Mutational analysis of mouse Wnt-1 identifies two temperature-sensitive alleles and attributes of Wnt-1 protein essential for transformation of a mammary cell line.
The proto-oncogene Wnt-1 encodes a cysteine-rich, secretory glycoprotein implicated in virus-induced mouse mammary cancer and intercellular signaling during vertebrate neural development. To attempt to correlate structural motifs of Wnt-1 protein with its function, 12 mutations were introduced singly and in several combinations into the coding sequence of Wnt-1 cDNA by site-directed mutagenesis. Mutant alleles in a retroviral vector were tested for their ability to transform the mouse mammary epithelial cell line C57MG in two ways: by direct infection of C57MG cells and by infection of NIH3T3 cells that serve as donors of Wnt-1 protein to adjacent C57MG cells in a secretion-dependent (paracrine) assay. In addition, the synthesis and secretion of mutant proteins were monitored in multiple cell types by immunological assays. Deletion of the signal peptide demonstrated that transformation in both direct and paracrine assays depends upon entry of Wnt-1 protein into the endoplasmic reticulum. Changes in potential proteolytic processing sites (two basic dipeptides and a probable signal peptidase cleavage site) did not adversely impair biological activity or protein processing and uncovered a second site for cleavage by signal peptidase. Replacement of each of the four asparagine-linked glycosylation sites did not affect transforming activity at normal temperatures, but one glycosylation site mutant was found to be temperature-sensitive for transformation. An allele encoding a protein that lacks all four glycosylation sites was also transformation competent. In two of four cases, substitution of serine for a cysteine residue impaired transforming activity at the usual temperature, and transformation was temperature sensitive in a third case, implying that at least some of the highly conserved cysteine residues are important for Wnt-1 function.
Mason
J O
JO
Department of Microbiology and Immunology, University of California, San Francisco 94143-0502.
Kitajewski
J
J
Varmus
H E
HE
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003545:Cysteine / Q000235:genetics / Q000502:physiology; D004252:DNA Mutational Analysis; D006031:Glycosylation; D006358:Hot Temperature; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014170:Transformation, Genetic / Q000235:genetics / Q000502:physiology*; D051153:Wnt Proteins; D051155:Wnt1 Protein; D029961:Zebrafish Proteins
D011518:Proto-Oncogene Proteins; D051153:Wnt Proteins; D051155:Wnt1 Protein; C495246:Wnt1 protein, mouse; D029961:Zebrafish Proteins; D003545:Cysteine