Screening And Functional Analysis Of Crk1 Interacting Proteins

Crk1, a Cdc2-related protein kinase from the human pathogenic fungusCandida albicans, plays an important role in hyphal development and virulence. Inorder to study its functional mechanism, C. albicans genomic DNA was digestedwith Sau3AI and fused to activating domain(AD) of LexA to construct an AD-fusedC. albicans genomic DNA expression library. The library contained 7.7 104independent clones and included 1.2 105 kb DNA which was 10 fold of the C.albicans whole genomic DNA. The Crk1 fused to DNA binding domain of LexA wasused as the bait to screen the LexA library. Among 9 105 transformants, eight Leuand LacZ double positive clones were selected. With restriction enzyme analysis,three kinds of fragments were identified. The clones NJ1, NJ2, NJ3 were sequencedand analyzed with BLAST program. It was found that NJ1, NJ2, NJ3 were homologsof S. cerevisiae gene STI1, RET2 and NFI1, respectively. The kinase domain ofCrk1(Crk1N) interacted weakly with protein encoded by NJ1, while the noncatalyticdomain of Crk1(Crk1C) interacted strongly with proteins encoded by NJ2 and NJ3.These proteins may be involved in maturation, transport, localization, and activityregulation of Crk1.We also screened the library with Crk1N(1 370 aa) andCrk1C(350 702 aa). A CDC37 orthologue (CaCDC37) was cloned from the screening with the Crk1kinase domain as the bait. The CaCdc37 interacted preferentially with the kinasedomain of Crk1 (Crk1N) as shown by two-hybrid and immunoprecipitationexperiments. The CaCDC37 could complement a cdc37 thermosensitive mutant(cdc37-34) of Saccharomyces cerevisiae. Importantly, Crk1 protein was hardlydetectable in the cdc37-34 mutant at restrictive temperature. However, upon III中国科学院上海生命科学研究院生物化学与细胞生物学研究所 博士学位论文 摘 要expression of CaCdc37 in the cdc37 mutant, Crk1 protein was detected even atrestrictive temperature. Our data suggested that the CaCdc37 was required for theproduction of Crk1 kinase. Like Cdc37 proteins of S. cerevisiae and highereukaryotes, CaCdc37 might function as a molecular chaperone that stabilized Crk1and other protein kinases in C. albicans. In support of this, CaSTI1 was identifiedfrom a two-hybrid screen with the full length Crk1 as the bait. CaSti1 showed two-hybrid interactions with both Crk1 and the CaCdc37. We presented a model of Crk1maturation in accordance with this results.. Crk1 had the highest homology with Bur1 in S. Cerevisiae and Cdk9 in homosapain. The later two proteins were involved in phosphorylation of RNA polymeraseII 's C terminal domain (CTD). The crk1 deletion was sensitive to 6-azauradine,which indicated Crk1 had the same activity with Bur1 and Cdk9. But the interactionbetween Crk1 and CTD of RNA polymerase II was not detected in the two hybridsystem. GFP fused Crk1 was highly expressed to detect the location of Crk1 in C.albicans. Cln3 was one of G1 cyclins in Saccharomyces cerevisiae. In order to study thefunction of Cln3 in cell cycle and morphogenesis, we constructed a cln3 null mutantand analyzed its phenotype. Our results indicated that the cln3 null mutant was moresensitive to α pheromone, and arrested at G1 phase. The hypersensitivity to α-pheromone was not suppressed by overexpression of BUR1.The null mutant showeda different phenotype with that of the other two G1 cyclin mutants. Thefilamentous growth in diploid cells of cln3 mutant was stronger than that in wild typecells, while invasive growth of the haploid cells was partially inhibited. Theresults suggested that the Cln3 plays a unique function in morphogenesis under adifferent mechanism with that used by Cln1 and Cln2.