Fucosylation of Cripto is required for its ability to facilitate nodal signaling.

O-linked fucose modification is rare and has been shown to occur almost exclusively within epidermal growth factor (EGF)-like modules. We have found that the EGF-CFC family member human Cripto-1 (CR) is modified with fucose and through a combination of peptide mapping, mass spectrometry, and sequence analysis localized the site of attachment to Thr-88. The identification of a fucose modification on human CR within its EGF-like domain and the presence of a consensus fucosylation site within all EGF-CFC family members suggest that this is a biologically important modification in CR, which functionally distinguishes it from the EGF ligands that bind the type 1 erbB growth factor receptors. A single CR point mutation, Thr-88 --> Ala, results in a form of the protein that is not fucosylated and has substantially weaker activity in cell-based CR/Nodal signaling assays, indicating that fucosylation is functionally important for CR to facilitate Nodal signaling.

Reactivity of cysteines in the transmembrane region of the Na, K-ATPase alpha subunit probed with Hg(2+).

To gain insight into the structure and conformational coupling in the Na,K-ATPase, this study characterized the reaction of the alpha1 subunit transmembrane cysteines with a small probe. Intact HeLa cells expressing heterologous Na,K-ATPase were treated with (microm) HgCl(2) after placing the enzyme predominantly in either of two conformations, phosphorylated E2P.Na/E2P or dephosphorylated ATP.E1. K/ATP.E1. Under both conditions the treatment led to enzyme inactivation following a double exponential kinetic as determined by ouabain-sensitive K(+) uptake measurements. However, the rate constant of the slow reacting component was ten times larger when the protein was probed in a medium that would favor enzyme phosphorylation. Enzymes carrying mutations of cysteines located in the alpha1 subunit transmembrane region were used to identify the reacting-SH groups. Replacement Cys104Ser reduced enzyme inactivation by removing the slow reacting component under both treatment conditions. Replacement of Cys964 reduced the inactivation rate constant of the fast reacting component (79%) and removed the slow reacting component when the dephosphorylated enzyme was treated with Hg(2+). Moreover, Cys964Ser substituted enzyme was insensitive to Hg(2+) when treated under phosphorylation conditions. These results indicate that Cys964 is involved in the fast inactivation by Hg(2+). Although the double mutant Cys964, 104Ser was still partially inactivated by treatment under nonphosphorylating conditions, an enzyme devoid of transmembrane cysteines was insensitive to Hg(2+) under all treatment conditions. Thus, this enzyme provides a background where accessibility of engineered transmembrane cysteines can be tested.

Functional role of cysteine residues in the (Na,K)-ATPase alpha subunit.

The structural-functional roles of 23 cysteines present in the sheep (Na,K)-ATPase alpha1 subunit were studied using site directed mutagenesis, expression, and kinetics analysis. Twenty of these cysteines were individually substituted by alanine or serine. Cys452, Cys455 and Cys456 were simultaneously replaced by serine. These substitutions were introduced into an ouabain resistant alpha1 sheep isoform and expressed in HeLa cells under ouabain selective pressure. HeLa cells transfected with a cDNA encoding for replacements of Cys242 did not survive ouabain selective pressure. Single substitutions of the remaining cysteines yielded functional enzymes, although some had reduced turnover rates. Only minor variations were observed in the enzyme Na(+) and K(+) dependence as a result of these replacements. Some substitutions apparently affect the E1<-->E2 equilibrium as suggested by changes in the K(m) of ATP acting at its low affinity binding site. These results indicate that individual cysteines, with the exception of Cys242, are not essential for enzyme function. Furthermore, this suggests that the presence of putative disulfide bridges is not required for alpha1 subunit folding and subsequent activity. A (Na,K)-ATPase lacking cysteine residues in the transmembrane region was constructed (Cys104, 138, 336, 802, 911, 930, 964, 983Xxx). No alteration in the K(1/2) of Na(+) or K(+) for (Na,K)-ATPase activation was observed in the resulting enzyme, although it showed a 50% reduction in turnover rate. ATP binding at the high affinity site was not affected. However, a displacement in the E1<-->E2 equilibrium toward the E1 form was indicated by a small decrease in the K(m) of ATP at the low affinity site accompanied by an increase in IC(50) for vanadate inhibition. Thus, the transmembrane cysteine-deficient (Na,K)-ATPase appears functional with no critical alteration in its interactions with physiological ligands.