Bacterial lipopolysaccharide induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in macrophages.

S.L Weinstein,J.S Sanghera,K Lemke,A.L Defranco,S.L Pelech

doi:10.1016/s0021-9258(18)42133-3

Abstract

Bacterial lipopolysaccharide (LPS) is a potent activator of antibacterial responses by macrophages. Following LPS stimulation, the tyrosine phosphorylation of several proteins is rapidly increased in macrophages, and this event appears to mediate some responses to LPS. We now report that two of these tyrosine phosphoproteins of 41 and 44 kDa are isoforms of mitogen-activated protein (MAP) kinase. Each of these proteins was reactive with anti-MAP kinase antibodies and comigrated with MAP kinase activity in fractions eluted from a MonoQ anion-exchange column. Following LPS stimulation, column fractions containing the tyrosine phosphorylated forms of p41 and p44 exhibited increased MAP kinase activity. Inhibition of LPS-induced tyrosine phosphorylation of these proteins was accompanied by inhibition of MAP kinase activity. Additionally, induction of p41/p44 tyrosine phosphorylation and MAP kinase activity by LPS appeared to be independent of activation of protein kinase C, even though phorbol esters also induced these responses. These results demonstrate that LPS induces the tyrosine phosphorylation and activation of at least two MAP kinase isozymes. Since MAP kinases appear to modulate cellular processes in response to extracellular signals, these kinases may be important targets for LPS action in macrophages.

Highlights

Bacterial lipopolysaccharide (LPS) is a potent acti- arachidonic acid metabolites [3, 4]
We have investigated the early biochemical events that are triggered in macrophages by LPS
We reported that LPS increases protein tyrosine phosphorylation in murine macrophages and that thisearly signaling event appears inducedtyrosine phosphorylation of these proteiwnsas to mediate some downstream macrophage responses to LPS

Summary

Among the most prominent tyrosine phosphorylated bands in

The major outer membrane component of Gram-negative bacteria, lipopolysaccharide (LPS),’ is a potent activator of macrophage responses involved in the host defense against infection [1, 2]. We show that MAP kinase activity is increased following LPS treatment, and thisresponse appears to occur regulated kinase; ~ 4 4 ” ~ ‘4,4-kDa MAP kinase encoded by sea star as the result of tyrosine phosphorylation of at least two mpk gene; TBS, Tris-buffered saline; TBST, TBS containing 0.05%. The membrane was washed twice with TBS containing 0.05% Tween 20 (TBST) for 5 min before overnight incubation with rabbit polyclonal anti-MAP kinase antibodies or the mouse monoclonal PY-20antiphosphotyrosineantibody (in 1%gelatin-TBST; 1:lOOO dilution) or the mouse monoclonal 4G10 antiphosphotyrosine antibody (1:3 diluted culture supernatant in TBST). The membrane was stripped (2% SDS, 62.5 mM Tris, pH6.7, 100mM 2-mercaptoethanol, 50 'c, 30 m i d and reprobed witha mouse monoclonal anti-MAP kinaseantibody (1:5,000 dilution in TBST, overnight, 4 "C), followed by sheep antimouse IgG-horseradish peroxidase antibodies (1:15,000 dilution in TBST, 1h, 25 "C).

RESULTS

AcLtPivSation of MAP Kinases Macrophages

MonoQ MBP kinase peak

MonoQ fractlon number

DISCUSSION

LPS Activation of MAP Kinases inMacrophages