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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 Rac1. 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 Tiam1. Addition of the calcium ionophore ionomycin alone induced the translocation of Tiam1. 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 3-kinase by wortmannin had little effect on the translocation of Tiam1. The role of phosphorylation was further studied by comparing the phosphorylation pattern of Tiam1 in the membranes versus whole cell Tiam1. PDGF-induced phosphorylation of membrane-associated Tiam1 occurred more rapidly than that of the total Tiam1 pool, and CaMK II, but not PKC, played a significant role in this process. Furthermore, by using the p21-binding domain of PAK-3, we show that PDGF, but not lysophosphatidic acid, activates Rac1 in vivo and that this activation involves CaMK II and PKC, but not 3-phosphoinositides. Our results indicate that Tiam1 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.

Yeast two-hybrid screening of a human kidney cDNA library using the GTP-bound form of a class II ADP-ribosylation factor (ARF5) identified a novel ARF5-binding protein with a calculated molecular mass of 82.4 kDa, which was named arfophilin. Northern hybridization analysis showed high level arfophilin mRNA expression in human heart and skeletal muscle. Arfophilin bound only to the active, GTP-bound form of ARF5 and did not bind to GTP-ARF3, which is a class I ARF. The N terminus of ARF5 (1-17 amino acids) was essential for binding to arfophilin. The GTP-bound form of ARF5 with amino acid residues in the N terminus mutated to those in ARF4 (another class II ARF) also bound to arfophilin, suggesting it is a target protein for GTP-bound forms of class II ARFs. The binding site for ARF on arfophilin was localized to the C terminus (residues 612-756), which contains putative coiled-coil structures. Recombinant arfophilin overexpressed in CHO-K1 cells was localized in the cytosol and translocated to a membrane fraction in association with GTP-bound ARF5. ARF5 containing the N terminus of ARF3 did not promote translocation indicating that class II ARFs are specific carriers for arfophilin.

A number of guanine nucleotide exchange factors have been identified that activate Rho family GTPases, by promoting the binding of GTP to these proteins. We have recently demonstrated that lysophosphatidic acid and several other agonists stimulate phosphorylation of the Rac1-specific exchange factor Tiam1 in Swiss 3T3 fibroblasts, and that protein kinase C is involved in Tiam1 phosphorylation (Fleming, I. N., Elliott, C. M., Collard, J. G., and Exton, J. H. (1997) J. Biol. Chem. 272, 33105-33110). We now show, through manipulation of intracellular [Ca2+] and the use of protein kinase inhibitors, that both protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II are involved in the phosphorylation of Tiam1 in vivo. Furthermore, we show that Ca2+/calmodulin-dependent protein kinase II phosphorylates Tiam1 in vitro, producing an electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Significantly, phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II, but not by protein kinase C, enhanced its nucleotide exchange activity toward Rac1, by approximately 2-fold. Furthermore, Tiam1 was preferentially dephosphorylated by protein phosphatase 1 in vitro, and treatment with this phosphatase abolished the Ca2+/calmodulin-dependent protein kinase II activation of Tiam1. These data demonstrate that protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II phosphorylate Tiam1 in vivo, and that the latter kinase plays a key role in regulating the activity of this exchange factor in vitro.