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. 2015 Nov 1;128(21):3910–3921. doi: 10.1242/jcs.166686

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© 2015. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 1. — Mck1 phosphorylates Kip2 in vivo and in vitro. (A) Details of Kip2 variants described in this paper and abbreviations used in labels, as well as graphical representation of Kip2. Positions of the GSK-3, Cdc28 and Dbf2 consensus sites, residues S63 and T275 are shown in red. The SxIP Bim1/EB1-binding motif is marked in yellow. (B) Comparison of phosphorylation and expression levels between unphosphorylatable Kip2^13myc variants and Kip2^13myc in mck1Δ and rim11Δ cells. Phosphorylation of Kip2^13myc is reduced upon introduction of S63A mutation, in mck1Δ cells and marginally in rim11Δ cells. Shown is a western blot against the Myc epitope in whole-cell extracts. Variant labelling see (A), AA corresponds to Kip2^13myc-S63A T275A. Arc1 serves as loading control. (C) Depletion of Mck1 leads to decrease in Kip2 phosphorylation. Overexpression of Mck1 does not increase phosphorylation of hypo-phosphorylated Kip2^13myc variants mutated in the GSK-3 consensus site(s). ^HAMck1 expression from the GAL1-10 promoter (P_GAL) was induced by addition of galactose (+), depletion (−) by addition of glucose to the medium. Western blot shows Kip2^13myc phosphorylation and ^HAMck1 expression levels. Arc1 serves as loading control. (D,E) In vivo ³²P-phosphate labelling of Kip2^13myc variants and Kip2^13myc in different kinase mutants. Kip2 loading: western blot of the Kip2^13myc variants after immunoprecipitation from cells labelled with ³²P-phosphate, the same blot was used for the autoradiography shown. (F) Mck1 phosphorylates Kip2 in vitro. ^MBPKip2ΔC^GFP and displayed variants were pre-incubated with Cdk1 and cyclin B and/or ATP followed by incubation with Mck1^TAP and γ³²P-ATP as indicated. Autoradiography of the subsequent SDS-PAGE is shown. Mck1^TAP phosphorylates Kip2 only after pre-incubation with Cdk1 and phosphorylation is abolished after introduction of the S63A and S275A mutations. (G) Sequential phosphorylation of Kip2 N-terminus by Mck1. A fusion of the N-terminal 80 aa of Kip2 to GST (GST-Kip2^1-80) is phosphorylated only after pre-incubation with Cdk1. Mutation of S63 but also of the upstream T59 abrogated phosphorylation.