4-Hydroxynonenal activates Src through a non-canonical pathway that involves EGFR/PTP1B

Abstract

Src, a non-receptor protein tyrosine kinase involved in many biological processes, can be activated through both redox-dependent and independent mechanisms. 4-Hydroxy-2-nonenal (HNE) is a lipid peroxidation product that is increased in pathophysiological conditions associated with Src activation. This study examined how HNE activates human c-Src. In the canonical pathway Src activation is initiated by dephosphorylation of pTyr530 followed by conformational change that causes Src auto-phosphorylation at Tyr419 and its activation. HNE increased Src activation in both dose- and time-dependent manner, while it also increased Src phosphorylation at Tyr530 (pTyr530 Src), suggesting that HNE activated Src via a non-canonical mechanism. Protein tyrosine phosphatase 1B inhibitor (539741), at concentrations that increased basal pTyr530 Src, also increased basal Src activity and significantly reduced HNE-mediated Src activation. The EGFR inhibitor, AG1478, and EGFR silencing, abrogated HNE-mediated EGFR activation and inhibited basal and HNE-induced Src activity. In addition, AG1478 also eliminated the increase of basal Src activation by a PTP1B inhibitor. Taken together these data suggest that HNE can activate Src partly through a non-canonical pathway involving activation of EGFR and inhibition of PTP1B.

Keywords: EGFR; Src kinase; lipid peroxidation; protein tyrosine phosphatase; redox signaling; signal transduction.

Fig. 1

HNE activated Src in dose- and time-dependent manner. (A) HNE dose-dependently increased Src phosphorylation at Tyr419 and Tyr530; (B) Src phosphorylation was increased by HNE in a time-dependent manner. Cells were exposed to different HNE concentrations for 1 h (A) or to 10 μM HNE for indicated time (B), and pTyr419Src and pTyr530Src were determined by Western Blotting. *, P<0.05, **, P<0.01, compared with control, N= 3.

Fig. 2

Effect of 539741 on Src phosphorylation by HNE. Cells were pretreated with different concentrations of 539741for 1 h before being exposed to 10 μM HNE, Src phosphorylation at both Tyr530 and Tyr419 was then determined by Western Blotting. *, P<0.05, **, p<0.01, compared with vehicle control; #, p<0.05, ##, P<0.01, compared with control of same concentration of PTP1B inhibitor, N=3.

Fig. 3

EGFR is involved in HNE-mediated Src activation. (A) HNE caused EGFR Tyr845 phosphorylation; cells were treated with 10 μM HNE for indicated time and the phosphorylation of EGFR at Tyr845 was determined with Western Blotting. *, P<0.01 compared with starting time, N=3. (B) AG1478 abrogated HNE-mediated EGFR activation. Cells were pretreated with 10 μM AG1478 for 30 min before exposure to HNE for 1 h, and pTyr845 EGFR was determined. *, P<0.01 compared with vehicle control, N=3. (C) EGFR inhibitor AG1478 diminished Src activation by HNE. Cells were pretreated with 10 μM AG1478 for 30 min before being exposed to HNE for 1 h, and pTyr419Src was determined with Western Blotting. *, P<0.01 compared with vehicle control; #, P<0.01 compared with HNE, N=3.

Fig. 4

Effect of EGFR silencing on HNE-mediated Src activation. Cells were transiently transfected with EGFR siRNA (50 nM) for 72 h before being treated with HNE. (A) EGFR silencing with siRNA. *, P<0.01 compared with vehicle control, #, P<0.01 compared with HNE, N=3. (B) EGFR silencing reduced Src activation by HNE. *, P<0.01 compared with vehicle control, #, P<0.01 compared with HNE, N=3.

Fig. 5

EGFR is a PTP1B target. (A) EGFR was involved in PTP1B-mediated Src activation. Cells were pretreated with/without 10 μM AG1478 for 30 min before being exposed to 20 μM 539741 for 30 min, and pTyr419Src and total Src was determined with Western Blotting. *, P<0.01 compared with vehicle control, N=3. (B) PTP1B regulated EGFR phosphorylation at Tyr845 by HNE. Cells were pretreated with/without 20 μM 539741 for 30 min before being exposed to HNE for 1 h, and pEGFR845 was determined with Western blotting.

Fig. 6

Regulatory mechanism of Src activation by HNE. HNE could activate EGFR directly through forming conjugate or indirectly through inhibiting PTP1B, which dephosphorylates/inhibits pEGFR845. Active pEGFR then activates Src. PTP1B is also involved in dephosphorylation of pTyr530Src. Whether HNE inhibits PTPs involved in dephosphorylation of pTyr419Src is unknown.