The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is a critical factor regulating myeloid-cell activation. In a previous paper (Freedman et al., 2015) we showed that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation in macrophages, functioning as a rheostat regulating signaling. 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. 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 therefore builds cell specificity into the LynA checkpoint.

CskASc-Cbl+/- BMDMs were rested or treated 15 s with 3-IB-PP1, washed with ice-cold PBS, and lysed in 1% Lauryl Maltoside Buffer containing 150 mM NaCl, 0.01% sodium azide, and protease and phosphatase inhibitors (MSSAFE, Sigma-Aldrich, St. Louis, MO). After scraping the plates, cells and detergent were sonicated on a Diagenode biorupter for 5 min with a 50% duty cycle. The lysate was then cleared by centrifugation for 15 min at 14,000 rpm at 4°C in a tabletop centrifuge. Lysates were precleared for 30 min at 4°C with Protein G Sepharose beads (Sigma-Aldrich) and normal rabbit serum (Jackson ImmunoResearch, West Grove, PA). LynA-specific antibody (20) (C13F9, Cell Signaling) was pre-bound to Protein-G-Sepharose beads for at least 2 h. A bicinchoninic acid (BCA) protein assay was performed on the whole-cell lysates (Thermo Fisher) to ensure that equal amounts of protein from untreated and 3-IB-PP1-treated cells would be subjected to immunoprecipitation. Lysates were then mixed continuously with antibody and beads for 2 h at 4°C to immunoprecipitate LynA. Samples were applied to micro bio-spin chromatography columns (Bio-Rad, Hercules, CA), washed, and eluted with SDS Sample Buffer containing 125 mM Tris, 10% glycerol, 5% 2-mercaptoethanol, and 25% SDS. Samples were then concentrated to 40 µl using Ultracel-3K centrifugal spin columns (EMD Millipore). Each 40 µl immunoprecipitate sample was then resolved by gel electrophoresis. A calibration curve with a bovine serum albumin (BSA) standard curve was used to ensure that equal mass quantities of immunoprecipitated protein in the untreated and 3-IB-PP1-treated samples would be subjected to trypsin digest. For this calibration, we ran 0.05 - 20 µg BSA or the immunoprecipitate samples on a 10% Mini-PROTEAN TGX gel (Bio-Rad) at 170 V. Total protein was visualized without fixation using SimplyBlue Safestain (Thermo Fisher) according to manufacturer's instructions. Following staining, the gel was washed 2 x 1 h with water and imaged on a LI-COR Odyssey. The staining intensities were quantified by densitometry, and the BSA signals vs. mass quantities were fit to a linear function (GraphPad Prism). This function was then used to quantify the mass quantity of total protein in each immunoprecipitates. Gel pieces including the lower-MW (nonubiquitinated) Lyn species and higher-MW (polyubiquitinated) species were excised, spiked with the isotope-labeled LynA Y32 phosphopeptide [H]TI[pY]VRDP[13C515N1]TSNK[OH] (Sigma-Aldrich), and subjected to in-gel digestion with TPCK-treated sequencing-grade trypsin (Promega, Madison, WI) and STAGE Tip peptide cleanup as previously described (91) except that iodoacetamide was used as the alkylating reagent. Digested samples were submitted for identification at the University of Minnesota’s Center for Mass Spectrometry and Proteomics.

BMDM stimulations have been described previously (20). After resting as described above, 1 M live Jurkat cells, mast cells, or adherent BMDMs were treated at 37C in DMEM (myeloid cells) or PBS (Jurkat cells) with 10 µM 3-IB-PP1, a gift from K. Shokat (University of California, San Francisco). Signaling reactions were quenched by placing on ice and lysing cells in sodium dodecyl sulfate (SDS) buffer (128 mM Tris base, 10% glycerol, 4% SDS, 50 mM dithiothreitol (DTT), pH 6.8). Whole-cell lysates were prepared for immunoblotting by sonicating with a Bioruptor (Diagenode, Inc., Denville, NJ) for 3 min and boiling for 15 min. For immunoblotting 0.25 M cell equivalents were run in each lane of a 7% NuPage Tris-Acetate gel (Invitrogen, Carlsbad, CA) and then transferred to an Immobilon-FL PVDF membrane (EMD Millipore, Burlington, MA). REVERT Total Protein Stain (LI-COR Biosciences, Lincoln, NE) was used according to the standard protocol to quantify lane loading. After destaining, membranes were treated with Odyssey Blocking Buffer (TBS) for at least 1 h. Blotting was performed using standard procedures, and blots were imaged on an Odyssey CLx near-infrared imager (LI-COR).