In C. elegans most posteriorly directed cell and growth cone migrations require
vab-8, a gene that encodes at least two protein products known as VAB-8L and VAB-8S. VAB-8L is a 1066 amino acid protein that contains an N-terminal kinesin-like motor domain and functions in
vab-8-dependent growth cone migrations. VAB-8S is colinear with the C-terminal half of VAB-8L and lacks the kinesin-like motor domain. VAB-8S is necessary for certain
vab-8-dependent cell migrations. To identify VAB-8-interacting proteins, we conducted a yeast two-hybrid screen using full length VAB-8L as the bait. One protein identified was UNC-51, a serine/threonine kinase required for proper axon outgrowth (Ogura et al., 1994). UNC-51 was found to interact with another novel protein, UNC-14 (Ogura et al. 1997). We have found that both VAB-8 and UNC-14 bind the C-terminal 100 amino acids of UNC-51 both in yeast and in vitro. Several observations suggest that VAB-8 and UNC-51 also interact in C. elegans. First,
vab-8 and
unc-51 mutants have been shown to display axon outgrowth defects. We used a
ceh-23::GFP transgene to assay the axons of CAN neurons, which extend growth cones both anteriorly and posteriorly. In
vab-8 mutants, only posteriorly directed axon outgrowth was defective, whereas in
unc-51 mutants, both anteriorly and posteriorly directed outgrowth were defective. Second, both genes have been shown to be expressed in neurons that require
vab-8 function. Using the
ceh-23 promoter to drive
vab-8 or
unc-51 expression, we have shown that both genes act cell autonomously for CAN axon migrations. Finally, misexpression of the UNC-51-binding domain of VAB-8 under control of the
ceh-23 promoter resulted in a highly penetrant CAN posterior growth cone migration defect, a Vab-8 phenotype, presumably by interfering with UNC-51 binding to wild-type VAB-8L. Misexpression of the VAB-8-binding domain of UNC-51 also produced CAN axon outgrowth defects. The misexpression phenotypes are suppressed by simultaneous misexpression of both protein fragments. We propose that in this experiment the VAB-8 and UNC-51 fragments associate with each other, thereby allowing endogenous full-length VAB-8 and UNC-51 to interact and carry out their function in directing axon outgrowth. The protein interactions and similar mutant phenotypes suggest that VAB-8 and UNC-51 act in the same pathway. Overexpression of
vab-8 suppressed the posterior axon outgrowth defects of
unc-51 mutants, indicating a positive regulatory relationship between the two proteins. The same
vab-8 overexpression array also enhanced the anterior axon outgrowth defects of
unc-51. We are also taking biochemical approaches to determine the relevance of VAB-8 and UNC-51 interaction. We have demonstrated that VAB-8 is phosphorylated when co-expressed with UNC-51 in COS cells. We are testing whether VAB-8 is phosphorylated directly by UNC-51, and whether VAB-8 phosphorylation is important for its function in directing axon migration.