Novel role of PKC epsilon in mitotic spindle stability

Mitosis is a highly regulated process characterized by dramatic and coordinated
morphological changes to ensure the fidelity of chromosome segregation. Missegregation
of mitotic chromosomes leads to a condition that underlies chromosomal
instability(1), which is a hallmark of cancer. In order to assure symmetry and bipolarity
of the cell division process, mitotic spindle microtubules properly segregate
mitotic chromosomes (2). Among the several isoforms of serine/threonine kinases,
PKCε is one of the best understood for its role as a transforming oncogene, and it
has been found overexpressed in different types of tumors. In 2008, Saurin and colleagues
demonstrated the involvement of PKCε in the regulation of the late stage of
mitosis (3). Through its association with 14-3-3 at the midbody, PKCε is essential for
the successful completion of cytokinesis, and the inhibition of functional PKCε-14-3-
3 complex leads to abscission failure and multinucleated phenotype in cells. In this
study, we found that PKCε is involved in mitotic spindle stability. Using fluorescence
microscopy, we found that the active form of PKCε (phosphorylated at Ser-729), colocalizes
to the centrosome in cells in metaphase, where the mitotic spindle nucleation
occurs. Furthermore, experiments of co-immunoprecipitation revealed that, when
cells are synchronized in metaphase, PKCɛ is associated to ɣ-tubulin, a member of
the tubulin superfamily localized to the microtubule organizing centers and is essential
for microtubule nucleation from centrosomes. Consequently modulation of PKCɛ
expression affects spindle stability: PKCɛ downregulation by specific shRNA results
in mitotic spindle disorganization with a reduction of the amount of centrosomal and
mitotic ɣ-Tubulin and αβ-tubulin fluorescence. Mitotic spindle formation assays using
Nocodazole, known to interfere with the polymerization of microtubules, revealed
that cells lacking PKCɛ were unable to regrow microtubules after depolymerization.
These results reveal a novel role of PKCɛ in mitotic spindle stability, which likely
determinant for genome stability.