulin-treated oocyte extracts revealed two main peaks of S6 kinase activity. Fractions from the first peak displayed S6 peptide kinase activity that was essentially abolished in profiles from PTPase 1B-injected oocytes. Material from the second peak, which was best revealed by using 40S ribosomes as substrate and had comparatively little S6 peptide kinase activity, was minimally affected by PTPase 1B. These observations suggest that at least two distinct "S6 kinases" are involved in ribosomal protein S6 phosphorylation in vivo and that the activation pathways for these enzymes differ in their sensitivity to PTPase 1B.
Insights
Protein-tyrosine-phosphatase 1B (PTPase 1B) inhibits insulin signaling by blocking tyrosyl phosphorylation and S6 peptide kinase activation in Xenopus oocytes, revealing distinct S6 kinase pathways.
Area of Science:
- Cellular Biology
- Molecular Endocrinology
- Signal Transduction
Background:
- Protein-tyrosine-phosphatase 1B (PTPase 1B) dephosphorylates phosphotyrosyl residues.
- Insulin signaling involves tyrosine phosphorylation and activation of S6 kinases.
- Xenopus oocytes are a model system for studying early insulin responses.
Purpose of the Study:
- To investigate the role of PTPase 1B in insulin signaling pathways.
- To determine the effect of PTPase 1B on insulin-stimulated protein phosphorylation and S6 kinase activation.
- To elucidate the distinct mechanisms of S6 kinase activation in response to insulin.
Main Methods:
- Microinjection of PTPase 1B into Xenopus oocytes.
- Assessing insulin-stimulated tyrosine phosphorylation of endogenous proteins.
- Measuring S6 peptide kinase and S6 kinase activity using different substrates.
- Mono Q chromatography to separate S6 kinase isoforms.
Main Results:
- PTPase 1B microinjection increased PTPase activity and blocked insulin-stimulated tyrosyl phosphorylation.
- PTPase 1B inhibited the activation of an S6 peptide kinase but not an S6 kinase acting on 40S ribosomes.
- Mono Q chromatography revealed two distinct S6 kinase peaks with differential sensitivity to PTPase 1B.
Conclusions:
- PTPase 1B plays a role in regulating insulin signaling by dephosphorylating key substrates.
- At least two distinct S6 kinases are involved in ribosomal protein S6 phosphorylation.
- The activation pathways of these S6 kinases exhibit differential sensitivity to PTPase 1B, suggesting distinct regulatory mechanisms.