Adapters are typically viewed as molecules coordinating the recruitment of positive effectors of cell signaling. Herein, we report the identification of Dok-3, a novel adapter molecule belonging to the Dok family. Our studies show that Dok-3 is highly expressed in several hemopoietic cell types, including B cells and macrophages. It undergoes rapid tyrosine phosphorylation in response to immunoreceptor-mediated cellular activation, seemingly as a result of the action of Src family kinases. This phosphorylation induces the binding of Dok-3 to at least two inhibitory molecules, the 5 inositol phosphatase SHIP and the protein tyrosine kinase Csk. We also demonstrate that augmented expression of wild-type Dok-3 in a B-cell line results in an inhibition of immunoreceptor-mediated nuclear factor of activated T-cells (NFAT) activation and cytokine release, while introduction of a Dok-3 mutant with impaired ability to associate with SHIP and Csk enhances B-cell responsiveness. Taken together, these results indicate that Dok-3 is an adapter involved in the recruitment of inhibitory molecules and that it may play a significant role in the negative regulation of immunoreceptor signaling in hemopoietic cells such as B cells and macrophages.Immunoreceptors such as the T-cell receptor (TCR) for antigen, the B-cell receptor (BCR) for antigen, and a variety of receptors for the Fc portion of immunoglobulins (Ig), play central roles in antigen-specific and natural immunity (6,20,34,54,59,66). Typically, these receptors contain several chains, including ligand-binding subunits and subunits involved in signal transduction. Accumulating data show that immunoreceptors mediate their biological effects via the induction of intracellular protein tyrosine phosphorylation. While they lack intrinsic protein tyrosine kinase (PTK) activity, they possess within their cytoplasmic domain a motif termed the immunoreceptor tyrosine-based activation motif (ITAM), which has the ability to recruit and activate cytoplasmic PTKs.Two classes of cytoplasmic PTKs have been implicated in immunoreceptor-mediated signal transduction: the Src and Syk/Zap-70 families (11,15,16,59,63). Genetic and biochemical studies have shown that Src-related enzymes initiate immunoreceptor signaling through their capacity to phosphorylate two conserved tyrosines in the ITAMs. This phosphorylation permits the binding and activation of Syk/Zap-70-related PTKs, which amplify the immunoreceptor-induced signal. Together, Src and Syk/Zap family kinases activate downstream effectors, including phospholipase C (PLC)-␥, the guanine nucleotide exchange factor Vav, phosphatidylinositol (PI) 3Ј kinase, and Ras. These targets lead to reorganization of the cytoskeleton, transcriptional activation, and, ultimately, induction of immune functions.Intracellular signals delivered by PTKs such as Src and Syk/ Zap-70 family kinases are coordinated by a class of molecules termed "adapters" or "linkers" (49,51,56). Even though these polypeptides lack intrinsic catalytic activity, they possess mo...

Nephrin is a slit diaphragm protein critical for structural and functional integrity of visceral glomerular epithelial cells (podocytes) and is known to be tyrosine phosphorylated by Src family kinases. We studied the role of phosphoinositide 3-kinase (PI3K), activated via the phosphorylation of nephrin, in actin cytoskeletal reorganization of cultured rat podocytes. Phosphorylation of rat nephrin by the Fyn kinase markedly increased its interaction with a regulatory subunit of PI3K. Stable transfection of rat nephrin in the podocytes with podocin led to nephrin tyrosine phosphorylation, PI3K-dependent phosphorylation of Akt, increased Rac1 activity, and an altered actin cytoskeleton with decreased stress fibers and increased lamellipodia. These changes were reversed with an inhibitor of PI3K and not seen when the nephrin-mutant Y1152F replaced wild-type nephrin. Rac1 and Akt1 contributed to lamellipodia formation and decreased stress fibers, respectively. Finally, in the rat model of puromycin aminonucleoside nephrosis, nephrin tyrosine phosphorylation, nephrin-PI3K association, and glomerular Akt phosphorylation were all decreased. Our results suggest that PI3K is involved in nephrin-mediated actin reorganization in podocytes. Disturbed nephrin-PI3K interactions may contribute to abnormal podocyte morphology and proteinuria.

