Roles of IkappaB kinase alpha in centrosome duplication and skin carcinogenesis

IκB kinase α (IKKα) is one kinase subunit of the IKK complex that is responsible for NF-κB activation. Previous studies have shown that IKKα determines mouse keratinocyte terminal differentiation independent of the NF-κB pathway. Accumulating evidence suggests that IKKα functions as a tumor suppressor in skin carcinogenesis; however, the downstream pathways mediating this function are largely unknown. By using primary cultured keratinocytes, we found that Ikkα-/- cells developed aneuploidy and underwent spontaneous immortalization and transformation while wild type cells underwent terminal differentiation in the same culture condition. Using proteomic analysis we identified nucleophosmin (NPM), a centrosome duplication regulator, as an IKKα substrate. We further demonstrated that IKKα interacted with NPM and colocalized with NPM on the centrosome, suggesting that NPM is a physiological substrate of IKKα. Loss of IKKα reduced centrosome-bound NPM and promoted abnormal centrosome amplification, which contributed to aneuploidy development. Detailed analysis revealed that ablation of IKKα target site serine-125 of NPM induced destabilization of NPM hexamers, disrupted NPM association with centrosomes, and resulted in abnormal centrosome amplification. Re-introduction of IKKα rescued the defect in Ikkα-/- keratinocytes. Thus, IKKα is required for maintaining proper centrosome duplication by phosphorylating NPM. ^ UV is the major etiological agent for human skin cancer and UV-induced mouse skin carcinogenesis is one of the most relevant experimental models for human skin carcinogenesis. Thus, we further evaluated IKKα function in UV-induced skin carcinogenesis in Ikkα+/- mice. We demonstrated that IKKα is also critical in UV skin carcinogenesis, as evidenced by increased tumor multiplicity and reduced tumor latency in Ikkα+/- mice after chronic UVB treatment. Reduced expression of IKKα decreased UV-induced apoptosis and promoted accumulation of P53 mutations in the epidermis. This indicates that IKKα is critical for UV-induced apoptosis in vivo and thus prevents mutation accumulation that is important for tumor development. ^ Together, these findings uncover previously unknown in vivo functions of IKKα in centrosome duplication and apoptosis, thus providing a possible mechanism of how loss of IKKα may contribute to skin carcinogenesis. ^

IDENTIFICATION AND ANALYSIS OF A NOVEL ROLE FOR THE TOUSLED-LIKE KINASE IN REGULATING MITOTIC SPINDLE DYNAMICS

Deregulation of kinase activity is one example of how cells become cancerous by evading evolutionary constraints. The Tousled kinase (Tsl) was initially identified in Arabidopsis thaliana as a developmentally important kinase. There are two mammalian orthologues of Tsl and one orthologue in C. elegans, TLK-1, which is essential for embryonic viability and germ cell development. Depletion of TLK-1 leads to embryonic arrest large, distended nuclei, and ultimately embryonic lethality. Prior to terminal arrest, TLK-1-depleted embryos undergo aberrant mitoses characterized by poor metaphase chromosome alignment, delayed mitotic progression, lagging chromosomes, and supernumerary centrosomes. I discovered an unanticipated requirement for TLK-1 in mitotic spindle assembly and positioning. Normally, in the newly-fertilized zygote (P0) the maternal pronucleus migrates toward the paternal pronucleus at the posterior end of the embryo. After pronuclear meeting, the pronuclear-centrosome complex rotates 90° during centration to align on the anteroposterior axis followed by nuclear envelope breakdown (NEBD). However, in TLK-1-depleted P0 embryos, the centrosome-pronuclear complex rotation is significantly delayed with respect to NEBD and chromosome congression, Additionally, centrosome positions over time in tlk-1(RNAi) early embryos revealed a defect in posterior centrosome positioning during spindle-pronuclear centration, and 4D analysis of centrosome positions and movement in newly fertilized embryos showed aberrant centrosome dynamics in TLK-1-depleted embryos. Several mechanisms contribute to spindle rotation, one of which is the anchoring of astral microtubules to the cell cortex. Attachment of these microtubules to the cortices is thought to confer the necessary stability and forces in order to rotate the centrosome-pronuclear complex in a timely fashion. Analysis of a microtubule end-binding protein revealed that TLK-1-depleted embryos exhibit a more stochastic distribution of microtubule growth toward the cell cortices, and the types of microtubule attachments appear to differ from wild-type embryos. Additionally, fewer astral microtubules are in the vicinity of the cell cortex, thus suggesting that the delayed spindle rotation could be in part due to a lack of appropriate microtubule attachments to the cell cortex. Together with recently published biochemical data revealing the Tousled-like kinases associate with components of the dynein microtubule motor complex in humans, these data suggest that Tousled-like kinases play an important role in mitotic spindle assembly and positioning.