In vitro phosphorylation assay

For the phosphorylation assays, 25 μg of the wild-type and mutants His-SUMO-GSN proteins were incubated with 10 μg of His-PHO85-1 and 10 μg of His-PCL-1 proteins in reacting buffer (1 mM ATP, 25 mM MgCl₂, [γ-³²P]-ATP, activity 330 mCi/mmol, ∼1200 cpm/mmol) in 25 μL of reaction volume for 30 min at 30°C. Crude cellular extract of a N. crassa mutant strain (Δgsn), which does not synthesize glycogen synthase, was also used as a source of protein kinases. After incubation, His-SUMO-GSN proteins were immobilized on Ni-NTA agarose beads (Qiagen, Hilden, Germany) under low agitation. The beads were collected by centrifugation, washed three times in washing buffer (50 mM Tris, pH 8.0, 30 mM imidazole, 500 mM NaCl) and suspended in elution buffer (50 mM Tris, pH 8.0, 500 mM imidazole, 500 mM NaCl). After protein elution, Laemmli buffer (Laemmli, 1970) was added, the protein samples were boiled for 5 min and separated on a 12% SDS-PAGE gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue (CBB), dried and exposed to X-ray film.