Figure 2.

SUR is a substrate of PLK1 in dividing cells. Ser20 and Thr21 of SUR are substrates of PLK1. Bacterially recombinant GST–SUR fusion proteins were phosphorylated in vitro using [γ-³²P]-ATP and active PLK1 as described under ‘Materials and methods’. Samples were separated by SDS–PAGE. (A) CB-stained gel of samples of wild type GST–SUR plus PLK1 (GST–SUR + PLK1), single and double mutant S20A/T21A GST–SUR plus PLK1 (GST–SUR^S20A/T21A + PLK1). (B) The same gel was dried and subsequently incubated with X-ray film. Note that there was dramatic incorporation of ³²P into wild type, single site mutants (SUR^S20A and SUR^T21A) but little into the double mutant SUR^S20A/T21A or GST. (C) SUR is a cognate substrate of PLK1 in vivo. Extracts of nocodazole-synchronized mitotic HeLa cells were precipitated using an anti-SUR antibody. The precipitates were then fractionated by SDS–PAGE followed by immunoblotting of SUR (top panel) and serine phosphorylation (lower panel). Note that the SUR phosphorylation is a function of PLK1 but not CDK1. (D) Ser20 of SUR is phosphorylated in mitosis. GFP–SUR^wt, GFP–SUR^S20A and SUR^T21A were immunoprecipitated and isolated proteins were analyzed by western blotting. The membrane was first probed for GFP–SUR (upper panel) followed by detection of phosphoserine (lower panel). Lane 1, GFP–SUR^wt i.p.; lane 2, GFP–SUR^T21A i.p.; lane 3, GFP–SUR^S20A i.p. Note that GFP–SUR^S20A was not detected by phosphoserine antibody. (E) Association of PLK1 with SUR is independent of phosphorylation. Aliquots of HeLa cells were transiently transfected to express various GFP-tagged SUR plasmids with FLAG–PLK1. Thirty-six hours after transfection, these cells were lysed and clarified cell lysates were precipitated with FLAG-M2 agarose as described in ‘Material and methods’ section. Note that GFP–SUR^S20A weakened its association with PLK1.