Sammendrag
Funksjonen til CKS2 proteinet har ikke blitt fullstendig klarlagt, men proteinet antas å spille en rolle i celle regulering på grunn av sin interaksjon med CDK proteiner. Målet med dette prosjektet var å etablere en Y2H metode for å vurdere CKS2 interaksjoner med CDK1 og CDK2. For å simulere defosforylert tilstand in vivo, ble CDK1AF, CDK2AF og CDK2F brukt som templat. CDK1AF og CDK2AF inneholder mutasjoner i gensekvensen på tyrosin 14 (T14A) og treonin 15 (Y15F), mens CDK2F kun inneholder mutasjon på treonin 15. For CKS2 ble normal cDNA brukt som templat. PCR ble brukt for å amplifisere cDNA til CKS2, CDK1AF, CDK2AF og CDK2F. Fusjons plasmider ble konstruert ved å lime hver gen sekvens inn i de to vektorene, pGBKT7 og pGADT7. Plasmidene ble så transformert inn i E.coli, sekvensert og deretter transformert inn i S. cerevisiae celler i passende kombinasjoner. CKS2 interaksjoner med CDK1AF, CDK2AF og CDK2F ble målt ved bruk av en dråpetest assay. For hver protein-protein interaksjon, ble det testet to paralleller, både når CKS2 var ’bait’ protein (klonet i pGBKT7) og ’prey’ protein (klonet i pGADT7). Resultatene viste at CKS2 interagerte med CDK1AF, CDK2AF og CDK2F i Y2H systemet, uavhengig av om CKS2 eller en CDK var klonet som ’bait’ protein. Konklusjonen er derfor at Y2H systemet kan brukes til å vurdere CKS2 interaksjoner med CDK proteiner, og dette er det første skrittet mot å etablere en Y2H screen for CKS2. En slik screen kan enten inneholde et cDNA ’bibliotek’ for å identifisere andre proteiner som interagerer med CKS2, eller et ’bibliotek’ over små-molekyler for å identifisere forbindelser som hemmer CKS2 interaksjoner.
The function of CKS2 proteins is not clarified, but they are assumed to play a direct role in cell regulation because of interactions with CDKs. The aim of this project was to establish the Y2H method for assessing the CKS2 interactions with CDK1 and CDK2. In order to simulate the dephosphorylated state of CDK in vivo, CDK1AF and CDK2AF sequences containing T14A and Y15F mutations, were used as templates for the CDKs. CDK2F, a template with only a single mutation (Y15F) was also tested. For CKS2, normal cDNA was used as template. Primers were designed and used in PCR to amplify cDNA of CKS2, CDK1AF, CDK2AF and CDK2F. Fusion plasmids were constructed by ligating each gene sequence into both yeast expression vectors pGBKT7 and pGADT7. The plasmid constructs were transformed into XL 10-Gold Ultracompetent E. coli cells to produce many replicates of the plasmids. For CKS2 and CDK1AF, direct cloning into pGBKT7 and pGADT7 was not successful, and subcloning into pGEM-T Easy vectors was performed. Plasmid constructs were verified by sequencing, and different combinations of plasmids were transformed into AH109 S. cerevisiae cells. CKS2 interactions with CDK1AF, CDK2AF and CDK2F were measured by growth in S. cerevisiae two-hybrid analyses. For each PPI, two parallels were tested both when CKS2 was used as bait (cloned in pGBKT7) and prey (cloned in pGADT7). The results showed that CKS2 was interacting with CDK1AF, CDK2AF and CDK2F in the Y2H system, regardless of whether CKS2 or the CDK was cloned as the bait protein. We therefore conclude that the Y2H system can be used to assess CKS2 interactions with CDKs. This is the first step towards establishment of Y2H screens for CKS2. Such screens can either contain a cDNA library to identify other CKS2 interacting proteins, or a library of small molecule compounds to find inhibitors of the CKS2 interactions.