| Summary: | 碩士 === 國立中山大學 === 生物科學系研究所 === 99 === Nuclear protein KLIP1 cooperates with myeloid leukemia factor 1 (MLF1) to inhibit the programmed cell death resulting in tumor formation. It also inhibits the activity of thymidine kinase promoter of Kaposi’s sarcoma-associated Herpes Virus. KLIP1 functions as a centromere protein, hence acquires its name as CENP-U or CENP-50, to regulate the separation of sister-chromatids during mitosis. These results indicate that KLIP1 plays important roles in regulation of transcription and cell cycle. In this study, six potential SUMO modification sites, K33, K63, K126, K127, K185 and K210, were identified bioinformatically using SUMOplot. Many reports address that SUMO modification alters the transcriptional activity, protein-protein interaction, the subcellular localization and stability of its target protein. Recent data suggest that SUMO is required for centromere binding protein to mediate proper mitotic spindle attachment to the kinetochore, and previous research suggest that there has a SUMO-interaction motif (SIM) in KLIP1 protein sequence. To reveal the interaction between KLIP1 and SUMO-1, and study its effects on KLIP1 function, we co-express GFP-KLIP1 and His-tagged SUMO-1 in HEK 293 cells. After affinity purification of SUMOylated proteins from transfected cells using nickel conjugated beads and subsequent western blotted with anti-GFP. The results indicated the interaction between KLIP1 and SUMO-1 in co-transfected cells. Our confocal microscopy imaging also found colocalization of GFP-KLIP1 with RFP-SUMO-1 nuclear foci. In addition, we failed to detect the interaction between SUMO-1 and mutant KLIP1-M6 ,whose six potential SUMO modified lysine residues were mutated to arginine. Furthermore, we found a distinct nuclear localization of GFP-KLIP1-M6 as compared to the image of wildtype GFP-KLIP1, which show a significant higher frequency of colocalization with RFP-SUMO-1 foci. Taken together, our data suggest the interaction between KLIP1 and SUMO-1 may be related to these six potential lysine residues, which upon mutation blocks its colocalization with SUMO-1 in nuclear foci. The biological significance of their interaction are awaits for further investigation.
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