The use of the Yeast Two Hybrid system to detect CKS2 dimerization and interactions with CDK1 and CDK2

Abstract

Elevated expression of CKS2 protein has been detected in various types of cancer. Emerging evidence suggests that CKS2 affects cell cycle regulation through interactions with CDK1 and CDK2, and discovery of compounds that inhibit this interaction may be used to develop a novel cancer medicine. The aim of this thesis was to establish the yeast two hybrid (Y2H) system for detection of CKS2 interaction with CDK1, CDK2 and itself, using the URA3 reporter gene, as the system can be reversed and used to screen for compounds that dissociate the interactions. The HIS3 reporter gene was also used. The former two interactions have been detected with this reporter in a previous Y2H study. Versions of CDK1 and CDK2 that mimick the dephosphorylated, active state of the proteins were used. These proteins contained substitutions at amino acid 14 and 15, and are referred to as CDK1 AF and CDK2 AF. Primers were designed for use in cDNA amplification of CKS2, CDK1 AF and CDK2 AF by PCR. Each gene sequence was fused into the pENTR™/D-TOPO® vector, generating entry clones by a topoisomerase based cloning methodology. The gene sequences were transferred to the yeast expression vectors pDESTTM32 and pDESTTM22 in a site-specific recombination reaction. These vectors contained the CEN6/ARS4 sequence for replication which maintained low expression levels of the fusion protein. The expression vector constructs were verified through sequencing, and combinations of the constructs were transformed into S. cerevisiae MaV203. The CKS2 interactions were measured by plating onto specific media. Transcription of the HIS3 reporter gene was activated in the S. cerevisiae MaV203 cells when the interactions between CKS2 and the CDKs were investigated, providing further support to previous findings. Transcription of the URA3 reporter gene was, however, not activated, probably because the strength of the interactions was too low to be detected through URA3. An attempt to activate the URA3 reporter by using high copy-number plasmids from a different system did not show activation of URA3 either. The CKS2-CKS2 interaction was not detected either by use of the HIS3 or the URA3 reporter gene. Further work has to be performed in order to counteract the obstacles of URA3 activation.