Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
* Author for correspondence (e-mail: garriga{at}berkeley.edu)
Accepted 22 September 2004
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SUMMARY |
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Key words: C. elegans, UNC-14, UNC-51, VAB-8, Axon guidance
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Introduction |
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While UNC-6/netrin and its receptors guide cells and growth cones along the
dorsoventral axis in many organisms, an equivalent global guidance system for
anteroposterior (AP) migrations has not been identified. Several molecules
involved in AP guidance, however, have been described. An AP gradient of Wnt4
was recently shown to guide commissural axons anteriorly along the rat spinal
cord (Lyuksyutova et al.,
2003). In C. elegans, two molecules have been implicated
in AP guidance. The anteriorly directed migrations of the right Q neuroblast
and its descendants require MIG-13, a conserved cell surface molecule. While
loss of MIG-13 is specific in its effects, expression of mig-13 from
a heat-shock promoter shifts the final positions of many migratory cells
anteriorly, whether they normally migrate anteriorly or posteriorly
(Sym et al., 1999
). C.
elegans VAB-8, by contrast, is both necessary and sufficient for
posteriorly directed migrations (Wightman
et al., 1996
; Wolf et al.,
1998
). vab-8 encodes two novel intracellular proteins,
VAB-8L and VAB-8S (Wolf et al.,
1998
). VAB-8L is 1066 amino acids long and contains an N-terminal
kinesin-like motor domain and a novel C terminus. VAB-8S lacks the
kinesin-like motor domain. VAB-8L functions in all vab-8-dependent
axon migrations, as well as in some cell migrations. VAB-8S functions in a
subset of vab-8-dependent cell migrations. Both forms of VAB-8 can
function cell autonomously in cell migration and axon guidance
(Wolf et al., 1998
). Based on
the sequence of its motor domain, VAB-8 has been placed in a subfamily of
divergent kinesin-like molecules (Miki et
al., 2001
).
How does vab-8 carry out its function in directing posterior
migrations? To identify proteins that interact with VAB-8, we conducted a
yeast two-hybrid screen using VAB-8L as bait and identified UNC-51.
unc-51 mutants exhibit axon outgrowth defects, though the defects are
not restricted to posteriorly directed axons
(Hedgecock et al., 1985;
McIntire et al., 1992
).
unc-51 mutant axons also have unusually large varicosities, and
electron microscopy revealed abnormal vesicles and cisternae-like structures
within the axons (McIntire et al.,
1992
).
The gene unc-51 encodes a serine/threonine kinase that is
expressed in all C. elegans neurons
(Ogura et al., 1994). Mouse
homologs of UNC-51, Unc51.1 and Unc51.2, are expressed in the developing and
mature cerebellum, as well as in cultured granule cells
(Tomoda et al., 1999
).
Transfection of a dominant negative form of Unc51.1 in cultured granule cells
inhibited neurite formation and extension, suggesting that the function of
unc-51 in neurite outgrowth is conserved
(Tomoda et al., 1999
).
UNC-51 can also bind to UNC-14, a novel C. elegans protein
involved in axon outgrowth (Ogura et al.,
1997). unc-51 and unc-14 mutants display similar
axon defects, although the phenotypes of unc-14 mutants are less
severe than those of unc-51 mutants
(McIntire et al., 1992
). Like
unc-51, unc-14 is expressed in most neurons
(Ogura et al., 1997
).
In this study, we describe the physical interactions between VAB-8 and UNC-51, and provide genetic evidence that the interactions of UNC-51 with VAB-8 and UNC-14 are necessary for posteriorly directed axon outgrowth. We also show that VAB-8 and UNC-14 can be substrates for UNC-51 kinase activity. We propose that UNC-51 regulates VAB-8 and UNC-14 to direct axon outgrowth posteriorly.
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Materials and methods |
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About 500,000 clones were screened using the full-length VAB-8 as bait. 46 clones were positive for both growth on histidine and ß-galactosidase activity. Only three out of the 46 clones required the presence of the library prey plasmids to activate the GAL4 promoter.
For the yeast two-hybrid binding assay, sequences of various domains of
UNC-51 were generated by PCR using pBLO (vector containing unc-51
cDNA) (described by Ogura et al.,
1994) as template. The resulting PCR products were subcloned into
the pACTII vector (Clontech).
In vitro binding
Sequences of various domains of VAB-8 were generated by PCR using pV8SL and
subcloned into pCITE-4a(+) vector (Novagen). unc-14 sequences were
amplified by PCR from pR4BK1, a plasmid containing the full-length
unc-14 cDNA (Ogura et al.,
1997) and subcloned into the pCITE-4a(+) vector. unc-51
sequences were amplified by PCR from pBLO and subcloned into pGEX-4T-2 vector
to generate GST fusion proteins (Pharmacia Biotech).
