Identification of Drosophila Myt1 kinase and its role in Golgi during mitosis.
Entry into mitosis is regulated by inhibitory phosphorylation of cdc2/cyclin B, and these phosphorylations can be mediated by the Wee kinase family. Here, we present the identification of Drosophila Myt1 (dMyt1) kinase and examine the relationship of Myt1 and Wee1 activities in the context of cdc2 phosphorylation. dMyt1 kinase was found by BLAST-searching the complete Drosophila genome using the amino acid sequence of human Myt1 kinase. A single predicted polypeptide was identified that shared a 48% identity within the kinase domain with human and Xenopus Myt1. Consistent with its putative role as negative regulator of mitotic entry, overexpression of this protein in Drosophila S2 cells resulted in a reduced rate of cellular proliferation while the loss of expression via RNA interference (RNAi) resulted in an increased rate of proliferation. In addition, loss of dMyt1 alone or in combination with Drosophila Wee1 (dWee1) resulted in a reduction of cells in G2/M phase and an increase in G1 phase cells. Finally, loss of dMyt1 alone resulted in a significant reduction of phosphorylation of cdc2 on the threonine-14 (Thr-14) residue as expected. Surprisingly however, a reduction in the phosphorylation of cdc2 on the tyrosine-15 (Tyr-15) residue was only observed when both dMyt1 and dWee1 expression was reduced via RNAi and not by Wee1 alone. Most strikingly, in the absence of dMyt1, Golgi fragmentation during mitosis was incomplete. Our findings suggest that dMyt1 and dWee1 have distinct roles in the regulation of cdc2 phosphorylation and the regulation of mitotic events.
Cornwell
William D
WD
Department of Oncology Research, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, Mail Code UW 2532, King of Prussia, PA 19406, USA.
Kaminski
Paula J
PJ
Jackson
Jeffrey R
JR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002455:Cell Division; D002460:Cell Line; D004330:Drosophila / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D029721:Drosophila Proteins / Q000235:genetics* / Q000502:physiology*; D006056:Golgi Apparatus / Q000201:enzymology* / Q000648:ultrastructure; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins; D008938:Mitosis; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011494:Protein Kinases / Q000235:genetics* / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology*; D012313:RNA / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012313:RNA; D011494:Protein Kinases; C458479:Myt1 protein, Drosophila; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; C490679:PKMYT1 protein, human; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase