Phosphorylation and Specific Ubiquitin Acceptor Sites Are Required for Ubiquitination and Degradation of the IFNAR1 Subunit of Type I Interferon Receptor

Abstract

Ubiquitination, endocytosis, and lysosomal degradation of the IFNAR1 (interferon ␣ receptor 1) subunit of the type I interferon (IFN) receptor is mediated by the SCF ␤-Trcp (Skp1-Cullin1-F-box protein ␤ transducin repeat-containing protein) E3 ubiquitin ligase in a phosphorylation-dependent manner. In addition, stability of IFNAR1 is regulated by its binding to Tyk2 kinase. Here we characterize the determinants of IFNAR1 ubiquitination and degradation. We found that the integrity of two Ser residues at positions 535 and 539 within the specific destruction motif present in the cytoplasmic tail of IFNAR1 is essential for the ability of IFNAR1 to recruit ␤-Trcp as well as to undergo efficient ubiquitination and degradation. Using an antibody that specifically recognizes IFNAR1 phosphorylated on Ser 535 we found that IFNAR1 is phosphorylated on this residue in cells. This phosphorylation is promoted by treatment of cells with IFN␣. Although the cytoplasmic tail of IFNAR1 contains seven Lys residues that could function as potential ubiquitin acceptor sites, we found that only three (Lys 501 , Lys 525 , and Lys 526 ), all located proximal to the destruction motif, are essential for ubiquitination and degradation of IFNAR1. Expression of Tyk2 stabilized IFNAR1 in a manner that was dependent neither on its binding to ␤-Trcp nor IFNAR1 ubiquitination. We discuss the complexities and specifics of the ubiquitination and degradation of IFNAR1, which is a ␤-Trcp substrate that undergoes degradation via a lysosomal pathway.Ubiquitin-mediated proteolysis plays an important regulatory role in many biological processes including cell cycle progression, transcription, and signal transduction (1). The process of protein ubiquitination is catalyzed by three distinct enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligases (E3). The E1 enzyme activates ubiquitin in an ATP-dependent reaction and then transfers ubiquitin to an E2. The E2 enzyme then cooperates with an E3 ligase to catalyze the formation of an isopeptide bond between ubiquitin and substrate. E3 ligases, which bind both cognate E2 and the substrates, are responsible for conferring substrate specificity in the ubiquitination reaction. Proteins polyubiquitinated by these enzymes are degraded by the 26 S proteasome (1, 2).Alternately, oligo-or monoubiquitinated transmembrane proteins are endocytosed and degraded via the lysosomal pathway (3-6). E3 ligases that mediate the ubiquitination of cell surface receptors include c-Cbl/Hakai and Rsp5/NEDD4 family members (7,8). Recently we have shown that the SCF ␤-Trcp E3 ubiquitin ligase mediates ligand-induced ubiquitination of the IFNAR1 subunit of the type I interferon receptor complex. SCF ␤-Trcp -mediated ubiquitination of IFNAR1 1 leads to endocytosis of this receptor complex, followed by its degradation via lysosomal pathway, ultimately resulting in the down-regulation of the Type I IFN receptor. This mechanism plays a pivotal role in the negative regulation of IFN signaling (9).SC...

Identification of a consensus site for TRAF6/p62 polyubiquitination

“…Some structural preferences exist for ubiquitin ligation of the targeted proteins such as preferred choice of lysines in α-helices, and then for easily accessible lysines in the loop regions. Our findings add to the growing list that indicates a bias towards a consensus sequence motif for ubiquitination by a given E3 [19][20][21][22][23][24][25]. Moreover, it appears that the E3 targets an accessible surface residue providing the selection process with a conformational recognition mechanism.…”

Effects of protein phosphorylation on ubiquitination and stability of the translational inhibitor protein 4E-BP1

“…It is possible that other components of the cell's proteolytic machinery are involved in the degradation of polyubiquitinated 4E-BP1. For example, lysosomes have been demonstrated to degrade a variety of polyubiquitinated proteins (Hicke, 2001;Kumar et al, 2004). However, treatment of Jurkat cells with the lysosomal inhibitor chloroquine did not prevent the calyculin A-induced loss of normal-sized 4E-BP1 or cause any further accumulation of the larger forms (data not shown).…”

“…Moreover, the susceptibility of such proteins to be processed by this pathway is often controlled by protein phosphorylation. In addition to the now classical cases of the inhibitor of nuclear factor kB (IkB) family (Karin and Ben-Neriah, 2000;Chen, 2005;Krappmann and Scheidereit, 2005) and cyclins (Lin et al, 2006a), other examples of proteins whose (poly)ubiquitination is stimulated by their phosphorylation include the type I interferon receptor IFNAR1 (Kumar et al, 2004), the transcription factor STAT1 (Kim and Maniatis, 1996), the antiapoptotic protein Bcl-2 (Lin et al, 2006b) and bcatenin (Aberle et al, 1997;Orford et al, 1997). On the other hand, phosphorylation can inhibit the ubiquitination of other proteins such as the proto-oncogenes c-jun (Musti et al, 1997;Fuchs et al, 1998), c-fos (Okazaki and Sagata, 1995) and c-mos (Nishizawa et al, 1993).…”

Ligand-independent pathway that controls stability of interferon alpha receptor

“…Antibodies against Tyk2 [14], as well as antibodies recognizing total IFNAR1 (EA12 and GB8) or IFNAR1 phosphorylated on Ser535 (in human receptor) or Ser526 (in murine receptor) [10,17] were described previously. Antibody against ubiquitin (FK2 mAb) was from Biomol.…”

“…Turnover of IFNAR1 requires its ubiquitination by the SCF β-Trcp/HOS E3 ubiquitin ligase [8], which recognizes the conserved phosphorylated 534 DSGNYS destruction motif [9]. Previously we reported that phosphorylation of IFNAR1 on Ser535 within this motif (essential for recruitment of β-Trcp) is increased upon stimulation of cells with IFNα [10], and catalytic activation of Tyk2 is required for such an increase [11]. Here we describe ligand-and Tyk2-independent pathway that regulates phosphorylation of IFNAR1 on Ser535 as well as IFNAR1 ubiquitination and degradation.…”