Multiple localization of endogenous MARK4L protein in human glioma.
BACKGROUND
We have previously shown that the sustained expression of MARK4L transcripts in glioma and neural progenitors (NHNPs) declines after exposure to antisense MARK4L oligonucleotides in glioblastoma cell lines. Array-CGH confirmed the genomic duplication of MARK4L identified by FISH in a glioblastoma cell line. This background together with literature data on the exogenous association of MARK4 with interphase centrosome prompted us to investigate the sub-cellular localization of the endogenous MARK4L protein aiming at achieving insights on its possible role in the pathomechanisms of glioma.


METHODS
Immunodetection was carried out to validate the specificity of MARK4L antibody in gliomas and NHNPs. Mass spectrometry was applied for MARK4L protein identification in a representative glioblastoma cell line. Combined biochemical fractionation and immunodetection analyses were performed to confirm the sub-cellular localization of MARK4L achieved by immunofluorescence in glioma cell lines.


RESULTS
By assigning MARK4L protein within the band immunoprecipitated by the specific antibody we validated our anti-MARK4L antibody. We demonstrated that the endogenous MARK4L: (i) colocalizes with centrosomes at all mitotic stages and resides in centrosome-enriched fractions; (ii) associates with the nucleolus and the midbody and respective fractions, and (iii) co-stains the aberrant centrosome configurations observed in glioma cell lines.


CONCLUSIONS
The overall data merge on the multiplex entry of MARK4L into the cell cycle and link it to the aberrant centrosomes in glioma cell lines suggesting a possible role of this kinase in the abnormal mitotic processes of human glioma.
Magnani
Ivana
I
Dipartimento di Medicina, Chirurgia e Odontoiatria, Sezione di Genetica Medica, Università di Milano, Milan, Italy.
Novielli
Chiara
C
Bellini
Melissa
M
Roversi
Gaia
G
Bello
Lorenzo
L
Larizza
Lidia
L
D000906:Antibodies / Q000276:immunology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D018385:Centrosome / Q000378:metabolism; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D047468:Immunoprecipitation; D007399:Interphase; D013058:Mass Spectrometry; D008938:Mitosis; D008970:Molecular Weight; D009474:Neurons / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D013234:Stem Cells / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D000906:Antibodies; C483047:MARK4 protein, human; D017346:Protein Serine-Threonine Kinases