A New Identity for MLK3 as a NIMA-Related, Cell Cycle Regulated Kinase that is Localized Near Centrosomes and Influences Microtubule Organization

Although conserved counterparts for most proteins involved in the G 2 /M transition of the cell cycle have been found in all eukaryotes, a notable exception is the essential but functionally enigmatic fungal kinase NIMA. While a number of vertebrate kinases have been identified with catalytic domain homology to NIMA, none of these resemble NIMA within its extensive non-catalytic region, a region critical for NIMA function in Aspergillus nidulans. We used a bioinformatics approach to search for proteins with homology to the non-catalytic region of NIMA and identified mixed lineage kinase 3 (MLK3). MLK3 has been proposed to serve as a component in MAP kinase cascades, particularly those resulting in the activation of the c-Jun N-terminal kinase (JNK). Here we describe the first in-depth study of endogenous MLK3 and report that, like NIMA, MLK3 phosphorylation and activity are enhanced during G2/M, whereas JNK remains inactive. Coincident with the G2/M transition, a period marked by dramatic reorganization of the cytoplasmic microtubule network, endogenous MLK3 transiently disperses away from the centrosome and centrosomal-proximal sites where it is localized during interphase. Furthermore, when overexpressed, MLK3, like NIMA, localizes to the centrosomal region, induces profound disruption of cytoplasmic microtubules and a nuclear distortion phenotype that differs from mitotic chromosome condensation. Cellular depletion of MLK3 protein using siRNA technology results in an increased sensitivity to the microtubule-stabilizing agent taxol. Our studies suggest a new role for MLK3, separable from its function in the JNK pathway, that may contribute to promoting microtubule instability, a hallmark of M phase entry.