NIK, a recently identified Nck-interacting kinase, acts upstream of the MEK kinase MEKK1 to activate the c-Jun N-terminal kinase JNK. We now show that NIK binds to and divergently activates the plasma membrane Na ؉ -H ؉ exchanger NHE1. In a genetic screen, NHE1 interacted with NIK at a site N-terminal (amino acids 407-502) to the Nck-binding domain, and this site is critical for its association with NHE1 in vivo. NIK also phosphorylates NHE1; however, the phosphorylation sites, which are distal to amino acid 638, are distinct from the NIK-binding site on NHE1 (amino acids 538 -638). Expression of wild-type, but not a kinase-inactive, NIK in fibroblasts increased NHE1 phosphorylation and activity. The kinase domain of NIK, however, was not sufficient for this response in vivo. Full phosphorylation and activation of NHE1 required both the kinase and the NHE1-binding domains of NIK, suggesting that the NHE1-binding site functions as a targeting signal. The functional significance of an interaction between NIK and NHE1 was confirmed by the ability of a kinase-inactive NIK to selectively inhibit activation of NHE1 by platelet-derived growth factor but not by thrombin. Moreover, although NIK activates JNK through a mechanism dependent on MEKK1, it phosphorylated and activated NHE1 independently of MEKK1. These findings indicate that NIK acts downstream of platelet-derived growth factor receptors to phosphorylate and activate NHE1 divergently of its activation of JNK.

Somatostatin regulates multiple biological functions by acting through a family of five G protein-coupled receptors, somatostatin receptors (SSTRs) 1-5. Although all five receptor subtypes inhibit adenylate cyclase activity and decrease intracellular cAMP levels, specific receptor subtypes also couple to additional signaling pathways. In CCL39 fibroblasts expressing either human SSTR1 or SSTR2, we demonstrate that activation of SSTR1 (but not SSTR2) attenuated both thrombin-and integrin-stimulated Rho-GTP complex formation. The reduction in Rho-GTP formation in the presence of somatostatin was associated with decreased translocation of Rho and LIM kinase to the plasma membrane and fewer focal contacts. Activation of Rho resulted in the formation of intracellular actin stress fibers and cell migration. In CCL39-R1 cells, somatostatin treatment prevented actin stress fiber assembly and attenuated thrombin-stimulated cell migration through Transwell membranes to basal levels. To show that native SSTR1 shares the ability to inhibit Rho activation, we demonstrated that somatostatin treatment of human umbilical vein endothelial cells attenuated thrombin-stimulated Rho-GTP accumulation. These data show for the first time that a G protein-coupled receptor, SSTR1, inhibits the activation of Rho, the assembly of focal adhesions and actin stress fibers, and cell migration.The low molecular mass GTPase Rho plays a central role in regulating organization of the actin-based cytoskeleton in mammalian cells. Activated, GTP-bound Rho promotes the formation of contractile actin filaments into stress fibers and the assembly of cell adhesion complexes (1, 2). Through its coordinate regulation of actin filaments, contractility, and cell adhesion, Rho also plays a critical role in cell migration (3, 4) and in tumor invasion (5, 6). Rho is activated by transmembrane receptors, including the integrin family of adhesion receptors (7) and a subset of heptahelical G protein-coupled receptors (GPCRs) 1 (8). Although the signaling pathway linking integrin receptors to Rho has not been determined, GPCRs, including those for lysophosphatidic acid (9 -11) and thrombin (11-13), activate Rho through the heterotrimeric GTPases G 12 and G 13 . However, GPCRs linked to the inhibition of Rho and downstream cytoskeletal reorganization have not been identified. We now report that somatostatin (SST), acting at the GPCR subtype SSTR1, inhibits Rho activity, attenuates the assembly of actin stress fibers and focal adhesions, and inhibits cell migration. Five distinct SSTR subtypes that are activated by SST have been identified, and these receptors generally have potent inhibitory effects on diverse cell functions such as hormone secretion, neurotransmitter release, smooth muscle contractility, and cell proliferation (14, 15). Effector pathways regulated by SSTRs, including inhibition of adenylate cyclase and Ca 2ϩ channel activity and stimulation of K ϩ channel and phosphatase activity, are mediated by pertussis toxin (PTX)-sensitive mechanisms, most lik...

Intravesical bacillus Calmette-Guérin (BCG) has been shown to be an effective treatment for superficial transitional cell carcinoma of the bladder. The mechanisms by which BCG achieves this effect remain unclear. Reports have attributed an important role to fibronectin both in the initial attachment of BCG to bladder surfaces and in the limitation of tumor cell motility. In the present study, using limited protease cathepsin B degradation followed by Western blot analyses with antibodies to various domains of the fibronectin molecule, we showed that BCG appears to bind to fibronectin near the carboxyl terminal and adjacent to the heparin binding domain. Furthermore a 51-chromium release assay with human bladder cancer cell line T24 as target cells and lymphokine activated killer (LAK) cells as effector cells showed that fibronectin was needed for tumor cytotoxicity by the LAK cells. By using antibodies and peptides to various domains of the fibronectin molecule, the heparin binding domain, but not the cell binding domain, carboxyl terminal region, or the amino terminal region of the fibronectin molecule, was identified as essential to tumor cell lysis by the LAK cells. Flow cytometric analysis showed that both peripheral blood lymphocytes and the LAK cells express fibronectin receptors VLA-3, VLA-4 and VLA-5 on their surfaces. However, the numbers of receptors are not significantly different in the two cell populations. We conclude that, by binding near the carboxyl terminal region and adjacent to the heparin-binding domain of the fibronectin molecule, BCG may protect this region of the molecule from tumor proteases, and may thus allow the antitumor activity of the host immune cells to take place.

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