An orthogonal c-Cbl recognition mode targets LynA for rapid degradation and builds specificity into the LynA checkpoint

Abstract

11The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based 12 activation motifs (ITAMs), is critical factor regulating myeloid-cell activation. In a previous paper 13 (Freedman et al., 2015) we showed in macrophages that the SFK LynA is uniquely susceptible to rapid 14 ubiquitin-mediated degradation, functioning as a rheostat regulating ITAM signaling. We now report the 15 mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into 16 the LynA rheostat. Using genetic and biochemical analysis, we found that the E3 ubiquitin ligase c-Cbl 17 preferentially targets LynA via tyrosine 32 in its unique insert region. This orthogonal mode of c-Cbl 18 recognition depresses the steady-state level of macrophage LynA. Mast cells, however, express little c- 19Cbl and have correspondingly high steady-state levels of LynA. Upon activation, mast-cell LynA is not 20 rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl 21 expression therefore builds cell specificity into the LynA checkpoint. 2245 macrophage activation (20). The mechanism preferentially targeting LynA for polyubiquitination and the 46 molecular basis of this selectivity have not been elucidated previously. 47Unlike Dectin-1 and FcγR signaling in macrophages, which is typically triggered in the context of 48 pathogen-induced µm-scale clusters of receptors (5, 9, 20), mast-cell FcεR signaling has a low 49 threshold for activation--small or even monovalent antigen-IgE complexes are sufficient to induce a 50 signaling response (21, 22). Like macrophages, mast cells express LynA, and this disparity in receptor 51 sensitivity has not previously been explained. This paper describes the mechanism by which active LynA is selectively recognized and rapidly 53 degraded, tuning its activation kinetics and its steady-state protein levels in a cell-specific manner. To 54 reveal the requirements for LynA degradation, we synchronized receptor-independent SFK activation 55 using the designer inhibitor 3-IB-PP1 (23-25), which specifically inhibits a variant of the SFK-inhibitory 56 kinase Csk (Csk AS ) (20, 25, 26). Csk is responsible for phosphorylating a key inhibitory tyrosine in the 57 C-terminus of all the SFKs. Inhibiting Csk AS leads to rapid and robust SFK activation due to the loss of 58 the dynamic equilibrium between Csk and the phosphatases CD45 and CD148 (27, 28). We showed 59 previously that SFK activation induced via 3-IB-PP1 treatment leads to the phosphorylation of the E3 60 ubiquitin ligase c-Cbl and the preferential polyubiquitination and degradation of the SFK LynA, but it 61 was not clear whether these two events were linked. Using knockdown, overexpression, and genetic 62 models, we now demonstrate that c-Cbl, but not its homolog Cbl-b, controls the steady-state expression 63 of LynA and mediates its rapid degradation upon activation in macrophages. LynA is targeted by c-Cbl 64 via a phosphorylation site, Tyr32 within its unique r...