Cdk1 phosphorylation sites on Cdc27 are required for correct chromosomal localisation and APC/C function in syncytial Drosophila embryos.
Anaphase-promoting complex or cyclosome (APC/C) controls the metaphase-to-anaphase transition and mitosis exit by triggering the degradation of key cell cycle regulators such as securin and B-type cyclins. However, little is known about the functions of individual APC/C subunits and how they might regulate APC/C activity in space and time. Here, we report that two potential Cdk1 kinase phosphorylation sites are required for the chromosomal localisation of GFP::Cdc27 during mitosis. Either or both of the highly conserved proline residues in the Cdk1 phosphorylation consensus sequence motifs were mutated to alanine (Cdc27 P304A or P456A). The singly mutated fusion proteins, GFP::Cdc27P304A and GFP::Cdc27P456A, can still localise to mitotic chromosomes in a manner identical to wild-type GFP::Cdc27 and are functional in that they can rescue the phenotype of the cdc27L7123 mutant in vivo. However, when both of the Cdk1 phosphorylation sequence motifs were mutated, the resulting GFP::Cdc27P304A,P456A construct was not localised to the chromosomes during mitosis and was no longer functional, as it failed to rescue mutant phenotypes of the cdc27L7123 gene. High levels of cyclin B and cyclin A were detected in mutant third instar larvae brain samples compared with its wild-type control. These results show for the first time that the two potential Cdk1 phosphorylation sites on Drosophila Cdc27 are required for its chromosomal localisation during mitosis and imply that these localisations specific to Cdc27 are crucial for APC/C functions.
Huang
Jun-Yong
JY
Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, University of Newcastle upon Tyne, Catherine Cookson Building, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. junyong.huang@ncl.ac.uk
Morley
Gary
G
Li
Deyu
D
Whitaker
Michael
M
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism; D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002875:Chromosomes / Q000378:metabolism*; D003078:Colchicine / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000033:anatomy & histology / Q000196:embryology* / Q000378:metabolism; D015726:Giant Cells / Q000166:cytology / Q000378:metabolism; D008938:Mitosis / Q000502:physiology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism; D050257:Tubulin Modulators / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*
D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; C577365:Cdc27 protein, Drosophila; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D011993:Recombinant Fusion Proteins; D050257:Tubulin Modulators; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D016203:CDC2 Protein Kinase; D003078:Colchicine