Human EML4, a novel member of the EMAP family, is essential for microtubule formation.
Human EML4 (EMAP-like protein 4) is a novel microtubule-associated WD-repeat protein of 120 kDa molecular weight, which is classified as belonging to the conserved family of EMAP-like proteins. Cosedimentation assays demonstrated that EML4 associates with in vitro polymerized microtubules. Correspondingly, immunofluorescence stainings and transient expression of EGFP-labeled EML4 revealed a complete colocalization of EML4 with the interphase microtubule array of HeLa cells. We present evidence that the amino-terminal portion of EML4 (amino acids 1-249) is essential for the association with microtubules. Immunoprecipitation experiments revealed that EML4 is hyperphosphorylated on serine/threonine residues during mitosis. In addition, immunofluorescence stainings demonstrated that hyperphosphorylated EML4 is associated with the mitotic spindle, suggesting that the function of EML4 is regulated by phosphorylation. siRNA-mediated knockdown of EML4 in HeLa cells led to a significant decrease in the number of cells. In no case mitotic figures could be observed in EML4 negative HeLa cells. Additionally, we observed a significant reduction of the proliferation rate and the uptake of radioactive [3H]-thymidine as a result of EML4 silencing. Most importantly, EML4 negative cells showed a completely modified microtubule network, indicating that EML4 is necessary for correct microtubule formation.
Pollmann
Marc
M
Department of Hematopathology and Lymph Node Registry, Founded by the German Association of Pathologists at the University of Kiel, Michaelisstr, 11, 24105 Kiel, Germany.
Parwaresch
Reza
R
Adam-Klages
Sabine
S
Kruse
Marie-Luise
ML
Buck
Friedrich
F
Heidebrecht
Hans-Juergen
HJ
D000818:Animals; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008807:Mice, Inbred BALB C; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000502:physiology*; D008870:Microtubules / Q000502:physiology* / Q000648:ultrastructure; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D018797:Cell Cycle Proteins; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C415585:EML4 protein, human; D012697:Serine Endopeptidases