NEW INSIGHTS INTO THE ROLE OF THE CENTROSOMAL MARK4 KINASE IN REGULATING THE DYNAMICS AND REMODELLING OF CYTOSKELETON FROM OVEREXPRESSION STUDIES OF ITS TWO ISOFORMS IN NORMAL AND TUMOR CELLS

MAP/Microtubule Affinity Regulating Kinase 4 (MARK4) belongs to a highly conserved family of serine–threonine kinases (MARKs) that are able to phosphorylate the microtubule associated proteins (MAPs), and cause these proteins to detach from microtubules (MTs) increasing microtubules dynamics. MARKs kinases represent the mammalian homologues of PAR-1, a protein involved in establishment of the cell shape and polarity in lower eukaryotes. The MARK4 gene is located at 19q13.2 and encodes at least two alternatively spliced isoforms, MARK4L and MARK4S, which have an identical protein structure apart from the C-terminal region. The two isoforms are differentially expressed in human tissues, particularly in the central nervous system (CNS). Several studies reported that MARK4S is expressed in normal brain tissue and neurons, suggesting that this isoform has a role in neuronal differentiation. Conversely, MARK4L is up-regulated in glioma and neural progenitor cells, pointing to a possible role of this isoform in cell proliferation. Recently, we highlighted an increasingly subverted MARK4L/MARK4S ratio, with prevalence of MARK4L, in glioblastoma and glioblastoma-derived cancer stem cells, that recapitulate the expression profiling of neural stem cells. These findings suggest that the expression of the two MARK4 isoforms is tightly regulated during the proliferation/differentiation of neural stem cells and changes in their expression levels may be a molecular marker of tumour transformation. Unlike the other members of the family (MARK1, MARK2 and MARK3), that exhibit uniform cytoplasmic localisation, both MARK4 isoforms localise at the centrosomes and in the midbody, supporting their involvement in mitotic division and cytokinesis. To elucidate the role played by MARK4 isoforms in cell cycle progression and in the regulation of the cytoskeleton, we monitored the activation status of MARK4 during the cell cycle and performed overexpression experiments in fibroblasts and glioma cell lines. We showed that despite MARK4 is expressed across all the cell cycle phases, its active form, phosphorylated at the Thr214 residue, is prevalent in mitosis. Phospho-MARK4 is detected in centrosomes at all mitotic stages and in the midbody during cytokinesis. Conversely, only a fraction of interphase centrosomes show phospho-MARK4 positive signals. Overexpression experiments on fibroblasts and glioma cell lines demonstrated the role of MARK4 in the regulation of cytoskeleton dynamics. Indeed overexpression of MARK4L or MARK4S led to a sharp decrease in microtubule density in both the cell systems, as monitored by immunofluorescence experiments. By contrast, overexpression of catalytically inactive MARK4L/MARK4S mutants, did not affect the microtubule network, indicating that the effects on MTs are dependent on the kinase activity of MARK4 and likely linked to MAPs phosphorylation. Besides the effect on MT array, overexpressed MARK4L in fibroblasts showed a filamentous staining pattern, co-localising with vimentin, the core component of cytoskeleton intermediate filaments. In contrast, overexpressed MARK4S co-localised with vimentin to a lesser extent and only in few cells. The MARK4L-vimentin co-localisation was particularly evident in the perinuclear zone and in some overexpressing cells, the filaments appeared reshaped as compared to those in GFP-transfected cells, and showed the formation of bundle structures. These alterations seem to be due to the kinase activity of MARK4L since overexpression of kinase dead mutants did not remodel the intermediate filaments. The overall data highlight MARK4 as a key component in the regulation of microtubules dynamics, and indicate vimentin as a plausible target of MARK4L activity, suggesting a wide-ranging influence of MARK4 on cytoskeleton. Moreover the dynamic involvement of active MARK4 in structure like centrosomes and midbody, crucial for mitosis and cytokinesis, point to a fundamental role for this kinase in the cell cycle.