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CKAP2 phosphorylation by CDK1/cyclinB1 is crucial for maintaining centrosome integrity.

ABSTRACT: Previously, we have reported that CKAP2 is involved in the maintenance of centrosome integrity, thus allowing for proper mitosis in primary hepatocytes. To understand this biological process, we identified the mitosis-specific phosphorylation sites in mouse CKAP2 and investigated CKAP's possible role in cell cycle progression. Because we observed mouse CKAP2 depletion in amplified centrosomes and aberrant chromosomal segregation, which was rescued by ectopic expression of wild-type CKAP2, we focused on the centrosome duplication process among the various aspects of the cell cycle. Among the identified phosphorylation sites, T603 and possibly S608 were phosphorylated by CDK1-cyclin B1 during mitosis, and the ectopic expression of both T603A and S608A mutants was unable to restore the centrosomal abnormalities in CKAP2-depleted cells. These results indicated that the phosphorylation status of CKAP2 during mitosis is critical for controlling both centrosome biogenesis and bipolar spindle formation.

SUBMITTER: Yoo BH

PROVIDER: S-EPMC5565958 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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CKAP2 phosphorylation by CDK1/cyclinB1 is crucial for maintaining centrosome integrity.

Yoo Bum Ho BH Kang Du-Seock DS Park Chi-Hu CH Kang Kyeongjin K Bae Chang-Dae CD

Experimental & molecular medicine 20170714 7

Previously, we have reported that CKAP2 is involved in the maintenance of centrosome integrity, thus allowing for proper mitosis in primary hepatocytes. To understand this biological process, we identified the mitosis-specific phosphorylation sites in mouse CKAP2 and investigated CKAP's possible role in cell cycle progression. Because we observed mouse CKAP2 depletion in amplified centrosomes and aberrant chromosomal segregation, which was rescued by ectopic expression of wild-type CKAP2, we foc ...[more]

PMID: 28706298

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Project description:The storage of rAAV vectors for gene therapy applications is critical for ensuring a constant product quality and defined amount of medication at the time of administration. Therefore, we determined the influence of different storage conditions on the physicochemical and biological properties of rAAV8 and rAAV9 preparations. Particular attention was paid to short-term storage, which plays a crucial role in both the manufacturing process and in clinical applications. Additionally, we addressed the question, of viability of rAAV8 and rAAV9 when subjected to very low-temperature storage conditions (below -65 °C) or lyophilization. To determine the impact on rAAV vectors, various analyses were used, including the quantification of capsid and genome titers, as well as biopotency assessments, which are pivotal determinants in characterizing vector behavior and efficacy. Our data showed that freeze/thaw cycles hardly affected the functionality of rAAV9-aGAL vectors. In contrast, prolonged storage at room temperature for several days, resulted in a discernible decrease in biopotency despite consistent capsid and genome titers. When the storage temperature was further increased, the rAAV8-aGAL decay accelerated. For example, a short-term exposure of + 40 °C and more, led to a reduction in the physical viral titer and to an even faster decline in efficacy determined by biopotency. However, the addition of sucrose and sorbitol to the rAAV9-aGAL and rAAV9-GAA preparations reduced the temperature sensitivity of rAAV and improved its stability. Furthermore, exposure of rAAV9-aGAL to highly acidic conditions (pH 2.5) dramatically reduced its biopotency by 70% or more. Most interestingly, a long-term storage of rAAV9-aGAL and rAAV8-FVIII vectors over 12 and 36 months, respectively, demonstrated exceptional stability at storage temperatures below -65 °C. Also, lyophilization conserved functionality for at least 10 months. Our data showed how to maintain rAAV biopotency levels over the time without substantial loss. Storage at very low temperatures (below -65 °C) preserved its effectiveness over years. Overall, pH and temperature conditions during the manufacturing process, storage and clinical application are worth considering. Consistency in the rAAV capsid titer determination did not necessarily indicate the preservation of biopotency. In conclusion, our approach determined several options for maximizing rAAV stability.

Dataset Information

CKAP2 phosphorylation by CDK1/cyclinB1 is crucial for maintaining centrosome integrity.

Publications

CKAP2 phosphorylation by CDK1/cyclinB1 is crucial for maintaining centrosome integrity.

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