Figure 1. Binding and Phosphorylation of RP1 by CK2.

1A Identification of CK2 phosphorylation site - RP1-sequence (amino acid), three potential CK2 kinase sites S59, S72, S236 (underlined) were identified by prosite scan (www.expasy.ch). The peptides used for in vitro experiments (1C) are marked in bold. S236 the actual CK2 phosphorylation site is shown in red. 1B Interaction–assay RP1/CK2 - Endogenous RP1 (first panel) was co-precipitated with its potential binding partners. RP1/CK2 kinase interaction could be detected by specific α/ß CK2 subunit antibodies. The black wedges in this panel indicate increasing stringency of washing procedure (% Tween20/PBS). In a reverse experiment (right side panel), endogenous RP1 was verified as genuine CK2 binding substrate. By using CK2 subunits as baits, RP1 could be detected in the pulldowns by its specific RP1 antibody (right panel). No signal was seen when an insignificant IgG antibody was used. On the far right 1/10 of cell lysate of the foregoing experiments is depicted as an input control. The black wedges in this panel indicate stringency of the washing procedure (0.01% and 0.3% Tween/PBS). 1C Biotinylated peptides (A: aa54–65, B: aa70–80, C: aa229–240) containing the potential CK2 phosphorylation sites S⁵⁹, S⁷², S²³⁶ were synthesized and tested as CK2 phosphorylation substrates (A, B, C, 3 µg each) in an in vitro phosphorylation assay. A known positive CK2 kinase site peptide (DDDDSDDDDD, 3 µg) served as a control. The black wedge indicates incubation times (minutes). 1D CK2 kinase assay - Recombinant CK2 and ³³P-gamma-ATP were incubated in vitro with different amounts of RP1-wt protein (first panel shows a coomassie stain of his-tagged purified RP1 protein used for the assay) and phosphorylation was measured by autoradiography (middle panel). The amounts of RP1 protein used are indicated above the middle panel. Autophosphorylation of CK2 at its subunit ß served as positive control RP1-ALA236 mutated protein (ALA) was almost non-phosphorylated in comparison to the wild type protein (right side upper panel). The lower panel on the right side shows a coomassie stain representing the amount of RP1 used for this experiment.