We have shown that ouabain activates Src, resulting in subsequent tyrosine phosphorylation of multiple effectors. Here, we tested if the Na ؉ /K ؉ -ATPase and Src can form a functional signaling complex. In LLC-PK1 cells the Na ؉ /K ؉ -ATPase and Src colocalized in the plasma membrane. Fluorescence resonance energy transfer analysis indicated that both proteins were in close proximity, suggesting a direct interaction. GST pulldown assay showed a direct, ouabain-regulated, and multifocal interaction between the ␣1 subunit of Na ؉ /K ؉ -ATPase and Src. Although the interaction between the Src kinase domain and the third cytosolic domain (CD3) of ␣1 is regulated by ouabain, the Src SH3SH2 domain binds to the second cytosolic domain constitutively. Functionally, binding of Src to either the Na ؉ /K ؉ -ATPase or GST-CD3 inhibited Src activity. Addition of ouabain, but not vanadate, to the purified Na ؉ /K ؉ -ATPase/Src complex freed the kinase domain and restored the Src activity. Consistently, exposure of intact cells to ouabain apparently increased the distance between the Na ؉ /K ؉ -ATPase and Src. Concomitantly, it also stimulated tyrosine phosphorylation of the proteins that are associated with the Na ؉ /K ؉ -ATPase. These new findings illustrate a novel molecular mechanism of signal transduction involving the interaction of a P-type ATPase and a nonreceptor tyrosine kinase.

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Recent studies have ascribed many non-pumping functions to the Na/K-ATPase. Here, we present experimental evidence demonstrating that over half of the plasma membrane Na/KATPase in LLC-PK1 cells is performing cellular functions other than ion pumping. This "non-pumping" pool of Na/K-ATPase, like the pumping pump, binds ouabain. Depletion of either cholesterol or caveolin-1 moves some of the "non-pumping" Na/KATPase into the pumping pool. Graded knock-down of the ␣1 subunit of the Na/K-ATPase eventually results in loss of this "non-pumping" pool while preserving the pumping pool. Our prior studies indicate that a loss of the non-pumping pool is associated with a loss of receptor function as evidenced by the failure of ouabain administration to induce the activation of Src and/or ERK. Therefore, our new findings suggest that a substantial amount of surface-expressed Na/K-ATPase, at least in some types of cells, may function as non-canonical ouabain-binding receptors.

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An overview is presented of some of the major insights that have come from studies of the structure, stability, and folding of T4 phage lysozyme. A major purpose of this review is to provide the reader with a complete tabulation of all of the variants that have been characterized, including melting temperatures, crystallographic data, Protein Data Bank access codes, and references to the original literature. The greatest increase in melting temperature (T m ) for any point mutant is 5.1°C for the mutant Ser 117 fi Val. This is achieved in part not only by hydrophobic stabilization but also by eliminating an unusually short hydrogen bond of 2.48 Å that apparently has an unfavorable van der Waals contact. Increases in T m of more than 3-4°C for point mutants are rare, whereas several different types of destabilizing substitutions decrease T m by 20°C or thereabouts. The energetic cost of cavity creation and its relation to the hydrophobic effect, derived from early studies of ''large-tosmall'' mutants in the core of T4 lysozyme, has recently been strongly supported by related studies of the intrinsic membrane protein bacteriorhodopsin. The L99A cavity in the C-terminal domain of the protein, which readily binds benzene and many other ligands, has been the subject of extensive study. Crystallographic evidence, together with recent NMR analysis, suggest that these ligands are admitted by a conformational change involving Helix F and its neighbors. A total of 43 nonisomorphous crystal forms of different monomeric lysozyme mutants were obtained plus three more for synthetically-engineered dimers. Among the 43 space groups, P2 1 2 1 2 1 and P2 1 were observed most frequently, consistent with the prediction of Wukovitz and Yeates.

Using an inverse mantle convection model that assimilates seismic structure and plate motions, we reconstruct Farallon plate subduction back to 100 million years ago. Models consistent with stratigraphy constrain the depth dependence of mantle viscosity and buoyancy, requiring that the Farallon slab was flat lying in the Late Cretaceous, consistent with geological reconstructions. The simulation predicts that an extensive zone of shallow-dipping subduction extended beyond the flat-lying slab farther east and north by up to 1000 kilometers. The limited region of flat subduction is consistent with the notion that subduction of an oceanic plateau caused the slab to flatten. The results imply that seismic images of the current mantle provide more constraints on past tectonic events than previously recognized.

Use of ACE inhibitors or ARBs in people with CKD reduces the risk for kidney failure and cardiovascular events. ACE inhibitors also reduced the risk for all-cause mortality and were possibly superior to ARBs for kidney failure, cardiovascular death, and all-cause mortality in patients with CKD, suggesting that they could be the first choice for treatment in this population.

SHP-1 is a cytosolic protein-tyrosine phosphatase that behaves as a negative regulator in eukaryotic cellular signaling pathways. To understand its regulatory mechanism, we have determined the crystal structure of the C-terminal truncated human SHP-1 in the inactive conformation at 2.8-Å resolution and refined the structure to a crystallographic R-factor of 24.0%. The three-dimensional structure shows that the ligand-free SHP-1 has an auto-inhibited conformation. Its N-SH2 domain blocks the catalytic domain and keeps the enzyme in the inactive conformation, which supports that the phosphatase activity of SHP-1 is primarily regulated by the N-SH2 domain. In addition, the C-SH2 domain of SHP-1 has a different orientation from and is more flexible than that of SHP-2, which enables us to propose an enzymatic activation mechanism in which the C-SH2 domains of SHPs could be involved in searching for phosphotyrosine activators.Tyrosine phosphorylation is a key mechanism for regulating eukaryotic cellular signaling pathways. The protein tyrosine phosphorylation level is precisely regulated by two types of enzymes: protein-tyrosine kinases (PTKs) 1 and protein-tyrosine phosphatases (PTPs), in which PTPs act to counter-balance the process through dephosphorylation of the phosphorylated tyrosines (1, 2). PTPs can be divided into two groups, receptor protein-tyrosine phosphatases and cytosolic proteintyrosine phosphatases. The SH2 domain-containing PTPs, SHP-1 and SHP-2, are both cytosolic PTPs and share many structural and regulatory features. They both have two tandem SH2 domains at the N terminus followed by a single catalytic domain and an inhibitory C-terminal tail. However, irrespective of similar structural and regulatory characteristics, these two enzymes have different biological function in vivo.Different from SHP-2, which is expressed in all kinds of tissues, SHP-1 is predominantly expressed in hematopoietic and epithelial cells and behaves mainly as a negative regulator of signaling pathways in lymphocytes (1, 2). SHP-1 is dormant in the cytosol, with its phosphatase activity inhibited by both the SH2 domains and the C-terminal tail (1,(3)(4)(5). In response to an activation signal, SHP-1 is recruited to membrane-bound inhibitory receptors via the binding of its SH2 domains to the tyrosine-phosphorylated immunoreceptor tyrosine-based inhibitory motif within the cytoplasmic domain of a receptor (6 -8). During this process, SHP-1 undergoes a structural rearrangement, exposes its active site, and binds to the downstream substrates, thereby dephosphorylating the substrates to turn off the cellular signals.SHP-1 also presents in several types of non-hematopoietic cells (9 -12). Overexpression of a catalytically inactive SHP-1 mutant in these cells strongly suppressed mitogen-activated pathways, reducing signal transduction and activation of transcription; these findings demonstrate that SHP-1 has a positive effect on mitogenic signaling in these non-hematopoietic cells (10, 11). Thus, SHP-1 probably has both the nega...