UNC-5 function requires phosphorylation of cytoplasmic tyrosine 482, but its UNC-40-independent functions also require a region between the ZU-5 and death domains.
Members of the UNC-5 protein family are transmembrane receptors for UNC-6/netrin guidance cues. To analyze the functional roles of different UNC-5 domains, we sequenced mutations in seven severe and three weak alleles of unc-5 in Caenorhabditis elegans. Four severe alleles contain nonsense mutations. Two weak alleles are truncations of the cytodomain, but one is a missense mutation in an extracellular immunoglobulin domain. To survey the function of different regions of UNC-5, wild-type and mutant unc-5::HA transgenes were tested for their ability to rescue the unc-5(e53) null mutant. Our data reveal partial functional requirements for the extracellular domains and identify a portion of the cytoplasmic juxtamembrane (JM) region as essential for rescue of migrations. When nine cytodomain tyrosines, including seven in the JM region, are mutated to phenylalanine, UNC-5 function and tyrosine phosphorylation are largely compromised. When F482 in the JM region of the mutant protein is reverted to tyrosine, UNC-5 tyrosine phosphorylation and in vivo function are largely recovered, suggesting that Y482 phosphorylation is critical to UNC-5 function in vivo. Our data also show that part of the ZU-5 motif is required for UNC-40-independent signaling of UNC-5.
Killeen
Marie
M
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.
Tong
Jeifei
J
Krizus
Aldis
A
Steven
Robert
R
Scott
Ian
I
Pawson
Tony
T
Culotti
Joseph
J
D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000502:physiology*; D002465:Cell Movement; D003593:Cytoplasm / Q000737:chemistry; D010766:Phosphorylation; D011956:Receptors, Cell Surface / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D015815:Cell Adhesion Molecules; D011956:Receptors, Cell Surface; C102174:UNC-40 protein, C elegans; C077588:UNC-5 protein, C elegans; D014443:Tyrosine