GST fusion proteins were expressed in E. coli, strain
BL21(pLys) (Studier et
al., 1990), and purified and bound to glutathione-Sepaharose 4B
beads according to the manufacturer's protocol (Pharmacia Biotech). The
various bound fusion proteins were quantified by comparison to known proteins
on SDS-PAGE gels stained with GelCode Blue Stain Reagent (PIERCE). 10-20 µg
of bead-conjugated GST fusion proteins were exchanged into binding buffer [20
mM Hepes, 50 mM KCl, 2.5 mM MgCl2, 10% glycerol, 1 mM DTT, 0.2%
Triton X-100, 1% BSA, protease inhibitors (Calbiochem, 539134), pH 7.6] and
rocked for at least 30 minutes at 4°C before addition of in vitro
transcribed and translated protein fragments. In vitro transcription and
translation of VAB-8 protein fragments were carried out using the TnT Quick
Reticulocyte Lysate System (Promega, L1170). To the lysate system, 1 µg of
pCITE-VAB-8 plasmid was added as transcription template, and 20 µCi of
[35S]methionine (>1000 Ci/mmole) was added to label the protein
products. Freshly synthesized protein fragments were added to bead-bound GST
fusion proteins and allowed to bind overnight at 4°C. The beads were then
washed four times in wash buffer (10 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 0.2%
Triton X-100, pH 7.5) and resuspended in 2x sample buffer (125 mM Tris
pH 6.9, 20% glycerol, 4.2% SDS, 3% 2-mercaptoethanol, 1% bromophenol
blue). Proteins were denatured at 85-90°C for 10 minutes and
separated on 12% or 18% SDS-PAGE gels. The gels were dried onto Whatman paper
and exposed to film (Kodak Biomax MR) overnight.
C. elegans strains
Strains were maintained at 20°C as described by Brenner
(Brenner, 1974). The mutations
used in this work were: unc-14(e866)I
(Ogura et al., 1997
),
vab-8(ev411)V (Wightman et al.,
1996
; Wolf et al.,
1998
), unc-51(e369)V
(Brenner, 1974
;
Ogura et al., 1994
), and
unc-51(e1120)V (Ogura et al.,
1994
).
The promoter::gene fusion DNA constructs were injected into adult
hermaphrodites and maintained as extrachromosomal arrays
(Mello et al., 1991).
ceh-23 promoter constructs
GFP is expressed in the CAN cells of animals bearing the
Pceh-23::gfp transgene (pTF1). pTF1 was constructed by cutting a 7 kb
ceh-23 promoter region with SphI and SmaI and
subcloning the fragment into pPD95.77 (Fire lab 1995 vector kit). A solution
containing 50 ng/µl of pTF1 and 50 ng/µl of pRF4
[rol-6(su1006)] was injected into the wild type to produce the
extrachromosomal array gmEx217.
All the other ceh-23 promoter constructs were generated by PCR amplification and by subcloning into the SmaI site of pTF1.
Cell autonomy experiments
A solution containing 1 ng/µl pceh-23::vab-8L::gfp and 100 ng/µl pTF1
was injected into vab-8 (ev411) to produce the extrachromosomal array
gmEx294. A solution containing 1 ng/µl pceh-23::unc-51::gfp and
100 ng/µl pTF1 was injected into unc-51(e369) to generate the
extrachromosomal array gmEx278.
Peptide expression experiments
pceh-23::vab-8(332-514)::gfp contains the cDNA sequences of VAB-8 that
encode amino acids 332-514. These sequences were subcloned into pTF1 as
described above. A solution containing 50 ng/µl
pceh-23::vab-8(332-514)::gfp, 50 ng/µl pTF1 and 10 ng/µl pmyo-2::gfp was
injected into wild-type hermaphrodites to generate the extrachromosomal array
gmEx266. pceh-23::unc-51(451-856)::gfp contains sequences
corresponding to amino acids 451 to the C-terminal end (aa 856) of UNC-51. A
solution containing 100 ng/µl pceh-23::unc-51(451-856)::gfp, 50 ng/µl
pTF1 and 20 ng/µl pflp-1::gfp (a gift from Chris Li) was injected into wild
type to generate extrachromosomal array gmEx264.
vab-8 overexpression
To overexpress vab-8, a solution containing 100 ng/µl pFWV8LG,
100 ng/µl pTF1 and pRF4 [rol-6(su1006)] DNA was injected into wild
type to generate the extrachromosomal array gmEx230. pFWV8LG is a
gene-cDNA fusion of vab-8 that expresses the VAB-8 long form from the
vab-8 promoter (Wolf et al.,
1998).
