The signaling routes linking G-protein-coupled receptors to mitogen-activated protein kinase (MAPK) may involve tyrosine kinases, phosphoinositide 3-kinase gamma (PI3Kgamma), and protein kinase C (PKC). To characterize the mitogenic pathway of bradykinin (BK), COS-7 cells were transiently cotransfected with the human bradykinin B(2) receptor and hemagglutinin-tagged MAPK. We demonstrate that BK-induced activation of MAPK is mediated via the alpha subunits of a G(q/11) protein. Both activation of Raf-1 and activation of MAPK in response to BK were blocked by inhibitors of PKC as well as of the epidermal growth factor (EGF) receptor. Furthermore, in PKC-depleted COS-7 cells, the effect of BK on MAPK was clearly reduced. Inhibition of PI3-Kgamma or Src kinase failed to diminish MAPK activation by BK. BK-induced translocation and overexpression of PKC isoforms as well as coexpression of inactive or constitutively active mutants of different PKC isozymes provided evidence for a role of the diacylglycerol-sensitive PKCs alpha and epsilon in BK signaling toward MAPK. In addition to PKC activation, BK also induced tyrosine phosphorylation of EGF receptor (transactivation) in COS-7 cells. Inhibition of PKC did not alter BK-induced transactivation, and blockade of EGF receptor did not affect BK-stimulated phosphatidylinositol turnover or BK-induced PKC translocation, suggesting that PKC acts neither upstream nor downstream of the EGF receptor. Comparison of the kinetics of PKC activation and EGF receptor transactivation in response to BK also suggests simultaneous rather than consecutive signaling. We conclude that in COS-7 cells, BK activates MAPK via a permanent dual signaling pathway involving the independent activation of the PKC isoforms alpha and epsilon and transactivation of the EGF receptor. The two branches of this pathway may converge at the level of the Ras-Raf complex.

Insulinoma-associated protein (IA)-2␤, also known as phogrin, is an enzymatically inactive member of the transmembrane protein tyrosine phosphatase family and is located in dense-core secretory vesicles. In patients with type 1 diabetes, autoantibodies to IA-2␤ appear years before the development of clinical disease. The genomic structure and function of IA-2␤, however, is not known. In the present study, we determined the genomic structure of IA-2␤ and found that both human and mouse IA-2␤ consist of 23 exons and span ϳ1,000 and 800 kb, respectively. With this information, we prepared a targeting construct and inactivated the mouse IA-2␤ gene as demonstrated by lack of IA-2␤ mRNA and protein expression. The IA-2␤ ؊/؊ mice, in contrast to wild-type controls, showed mild glucose intolerance and impaired glucose-stimulated insulin secretion. Knockout of the IA-2␤ gene in NOD mice, the most widely studied animal model for human type 1 diabetes, failed to prevent the development of cyclophosphamide-induced diabetes. We conclude that IA-2␤ is involved in insulin secretion, but despite its importance as a major autoantigen in human type 1 diabetes, it is not required for the development of diabetes in NOD mice.

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Nitric oxide (NO) plays a key role in vascular function, cell proliferation, and apoptosis. Proper subcellular localization of endothelial NO synthase (eNOS) is crucial for its activity; however, the role of eNOS trafficking for NO biosynthesis remains to be defined. Overexpression of NOS-interacting protein (NOSIP) induces translocation of eNOS from the plasma membrane to intracellular compartments, thereby impairing NO production. Here we report that endogenous NOSIP reduces the enzymatic capacity of eNOS, specifically in the G 2 phase of the cell cycle by targeting eNOS to the actin cytoskeleton. This regulation is critically dependent on the nucleocytoplasmic shuttling of NOSIP and its cytoplasmic accumulation in the G 2 phase. The predominant nuclear localization of NOSIP depends on a bipartite nuclear localization sequence (NLS) mediating interaction with importin ␣. Mutational destruction of the NLS abolishes nuclear import and interaction with importin ␣. Nuclear export is insensitive to leptomycin B and hence different from the CRM1-dependent default mechanism. Inhibition of NOSIP expression by RNA interference completely abolishes G 2 -specific cytoskeletal association and inhibition of eNOS. These findings describe a novel cell cycle-dependent modulation of endogenous NO levels that are critical to the cell cycle-related actions of NO such as apoptosis or cell proliferation.Endothelium-derived nitric oxide (NO) plays a key role in vascular function including angiogenesis, cell proliferation, and apoptosis (30). The short-lived character and the variety of biological targets of NO urge for tight temporal and spatial control of NO biosynthesis, reflected in the complex machinery modulating endothelial nitric oxide synthase (eNOS) activity (14). While the mechanisms of direct eNOS activation are quite well understood, the subcellular targeting of eNOS and its implication for NO bioavailability are subject to controversial discussions. Conventional wisdom holds that eNOS becomes activated at the plasma membrane, e.g., in response to vascular endothelial growth factor or shear stress (14, 30). It has been suggested, but is not generally accepted, that upon activation eNOS dissociates from the plasma membrane and translocates to the cytosol (14). Besides the plasma membrane, the Golgi complex represents a major target site for eNOS; however, it is unclear whether it also contains active eNOS, either as a consequence of activation in situ or due to the translocation of activated eNOS from plasma membrane to the Golgi (15). Circumstantial experimental evidence indicates that eNOS may also be targeted to the cytoskeleton and to intercellular junctions; however, the implications of these localizations for eNOS activity are also unclear. The detergentinsoluble pool, commonly considered to represent the cytoskeleton, has been claimed to be an "inactive" compartment for eNOS (18,30). In contrast, eNOS has been reported to translocate to intercellular junctions and to associate with the actin cytoskeleton in respon...