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Assembly of protein kinase CK2: investigation of complex formation between catalytic and regulatory subunits using a zinc-finger-deficient mutant of CK2beta.

ABSTRACT: Protein kinase CK2 is a tetrameric enzyme comprised of two regulatory subunits (CK2beta) and two catalytic subunits (CK2alpha and/or CK2alpha'). The crystal structure of dimeric CK2beta demonstrated that a zinc finger mediates CK2beta dimerization, therefore we constructed a mutant in which cysteine residues 109 and 114 were mutated to serine. Our objectives were to examine the effects of disrupting the zinc finger of the regulatory CK2beta subunit on CK2 tetramer assembly. Examination of this zinc-finger-deficient mutant of CK2beta using a yeast two-hybrid assay demonstrates that the mutant fails to form CK2beta homodimers. In order to extend these studies, we co-transfected COS-7 cells with epitope-tagged constructs and performed co-immunoprecipitation assays. The results from these studies demonstrate that the mutant fails to form CK2beta homodimers and fails to interact with catalytic CK2 subunits. Furthermore, we demonstrate that the mutant CK2beta is not appreciably phosphorylated in cells. Using in vitro binding assays, we demonstrated that the mutant CK2beta protein fails to interact with glutathione S-transferase-CK2alpha'. Finally, we demonstrate that the mutant is translated at an equivalent rate to wild-type CK2beta, but is degraded much more rapidly. Overall, our results are consistent with the model that beta-beta dimerization precedes incorporation of catalytic subunits into tetrameric CK2 complexes, and that beta-beta dimerization is a prerequisite for the stable incorporation of catalytic subunits into CK2 complexes.

SUBMITTER: Canton DA

PROVIDER: S-EPMC1222035 | biostudies-other | 2001 Aug

REPOSITORIES: biostudies-other

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Project description:BackgroundTranscription factors, regulating the expression inventory of a cell, interact with its respective DNA subjugated by a specific recognition pattern, which if well exploited may ensure targeted genome engineering. The mostly widely studied transcription factors are zinc finger proteins that bind to its target DNA via direct and indirect recognition levels at the interaction interface. Exploiting the binding specificity and affinity of the interaction between the zinc fingers and the respective DNA can help in generating engineered zinc fingers for therapeutic applications. Experimental evidences lucidly substantiate the effect of indirect interaction like DNA deformation and desolvation kinetics, in empowering ZFPs to accomplish partial sequence specificity functioning around structural properties of DNA. Exploring the structure-function relationships of the existing zinc finger-DNA complexes at the indirect recognition level can aid in predicting the probable zinc fingers that could bind to any target DNA. Deformation energy, which defines the energy required to bend DNA from its native shape to its shape when bound to the ZFP, is an effect of indirect recognition mechanism. Water is treated as a co-reactant for unfurling the affinity studies in ZFP-DNA binding equilibria that takes into account the unavoidable change in hydration that occurs when these two solvated surfaces come into contact.ResultsAspects like desolvation and DNA deformation have been theoretically investigated based on simulations and free energy perturbation data revealing a consensus in correlating affinity and specificity as well as stability for ZFP-DNA interactions. Greater loss of water at the interaction interface of the DNA calls for binding with higher affinity, eventually distorting the DNA to a greater extent accounted by the change in major groove width and DNA tilt, stretch and rise.ConclusionMost prediction algorithms for ZFPs do not account for water loss at the interface. The above findings may significantly affect these algorithms. Further the sequence dependent deformation in the DNA upon complexation with our prototype as well as preference of bases at the 2nd and 3rd position of the repeating triplet provide an absolutely new insight about the indirect interactions undergoing a change that have not been probed yet.

Project description:The long-term success of coronary stent implantation is limited by in-stent restenosis (ISR). In spite of a broad variety of animal models available, an ideal high-throughput model of ISR has been lacking. Apolipoprotein E (apoE) deficient rats enable the evaluation of human-sized coronary stents while at the same time providing an atherogenic phenotype. Whereas apoE deficient rats have been proposed as animal model of atherosclerosis, to date it is unknown whether they also develop pronounced ISR. We sought to assess ISR after abdominal aorta stent implantation in apoE deficient rats. A total of 42 rats (16 wildtype, 13 homozygous apoE-/- and 13 heterozygous apoE+/- rats) underwent abdominal aorta stent implantation. After 28 days blood samples were analyzed to characterize lipid profiles. ISR was assessed by histomorphometric means. Homozygous apoE-/- rats exhibited significantly higher total cholesterol and low-density cholesterol levels than wildtype apoE+/+ and heterozygous apoE+/- rats. ISR was significantly pronounced in homozygous apoE-/- rats as compared to wildtype apoE+/+ (p?=?<0.0001) and heterozygous apoE+/- rats (p?=?0.0102) on western diet. Abdominal aorta stenting of apoE-/- rats is a reliable model to investigate ISR after stent implantation and thus can be used for the evaluation of novel stent concepts. Apolipoprotein E (apoE) deficient rats have been proposed as animal model of atherosclerosis. We investigated the development of restenosis 28 days after stent implantation into the abdominal aorta of wildtype apoE+/+, homozygous apoE-/- and heterozygous apoE+/- rats, respectively. Homozygous apoE-/- rats exhibited significantly higher LDL and significantly lower HDL cholesterol levels compared to wildtype apoE+/+ and heterozygous apoE+/- rats. Restenosis after stent implantation was significantly pronounced in western-diet-fed homozygous apoE-/- rats, accompanied by a significantly increased neointimal thickness. Thus, apoE knockout rats exhibit elevated restenosis and might provide a novel tool for testing of innovative stent concepts.

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Assembly of protein kinase CK2: investigation of complex formation between catalytic and regulatory subunits using a zinc-finger-deficient mutant of CK2beta.

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