Src family kinases (SFKs) are a family of protein tyrosine kinases containing 
nine members: Src, Lyn, Fgr, Hck, Lck, Fyn, Blk, Yes, and Ylk. Although SFK 
activation is a major immediate signaling event in LPS/Toll-like receptor 4 
(TLR4) signaling, its precise role has remained elusive due to various 
contradictory results obtained from a certain SFK member-deficient mice or 
cells. The observed inconsistencies may be due to the compensation or redundancy 
by other SFKs upon a SFK deficiency. The chemical rescuing approach was 
suggested to induce temporal and precise SFK activation in living cells, thereby 
limiting the chance of cellular adaption to a SFK-deficient condition. Using the 
rescuing approach, we demonstrate that restoring SFK activity not only induces 
tyrosine phosphorylation of TLR4, but also inhibits LPS-induced NFκB and JNK1/2 
activation and consequently suppresses LPS-induced cytokine production. TLR4 
normally recruits TIR domain-containing adaptors in response to LPS, however, 
temporally restored SFK activation disrupts the LPS-induced association of MyD88 
and Mal/Tirap with TLR4. Additionally, using kinase-dead SFK-Lyn (Y397/508F) and 
constitutively active SFK-Lyn (Y508F), we found that the kinase-dead SFK 
inhibits TLR4 tyrosine phosphorylation with reduced binding affinity to TLR4, 
while the kinase-active SFK strongly binds to TLR4 and promotes TLR4 tyrosine 
phosphorylation, suggesting that SFK kinase activity is required for TLR4 
tyrosine phosphorylation and TLR4-SFK interaction. Together, our results 
demonstrate that SFK activation induces TLR4 tyrosine phosphorylation, 
consequently dissociating MyD88 and Mal/Tirap from TLR4 and inhibiting 
LPS-induced inflammatory responses, suggesting a negative feedback loop 
regulated by SFK-induced tyrosine phosphorylation in TLR4.