We recently discovered a novel signaling phenomenon involving a rapid and transient rise in intracellular low molecular weight iron complex(es) in activation of IkappaB kinase (IKK) in hepatic macrophages. We also showed direct treatment with ferrous iron substitutes for this event to activate IKK. The present study used this model to identify upstream kinases responsible for IKK activation. IKK activation induced by iron is abrogated by overexpression of a dominant negative mutant (DN) for transforming growth factor beta-activated kinase-1 (TAK1), NF-kappaB-inducing kinase, or phosphatidylinositol 3-kinase (PI3K) and by treatment with the mitogen-activated protein kinase (MAPK) kinase-1 (MEK1) inhibitor. Iron increases AKT phosphorylation that is prevented by DNTAK1 or DNp21ras. Iron causes ERK1/2 phosphorylation that is attenuated by DN-PI3K, prevented by DNp21ras, but unaffected by DNTAK1. Iron-induced TAK1 activity is not affected by the PI3K or MEK1 inhibitor, suggesting TAK1 is upstream of PI3K and MEK1. Iron increases interactions of TAK1 and PI3K with p21ras as demonstrated by co-immunoprecipitation and co-localization of these proteins with caveolin-1 as shown by immunofluorescent microscopy. Finally, filipin III, a caveolae inhibitor, abrogates iron-induced TAK1 and IKK activation. In conclusion, MEK1, TAK1, NF-kappa-inducing kinase, and PI3K are required for iron-induced IKK activation in hepatic macrophages and TAK1, PI3K, and p21ras physically interact in caveolae to initiate signal transduction.