Figure 2.

Autophosphorylation of Sln1-HK. (a) Schematic showing two alternate routes for working with resin-bound GST-Sln1-HK. The Sln1-HK domain can be cleaved from the GST tag and eluted from the resin using thrombin. The isolated Sln1-HK domain can then be autophosphorylated using radiolabeled ATP. This strategy is best for working with Sln1-HK mutants. A convenient alternative is to autophosphorylate GST-Sln1-HK while bound to resin. Bead-bound phospho-GST-Sln1-HK can then be used directly as a phosphodonor for in vitro phosphotransfer assays. (b) A representative autophosphorylation time-course for soluble Sln1-HK (reproduced from Tao et al., 2002). Sln1-HK (2 μg) was incubated with [γ-³²P]ATP in reaction buffer and aliquots were removed at the times indicated and quenched in SDS loading buffer. Maximal levels of autophosphorylation are typically reached in 30–40 minutes (c) An example of phosphotransfer from bead-bound GST-Sln1-HK to the Sln1-Rec domain (reproduced from Ault et al., 2002). ³²P-labeled GST-Sln1-HK (2.5 μg) was incubated alone (lane 1) or with 1 μg Sln1-Rec for 5, 15, 30, or 60 minutes (lanes 2–5, respectively). Most of the radiolabel is transferred within 5 minutes and the gradual loss of radiolabel from Sln1-Rec is due to its intrinsic rate of dephosphorylation.