Src phosphorylation of the ADAM17 C-terminus is required for its activation by HG. (A) Src inhibitors SU6656 or PP2 blocked HG (1 h)-induced ADAM17 activation (n=7, ^‡P<0.001 versus all others). (B) Mesangial cells were treated with HG for the indicated times. ADAM17 was then immunoprecipitated. Immunoblotting shows HG induces ADAM17–Src association (n=3). (C) HG induces a time-dependent increase in ADAM17 Y702 phosphorylation (pADAM17 Y702) (n=2). (D) This was blocked by the Src inhibitors PP2 and SU6656 after 1 h of HG (n=6, *P<0.05 versus all others). (E) ADAM17 KO MEFs were transfected with WT or ΔC ADAM17, treated with HG for 1 h, then ADAM17 was immunoprecipitated using its HA tag. HG-induced Src/ADAM17 association was seen only in cells expressing WT ADAM17 (n=3, ^†P<0.01 versus all others). Note that the HA blot is the same as that used in Fig. 3I as this originates from the same experiment, with reprobing done to detect FAK and Src. (F) ADAM17 KO MEFs were transfected with empty vector pcDNA, WT ADAM17 or Y702A ADAM17. HG induced ADAM17 activation only in cells expressing WT ADAM17 (n=4; *P<0.05, ^†P<0.01, ^‡P<0.001, ^#P<0.001 versus both pcDNA groups).

FAK is required for ADAM17 activation and Src enables FAK–ADAM17 interaction in response to HG. (A) Mesangial cells were treated for the indicated times with HG. FAK activation, assessed by its phosphorylation on Y397 (pFAK), was increased by HG (n=2). (B) FAK was immunoprecipitated from whole cell lysate after treatment with HG. Immunoblotting shows Src association with FAK increases in response to HG (n=3). (C) ADAM17 activation by HG (1 h) is prevented by the FAK inhibitor PF573228 (n=6, ^‡P<0.001 versus others). (D) Association of ADAM17 and Src in response to HG (1 h) was prevented by PF573228, as assessed by coimmunoprecipitation (n=2). (E) ADAM17 phosphorylation at Y702 was also prevented by FAK inhibition with PF573228 (n=2). (F) ADAM17 was immunoprecipitated from whole cell lysate after treatment with HG for the indicated times. Immunoblotting shows FAK association with ADAM17 increases in response to HG (n=4). (G) Inhibition of FAK activity with PF573228 prevents ADAM17/FAK association in response to HG (1 h) (n=4). (H) ADAM17 interaction with FAK in response to HG requires Src activity, since this was prevented by the two Src inhibitors SU6656 and PP2 (n=3). (I) ADAM17 KO MEFs were transfected with WT or ΔC ADAM17, treated with HG for 1 h, then ADAM17 was immunoprecipitated using its HA tag. HG-induced FAK–ADAM17 association was seen only in cells expressing WT ADAM17 (n=3, *P<0.05 versus other groups). The HA blot is the same as that used in Fig. 2E as this originates from the same experiment, with reprobing done to detect FAK. (J) ADAM17 KO MEFs were transfected with WT or Y702A ADAM17, treated with HG for 1 h, then ADAM17 was immunoprecipitated using its HA tag. No HG-induced association between ADAM17 and FAK was seen in cells expressing Y702A (n=3). IgG control immunoprecipitation of HG-treated cells with WT ADAM17 shows some nonspecific pull-down of FAK. HC, antibody heavy chain.

PI3K mediates FAK–ADAM17 association and ADAM17 activation by HG. (A) PI3K inhibitors LY294002 or wortmannin inhibited HG (1 h)-induced ADAM17 activation (n=4, ^‡P<0.001 versus other groups, ^†P<0.01, ^#P<0.01 versus wort). (B) FAK activation, as assessed by its Y397 autophosphorylation, was unaffected by PI3K inhibitors (n=3). (C) Similarly, Src activation, assessed by its Y416 phosphorylation, was also unaffected by PI3K inhibitors (n=3). (D) Mesangial cells were treated with both PI3K inhibitors prior to HG for 1 h, after which ADAM17 was immunoprecipitated. FAK–ADAM17 association was prevented by both inhibitors (n=3).

