Fibroblast growth factor receptor (FGFR) activation leads to receptor autophosphorylation and increased tyrosine phosphorylation of several intra cellular proteins. We have previously shown that autophosphorylated tyrosine 766 in FGFR1 serves as a binding site for one of the SH2 domains of phospholipase Cy and couples FGFR1 to phosphatidylinositol hydrolysis in several cell types. In this report, we describe the identification of six additional autophosphorylation sites (Y-463, Y-583, Y-585, Y-653, Y-654 and Y-730) on FGFR1. We demonstrate that autophosphorylation on tyrosines 653 and 654 is important for activation of tyrosine kinase activity of FGFR1 and is therefore essential for FGFR1-mediated biological responses. In contrast, autophosphorylation of the remaining four tyrosines is dispensable for FGFR1-mediated mitogen-activated protein kinase activation and mitogenic signaling in L-6 cells as well as neuronal differentiation of PC12 cells. Interestingly, both the wild-type and a mutant FGFR1 (FGFR1-4F) are able to phosphorylate Shc and an unidentified Grb2-associated phosphoprotein of 90 kDa (pp90). Binding of the Grb2/Sos complex to phosphorylated Shc and pp90 may therefore be the key link between FGFR1 and the Ras signaling pathway, mito-genesis, and neuronal differentiation.

Mitogen-activated protein (MAP) kinases are critical mediators of innate immune responses. In response to lipopolysaccharide (LPS), MAP kinases are rapidly activated and play an important role in the production of proinflammatory cytokines. Although a number of MAP kinase phosphatases (MKPs) have been identified, their roles in the control of cytokine production have not been well defined. In the present report, we investigated the role of MKP-1 in alveolar macrophages stimulated with LPS. We found that LPS triggered transient activation of three MAP kinase subfamilies, ERK, JNK, and p38, in both immortalized and primary murine alveolar macrophages. MKP-1 was rapidly induced by LPS, and its induction correlated with the dephosphorylation of these MAP kinases. Blocking MKP-1 with triptolide prolonged the activities of both JNK and p38 in immortalized alveolar macrophages. Stimulation of primary alveolar macrophages isolated from MKP-1-deficient mice with LPS resulted in a prolonged p38 phosphorylation compared with wild type alveolar macrophages. Accordingly, these MKP-1-deficient alveolar macrophages also mounted a more robust and rapid tumor necrosis factor α production than their wild type counterparts. Adenovirus-mediated MKP-1 overexpression significantly attenuated tumor necrosis factor α production in immortalized alveolar macrophages. Finally, MKP-1 was induced by a group of corticosteroids frequently prescribed for the treatment of inflammatory lung diseases, and the anti-inflammatory potencies of these drugs closely correlated with their abilities to induce MKP-1. Our studies indicated that MKP-1 plays an important role in dampening the inflammatory responses of alveolar macrophages. We speculate that MKP-1 may represent a novel target for therapeutic intervention of inflammatory lung diseases.

The carboxylic acid ionophore monensin, known as an electroneutral Na⁺ ionophore, an anticoccidial agent, and a growth-promoting feed additive in agriculture, is shown to be highly efficient as an ionophore for Pb²⁺ and to be highly selective for Pb²⁺ compared with other divalent cations. Monensin transports Pb²⁺ by an electroneutral mechanism in which the complex PbMonOH is the transporting species. Electrogenic transport via the species PbMon⁺ may also be possible. Monensin catalyzed Pb²⁺ transport is little affected by Ca²⁺, Mg²⁺, or K⁺concentrations that are encountered in living systems. Na⁺is inhibitory, but its effectiveness at 100 mm does not exceed ∼50%. The poor activity of monensin as an ionophore for divalent cations other than Pb²⁺ is consistent with the pattern of complex formation constants observed in the mixed solvent 80% methanol/water. This pattern also explains why Ca²⁺, Mg²⁺, and K⁺ are ineffective as inhibitors of Pb²⁺ transport, but it does not fully explain the actions of Na⁺, where kinetic features of the transport mechanism may also be important. When given to rats at 100 ppm in feed together with Pb²⁺ at 100 ppm in drinking water, monensin reduces Pb accumulation in several organs and tissues. It also accelerates the excretion of Pb that was accumulated previously and produces this effect without depleting the organs of zinc or copper. Monensin, used alone or in combination with other agents, may be useful for the treatment of Pb intoxication.

Endothelin‐1 (ET‐1) induces cell proliferation and differentiation through multiple G‐protein‐linked signaling systems, including p21^ras activation. Whereas p21^ras activation and desensitization by receptor tyrosine kinases have been extensively investigated, the kinetics of p21^ras activation induced by engagement of G‐protein‐coupled receptors remains to be fully elucidated. In the present study we show that ET‐1 induces a biphasic activation of p21^ras in rat glomerular mesangial cells. The first peak of activation of p21^ras, at 2–5 min, is mediated by immediate association of phosphorylated Shc with the guanosine exchange factor Sos1 via the adaptor protein Grb2. This initial activation of p21^ras results in activation of the extracellular signal‐regulated kinase (ERK) cascade. We demonstrate that ET‐1 signaling elicits a negative feedback mechanism, modulating p21^ras activity through ERK‐dependent Sos1 phosphorylation, findings which were confirmed using an adenovirus MEK construct. Subsequent to p21^ras and ERK deactivation, Sos1 reverts to the non‐phosphorylated condition, enabling it to bind again to the Grb2/Shc complex, which is stabilized by persistent Shc phosphorylation. However, the resulting secondary activation of p21^ras at 30 min does not lead to ERK activation, correlating with intensive, ET‐1‐induced expression of MAP kinase phosphatase‐1, but does result in increased p21^ras‐associated phosphatidylinositol 3‐kinase activity. Our data provide evidence that ET‐1‐induced biphasic p21^ras activation causes sequential stimulation of divergent downstream signaling pathways.

The intracellular mechanisms involved in the activation of extracellular signal-regulated kinase (ERK) are relatively well understood. However, the intracellular signaling pathways which regulate the termination of ERK activity remain to be elucidated. Mitogen-activated protein kinase phosphatase 1 (MKP-1) has been shown to dephosphorylate and inactivate ERK in vitro and in vivo. In the present study, we show in NIH3T3 fibroblasts that activation of the stress-activated protein kinase (SAPK) pathway by either specific extracellular stress stimuli or via induction of MEKK, an upstream kinase of SAPK, results in MKP-1 gene expression. In contrast, selective stimulation of the ERK pathway by 12-O-tetradecanoylphorbol-13-acetate or following expression of constitutively active MEK, the upstream dual specificity kinase of ERK did not induce the transcription of MKP-1. Hence, these findings demonstrate the existence of cross-talk between the ERK and SAPK signaling cascades since activation of SAPK induced the expression of MKP-1 that can inactivate ERK. This mechanism may modulate the cellular response to stimuli which employ the SAPK signal transduction pathway.