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Tyrosine and threonine phosphorylation of an immunoaffinity-purified 44-kDa MAP kinase
J C Scimeca1, R Ballotti, T T Nguyen
1INSERM U145, Faculté de Médecine, Nice, France.
Abstract:
We have approached the functioning of a MAP kinase, which is thought to be a "switch kinase" in the phosphorylation cascade initiated from various receptor tyrosine kinases including the insulin receptor. To do so, antipeptide antibodies were raised against the C-terminal portion of ERK1 (extracellular signal-regulated kinase 1), a protein kinase belonging to the family of MAP kinases. With these antipeptide antibodies, we observed the following: (i) a 44-kDa protein can be specifically recognized both under native and denaturing conditions; (ii) a 44-kDa phosphoprotein can be revealed in 32P-labeled cells; its phosphorylation is stimulated by insulin, sodium orthovanadate, and okadaic acid; (iii) a MBP kinase activity can be precipitated, which phosphorylates MBP on threonine residues, and which is stimulated by insulin, sodium orthovanadate, okadaic acid, and fetal calf serum; (iv) this MBP kinase activity appears to be correlated with the in vivo induced phosphorylation of the 44-kDa protein. We next studied the in vitro phosphorylation of this 44-kDa/ERK1-immunoreactive protein. A time- and manganese-dependent phosphorylation was stimulated by the in vitro addition of sodium orthovanadate. Phosphoamino acid analysis of the in vitro phosphorylated 44-kDa protein revealed both threonine and tyrosine phosphorylation. Importantly, this in vitro phosphorylation of MAP kinase results in activation of phosphorylation of added MBP substrate. As a whole, our data indicate that the 44-kDa phosphoprotein identified by our antipeptide antibodies very likely corresponds to a MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Researchers identified a 44-kDa protein as a MAP kinase (extracellular signal-regulated kinase 1) using antipeptide antibodies. This protein
Area of Science:
- Cellular signaling
- Protein phosphorylation
- Enzymology
Background:
- MAP kinases (Mitogen-Activated Protein kinases) are crucial in signal transduction pathways.
- ERK1 (extracellular signal-regulated kinase 1) is a key MAP kinase involved in cellular responses.
- Receptor tyrosine kinases, like the insulin receptor, initiate signaling cascades involving MAP kinases.
Purpose of the Study:
- To characterize the function of a specific MAP kinase, ERK1, as a
- switch kinase
- ,
- Investigate the role of ERK1 in the phosphorylation cascade initiated by receptor tyrosine kinases, including the insulin receptor.
- ,
- Develop and utilize antipeptide antibodies for specific identification and functional analysis of ERK1.
Main Methods:
- Raised antipeptide antibodies against the C-terminal portion of ERK1.
- Utilized immunoprecipitation and Western blotting to detect and characterize the 44-kDa protein.
- Performed in vitro kinase assays using MBP (myelin basic protein) as a substrate.
- Analyzed phosphorylation patterns using 32P labeling and phosphoamino acid analysis.
Main Results:
- A 44-kDa protein, recognized by antipeptide antibodies, was identified under native and denaturing conditions.
- This 44-kDa protein is a phosphoprotein, with its phosphorylation stimulated by insulin, sodium orthovanadate, and okadaic acid.
- MBP kinase activity, phosphorylating MBP on threonine, was precipitated and correlated with the in vivo phosphorylation of the 44-kDa protein.
- In vitro phosphorylation of the 44-kDa protein by sodium orthovanadate led to MBP phosphorylation, indicating kinase activation.
Conclusions:
- The 44-kDa phosphoprotein identified is highly likely to be a MAP kinase, specifically ERK1.
- Insulin and other stimuli modulate the phosphorylation and activity of this MAP kinase.
- The study provides evidence for the role of this MAP kinase in signal transduction pathways initiated by receptor tyrosine kinases.