A mechanism underlying NOTCH-induced and ubiquitin-mediated JAK3 degradation.

Although NOTCH signaling is well known to regulate lymphopoiesis, Janus kinase 3 (JAK3) also plays a critical role in promoting lymphocyte development. We have previously found that NOTCH signaling leads to the degradation of JAK3 in B lineage cells, suggesting that NOTCH signaling exerts its biological effect on lymphopoiesis through modulating JAK3 levels. Here, we delineate the biochemical mechanisms involved in NOTCH-induced JAK3 ubiquitination and degradation. NOTCH signaling is known to transcriptionally activate the genes encoding ASB2 (ankyrin-repeat SOCS box containing protein 2) and SKP2 (S-phase kinase-associated protein 2). We show that not only NOTCH but also ASB2 and SKP2 can promote the ubiquitination and degradation of JAK3. Both ASB2 and SKP2 can interact with JAK3 through different domains; the FERM and pseudo-kinase domains each had high affinities for ASB2, whereas the kinase domain primarily associated with SKP2. ASB2 and SKP2 previously have been shown to associate with each other to bridge the formation of a non-canonical Cullin1 and Cullin5-containing dimeric E3 ligase complex. Interestingly, the R980W mutant of JAK3 exhibited diminished interaction with SKP2 and resistance to NOTCH or ASB2-induced degradation. Furthermore, dominant-negative mutants of either Cullin1 or Cullin5, which lack the C terminus responsible for recruiting the E2 enzymes, were able to prevent JAK3 degradation induced by both ASB2/SKP2 and NOTCH signaling. Together, these results suggest that JAK3 ubiquitination involves the non-canonical dimeric E3 ligase complex, and the R980W mutant will serve as an excellent tool for investigating the biological significance of NOTCH-mediated JAK3 turnover.