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First published online 22 May 2007
doi: 10.1242/jcs.006833

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Cdk1 phosphorylation sites on Cdc27 are required for correct chromosomal localisation and APC/C function in syncytial Drosophila embryos

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

View larger version (57K): [in this window] [in a new window]   Fig. 1. Confocal images showing small areas of Drosophila syncytial embryos that were taken from GFP::Cdc27 (A) or GFP::Cdc16 (B) transgenic flies with or without treatment with a 2.5 mM final intracellular concentration of colchicine. Drosophila GFP::Cdc27 associates with mitotic chromosomes (A, panels 1 and 7, 2 and 8, arrows), but GFP::Cdc16 does not (B, panels 1 and 7, 2 and 8, arrows indicate shadow regions), both GFP::Cdc27 and GFP::Cdc16 associate with nuclear envelope membrane (open arrowheads in A,B panels 1 and 7). Colchicine treatment clearly increases the association of GFP::Cdc27 with mitotic chromosomes (A, panels 3 and 9 arrows) compared with the metaphase control (A, panels 1 and 7, arrows). The localisation of the Drosophila GFP::Cdc16 is not affected by the same treatment (compare B, panels 3 and 9 with 1 and 7). A,B panels 1-3:merge images (GFP::Cdc27 or GFP::Cdc16 in green, chromosomes in red), A,B panels 4-6: Rhodamine-H1-labelled chromosomes in white; A,B panels 7-9: GFP::Cdc27 or GFP::Cdc16 in white; A,B, panels 7 and 8: non-colchicine treated embryos; A,B, panel 9: colchicine-treated embryos. The developmental stage of syncytial embryos for each experiment was about nuclear division cycle 8 or 9. Bars, 10 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 2. There are two potential phosphorylation sites for Cdk1 protein kinase in the Drosophila Cdc27 protein amino acid sequence. (A) Two consensus phosphorylation motifs for Cdk1 kinase (T-303 SSGTPFR and S-455 QPRSPPR) in the Drosophila Cdc27 amino acid sequence were identified using web site: http://scansite.mit.edu/. Highly conserved prolines (bold) from each motif were mutated to Alanine individually or together to create three mutated forms: P304A, P456A and double point mutations (P304A, P456A). All the constructs were fused with GFP at their N-terminus and used to generate transgenic Drosophila lines by the standard P-element-mediated transformation methods (see Materials and Methods). (B) Mutant Cdc27 proteins are still capable of incorporation into an APC/C complex. Embryo extracts made from control W67 or various GFP::Cdc27 fusion proteins as listed below, were immunoblotted with anti-Drosophila Cdc27 antibody (Top) or anti-Drosophila Cdc16 antibody (bottom) following co-immunoprecipitation (co-IP) of GFP fusion proteins with GFP antibody conjugated Dynabeads. Embryo extracts were made from an overnight collection of the flowing fly lines: lane 1, W67 (control); lane 2, gfp::cdc27/gfp::cdc27; lane 3, gfp::cdc27P304A,P456A/gfp::cdc27P304A,P456A; lane 4, gfp::cdc27P456A/gfp::cdc27P456A; lane 5, gfp::cdc27P304A/gfp::cdc27P304A. The amounts of immunoprecipitated mutant proteins were between 91-97% of those immunoprecipitated in wild-type controls. (C) Two potential Cdk1 phosphorylation sites contribute to the phosphorylation of Cdc27 in vitro by Cdk1. Samples were made from transgenic syncytial embryos. Lane 1, GFP; lane 2, GFP::Cdc27; lane 3, GFP::Cdc27P304A,P456A. Asterisk indicates GFP breakdown products from GFP::Cdc27P304A,P456A fusions. (D) Histone 1 kinase assay with Cdk1/cyclin B kinase in vitro as positive control.

View larger version (90K): [in this window] [in a new window]   Fig. 3. Both CDK1 phosphorylation sites are required for localisation of Cdc27 to mitotic chromosomes. (A,B) Confocal images from GFP::Cdc27P304A transgenic syncytial embryos at various stages of mitosis (B) showed no significant differences in dynamic localisation to the wild-type GFP::Cdc27 transgenic line (A). An identical result was seen with the GFP::Cdc27P456A single point mutant (not shown). However the fusion protein containing mutations in both potential Cdk1 phosphorylation sites (GFP::Cdc27P304A,P456A) was no longer found localised to the chromosomes during mitosis (C). White open arrowheads indicate the nuclear envelope membrane localised with fusion proteins (bright ring structures) in A,B,D in prophase and metaphase images. It is less abundant in C, white open arrows indicate the mitotic chromosomes regions; GFP fusion proteins in white associate with chromosomes in A and B; and do not associate with chromosomes (shadow regions) in C,D. Double white open arrowheads show the mid-body regions at anaphase and telophase. A, panels 1-4, merged confocal images (GFP::Cdc27 in green, Rhodamine-H1-labelled chromosomes in red); A, panels 5-8: Rhodamine-H1-labelled chromosomes in white. Bar, 10 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 4. GFP::Cdc27 and GFP::Cdc27P304A can support healthy embryonic development in established stable p340 (GFP::Cdc27) and p341 (GFP::Cdc27P304A) transgenic cdc27L7123 rescue lines, but that the GFP::Cdc27P304A,P456A double mutant fusion protein does not. (A) The endogenous Cdc27 protein or GFP::Cdc27 and GFP::Cdc27P304A fusion proteins are indicated by arrows in lanes 1-3 respectively. Some (11%) of endogenous Cdc27 is still present in lanes 2 and 3 with the embryo samples collected from parental fly genotypes of gfp::cdc27/gfp::cdc27; cdc27L7123/cdc27L7123 and gfp::cdc27P304A/gfp::cdc27P304A; cdc27L7123/cdc27L7123 respectively. These flies carry a mutation in the cdc27gene on the third chromosome. The mutation is caused by a P-element insertion at 519 bp upstream of the ATG start site of cdc27 (http://flybase.bio.indiana.edu/.bin/fbidq.html?FBal0087027#FBrf0111595) (Huang and Raff, 2002). Actin was detected as a loading control. (B) Western blot of late third instar larvae brain samples: lane 1, W67 control; lane 2, Cdc27L7123:cdc27L7123/cdc27L7123 homozygous original mutant derived from the cdc27L7123/TM6B Tb mutant fly line; lane 3, GFP::Cdc27P304A,P456A the attempted rescue homozygous mutant (gfp::cdc27P304A,P456A/gfp::cdc27P304A,P456A; cdc27L7123/cdc27L7123) derived from the cross gfp::cdc27P304A,P456A/gfp::cdc27P304A,P456A; cdc27L7123/TM6B Tb. Endogenous Cdc27 or GFP::Cdc27 was revealed by probing with anti-Drosophila Cdc27 antibody (top panel); cyclin A and cyclin B levels are shown in the middle panel using anti-Drosophila cyclin A or cyclin B antibodies respectively; Actin (bottom panel) was detected as a loading control. (C) Relative expression levels of endogenous Cdc27 or GFP::Cdc27 in third instar larvae brains from wild-type control samples (W67), Cdc27L7123 (cdc27L7123/cdc27L7123 homozygous mutant) and GFP::Cdc27P304A,P456A (the attempted rescue homozygous mutant: gfp::cdc27P304A,P456A/gfp::cdc27P304A,P456A; cdc27L7123/cdc27L7123) were quantified from the western blot results present in the top panel of B in lanes 1-3 of the endogenous Cdc27 bands and the GFP:: Cdc27P304A,P456A band. The endogenous Cdc27 band intensity in wild-type sample is set to 100% as the comparison.