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We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1,
CDK8/CycC and CDK9/CycT1. We attach a His6-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni2+-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating
kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero
et al. 2004, Eur J Biochem 271:1004–14). Our protocol provides a novel systematic approach for the purification of these three
(and possibly other) recombinant CDKs.
Published: August 18, 2004. 相似文献
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Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover 总被引:1,自引:0,他引:1
Notch signaling releases the Notch receptor intracellular domain (ICD), which complexes with CBF1 and Mastermind (MAM) to activate responsive genes. We previously reported that MAM interacts with CBP/p300 and promotes hyperphosphorylation and degradation of the Notch ICD in vivo. Here we show that CycC:CDK8 and CycT1:CDK9/P-TEFb are recruited with Notch and associated coactivators (MAM, SKIP) to the HES1 promoter in signaling cells. MAM interacts directly with CDK8 and can cause it to localize to subnuclear foci. Purified recombinant CycC:CDK8 phosphorylates the Notch ICD within the TAD and PEST domains, and expression of CycC:CDK8 strongly enhances Notch ICD hyperphosphorylation and PEST-dependent degradation by the Fbw7/Sel10 ubiquitin ligase in vivo. Point mutations affecting conserved Ser residues within the ICD PEST motif prevent hyperphosphorylation by CycC:CDK8 and stabilize the ICD in vivo. These findings suggest a role for MAM and CycC:CDK8 in the turnover of the Notch enhancer complex at target genes. 相似文献
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A M Lawrie P Tito H Hernandez N R Brown C V Robinson J A Endicott M E Noble L N Johnson 《Protein expression and purification》2001,23(2):252-260
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. 相似文献
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T-loop phosphorylation stabilizes the CDK7–cyclin H–MAT1 complex in vivo and regulates its CTD kinase activity 总被引:2,自引:0,他引:2
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Stphane Larochelle Jian Chen Ronald Knights Judit Pandur Patrick Morcillo Hediye Erdjument-Bromage Paul Tempst Beat Suter Robert P. Fisher 《The EMBO journal》2001,20(14):3749-3759
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Tat modifies the activity of CDK9 to phosphorylate serine 5 of the RNA polymerase II carboxyl-terminal domain during human immunodeficiency virus type 1 transcription
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Zhou M Halanski MA Radonovich MF Kashanchi F Peng J Price DH Brady JN 《Molecular and cellular biology》2000,20(14):5077-5086
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