CAN axon scoring
To score CAN axons, L4 hermaphrodites were immobilized in 5% sodium azide
and viewed under the 40x objective using a reticule (Zeiss).
Measurements were taken of: the distance between the CAN cell body and the
center of the vulva; the distance between the center of the vulva and the
PHA/B neurons; and the length of the CAN's posterior axon. Percentage of
migration was determined by dividing the CAN posterior axon length by the
distance between the CAN cell body and the PHA/B neurons. CAN cell bodies were
frequently misplaced anteriorly in many of the strains. To ensure that this
defect did not affect the scoring of axon length, only axons whose cell bodies
were located within 40 µm of the center of the vulva were scored as data
points. The axons were scored as having a misrouting defect if they had turned
away from their normal trajectory and migrated in the wrong direction.
Posterior axons extending 95-100% of the distance to the PHA/B neurons were considered wild type and were grouped together in the most posterior box of the scoring charts. We used a two-tailed Z test to determine if the percentages of wild-type axons were statistically different between two strains (two sample proportions, WebStat 3.0 program on http://www.webstatsoftware.com).
COS cell transfection and sample preparation
PCR-generated full-length vab-8 cDNA sequences were subcloned into
vector pcDNA/myc-HisB (Invitrogen) to generate pcDNA-vab-8-myc. PCR-generated
sequences of full-length unc-51 cDNA plus an extra C-terminal FLAG
tag were subcloned into vector pFLAG-CMV-5a (Sigma) to generate
pCMV-unc-51-FLAG. PCR-generated sequences of full-length unc-14 cDNA
plus a C-terminal HA tag were subcloned into vector pcDNA3 (Invitrogen) to
generate pcDNA-unc-14-HA.
COS cells were grown in Dulbecco's modified Eagle's medium (DMEM) + 10%
fetal calf serum at 37°C. Transfections were carried out using the
LipofectAminePlus Reagent (Invitrogen, 10964-013). For each 10-cm diameter
dish, 5 µg of pcDNA-vab-8-myc, 5 µg pcDNA-unc-14-HA, 3 µg
pCMV-unc-51-FLAG, 3 µg pCMV-unc-51(K39R)-FLAG, or 3-6 µg
pCMV-unc-51(AIKAI)-FLAG was used. Cells were harvested about 24 hours
after transfection. Cells were detached from dishes by treatment with trypsin,
washed with 1x PBS, resuspended in 0.7-1 ml cold 1% Triton X-100 lysis
buffer (1% Triton X-100, 50 mM Tris, 300 mM NaCl, 5 mM EDTA, protease
inhibitors, pH 8.0 at 4°C), and allowed to lyse on ice for 15 minutes.
To immunoprecipitate (IP) the UNC-51-FLAG fusion protein, 2.5-3 µl of anti-FLAG M2 monoclonal antibody (Stratagene, 200472) was added per ml of pre-cleared cell extract. To IP the VAB-8-Myc fusion protein, 12 µl of monoclonal anti-Myc antibody (Covance, MMS-150R) was added. To IP the UNC-14-HA fusion protein, 12 µl of monoclonal anti-HA antibody (Covance, MMS-101R) was added. Samples were nutated at 4°C for 2 hours to allow antibody binding, and then 40-120 µl resuspended protein G-Sepharose beads (Pharmacia Biotech) were added to capture the antibody-protein complex. The mixture was nutated for 30 minutes at 4°C. The beads were washed with 0.1% Triton wash buffer (0.1% Triton X-100, 50 mM Tris, 300 mM NaCl, 5 mM EDTA, pH 8.0 at 4°C) and exchanged into appropriate buffers for the subsequent reactions.
In vitro phosphatase and kinase reactions
IP bead samples were washed with phosphatase (PPase) wash buffer (50 mM
Tris, 0.1 mM EDTA, pH 7.2), transferred to 1x PPase reaction
buffer (50 mM Tris-HCl, 0.1 mM Na2EDTA, 5 mM dithiothreitol, 0.01%
Brij 35, 2 mM MnCl2, pH 7.5), and divided into four aliquots of 50
µl. 1 µl
PPase (NEB, P0753S) +/ PPase inhibitor (10 mM
Na3VO4 or 50 mM EDTA) was added to the IP protein
samples and incubated at 30°C for 15 minutes.
