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In vitro tyrosine phosphorylation studies on RAS proteins and calmodulin suggest that polylysine-like basic peptides
Y Fujita-Yamaguchi1, S Kathuria, Q Y Xu
1Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Abstract:
We have investigated the in vitro tyrosine phosphorylation of the HRAS and KRAS proteins by human placental insulin receptor kinase. Purified HRAS proteins are not phosphorylated by purified insulin receptor kinase. Since the tyrosine phosphorylation of calmodulin by the insulin receptor kinase in vitro requires cofactors such as protamine and poly(L-lysine), we examined the possibility that poly(L-lysine) may also potentiate the interaction between RAS proteins and the insulin receptor. We found that purified HRAS proteins are indeed phosphorylated by purified insulin receptor kinase in the presence of poly(L-lysine). In contrast, the KRAS protein, which carries an extremely basic domain (residues 172-182, Lys-Asp-Glu-Lys6-Ser-Arg), is phosphorylated by the receptor kinase without the addition of basic proteins. We then determined whether the KRAS basic domain peptide plays a role similar to that of poly(L-lysine) and found that both the HRAS protein and calmodulin are phosphorylated by the receptor kinase in the presence of the KRAS basic domain peptide. Further examination of the role of poly(L-lysine) in potentiating tyrosine phosphorylation of the HRAS protein and calmodulin by purified insulin receptor kinase indicates that poly(L-lysine) affects the conformation of these protein substrates as well as that of the receptor kinase domain. These studies suggest that polylysine-like basic proteins or domains are required to establish the interaction between insulin receptor kinase and its substrate.
Insights
Basic proteins like poly(L-lysine) are essential for the insulin receptor kinase to phosphorylate HRAS proteins in vitro. KRAS proteins, however, can be phosphorylated without these cofactors due to their intrinsic basic domain.
Area of Science:
- Biochemistry
- Molecular Biology
- Signal Transduction
Background:
- Insulin receptor kinase plays a crucial role in cellular signaling pathways.
- Tyrosine phosphorylation is a key post-translational modification regulating protein function.
- RAS proteins (HRAS, KRAS) are critical in cell growth and differentiation.
Purpose of the Study:
- To investigate the in vitro tyrosine phosphorylation of HRAS and KRAS proteins by human placental insulin receptor kinase.
- To determine the role of cofactors, such as poly(L-lysine), in this phosphorylation process.
- To elucidate the mechanism by which the insulin receptor kinase interacts with its substrates.
Main Methods:
- In vitro kinase assays using purified human placental insulin receptor kinase.
- Purified HRAS and KRAS proteins as substrates.
- Use of cofactors like poly(L-lysine) and a KRAS basic domain peptide.
- Analysis of protein phosphorylation patterns.
Main Results:
- Purified HRAS proteins were not phosphorylated by insulin receptor kinase alone.
- Poly(L-lysine) potentiated the tyrosine phosphorylation of HRAS by insulin receptor kinase.
- KRAS protein, possessing a basic domain, was phosphorylated without added cofactors.
- The KRAS basic domain peptide mimicked poly(L-lysine) in promoting HRAS and calmodulin phosphorylation.
- Poly(L-lysine) was found to affect the conformation of both substrates and the kinase domain.
Conclusions:
- Poly(L-lysine)-like basic proteins or domains are required for the interaction between insulin receptor kinase and its substrates, including HRAS and calmodulin.
- The intrinsic basic domain of KRAS facilitates its interaction with the insulin receptor kinase.
- These findings highlight the importance of substrate-specific interactions mediated by basic domains in regulating tyrosine kinase activity.