The putative tumor metastasis suppressor nm23H1 was originally identified in murine melanomas by subtraction cloning. It displays nucleoside diphosphate kinase activity and regulates cellular events, including growth and development. Recently nm23H1 has been reported to also act as a GTPase-activating protein of the Ras-related GTPase Rad. We attempted to determine whether nm23H1 also regulates Rho-family GTPases. Although we were unable to detect a direct association between nm23H1 and Rhofamily GTPases, nm23H1 was shown to be associated with a Rac1-specific nucleotide exchange factor, Tiam1, by interaction with its amino-terminal region in extracts from the cells expressing exogenous Tiam1 and from native tissue. Overexpression of nm23H1 inhibited the Tiam1-induced production of GTP-bound Rac1 and activation of c-Jun kinase. On the other hand, forced overexpression of the wild type, but not the kinase-inactivated mutant of nm23H1, converted the GDP-bound forms of Rac1, Cdc42, and RhoA to their GTP-bound forms in vitro by its nucleoside diphosphate kinase activity, but nm23H1 alone apparently did not produce the GTP-bound form of these GTPases in vivo. These results suggest that nm23H1 negatively regulates Tiam1 and inhibits Rac1 activation in vivo. Moreover, adhesion-stimulated membrane ruffles of Rat1 fibroblasts were reduced by overexpression of nm23H1. Based on these observations, we concluded that we had identified a function of nm23H1 as a regulator of Rac1 and that it may be related to the effect of nm23H1 as a tumor metastasis suppressor.

p21-activated kinase (PAK) is a common e ector protein of the small GTPases Cdc42 and Rac, leading to the activation of downstream mitogen activated protein kinases. PAK also mediates polarized cytoskeletal changes induced by these GTPases. The recently identi®ed PAK-interacting exchange factor (PIX) acts as a guanine nucleotide exchange factor on Rac, and colocalizes with PAK in a focal complex, but little is known about the associated signaling cascades, including upstream activators of PIX. In this study, we show that one of the isoforms of PIX, aPIX, is activated by signaling cascades from the platelet-derived growth factor (PDGF) receptor and EphB2 receptor, and from integrin-induced signaling through phosphatidylinositol 3-kinase (PI3-kinase). aPIX is activated by forming a complex with these receptors either via association with PAK and Nck, or direct association with the p85 regulatory subunit of PI3-kinase. Synthetic phosphoinositide and membrane targeted PI3-kinase augmented the aPIX activity in vivo. In Xenopus, aggregates of mesodermal cells derived from embryos microinjected with aPIX signi®cantly increased the peripheral spreading on ®bronectin substrate in response to PDGF through PI3-kinase. These results indicate that aPIX is activated by PI3-kinase, and is involved in the receptor mediated signaling leading to the activation of the kinase activity of PAK, and the migration of mesodermal cells on extracellular matrix.

These findings suggest that not only the expression of EPHB2, but the expression of its ligand EFNB1 may have some relation with the oncogenesis of gastric cancer.

Benzo[a]pyrene [B(a)P], a potent procarcinogen found in combustion products such as diesel exhaust and cigarette smoke, has been recently shown to activate the c-Jun NH 2 -terminal kinase 1 (JNK1) and induce caspase-3-mediated apoptosis in Hepa1c1c7 cells. However, the molecules of the signaling pathway that control the mitogen-activated protein kinase cascades induced by B(a)P and the interaction between those and apoptosis by B(a)P have not been well defined. We report here that B(a)P promoted Cdc42/Rac1, p21-activated kinase 1 (PAK1), and JNK1 activities in 293T and HeLa cells. Moreover, alpha-PAK-interacting exchange factor (␣ PIX) mRNA and its protein expression were upregulated by B(a)P. While overexpression of an active mutant of ␣ PIX (⌬CH) facilitated B(a)P-induced activation of Cdc42/Rac1, PAK1, and JNK1, overexpression of mutated ␣PIX (L383R, L384S), which lacks guanine nucleotide exchange factor activity, SH3 domaindeleted ␣PIX (⌬ SH3), which lacks the ability to bind PAK, kinase-negative PAK1 (K299R), and kinasenegative SEK1 (K220A, K224L) inhibited B(a)P-triggered JNK1 activation. Interestingly, overexpression of ␣PIX (⌬ CH) and a catalytically active mutant PAK1 (T423E) accelerated B(a)P-induced apoptosis in HeLa cells, whereas ␣PIX (⌬ SH3), PAK1 (K299R), and SEK 1 (K220A, K224L) inhibited B(a)P-initiated apoptosis. Finally, a preferential caspase inhibitor, Z-Asp-CH2-DCB, strongly blocked the ␣PIX (⌬ CH)-enhanced apoptosis in cells treated with B(a)P but did not block PAK1/JNK1 activation. Taken together, these results indicate that ␣PIX plays a crucial role in B(a)P-induced apoptosis through activation of the JNK1 pathway kinases.