Protein: DP03344 (UniProt P36955)
Region: residues 22	to 26
Annotation: molecular function regulator (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
The fully CK2 phosphorylation site mutant S24, 114E abolished PEDF neurotrophic activity but enhanced its antiangiogenic activity, while the PKA phosphorylation site mutant S227E reduced PEDF antiangiogenic activity. This is a novel role of extracellular phosphorylation that is shown here to completely change the nature of PEDF from a neutrophic to an antiangiogenic factor.
We found that PEDF is functionally modulated by extracellular phosphorylation. The CK2 phosphorylated PEDF had a reduced neurotrophic activity, while its antiangiogenic activity was significantly increased.
Because of the differences in ERK activation between plPEDF and rPEDF, we used this system to examine whether the phosphorylation mutants indeed mimic the effect of phosphorylation on PEDF activity. When used to stimulate HUVECs, the CK2 phosphorylation site mutants S24A and S24E did not have a significant effect, while S114A and S114E mutants demonstrated slightly reduced ability to stimulate ERK phosphorylation (Figure 4C). However, significant effects were found with the double mutants, as S24, 114A had a reduced effect, while S24, 114E enhanced ERK phosphorylation. These effects were even stronger than the effects of rPEDF or plPEDF. The higher activity of S24, 114E suggests that the 2 Glu residues indeed mimic the activity of phosphorylated PEDF.
The CK2 phosphorylation site mutants S24E/S24A and S114E/S114A had only small effects, as they all induced neuronal differentiation of the Y-79 cells. However, much fewer neurite-like processes and cell aggregates were observed when cells were treated with the S24, 114E mutant. With this mutant, the cells formed small coronalike structures but were very compact without any sprouts projecting from the cells, and this inhibitory effect was stronger than that of plPEDF. On the other hand, cells treated with the S24, 114A mutant exhibit neurite outgrowth and big aggregates similar to rPEDF.
When incubated together with bFGF, the CK2 nonphosphorylated double mutant, S24, 114A, exhibited an antiangiogenic activity that was similar to or slightly less than that of rPEDF, where rearrangement toward vessels could be seen, but clear
vessels did not form. On the other hand, the CK2 phosphorylated mutant, S24, 114E, appeared to be a very significant antiangiogenic factor, even stronger than plPEDF, as it did not allow any vessel formation.
We therefore conclude that phosphorylation of PEDF on its CK2 sites significantly enhanced the antiangiogenic activity of PEDF, while the phosphorylation on its PKA site may slightly reduce its antiangiogenic activity.
As shown in the aortic ring assay, the S24, 114E mutant had even stronger antiangiogenic activity relative to plPEDF, as plugs treated with this mutant had very little angiogenic response. In contrast, plugs treated with bFGF and S227E had much less antiangiogenic activity reflected in many infiltrating vessels. In addition plugs treated with bFGF and S24, 114A mutant or S227A mutant appeared similar to those treated with bFGF and rPEDF (not shown). These results further support that CK2-phosphorylated PEDF enhanced the antiangiogenic activity of PEDF, while the phosphorylation on its PKA site may reduce this activity.
Thus, we observed a CK2-dependent difference in the ability of PEDF to induce neuronal differentiation in retinoblastoma cells where both rPEDF and S24, 114A mutants induced neuronal differentiation, while the S24, 114E had almost no neurotrophic effect.
Thus, S24, 114E appeared to be a very significant antiangiogenic factor, and its inhibitory effect was more pronounced when compared with plPEDF antiangiogenic activity.
Additional support for a functional change of PEDF that is phosphorylation dependent was demonstrated by the degree of ERK activation. A significant effect was found with the double mutants, as S24, 114A reduced ERK activation, while S24, 114E enhanced ERK activation to a higher degree than the stimulation achieved by plPEDF.
We found that both CK2 and PKA phosphorylations of PEDF markedly affect its physiologic function. The fully CK2 phosphorylation site mutant S24, 114E abolished PEDF neurotrophic activity and enhanced its antiangiogenic activity, while the PKA phosphorylation site mutant S227E reduced PEDF antiangiogenic activity.