Xenopus phospho-CDK7/cyclin H expressed in baculoviral-infected insect cells.
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.
Lawrie
A M
AM
Laboratory 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.
Tito
P
P
Hernandez
H
H
Brown
N R
NR
Robinson
C V
CV
Endicott
J A
JA
Noble
M E
ME
Johnson
L N
LN
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D042846:CDC2-CDC28 Kinases; D002460:Cell Line; D003001:Cloning, Molecular; D019925:Cyclin A / Q000378:metabolism; D056748:Cyclin H; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D018411:Spodoptera; D013912:Threonine; D029867:Xenopus Proteins; D014982:Xenopus laevis; D000095043:Cyclin-Dependent Kinase-Activating Kinase
C534751:CCNH protein, human; D019925:Cyclin A; D056748:Cyclin H; D016213:Cyclins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D013912:Threonine; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase