Transforming activity of the Rho family GTPase, Wrch-1, a Wnt-regulated Cdc42 homolog, is dependent on a novel carboxyl-terminal palmitoylation motif.
Wrch-1 is a Rho family GTPase that shares strong sequence and functional similarity with Cdc42. Like Cdc42, Wrch-1 can promote anchorage-independent growth transformation. We determined that activated Wrch-1 also promoted anchorage-dependent growth transformation of NIH 3T3 fibroblasts. Wrch-1 contains a distinct carboxyl-terminal extension not found in Cdc42, suggesting potential differences in subcellular location and function. Consistent with this, we found that Wrch-1 associated extensively with plasma membrane and endosomes, rather than with cytosol and perinuclear membranes like Cdc42. Like Cdc42, Wrch-1 terminates in a CAAX tetrapeptide (where C is cysteine, A is aliphatic amino acid, and X is any amino acid) motif (CCFV), suggesting that Wrch-1 may be prenylated similarly to Cdc42. Most surprisingly, unlike Cdc42, Wrch-1 did not incorporate isoprenoid moieties, and Wrch-1 membrane localization was not altered by inhibitors of protein prenylation. Instead, we showed that Wrch-1 is modified by the fatty acid palmitate, and pharmacologic inhibition of protein palmitoylation caused mislocalization of Wrch-1. Most interestingly, mutation of the second cysteine of the CCFV motif (CCFV > CSFV), but not the first, abrogated both Wrch-1 membrane localization and transformation. These results suggest that Wrch-1 membrane association, subcellular localization, and biological activity are mediated by a novel membrane-targeting mechanism distinct from that of Cdc42 and other isoprenylated Rho family GTPases.
Berzat
Anastacia C
AC
Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, 27599-7512, USA.
Buss
Janice E
JE
Chenette
Emily J
EJ
Weinbaum
Carolyn A
CA
Shutes
Adam
A
Der
Channing J
CJ
Minden
Audrey
A
Cox
Adrienne D
AD
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000737:chemistry; D015153:Blotting, Western; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D004952:Esters / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D041681:NIH 3T3 Cells; D019308:Palmitic Acid / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D004952:Esters; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D019308:Palmitic Acid; D001710:Biotin; C432668:RHOU protein, human; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D003545:Cysteine