Variable phosphorylation states of pigment-epithelium–derived factor differentially regulate its function

Abstract

The pigment epithelium-derived factor (PEDF) belongs to the family of noninhibitory serpins. Although originally identified in the eye, PEDF is widely expressed in other body regions including the plasma. This factor can act either as a neurotrophic or as an antiangiogenic factor, and we previously showed that the 2 effects of PEDF are regulated through phosphorylation by PKA and CK2. Here, we studied the interplay between the PKA and CK2 phosphorylation of PEDF, and found that a PEDF mutant mimicking the CK2-phosphorylated PEDF cannot be phosphorylated by PKA, while the mutant mimicking the PKA-phosphorylated PEDF is a good CK2 substrate. Using triple mutants that mimic the PKA-and CK2-phosphorylated and nonphosphorylated PEDF, we found that PEDF can induce several distinct cellular activities dependent on its phosphorylation. The mutant mimicking the accumulative PKA plus CK2 phosphorylation exhibited the strongest antiangiogenic and neurotrophic activities, while the mutants mimicking the individual phosphorylation site mutants had either a reduced activity or only one of these activities. Thus, differential phosphorylation induces variable effects of PEDF, and therefore contributes to the complexity of PEDF action. It is likely that the triple phosphomimetic mutant can be used to generate effective antiangiogenic or neurotrophic drugs. IntroductionThe pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitors (serpin) superfamily, but as of today was not found to exhibit inhibitory activity against any proteases. 1,2 It was first isolated based on its ability to convert dividing retinoblastoma cells into differentiated neurons, and thus was characterized as a neurotrophic factor. 1,3 Later, it was shown that besides its neurotrophic functions, PEDF is a potent natural inhibitor of angiogenesis in the eye, 4 where it inhibits stimulatory activity of several strong proangiogenic factors. This antiangiogenic potency has also been shown in several animal models in which PEDF was demonstrated as the factor responsible for the reduction of bloodvessel growth in the eye. [5][6][7][8][9] Although originally discovered in the culture medium of pigment epithelial cells obtained from the fetal human retina, 10 it is clear today that PEDF is expressed not only in the retina, but also at multiple sites in the adult eye, [11][12][13] as well as in the adult human brain, the spinal cord, 14,15 and human plasma. 16 Therefore, it is possible that PEDF has the potential to inhibit angiogenesis throughout the body.It is well established that protein phosphorylation plays a key role in the regulation of most intracellular processes. However, it is becoming increasingly evident that protein kinases can also regulate extracellular processes, as the kinases are present extracellularly as either ectoprotein or exoprotein kinases. 17,18 The ectoprotein kinases are membrane-bound enzymes whose catalytic activities are localized on the extracellular cell surface of a wide variety of cells. The exoprotein k...

Protein Kinase CK2 and Angiogenesis

“…It was also shown that PDGF, the other growth factor implicated in angiogenesis, is phosphorylated by CK2. The CK2 phosphorylated form of PDGF has an elevated anti-angiogenic activity [48,49]. Recently, it turned out that PDG induced the CK2a' subunit expression [50].…”

“…PEDF phosphorylated by CK2 exhibited a strong anti-angiogenic activity [49]. Most of these experiments were performed with glutamic acid mutants, which mimic CK2 phosphorylation indicating that a negative charge at the CK2 phosphorylation sites is sufficient for the anti-angiogenic activity.…”

Role of pigment epithelium-derived factor in the reproductive system

“…PEDF undergoes N-glycosylation that occurs at the Asn285 site. In addition, Maik-Rachline et al (2005) demonstrated that PEDF is a phosphoprotein, which has several phosphorylation sites that determine its function as either neurotrophic or anti-angiogenic (Maik-Rachline & Seger 2006). The neurotrophic activity is mediated by PKA that phosphorylates PEDF on Ser 227, whereas the anti-angiogenic activity is mediated by CK2 that phosphorylates PEDF on Ser 24 and Ser 114.…”

“…The neurotrophic activity is mediated by PKA that phosphorylates PEDF on Ser 227, whereas the anti-angiogenic activity is mediated by CK2 that phosphorylates PEDF on Ser 24 and Ser 114. In addition, induction of PKA followed by CK2 phosphorylation exhibits its most potent anti-angiogenic and neurotrophic activities (Maik-Rachline & Seger 2006). On top of its neurotrophic and anti-angiogenic activities (Simonovic et al 2001), PEDF was shown to function as an anti-inflammatory (Wang et al 2008) and anti-oxidative factor (Yamagishi et al 2005, Bar-Joseph et al 2014.…”

Downregulation of pigment epithelium-derived factor in condyloma acuminatum

“…2 It is ubiquitous in humans and is functionally involved in biological processes such as antiangiogenesis, endothelial cell migration and inhibition of differentiation of tumour and neural cells. [3][4][5][6] There are few studies regarding PEDF expression in the skin, 7,8 and none in condyloma acuminatum. The aim of the present study, therefore, was to investigate SERPINF1 mRNA and PEGF protein levels in condyloma acuminatum and in healthy skin, using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively.…”