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Figure 2


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.





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