AbstractTLRs activate immune responses by sensing microbial structures such as bacterial LPS, viral RNA, and endogenous “danger” molecules released by damaged host cells. MyD88 is an adapter protein that mediates signal transduction for most TLRs and leads to activation of NF-κB and MAPKs and production of proinflammatory cytokines. TLR4-mediated signaling also leads to rapid activation of PI3K, one of a family of kinases involved in regulation of cell growth, apoptosis, and motility. LPS stimulates phosphorylation of Akt, a downstream target of PI3K, in wild-type (WT) mouse macrophages. LPS-induced phosphorylation of Akt serine 473 was blunted in MyD88−/− macrophages and was completely TLR4-dependent. MyD88 and p85 were shown previously to co-immunoprecipitate, and a YXXM motif within the Toll-IL-1 resistance (TIR) domain of MyD88 was suggested to be important for this interaction. To test this hypothesis, we compared expressed MyD88 variants with mutations within the YXXM motif or lacking the TIR domain or death domain and measured their capacities to bind PI3K p85, MyD88, and TLR4 by co-immunoprecipitation analyses. The YXXM → YXXA mutant MyD88 bound more strongly to p85, TLR4, and WT MyD88 than the other variants, yet was significantly less active than WT MyD88, suggesting that sustained interaction of MyD88/PI3K with the TLR4 intracellular “signaling platform” negatively regulates signaling. We propose a hypothetical model in which sustained PI3K activity at the membrane limits the availability of the PI3K substrate, thereby negatively regulating signaling.