Abstract. Visceral glomerular epithelial cells (GEC) are critical for normal permselectivity of the kidney. Nephrin is a molecule that is expressed specifically in GEC in a structure called the slit diaphragm and is required for normal morphology and permselectivity of GEC. However, the mechanisms of action of nephrin are not understood precisely. The intracellular domain of nephrin has six conserved tyrosine residues. It was hypothesized that these tyrosine residues are phosphorylated by Srcfamily kinases and that this phosphorylation modulates the function of nephrin. A transient transfection system was used to study the role of tyrosine phosphorylation of the cytoplasmic domain of nephrin in its function. When nephrin was cotransfected with Src-family kinases Fyn or Src in Cos-1 cells, nephrin was strongly tyrosine phosphorylated by Fyn and less so by Src. The results with tyrosine-to-phenylalanine mutations suggested that multiple tyrosine residues contribute to phosphorylation mediated by Src-family kinases. The intracellular domain of nephrin is known to interact with another slit diaphragm protein, podocin. When nephrin and podocin were transfected with Fyn, the interaction between nephrin and podocin was augmented significantly. Podocin was not tyrosine phosphorylated by Fyn; thus, the increased interaction is likely to be secondary to tyrosine phosphorylation of nephrin. Fyn also significantly augmented the activation of the AP-1 promoter induced by nephrin and podocin. In summary, Fyn phosphorylates the cytoplasmic domain of nephrin on tyrosine, leading to enhanced association with podocin and downstream signaling of nephrin.

The development of methods for detecting and manipulating matter at the level of individual macromolecules represents one of the key scientific advancements of recent decades. These techniques allow us to get information that is largely unobtainable otherwise, such as the magnitudes of microscopic forces, mechanistic details of catalytic processes, macromolecular population heterogeneities, and time-resolved, step-by-step observation of complex kinetics. Methods based on optical, mechanical, and ionic-conductance signal transduction are particularly developed. However, there is scope for new approaches that can broaden the range of molecular systems that we can study at this ultimate level of sensitivity and for developing new analytical methods relying on single-molecule detection. Approaches based on purely electrical detection are particularly appealing in the latter context, since they can be easily combined with microelectronics or fluidic devices on a single microchip to create large parallel assays at relatively low cost. A form of electrical signal transduction that has so far remained relatively underdeveloped at the single-molecule level is the direct detection of the charge transferred in electrochemical processes. The reason for this is simple: only a few electrons are transferred per molecule in a typical faradaic reaction, a heterogeneous charge-transfer reaction that occurs at the electrode's surface. Detecting this tiny amount of charge is impossible using conventional electrochemical instrumentation. A workaround is to use redox cycling, in which the charge transferred is amplified by repeatedly reducing and oxidizing analyte molecules as they randomly diffuse between a pair of electrodes. For this process to be sufficiently efficient, the electrodes must be positioned within less than 100 nm of each other, and the analyte must remain between the electrodes long enough for the measurement to take place. Early efforts focused on tip-based nanoelectrodes, descended from scanning electrochemical microscopy, to create suitable geometries. However, it has been challenging to apply these technologies broadly. In this Account, we describe our alternative approach based on electrodes embedded in microfabricated nanochannels, so-called nanogap transducers. Microfabrication techniques grant a high level of reproducibility and control over the geometry of the devices, permitting systematic development and characterization. We have employed these devices to demonstrate single-molecule sensitivity. This method shows good agreement with theoretical analysis based on the Brownian motion of discrete molecules, but only once the finite time resolution of the experimental apparatus is taken into account. These results highlight both the random nature of single-molecule signals and the complications that it can introduce in data interpretation. We conclude this Account with a discussion on how scientists can overcome this limitation in the future to create a new experimental platform that can be generally useful ...

Ischemia-reperfusion injury (IRI) is a major cause of renal dysfunction in both native kidneys and renal allografts. To broaden our understanding of the inflammatory mediators involved in IRI, we used multi-probe RNase protection assays to examine the expression of 26 different cytokine genes in a murine model of renal IRI. We observed that, in addition to up-regulation of IL-1beta and to a lesser extent TNF-alpha, IRI was associated with an intense and sustained up-regulation of three gp130-signaling cytokines, IL-6, IL-11, and leukemia inhibitory factor (LIF), as well as with up-regulation of the neutrophil chemotactic and activating mediator macrophage inflammatory protein (MIP)-2. Macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein (MCP)-1 were also moderately up-regulated after IRI, whereas mRNA levels of several other inflammatory mediators including IL-1alpha, IL-2, IL-4, interferon (IFN)-gamma, GM-CSF, and RANTES were minimally increased or remained undetectable. These findings identify MIP-2 as an attractive target for inhibition of leukocyte recruitment in renal IRI and also suggest a potentially novel role for gp130-mediated signals in IRI.