The International Journal of Biochemistry & Cell Biology 33 (2001) 577 – 589 Phosphorylation of the Fas associated factor FAF1 by protein kinase CK2 and identification of serines 289 and 291 as the in vitro phosphorylation sites Hans H. Jensen, Majbrit Hjerrild, Barbara Guerra, Martin R. Larsen, Peter Højrup, Brigitte Boldyreff * Institut for Biokemi og Molekylær Biologi, Syddansk Uniersitet, Campusej 55, DK-5230 Odense, Denmark Received 1 December 2000; received in revised form 5 February 2001; accepted 9 February 2001 Abstract We previously identified the human Fas associated factor (FAF1) as one of the interacting partners of protein kinase CK2  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 (k m 100 M) and the isolated catalytic  subunit (k m 200 M). 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. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: FAF1; Phosphorylation; Protein kinase CK2; Apoptosis; Mass spectrometry www.elsevier.com/locate/ijbcb 1. Introduction The Fas associated factor (FAF1) was first identified by a two-hybrid screening using Fas as bait [1]. In this screening the mouse FAF1 was isolated. It has been shown that it potentiates Fas-induced apoptosis and is a putative positive regulator of apoptosis. The quail homologue has been found by a completely different approach. It was isolated in a search for mRNAs differentially expressed when blood islands are induced in the Abbreiations: bFGF, basic fibroblast growth factor; FAF1, Fas associated factor 1; IPTG, isopropyl thio-ß-D-galactoside; Ni – NTA, nickel – nitriloacetic acid; PVDF, poly (vinylidene fluoride); TCA, trichloroacetic acid; TFA, trifluoroacetic acid; UTR, untranslated region. * Corresponding author. Tel.: +45-6550-2342; fax: +45- 6550-2467. E-mail address: boldy@bmb.sdu.dk (B. Boldyreff). 1357-2725/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII:S1357-2725(01)00039-5