CK2 and PKA phosphorylate PEDF in vitro. (A) rPEDF and plPEDF were incubated with CK2 holoenzyme, [γ³²P]-ATP, and increasing concentrations of poly-L-lysine (PLL) as described in “Materials and methods.” As a control, rPEDF and plPEDF were incubated in the same mix in the absence of CK2. After 45 minutes at 30°C, the reaction was arrested by boiling for 5 minutes in sample buffer, and the samples were subjected to 10% SDS-PAGE. The gel was stained with Coomassie blue (Coom, bottom panel), dried, and subjected to autoradiography (Auto, top panel). (B) rPEDF and plPEDF (50 μg/mL) were incubated with CK2 holoenzyme (4 μg/mL), [γ³²P]-ATP (10 μM, 6 Ci/mmol [222 GBq/mmol]), poly-l-lysine (200 nM), and increasing concentrations of heparin (Hep). Phosphorylation and analysis were performed as in panel A. (C) rPEDF and plPEDF (50 μg/mL) were incubated with the pure catalytic subunit of PKA (2.5 μg/mL), heparin (50 μg/mL), and [γ³²P]-ATP (10 μM, 6 Ci/mmol [222 GBq/mmol]). Phosphorylation and analysis were conducted as in panel A. (D) rPEDF was digested with trypsin as described in “Materials and methods.” At the indicated times, aliquots were removed from the reaction mixture and centrifuged, and sample buffer was added to the supernatant. Samples were boiled and subjected to 12.5% SDS-PAGE followed by silver stain. Right panel: rPEDF was phosphorylated by CK2 and loaded on a G25 Sephadex column to remove the excess of [γ³²P]-ATP. The eluted fraction was then subjected to trypsin digestion and subjected to 12.5% SDS-PAGE followed by autoradiography. (E) Alignment of the tryptic peptides revealed by mass spectrometry and N-terminus sequence analysis that were obtained from the trypsin-digested fragments of rPEDF within a schematic representation of PEDF.