The Golgi ‘casein kinase’ Fam20C is a genuine ‘phosvitin kinase’ and phosphorylates polyserine stretches devoid of the canonical consensus

Abstract

Egg yolk phosvitins, generated through the fragmentation of vitellogenins, are among the most heavily phosphorylated proteins ever described. Despite the early discovery in 1900 that chicken phosvitin is a phosphoprotein and its subsequent employment as an artificial substrate for a number of protein kinases, the identity of the enzyme(s) responsible for its phosphorylation remained a matter of conjecture until present. Here we provide evidence that phosvitin phosphorylation is catalyzed by Fam20C, an atypical protein kinase recently identified as the genuine casein kinase and responsible for the phosphorylation of many other secreted proteins at residues specified by the S-x-E/pS consensus. Such a conclusion is grounded on the following observations: i) the levels of Fam20C and phosphorylated vitellogenin rise in parallel upon treatment of zebrafish with oestrogens; ii) Zebrafish phosvitin is readily phosphorylated upon coexpression in U2OS cells with Fam20C, but not with its catalytically inactive mutant; iii) a peptide reproducing a stretch of 12 serines, which are phosphorylated in chicken phosvitin despite lacking the C terminal priming motif S-x-E, is efficiently phosphorylated by both recombinant and native Fam20C. The last finding expands the repertoire of potential targets of Fam20C to include several proteins known to harbour (p-Ser)n clusters not specified by any known kinase consensus.

FAM20C‐Mediated Phosphorylation of MEPE and Its Acidic Serine‐ and Aspartate‐Rich Motif

“…This indicates that the kinase can phosphorylate rows of consecutive serine residues in MEPE, even though they are not positioned in the consensus sequence. Likewise, FAM20C has been shown to phosphorylate polyserine stretches devoid of Ser‐x‐Glu/pSer motifs in phosvitins ( 25 ) and serine residues in the ASARM of OPN not located in Ser‐x‐Glu motifs. ( 23 ) The 489 Arg‐Asp 508 ‐ myc /His peptide was also observed with two phosphorylations in MEPE expressed without FAM20C.…”

“…(22,23) MEPE contains many serine residues having FAM20C recognition motifs, and the C-terminal ASARM motif contains a polyserine stretch that potentially could be targeted by the kinase. (25) Heretofore, no studies have investigated the phosphorylation of MEPE and its ASARM peptide, either in vivo or in vitro. Previous work has used synthetic phosphorylated ASARM peptides to provide important functional information, (7,11,13,17,19,21) ; but no information is yet available on actual phosphorylation sites in MEPE.…”

Phosphoproteomic analysis of duck egg white and insight into the biological functions of identified phosphoproteins

“…In addition, we noted a striking similarity between our current study and a previous study from our lab (Yang et al, 2019) that mainly the phosphoproteins were present in the extracellular region of DEW and CEW, respectively. Fam20C is a protein kinase dedicated to the phosphorylation of extracellular proteins (Tagliabracci et al, 2012) and is suggested to be responsible for the phosphorylation of vitellogenins (VTGs) (Cozza et al, 2018). In the current study, we found the VTGs were highly phosphorylated proteins.…”

Thinking outside of the cell: Secreted protein kinases in bacteria, parasites, and mammals

“…Interestingly, one of the earliest recorded instances of phosphorylation and, in retrospect, the existence of protein kinases originated from secreted milk and egg yolk proteins . Following the discovery of phosphorylase kinase , the protein kinase field exploded, culminating in the publication of the “Human Kinome” .…”

“…The flagship member of the Fam20 family of kinases is Fam20C. Fam20C is the physiological GCK and, more recently, has been identified as the phosvitin kinase, an abundant egg yolk protein, which has been known to be highly phosphorylated since the beginning of the 20th century . Fam20C is a secreted protein whose activity can be purified from whey fractions of milk.…”