Toll-like receptors (TLRs) activate immune responses by sensing microbial structures such as bacterial lipopolysaccharides (LPS), viral RNA, and endogenous "danger" molecules released by damaged host cells. Myeloid differentiation primary response gene 88 (MyD88) is an adapter protein that mediates signal transduction for most TLRs and the IL-1 receptor family and leads to activation of NF-kappaB, MAP kinases, and production of proinflammatory cytokines. TLR4-mediated signaling also leads to rapid activation of phosphoinositide 3'-kinase (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 completely TLR4-dependent. MyD88 and p85 were previously shown to co-immunoprecipitate and an 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 (DD), and measured their capacities to bind PI3K p85, MyD88, and TLR4 by co-immunoprecipitation analyses and their ability to activate LPS-induced NF-kappaB. Additionally, we tested cell-permeable decoy peptides that correspond to the YXXM motif of MyD88 as an alternative way to analyze its role in TLR4 signaling. The cell permeable decoy peptides are presumed to interact with the target protein, thus precluding the target protein's interaction with the protein from which the peptide sequence was derived. 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 these three proteins (TLR4, MyD88, PI3K) interact simultaneously in the signaling platform and that sustained interaction of MyD88/PI3K with the TLR4 intracellular signaling platform negatively regulates signaling. In addition, the YXXM cell permeable peptides had a broad inhibitory effect on TLR4 signaling suggesting the importance of the MyD88 YXXM motif in formation of a fully functional TLR4 signaling platform. We propose a hypothetical model in which sustained PI3K activity at the membrane limits the availability of PI3K substrate, thereby negatively regulating signaling.