Fig 2: c-Cbl induces K63 Her2 polyUb redirecting for lysosomal targeted degradation(A) Effect of ATG9A on phosphorylation of c-Cbl in BT474-TR. Cells were transfected with vector and ATG9A-encoding plasmid in the presence of trastuzumab (20 µg/ml). Protein levels for ATG9A, c-Cbl total and phosphorylation sites Tyr 731 and 774, Her2 total and phosphorylated Tyr 1112 were quantified by Image J software. Bands were normalised to a-tubulin. (B) Immunoprecipitation of Her2 followed by western blotting for K63 and Her2 in BT474-TR cells transfected with siRNA against c-Cbl or siRNA, upon overexpressing ATG9A or control vector. IgG was used as control. (C) Immunofluorescence staining for LAMP1 and Her2 in BT474-TR cells upon transfection with vector or ATG9A-encoding plasmid in the presence of trastuzumab (20 µg/ml). Percentage of Her2/LAMP1 co-localisation was quantified using LEICA LAS AF lite software. (D) Immunofluorescence staining for GFP-ATG9A and LAMP1 in BT474 and BT474-TR transfected with ATG9A-GFP encoding plasmid in the presence of trastuzmab (20 µg/ml). Data is representative of three experiments. *P < 0.05, unpaired Student's t test.

Fig 3: Schematic model of ATG9A's role in trastuzumab resistant cellsATG9A promotes c-Cbl recruitment to the receptor kinase Her2 on T1112, a known c-Cbl docking site, leading to an increase in Her2 poly-ubiquitination. This ultimately results in a decrease in Her2 protein levels, Her2 transcriptional activity and Her2 downstream PI3K/AKT signaling.

Fig 4: SFK activity is required for rapid degradation of LynA.(A) Immunoblots showing expression of transfected wild-type (wt), substrate-binding-impaired (T410K), or activation-loop-mutated (Y397F) His6V5-tagged LynA protein coexpressed in JCaM1.6 cells with memCskAS and c-Cbl and treated with 3-IB-PP1. Interdomain-B-phosphorylated Zap70pY319 (pZap70), an SFK target and prerequisite for Zap70 activation, reflects the catalytic activity of LynA; ß-Actin is shown as a loading control. (B) Overexpression of LynA variants in Jurkat cells. pZap70 reflects 3-IB-PP1-induced Lck activation. (C) Quantification of V5-tagged LynA variants wt (black dotted), T410K (gray), and Y397F (orange) in Jurkat cells during 3-IB-PP1 treatment, corrected for total protein content (TPS) and reported relative to the steady-state level for each variant of LynA. SEM, n = 3. Sig. from ANOVA2-Tukey: [T410K vs. wt, gray asterisks], [Y397F vs. wt, orange asterisks]; **p=0.0015–0.0083. Other pairs ns. Refer to Figure 6—source data 1. Refer to Figure 6—figure supplement 1 for additional experiments with the catalytic-site mutant LynAK275R.10.7554/eLife.46043.033Figure 6—source data 1.Quantification of kinase-impaired LynA proteins expressed in Jurkat cells.

Fig 5: Cotransfected LynA, LynB, and Hck56 can induce rapid degradation of LynAT410K in trans.(A) Representative immunoblots showing levels of His6V5-tagged LynAT410K (#) cotransfected with empty vector or active SFK (co) in memCskAS- and c-Cbl-expressing JCaM1.6 cells. Refer to Figure 7—figure supplement 1 for an illustration of the experimental layout. Cotransfected SFKs include untagged LynA, untagged LynAY32A, His6V5-tagged LynB, the long and short isoforms of Hck (Hck59+56), the long isoform of Hck (Hck59), FynT (Fyn), FynB, and Fgr. FynT is also expressed endogenously. His6V5-tagged LynAT410K# (or wild-type LynA# in the positive control condition) was quantified from a total LynA blot (top panel). Colors correspond to later figure panels. Coexpressed SFKs are visualized via total SFK and pSFKinact (inhibitory-tail phosphorylated SFK, corresponding to SrcpY530) blots as indicated. pSFKact (activation-loop phosphorylated, corresponding to SrcpY416), pZap70, and p-c-Cbl blots are used to show 3-IB-PP1-induced activity of the cotransfected SFK; Erk1/2 is shown as a loading control. Refer to Figure 7—figure supplement 2A for immunoblots of LynB, Fgr, and Fyn cotransfections. When cotransfected with LynAT410K, untagged wild-type LynA was expressed at very low levels, but its presence is inferred based on induction of Zap70 and c-Cbl phosphorylation with 3-IB-PP1 treatment. (B) Comparison of the expression (expr) levels of cotransfected SFKs (SFKco) and their ability to induce phosphorylation of the LynAT410K activation loop (pLynAact, the highest band in the pSFKact blots), Zap70pY319 and c-CblpY731 (p-c-Cbl) following 3-IB-PP1 treatment. If expression could not be directly compared with the wild-type LynA in the positive-control sample, pSFKinact and/or pSFKact blots werecompared, with the caveat that the antibodies may not bind different SFKs equally well (marked with asterisks). Despite transfection, Fyn was expressed at endogenous levels (endog). Refer to Figure 7—figure supplement 2B for blots showing the effects of activating endogenous Lck. (C–D) Quantification of His6V5-tagged wild-type LynA alone (black dotted) or His6V5-tagged LynAT410K alone (gray dotted) or cotransfected with the kinase-active SFKs. All LynA quantifications (#) are corrected for total protein content (TPS) and reported relative to the steady-state level for each transfection condition. SEM, n = 5 for LynAT410K and LynA alone, SEM, n = 4 for all others. Sig. from ANOVA2-Tukey: [LynAT410K vs. LynA black asterisks], LynAT410K vs. LynAT410K+LynA, coral asterisks], [LynAT410K vs. LynAT410K+LynAY32A, dark orange asterisks], [LynAT410K vs. LynAT410K+LynB, gold asterisks], [LynAT410K vs. LynAT410K+Hck59+56, teal asterisks], [Lyn vs. LynAT410K+Fyn, dark purple asterisk]; ***p=0.0007, **p=0.0017, *p=0.0180–0.0466. Other pairs ns. (C) Quantification of His6V5-tagged LynAT410K cotransfected with SFKs that induce degradation of LynAT410K. These include: untagged wild-type LynA (coral), untagged LynAY32A (dark orange), His6V5-tagged LynB (gold dotted), and Hck59+56 (teal). (D) Quantification of His6V5-tagged LynAT410K cotransfected with SFKs that do not induce degradation of LynAT410K: Hck59 (tan), Fgr (blue dotted), Fyn (purple), and FynB (light purple). Refer to Figure 7—source data 1.10.7554/eLife.46043.037Figure 7—source data 1.Quantification of LynAT410K coexpressed in Jurkat cells with other SFKs.