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Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies
Published on: March 19, 2014
Xenopus phospho-CDK7/cyclin H expressed in baculoviral-infected insect cells
A M Lawrie1, P Tito, H Hernandez
1Laboratory of Molecular Biophysics and Oxford Centre for Molecular Sciences, Biochemistry Department, University of Oxford, Rex Richards Building, South Parks Road, Oxford OX1 3QU, United Kingdom.
Abstract:
The cyclin-dependent kinase-activating kinase (CAK) catalyzes the phosphorylation of the cyclin-dependent protein kinases (CDKs) on a threonine residue (Thr160 in human CDK2). The reaction is an obligatory step in the activation of the CDKs. In higher eukaryotes, the CAK complex has been characterized in two forms. The first consists of three subunits, namely CDK7, cyclin H, and an assembly factor called MAT1, while the second consists of phospho-CDK7 and cyclin H. Phosphorylation of CDK7 is essential for cyclin association and kinase activity in the absence of the assembly factor MAT1. The Xenopus laevis CDK7 phosphorylation sites are located on the activation segment of the kinase at residues Ser170 and at Thr176 (the latter residue corresponding to Thr160 in human CDK2). We report the expression and purification of X. laevis CDK7/cyclin H binary complex in insect cells through coinfection with the recombinant viruses, AcCDK7 and Accyclin H. Quantities suitable for crystallization trials have been obtained. The purified CDK7/cyclin H binary complex phosphorylated CDK2 and CDK2/cyclin A but did not phosphorylate histone H1 or peptide substrates based on the activation segments of CDK7 and CDK2. Analysis by mass spectrometry showed that coexpression of CDK7 with cyclin H in baculoviral-infected insect cells results in phosphorylation of residues Ser170 and Thr176 in CDK7. It is assumed that phosphorylation is promoted by kinase(s) in the insect cells that results in the correct, physiologically significant posttranslational modification. We discuss the occurrence of in vivo phosphorylation of proteins expressed in baculoviral-infected insect cells.
Insights
The cyclin-dependent kinase-activating kinase (CAK) complex, comprising CDK7 and cyclin H, was successfully expressed and purified. This complex demonstrated phosphorylation activity on CDK2, indicating functional post-translational modifications in insect cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- The cyclin-dependent kinase-activating kinase (CAK) is crucial for activating cyclin-dependent protein kinases (CDKs) through phosphorylation.
- CAK exists in higher eukaryotes as a ternary complex (CDK7/cyclin H/MAT1) or a binary complex (phospho-CDK7/cyclin H).
- Phosphorylation of CDK7 itself is vital for cyclin association and kinase activity, especially without the MAT1 assembly factor.
Purpose of the Study:
- To express and purify the Xenopus laevis CDK7/cyclin H binary complex.
- To investigate the kinase activity of the purified complex.
- To confirm the occurrence and significance of in vivo phosphorylation of CDK7 in insect cells.
Main Methods:
- Coinfection of insect cells with recombinant baculoviruses encoding X. laevis CDK7 and cyclin H.
- Purification of the CDK7/cyclin H binary complex.
- In vitro kinase assays using CDK2, CDK2/cyclin A, histone H1, and peptide substrates.
- Mass spectrometry analysis to identify phosphorylation sites on CDK7.
Main Results:
- The X. laevis CDK7/cyclin H binary complex was successfully expressed, purified, and obtained in quantities suitable for crystallization.
- The purified complex phosphorylated CDK2 and CDK2/cyclin A but not histone H1 or specific peptide substrates.
- Mass spectrometry confirmed phosphorylation of Ser170 and Thr176 on CDK7, corresponding to known physiological sites.
Conclusions:
- Baculoviral expression in insect cells facilitates the production of a functional X. laevis CDK7/cyclin H complex.
- In vivo phosphorylation by insect cell kinases results in physiologically relevant post-translational modifications of CDK7.
- This system provides a valuable tool for studying CAK function and regulation.
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