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Updated: Aug 11, 2026
A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells
Published on: July 31, 2012
Expression, purification, and initial characterization of human Yes protein tyrosine kinase from a bacterial
1Department of Neuro-Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Abstract:
Protein tyrosine kinase Yes is a cellular homolog of v-Yes, the oncogenic protein product of avian sarcoma virus Y73. Yes is a member of the Src family and its activation has been associated with several types of human cancer. Human Yes has not been previously characterized enzymatically. To carry out biochemical characterizations of this enzyme, we expressed it as a fusion protein with glutathione S-transferase in Escherichia coli, to allow purification in a single step. The affinity-purified GST-Yes has a specific activity of 1.3 nmol min-1 mg-1 with polyE4Y as substrate and Km values of 100 microg ml-1 for polyE4Y and 70 microM for ATP-Mg. The enzyme has a preference for magnesium over manganese ion for maximal activity. The divalent metal cation serves two essential functions for the activity of Yes: one as a part of the phosphate-donating substrate ATP-Mg and the other as an essential activator. The enzyme undergoes autophosphorylation without apparent activation. Finally, we show that the enzyme is inactivated by incubation with protein tyrosine kinase Csk in an ATP-Mg-dependent manner, indicating that cellular Yes can be regulated by Csk phosphorylation. These represent the first biochemical characterization of human Yes protein tyrosine kinase.
Insights
This study biochemically characterizes human Yes protein tyrosine kinase, a Src family member implicated in cancer. Researchers found Yes undergoes autophosphorylation and is regulated by Csk kinase activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Protein tyrosine kinase Yes (Yes) is a cellular homolog of the oncogenic v-Yes.
- Yes is a member of the Src family, and its activation is linked to human cancers.
- Previous enzymatic characterization of human Yes was lacking.
Purpose of the Study:
- To perform the first biochemical characterization of human Yes protein tyrosine kinase.
- To understand the enzymatic activity and regulation of human Yes.
Main Methods:
- Expressed human Yes as a glutathione S-transferase (GST) fusion protein in Escherichia coli for single-step purification.
- Determined specific activity, Michaelis constants (Km) for substrate (polyE4Y) and ATP-Mg.
- Investigated the role of divalent metal cations (Mg2+/Mn2+) and autophosphorylation.
- Assessed regulation by protein tyrosine kinase C-terminal Src kinase (Csk).
Main Results:
- Affinity-purified GST-Yes exhibited a specific activity of 1.3 nmol/min/mg with polyE4Y.
- Km values were 100 μg/mL for polyE4Y and 70 μM for ATP-Mg.
- Maximal activity required magnesium ions, which are essential for substrate binding and activation.
- Yes underwent autophosphorylation and was inactivated by Csk in an ATP-Mg-dependent manner.
Conclusions:
- This study provides the first biochemical characterization of human Yes protein tyrosine kinase.
- Human Yes activity is dependent on Mg2+ and can be regulated by Csk-mediated phosphorylation.
- These findings offer insights into the role of Yes in cellular signaling and cancer.