To assay for UNC-51 autophosphorylation, immunoprecipitated UNC-51,
UNC-51(K39R) and UNC-51(AIKAI) were exchanged into kinase buffer (50 mM
Hepes, 10 mM MgCl2, pH 7.5). The reaction was carried out in a 50
µl volume of kinase buffer plus 0.2 mM ATP, 10 mM sodium orthovanadate,
protease inhibitors and 10 µCi [
-32P]ATP (3000
Ci/mmol). Samples were incubated at 25°C for about 30 minutes and
washed 4x with 0.1% Triton wash buffer. The bead pellets were
resuspended with 40 µl 2x sample buffer.
To assay for UNC-14 phosphorylation, GST-UNC-14 fusion protein (encoded by
pGEX-unc-14-HA) was expressed and purified as described above and washed
3x with kinase buffer. UNC-51-FLAG protein was immunoprecipitated from
COS cell extract as described above and eluted with two washes of 120 µl of
1 mg/ml 3x FLAG peptide in elution buffer (20 mM Hepes, 100 mM KCl, 0.2
mM EDTA, 0.1% NP40, protease inhibitors, pH 7.7) at room temperature for 25
minutes each. Kinase reactions were carried out with 70 µl of UNC-51-FLAG
elute plus 0.2 mM ATP, 10 mM MgCl2 and 20 µCi
[-32P]ATP. Samples were incubated at 25°C for about 30
minutes and washed 4x with 0.1% Triton wash buffer.
Protein samples were denatured at 85-90°C for 10 minutes, and separated
on 8% SDS-PAGE gel. The gel was dried onto Whatman paper and signals were
processed with a phospho-imager (Molecular Dynamics, Storm 820). Incorporation
of [-32P]ATP was determined by measuring band intensity
using the Image Quant 5.2 software (Molecular Dynamics).
unc-51 RNAi
unc-51 cDNA sequences corresponding to aa 1-410 were subcloned
into the L4440 vector (containing two T7 transcription start sites, Fire lab
1999 vector kit). The resulting construct was L4440-U51RNAi1. dsRNA was
prepared and injected using standard procedures (Fire Lab RNAi protocol,
Version 1.0,
www.ciwemb.edu/pages/firelab.html).
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Results |
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Using a yeast two-hybrid binding assay, we defined the C-terminal 106 amino
acids of UNC-51 as sufficient to bind VAB-8
(Fig. 1A). Using a GST pulldown
assay, we showed that the interaction between VAB-8 and UNC-51 was direct and
defined two regions of VAB-8 that can bind UNC-51
(Fig. 1B,C). Ogura et al.
(Ogura et al., 1997) had shown
that the C-terminal 401 amino acids of UNC-51 bound UNC-14, and we found that
the same 106 amino acid region of UNC-51 that bound VAB-8 also bound UNC-14
(Fig. 1D; data not shown).
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We analyzed vab-8(ev411) unc-51(e369) double mutants and observed
that the CAN posterior axon defect was worse in the double than in either
single mutant (data not shown). The e369 mutation does not appear to
eliminate unc-51 activity as the phenotypes it caused could be
enhanced by unc-51 RNAi (data not shown). Thus, we could not use the
increased severity of the vab-8(ev411) unc-51(e369) mutant phenotypes
to determine whether VAB-8 and UNC-51 act in the same or separate pathways. We
also examined the AVK axons, which extend posteriorly along the ventral nerve
cord (White et al., 1986). In
vab-8 or unc-51 mutants, AVK axons stopped short at various
points along the ventral nerve cord (Wolf
et al., 1998
) (data not shown). In vab-8 unc-51 double
mutants, the AVK axon defect was more severe (data not shown).
vab-8 and unc-51 act cell autonomously
If the interaction between VAB-8 and UNC-51 is functionally important for
axon guidance, both proteins should be required in the same cell. To test
whether both genes function cell autonomously, we expressed the long form of
vab-8 in the CAN from the ceh-23 promoter and found that
this transgene partially rescued both the early termination and misrouting
defects of vab-8(ev411) posterior CAN axons
(Fig. 3; data not shown).
Expression of an unc-51 cDNA from the ceh-23 promoter
rescued both the anterior and posterior CAN axon extension defects, as well as
the axon varicosity defect of unc-51 mutants
(Fig. 3; data not shown). Even
though these transgenes also expressed vab-8 or unc-51 in
several sensory neurons, it seems unlikely that expression from these cells
was responsible for rescue of the CAN defects. First, these sensory neurons
project their axons along trajectories that are distinct from those of the CAN
axons. Second, neither VAB-8 nor UNC-51 is a secreted molecule that can act
over a long distance. Thus, we propose that both VAB-8 and UNC-51 function in
the CAN cells to promote the directed outgrowth of their axons.
