In vitro kinase assays

Kinase assays were carried out in a total volume of 25 μl containing 12 mM Hepes pH 7.5, 10 mM MgCl₂, 25 μM ATP, an amount of the appropriate substrate as indicated and 0.19 μM (0.5 μg) GST-SRPK1 or 0.07 μM (0.1 μg) Akt for 30 min at 30°C. Recombinant active Akt1 and Akt2 were purchased from Upstate Biotechnology (Catalog# 14–276, current supplier Millipore Ltd.) and Life Technologies (Catalog# PV3184), respectively. Histone H2B (Catalog No 223514) and myelin basic protein (MBP) were purchased from Boehringer Mannheim and Life Technologies, respectively. R0, R1 and R2 peptides were previously described [21]. Incorporation of radioactivity was measured by excising the radioactive bands from an SDS-PAGE gel and scintillation counting. Michaelis constant (K_m) was determined from double-reciprocal plots of 1/V against 1/S, where V is the rate of phosphorylation and S is the substrate concentration. For the determination of K_m values the concentration of ATP was raised to 100 μM. Kinase activities were determined at eight concentrations of GST-LBRNt(62–92) (0.096, 0.2, 0.4, 0.82, 1.224, 1.63, 2.45 and 3.26 μM). One unit of activity is the amount of enzyme required to catalyze the transfer of 0.6 pmol phosphate to 50 pmol (1.5 μg) GST-LBRNt(62–92) per min.