TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src

Abstract

Interleukin 1 (IL-1) has been implicated in the reorganization of the actin cytoskeleton. An expression vector encoding a PKB/Akt pleckstrin-homology domain fused to a fluorescent protein was used to detect phosphoinositide 3-kinase (PI 3-kinase) products. It was observed that PI 3-kinase was activated either by treatment with IL-1 or by expression of either TRAF6, Src, MyD88 or dominant-positive PI 3-kinase, and resulted in the formation of long filopodia-like cellular protrusions that appeared to branch at membrane sites consisting of clusters of phosphoinositide. This depended upon a TRAF6 polyproline motif and Src catalytic activity, and was blocked by inhibitors of PI 3-kinase, Src and Ras. Using both conventional and split fluorescent protein probes fused to expressed TRAF6 and Src in living cells, the polyproline sequence of TRAF6 and the Src-homology 3 (SH3) domain of Src were shown to be required for interaction between these two proteins. Interaction occurred within the cytoplasm, and not at either the cell membrane or cytoplasmic sequestosomes. In addition, co-transfection of vectors expressing fluorescent-protein-fused TRAF6 and non-fluorescent MyD88, IRAK1 and IRAK2 revealed an inverse correlation between increased sequestosome formation and activation of both PI 3-kinase and NF-κB. Although a key factor in TRAF6-dependent activation of PI 3-kinase, ectopic expression of Src was insufficient for NF-κB activation and, in contrast to NF-κB, was not inhibited by IRAK2.

Interleukin (IL) 1β Induction of IL-6 Is Mediated by a Novel Phosphatidylinositol 3-Kinase-dependent AKT/IκB Kinase α Pathway Targeting Activator Protein-1

“…In colon cancer cells harboring this variant IL-6 may be particularly effective in advancing the adenoma/ carcinoma sequence. Interestingly, a dominant-negative mutant of c-Jun exhibited a significant antitumor effect in colon cancer demonstrating the possibility of AP-1-based gene therapy as a novel treatment of colorectal cancer (77)(78)(79)(80). Understanding the regulation of NFB and AP-1 and their cross-talk in the regulation of their target genes such as IL-6 may lead to the development of novel therapeutics for the control of inflammatory diseases of the mucosa.…”

The diverse functions of Src family kinases in macrophages

“…Although a TRAF6 binding protein can disrupt the association of TRAF6 with CD40 and block downstream signaling, it does not interfere with Src activation, placing Src upstream of TRAF6. Src has been shown to interact directly with TRAF6 in overexpression studies (130,133), but this has not been observed in primary macrophages. SFKs have also been implicated in cell survival pathways downstream of TNF family receptors such as receptor activator of NF kappa B (RANK) in osteoclasts; expression of a truncated Src that lacks the kinase domain in osteoclasts of src −/− mice reduces Akt activation, decreases osteoclast survival and exacerbates the osteopetrotic phenotype of src −/− mice (159).…”

“…Surprisingly, tyrosine phosphorylation resulting from CpG stimulation of macrophages occurs in the absence of either TLR9 or MyD88 implying that SFKs are involved in a distinct pathway that appears to be important for actin cytoskeletal rearrangements (112). This pathway involves PI3K and Akt, and is required for downstream activation of NF kappa B. Tyrosine phosphorylation of MAPKs in response to LPS is lost in macrophages lacking TLR4, but is only delayed in MyD88-deficient macrophages (128) (130,132,133). Src has also been shown to associate with IKK when overexpressed in HEK293 cells (134).…”

“…Overexpression studies in HEK293 cells have demonstrated that the SH3 domain of Src can associate with TRAF6, a key molecule involved in downstream signaling pathways from TLRs (130) suggesting that TRAF6 could be the connection to SFKs. However, Mukundan et al report not being able to see an association between Src and TRAF6 in monocytes or macrophages (131).…”

“…However, Mukundan et al report not being able to see an association between Src and TRAF6 in monocytes or macrophages (131). Interactions between Src and TRAF6 have been observed downstream of the IL-1 receptor, which uses a similar signaling pathway to TLR4 utilizing MyD88, in other cell types including hepatocytes and osteoclasts (130,132,133). Src has also been shown to associate with IKK when overexpressed in HEK293 cells (134).…”