Figure 4.

Cdk1-mediated phosphorylation of serine 386 is an essential priming step for Plk1-mediated phosphorylation at serine 286 and 393. (a) Purified recombinant PTP1B-His (WT, S386A, S386E) (500 ng), or Cdc25C (500 ng), was incubated with recombinant Cdk1/cyclin B1 (200 ng), ATP (100 μM) and 2 μCi of [γ-³²P] ATP for 1 h at 30 °C. The reaction was terminated by the addition of Laemmli buffer to 1 × concentration. (b) Purified PTP1B-His (WT, or S386A, S286A, S393A, and S286A/S393A mutants) (500 ng) was incubated with ATP (100 μM), in the presence of Cdk1 (460 ng) at 30 °C for 1 h. The reaction was terminated by incubation at 65 °C for 15 min. The reaction samples were chilled on ice for 5 min, and then incubated with Plk1 (460 ng), 2 μCi of [γ-³²P] ATP and ATP (100 μM), for a subsequent kinase reaction at 30 °C for 1 h. The reaction was terminated by the addition of Laemmli buffer. Protein was resolved by SDS-PAGE, and [γ-³²P] incorporation was measured by autoradiography after gel drying. The amount of PTP1B was visualised by CBB staining. (c) The amount of [γ-³²P] incorporation was normalised to WT PTP1B levels, and expressed as normalised intensity of [γ-³²P] incorporation. Results are representative of the mean±S.E.M. of three independent experiments (n=3, *=P<0.05, **=P<0.01, using a paired T-test)