title: Functional analysis of centrosomal kinase substrates in Drosophila melanogaster reveals a new function of the nuclear envelope component otefin in cell cycle progression.
abstract: Phosphorylation is one of the key mechanisms that regulate centrosome biogenesis, spindle assembly, and cell cycle progression. However, little is known about centrosome-specific phosphorylation sites and their functional relevance. Here, we identified phosphoproteins of intact Drosophila melanogaster centrosomes and found previously unknown phosphorylation sites in known and unexpected centrosomal components. We functionally characterized phosphoproteins and integrated them into regulatory signaling networks with the 3 important mitotic kinases, cdc2, polo, and aur, as well as the kinase CkIIβ. Using a combinatorial RNA interference (RNAi) strategy, we demonstrated novel functions for P granule, nuclear envelope (NE), and nuclear proteins in centrosome duplication, maturation, and separation. Peptide microarrays confirmed phosphorylation of identified residues by centrosome-associated kinases. For a subset of phosphoproteins, we identified previously unknown centrosome and/or spindle localization via expression of tagged fusion proteins in Drosophila SL2 cells. Among those was otefin (Ote), an NE protein that we found to localize to centrosomes. Furthermore, we provide evidence that it is phosphorylated in vitro at threonine 63 (T63) through Aurora-A kinase. We propose that phosphorylation of this site plays a dual role in controlling mitotic exit when phosphorylated while dephosphorylation promotes G(2)/M transition in Drosophila SL2 cells.
authors: Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics, Berlin, Germany.
authors: Karin
authors: K
authors: Habermann
authors: Ekaterina
authors: E
authors: Mirgorodskaya
authors: Johan
authors: J
authors: Gobom
authors: Verena
authors: V
authors: Lehmann
authors: Hannah
authors: H
authors: Müller
authors: Katharina
authors: K
authors: Blümlein
authors: Michael J
authors: MJ
authors: Deery
authors: Irina
authors: I
authors: Czogiel
authors: Christoph
authors: C
authors: Erdmann
authors: Markus
authors: M
authors: Ralser
authors: Jens Peter
authors: JP
authors: von Kries
authors: Bodo M H
authors: BM
authors: Lange
mesh_terms: D000818:Animals; D064127:Aurora Kinases; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism; D047390:Casein Kinase II / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle; D002460:Cell Line; D018385:Centrosome / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism*; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference
chemical_list: D029721:Drosophila Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; C063595:Ote protein, Drosophila; C072004:polo protein, Drosophila; D064127:Aurora Kinases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase