Phosphorylation of the Fas associated factor FAF1 by protein kinase CK2 and identification of serines 289 and 291 as the in vitro phosphorylation sites.
We previously identified the human Fas associated factor (FAF1) as one of the interacting partners of protein kinase CK2 beta subunit. Since FAF1 is a phosphoprotein we investigated whether it is a substrate for CK2. Here, we report the full length human FAF1 cDNA sequence, expression of FAF1 in Escherichia coli and purification and characterization of FAF1 as a substrate for CK2. FAF1 as well as an N-terminal 40 kDa degradation product serve as substrates for both the recombinant CK2 holoenzyme (km 100 microM) and the isolated catalytic alpha subunit (km 200 microM). Despite the high k(m) values, we obtained evidence that CK2 is the major cellular kinase responsible for FAF1 phosphorylation, using tissue extracts as kinase sources. By MALDI-MS we identified the two serine residues at positions 289 and 291 as the major in vitro CK2 phosphorylation sites. These data may help us elucidate the functions of FAF1 and the involvement of CK2 mediated phosphorylation in processes such as apoptotic signaling, ubiquitination, nuclear translocation and embryonic development.
Jensen
H H
HH
Institut for Biokemi og Molekylaer Biologi, Syddansk Universitet, Campusvej 55, DK-5230, Odense, Denmark.
Hjerrild
M
M
Guerra
B
B
Larsen
M R
MR
Højrup
P
P
Boldyreff
B
B
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D047390:Casein Kinase II; D018076:DNA, Complementary; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012694:Serine / Q000378:metabolism; D014020:Tissue Extracts
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; C490780:FAF1 protein, human; D011994:Recombinant Proteins; D014020:Tissue Extracts; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases