Translocation of the Rac1 guanine nucleotide exchange factor Tiam1 induced by platelet-derived growth factor and lysophosphatidic acid

Several guanine nucleotide exchange factors for the Rho family of GTPases that induce activation by exchanging GDP for GTP have been identified. One of these is the tumor invasion gene product Tiam1, which acts on Rad. In this study, we demonstrate that platelet-derived growth factor (PDGF) and lysophosphatidic acid induce the translocation of Tiam1 to the membrane fraction of NIH 3T3 fibroblasts in a time-dependent manner. Previously, we have shown that Tiam1 is phosphorylated by protein kinase C (PKC) and calcium/calmodulin kinase II (CaMK II) after stimulation with agonists, Here we show, by pretreatment of cells with kinase inhibitors, that CaMK II, but not PKC, is involved in the membrane translocation of Tiaml, Addition of the calcium ionophore ionomycin alone induced the translocation of Tiaml, However, the cell-permeable diacylglycerol oleoylacetylglycerol was without effect and did not enhance the effect of ionomycin, These data further indicated a role for CaMK II and not PKC, Inhibition of phosphoinositide S-kinase by wortmannin had little effect on the translocation of Tiaml, The role of phosphorylation was further studied by comparing the phosphorylation pattern of Tiaml in the membranes versus whole cell Tiaml, PDGF-induced phosphorylation of membrane-associated Tiaml occurred more rapidly than that of the total Tiaml pool, and CaMK II, but not PKC, played a significant role in this process, Furthermore, by using the pal-binding domain of PAK-3, we show that PDGF, but not lysophosphatidic acid, activates Rad in vice and that this activation involves CaMK II and PKC, but not 3-phosphoinositides. Our results indicate that Tiaml is translocated to and phosphorylated at membranes after agonist stimulation and that CaMK II, but not PKC, is involved in this process. Also, these kinases are involved in the activation of Rac in vivo.