TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.
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-kappaB. Although a key factor in TRAF6-dependent activation of PI 3-kinase, ectopic expression of Src was insufficient for NF-kappaB activation and, in contrast to NF-kappaB, was not inhibited by IRAK2.
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Kent Z Q
KZ
Wang
Nawarat
N
Wara-Aswapati
Jason A
JA
Boch
Yasuhiro
Y
Yoshida
Chang-Deng
CD
Hu
Deborah L
DL
Galson
Philip E
PE
Auron
D000199:Actins / Q000378:metabolism; D020816:Amino Acid Motifs; D002460:Cell Line; D003599:Cytoskeleton / Q000502:physiology*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism* / Q000502:physiology; D011554:Pseudopodia / Q000502:physiology; D015398:Signal Transduction; D048029:TNF Receptor-Associated Factor 6 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014162:Transfection
D000199:Actins; D007375:Interleukin-1; D016328:NF-kappa B; D048029:TNF Receptor-Associated Factor 6; D019869:Phosphatidylinositol 3-Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D016283:Proto-Oncogene Proteins p21(ras)