ABSTRACT
Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase.
Subject(s)
Carboxylesterase/metabolism , Drosophila Proteins/metabolism , Esterases/metabolism , Wnt Proteins/chemistry , Wnt Proteins/metabolism , Wnt Signaling Pathway , Acylation , Animals , Binding Sites , Carboxylesterase/chemistry , Drosophila Proteins/chemistry , Esterases/chemistry , Esterases/genetics , Fatty Acids, Monounsaturated/metabolism , Glycosylphosphatidylinositols/metabolism , Glypicans/metabolism , Humans , Kinetics , Ligands , Mass Spectrometry , Models, Molecular , Protein BindingABSTRACT
CAAX proteins have essential roles in multiple signalling pathways, controlling processes such as proliferation, differentiation and carcinogenesis. The â¼120 mammalian CAAX proteins function at cellular membranes and include the Ras superfamily of small GTPases, nuclear lamins, the γ-subunit of heterotrimeric GTPases, and several protein kinases and phosphatases. The proper localization of CAAX proteins to cell membranes is orchestrated by a series of post-translational modifications of the carboxy-terminal CAAX motifs (where C is cysteine, A is an aliphatic amino acid and X is any amino acid). These reactions involve prenylation of the cysteine residue, cleavage at the AAX tripeptide and methylation of the carboxyl-prenylated cysteine residue. The major CAAX protease activity is mediated by Rce1 (Ras and a-factor converting enzyme 1), an intramembrane protease (IMP) of the endoplasmic reticulum. Information on the architecture and proteolytic mechanism of Rce1 has been lacking. Here we report the crystal structure of a Methanococcus maripaludis homologue of Rce1, whose endopeptidase specificity for farnesylated peptides mimics that of eukaryotic Rce1. Its structure, comprising eight transmembrane α-helices, and catalytic site are distinct from those of other IMPs. The catalytic residues are located â¼10 Å into the membrane and are exposed to the cytoplasm and membrane through a conical cavity that accommodates the prenylated CAAX substrate. We propose that the farnesyl lipid binds to a site at the opening of two transmembrane α-helices, which results in the scissile bond being positioned adjacent to a glutamate-activated nucleophilic water molecule. This study suggests that Rce1 is the founding member of a novel IMP family, the glutamate IMPs.
Subject(s)
Biocatalysis , Membrane Proteins/chemistry , Methanococcus/enzymology , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Prenylation , Proto-Oncogene Proteins p21(ras)/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Conserved Sequence , Crystallography, X-Ray , Cysteine/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Endopeptidases/chemistry , Endopeptidases/metabolism , Endoplasmic Reticulum/enzymology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Glutamic Acid/metabolism , Humans , Membrane Proteins/metabolism , Metalloendopeptidases/chemistry , Metalloendopeptidases/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Peptide Hydrolases/classification , Protein Structure, Tertiary , Proto-Oncogene Proteins p21(ras)/chemistry , Signal Transduction , Substrate SpecificityABSTRACT
Signaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins.
