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.