FUNCTIONAL STUDY OF MARK4, A GENE ENCODING FOR TWO PROTEIN ISOFORMS,IN GLIOMA AND NORMAL CELLS

MARK4 (MAP/Microtubule Affinity-Regulating Kinase 4) belongs to a family of serine-threonine kinases phosphorylating Microtubule Associated Proteins, causing their detachment from the microtubules (MTs) and thus increasing MTs dynamics. MARK proteins show high homology with PAR complex proteins family, involved in assessing cell polarity during embryogenesis, epithelial morphogenesis, neural differentiation, and cell migration. MARK proteins are thus implied in several processes involving MT network: cytoskeleton dynamics, cell polarity, centrosomes formation, chromosomal segregation, cytokinesis. The MARK4 gene (19q13.2) encodes at least two alternatively spliced isoforms, L and S, differentially expressed in human tissues. MARK4S is the predominant isoform in normal brain and post-mitotic neurons. MARK4L has been found up-regulated in glioma cell lines and neural progenitors, as well as in hepatocarcinoma cell lines, suggesting a role in cell proliferation. The dual nature of MARK4 isoforms has been pinpointed by their expression profile in glioblastoma-derived cell lines and neural stem cells (NSCs), other than glioma, and the balance of the two isoforms, favouring the L splicing variant in glial tumors, is being investigated as a potential target of dysregulation in gliomagenesis. A linked view is whether the predominant expression of MARK4L, isoform of a gene involved in the microtubule dynamics, may concur to mitotic errors during gliomagenesis. Both isoforms of MARK4 have been found associated to centrosomes and midbody, in glioma as well as in normal cells, suggesting that the kinase might have a role in all phases of the cell cycle. Moreover, MARK4L showed an additional nucleolar localization in glioma, raising the idea that, in tumors, the L variant has isoform specific functions and interactions with nucleolar components. To verify the functional impact of MARK4 gene in glioma and in normal cells and to define the role of MARK4S and L isoforms with respect to their subcellular localization, we set up a functional study by RNA interference in glioblastoma (GBM) cell lines and normal fibroblasts, with specific silencing of both MARK4S and MARK4S+L. We showed that MARK4 depletion determines heavy alterations in the cell shape of both G-32 GBM cell line and fibroblasts, corroborating MARK4 implication in cytoskeleton organization, and in accordance with the known role of MARK proteins in MTs dynamics. MARK4 silencing particularly affected the centrosome cycle. Silenced G-32 GBM cell line and fibroblasts presented most of cells with the duplicated centrosome, apical to the nucleus, as typical of G1/S transition, while differently, control cells displayed centrosomes in all phases of the centrosome cycle. Accordingly, after MARK4 silencing, cell cycle analysis showed in both tumor cells and fibroblasts, an increase of G1 cells fraction and a strong reduction of mitoses, most of which displayed aberrations of spindle poles. These findings indicate that MARK4 depletion targets the G1/S transition checkpoint, probably knocking down a positive regulator. It is worth noticing that the observed alterations of cell morphology, centrosome and cell cycle progression, and of mitosis were more pronounced when silencing MARK4S+L, as if the depletion of the sole MARKS might be partially compensated by MARK4L, being both isoforms localized at centrosomes and midbody. MARK4 silencing on nucleoli revealed that the L isoform of MARK4 is not a specific marker of tumor (glioma) cell lines, in that it was also detectable in fibroblasts. However, MARK4 depletion showed a nucleolar pattern different in G-32 GBM cells from that of fibroblasts: G-32 showed, after both anti-MARK4S and MARK4S+L siRNA, a pronounced intensity of MARK4L signal in several nucleoli, while some others appeared silenced. In contrast, silenced fibroblasts showed, particularly after anti-MARK4S+L siRNA, several nucleoli unlabelled with MARK4L. It’s currently unknown the molecular basis for this difference which may be more than a quantitative one due to the higher levels of MARK4L in the nucleolar compartment of tumor cells where it’s generally upregulated. The concomitant alterations of both centrosomal and nucleolar compartments highlighted by MARK4 silencing raise the hypothesis of a MARK4 role in the regulation of the Nucleus - (Nucleolus) - Centrosome (NC) axis. NC - axis is oriented and paired with the polarization and migration axis of many cell types, including fibroblasts and neural cells. Microtubules act as major actors in organizing a dynamic structure along NC - axis, regulating nuclear movement during cell migration and polarization. Since MARK4 cooperates with the microtubule network, its depletion may interfere with the correct orientation of the NC – axis.