Phosphorylation of ADAM17 at T735 is required for its activation by HG. (A) HG induces a time-dependent increase in ADAM17 T735 phosphorylation (pADAM17 T735) (n=6; *P<0.05 versus control, ^†P<0.01 versus control). (B) The MEK inhibitor U0126, which inhibits Erk activation, prevented ADAM17 activation by HG (1 h) (n=4, ^†P<0.01 versus others). (C) The p38 inhibitor SB203580 had no effect on ADAM17 activation by HG (n=3, ^†P<0.01 versus control). (D) HG (1 h)-induced ADAM17 T735 phosphorylation was inhibited by the PI3K inhibitors LY294002 (LY) and wortmannin (wort), and by the MEK inhibitors PD98059 (PD) and U0126 (n=3, ^†P<0.01 versus control and LY, and *P<0.05 versus wortmannin, PD98059 and U0126). (E) PI3K inhibitors LY294002 and wortmannin, and the FAK inhibitor PF5732008 (PF) inhibit HG (1 h)-induced Erk activation (n=3, ^†P<0.01 versus other groups). (F) ADAM17 KO MEFs were transfected with empty vector pcDNA, WT ADAM17 or T735A ADAM17. HG induced ADAM17 activation only in cells expressing WT ADAM17 (n=4, *P<0.05, ^†P<0.01, ^#P<0.001 versus both pcDNA groups). (G) ADAM17 KO MEFs were transfected with WT or T735A ADAM17, treated with HG for 1 h, then ADAM17 immunoprecipitated using its HA tag. No HG-induced association between ADAM17 and FAK was seen in cells expressing T735A (n=3, ^†P<0.01 versus other groups). IgG control immunoprecipitation of HG-treated cells with WT ADAM17 shows some nonspecific pull-down of FAK. LC, antibody light chain.

HG increases mature ADAM17 at the cell surface through furin-mediated processing. (A) Mesangial cells were treated with HG for the indicated times. Cell surface proteins were biotinylated and immunoprecipitated, and ADAM17 identified by immunoblotting. Two bands are seen in the input (cell lysate), at around 120 kDa and 100 kDa, representing the proform of ADAM17 (pro) at the higher molecular weight, and the mature form at the lower molecular weight. Only the mature form appears at the cell surface, which is increased by HG (n=4). PDGFR serves as the loading control for cell surface proteins. (B) Mesangial cells were treated with the furin inhibitors hexa-D-arginine (hexa) or decanoyl-RVKR-CMK (CMK) prior to HG for 1 h. Cell surface proteins were biotinylated and pulled down, and ADAM17 assessed by immunoblotting. Both inhibitors decrease the HG-induced increase in mature ADAM17 at the cell surface, with a concomitant increase in the pro-form (n=4; ^†P<0.01, *P<0.05). (C,D) Mesangial cells were incubated with increasing concentrations of either hexa-D-arginine or decanoyl-RVKR-CMK. There was a dose-dependent decrease in ADAM17 activation by HG (1 h) with both inhibitors (for both, n=4; ^†P<0.01, ^‡P<0.001).

HG-induced increase of mature ADAM17 at the cell surface is differentially regulated by T735 and Y702 phosphorylation. (A) ADAM17 KO MEFs were transfected with WT ADAM17, T735A ADAM17 or Y702A ADAM17. After HG for 1 h, cell surface proteins were isolated and constructs identified by their HA tag. HG-induced ADAM17 cell surface localization required both T735 and Y702 phosphorylation (n=4, ^‡P<0.001 versus its control). (B) PI3K inhibitors LY294002 (LY) and wortmannin (wort) prevented cell surface localization of mature ADAM17 (n=6, ^†P<0.01 versus other groups). (C) Src inhibitors SU6656 and PP2 also prevented cell surface localization of mature ADAM17 (n=4; ^†P<0.01 versus control, *P<0.05 versus HG).