Unique-region phosphorylation targets LynA for rapid degradation, tuning its expression and signaling in myeloid cells.

Ben F Brian,Pål Sætrom,Tanya S Freedman,Adrienne S Jolicoeur,Siv A Hegre,Justin M Drake,Zoi E Sychev,Kathryn L Schwertfeger,Nagy Habib,Candace R Guerrero,Myra G Nunez

doi:10.7554/elife.46043

Abstract

The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is a critical factor regulating myeloid-cell activation. We reported previously that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation in macrophages, functioning as a rheostat regulating signaling (Freedman et al., 2015). We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic, biochemical, and quantitative phosphopeptide analyses, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via a phosphorylated tyrosine (Y32) in its unique region. This distinct mode of c-Cbl recognition depresses steady-state expression of LynA in macrophages derived from mice. Mast cells, however, express little c-Cbl and have correspondingly high LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression thus builds cell specificity into the LynA checkpoint.

Highlights

Phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) by Src-family kinases (SFKs) is the first enzymatic step in the activation of an innate immune response during a pathogen encounter
To identify the E3 ubiquitin ligase that mediates this degradation, we tested the functions of c-Cbl and Cblb, known modulators of ITAM signaling in both adaptive and innate immune cells (Abram and Lowell, 2008; Lutz-Nicoladoni et al, 2015; Tang et al, 2019)
We bred CskASCbl-/- and CskASCblb-/mice from the existing strains Cbl-/- (Rafiq et al, 2014) and Cblb-/- (Chiang et al, 2000) and generated CskAS-expressing, c-Cbl-deficient (CskASc-CblKO) and CskAS-expressing, Cbl-bdeficient (CskASCbl-bKO) bone-marrow-derived macrophages (BMDMs). (For clarity we refer to BMDM subtypes using protein nomenclature.) Immunoblots of whole-cell lysates show the loss of expression of c-Cbl and Cbl-b in CskASc-CblKO and CskASCbl-bKO BMDMs, respectively, compared to CskAS BMDMs (Figure 1A–B)

Summary

Introduction

Phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) by Src-family kinases (SFKs) is the first enzymatic step in the activation of an innate immune response during a pathogen encounter. Neither was able to induce degradation of LynAT410K, Fgr and Lck were markedly different from Hck and FynB in that, in addition to activation-loop-phosphorylating LynAT410K, they were capable of inducing Zap and c-Cbl phosphorylation (Figure 7B,D, Figure 7—figure supplement 2A–B) This suggests that the failure of Lck and Fgr to mediate LynA degradation is due to substrate specificity rather than localization or lack of participation in ITAM signaling pathways. CskAS mast cells were treated with 3-IB-PP1 for up to 15 min to activate SFKs and assess LynA degradation and signaling potential as reported by pErk1/2 (Figure 9H) Even this modest increase in mast-cell c-Cbl expression increased LynA degradation in response to 3-IB-PP1 and suppressed downstream phosphorylation of Erk1/2 (Figure 9I–J). Working together with transcriptional regulation to tune myeloid-cell responsiveness to SFK-mediated signaling

Discussion

A CskAS peritoneal B cell mast cell

Materials and methods