The fate of metaphase kinetochores is weighed in the balance of SUMOylation during S phase

Abstract

Genetic evidence suggests that conjugation of Small Ubiquitin-like Modifier proteins (SUMOs) plays an important role in kinetochore function, although the mechanism underlying these observations are poorly defined. We found that depletion of the SUMO protease SENP6 from HeLa cells causes chromosome misalignment, prolonged mitotic arrest and chromosome missegregation. Many inner kinetochore proteins (IKPs) were mis-localized in SENP6-depleted cells. This gross mislocalization of IKPs is due to proteolytic degradation of CENP-I and CENP-H via the SUMO targeted Ubiquitin Ligase (STUbL) pathway. Our findings show that SENP6 is a key regulator of inner kinetochore assembly that antagonizes the cellular STUbL pathway to protect IKPs from degradation during S phase. Here, we will briefly review the implications of our findings and present new data on how SUMOylation during S phase can control chromosome alignment in the subsequent metaphase.

Figure 1

Schematic representation of STUbL degradation pathway. Enzymes of the SUMO conjugation pathway are indicated with s (sE1, sE2, sE3), while enzymes involved in ubiquitin conjugation are indicated with u (uE1, uE2) or as STUbL (E3 enzyme). SENPs are SUMO deconjugating enzymes, while DUBs are ubiquitin deconjugating enzymes.

Figure 2

Schematic representation of the genetic and molecular interactions of the different kinetochore proteins. Filled arrows indicate complete dependence and broken arrows indicate partial dependence.

Figure 3

Plk localization is indirectly disrupted after SENP6 depletion. we treated HeLa cells with Control, SENP-6, CENP-I and CENP-O siRNA for 48 hours, and simultaneously synchronized the cells using a double thymidine block protocol, as described. Ten hours after release from the second thymidine block, the cells were preextracted, fixed in 4% paraformaldehyde, and stained for centrosomes (Centrin 2 antisera; red) and Plk1 (green) Images (i–iv) represents control, SENP-6, CENP-I and CENP-O knockdown respectively. SENP6 depletion in HeLa cells resulted in loss of punctuate Plk1 signal at the metaphase plate (i and ii), which we have confirmed as centromeric Plk1 (data not shown). Similar loss of Plk1 was also observed in case of CENP-I and CENP-O knockdown (iii and iv). The arrows indicate cytoplasmic Plk1 aggregates.