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The posterior axon defects were partially suppressed by the simultaneous expression of both protein peptides (gmEx266 and gmEx264; Fig. 4B), which presumably bind to each other to allow more of the endogenous wild-type VAB-8 and UNC-51 proteins to interact. To rule out the possibility that this suppression was due to increased levels of the ceh-23 promoter in animals bearing both transgenes, we also constructed animals that had gmEx266 and an additional array that contained an equivalent amount of the ceh-23 promoter as gmEx264. Addition of this array had no effect on the CAN defects induced by gmEx266, ruling out the possibility that suppression was caused by the depletion of ceh-23 transcriptional activators (data not shown). Taken together, the peptide expression experiments suggest that the VAB-8 and UNC-51 interaction in the CAN neuron is required for the directed outgrowth of its posterior axon.
The VAB-8 peptide also disrupts the interaction between UNC-51 and UNC-14
If the peptides only disrupted the interaction between VAB-8 and UNC-51, we
reasoned that their expression should not enhance a mutant completely lacking
vab-8 or unc-51 function. Since no unc-51 null
allele exists (see above), we expressed the VAB-8 peptide in
vab-8(ev411), a mutant that lacks VAB-8L function
(Wightman et al., 1996;
Wolf et al., 1998
). We found
that expression of the VAB-8 peptide could further enhance the posterior CAN
axon defect of the mutant (Fig.
4B). One possible explanation for this enhancement is that this
peptide interfered with the binding of UNC-51 to molecules other than VAB-8.
One candidate molecule is UNC-14, which was shown by Ogura et al. to bind the
C-terminal half of UNC-51, (Ogura et al.,
1997
). We refined the UNC-14-binding region to the C-terminal 106
amino acids of UNC-51 (data not shown), the same region that binds to VAB-8.
We analyzed two alleles of unc-14, e866
(Fig. 4B) and e1119
(data not shown). Both alleles are likely to be nulls as they are nonsense
mutations near the beginning of the open reading frame. Both unc-14
mutations caused a CAN posterior but not an anterior truncation defect.
To test the possibility that the VAB-8 peptide disrupted interactions between UNC-51 and both VAB-8 and UNC-14, we asked whether expression of the VAB-8 peptide would enhance the posterior CAN axon defect of an unc-14(e866); vab-8(ev411) double mutant. unc-14(e866) is a nonsense mutation at amino acid 106, and thus eliminates the UNC-51-binding domain. As expected, the posterior CAN axon defect was more severe in the unc-14(e866); vab-8(ev411) double mutant than in either single mutant alone, demonstrating that VAB-8 and UNC-14 function in distinct processes required for axon outgrowth (Fig. 4B). Expression of the VAB-8 peptide, however, did not enhance the posterior axon defect of the double mutant (Fig. 4B). This result is consistent with the interpretation that the VAB-8 peptide disrupted the interaction of UNC-51 with both VAB-8 and UNC-14, and in the absence of both of these proteins, expression of the VAB-8 peptide had no effect. The lack of enhancement did not result from an inability to generate a more severe phenotype, as we have observed more severe axon defects in other mutant backgrounds (data not shown). It is noteworthy that the VAB-8 peptide effect was weakly suppressed in the unc-14 mutant background (Fig. 4B). This result could be explained if the interaction of UNC-51 with VAB-8 plays a larger role in CAN axon outgrowth, so that in the absence of UNC-14, more UNC-51 protein is available to interact with VAB-8.
Overexpression of vab-8 suppresses the posterior CAN axon defect of unc-51 mutants
Our results indicate that the physical interaction between VAB-8 and UNC-51
is important for the functions of these proteins in axon outgrowth. One of
these proteins could activate the other, or alternatively, one could inhibit
the function of the other. To distinguish between these possibilities, we
overexpressed vab-8 in unc-51 mutants and found that
vab-8 overexpression suppressed the posterior CAN axon defect of
unc-51(e369) and unc-51(e1120) mutants
(Fig. 5). The vab-8
mini gene used in these experiments was previously shown to result in VAB-8
overexpression when present in extrachromosomal arrays
(Wolf et al., 1998). Our
result suggests that vab-8 and unc-51 act in a positive
regulatory pathway. Ordinarily, we would be unable to order the genes based on
this genetic result because neither of the unc-51 alleles appears to
be null. None of the characterized unc-51 mutations is a large
deletion, early nonsense or frameshift mutation, or is in the kinase domain
(Ogura et al., 1994
).
Furthermore, unc-51 RNAi was able to enhance the CAN axon defect of
unc-51(e369) (data not shown), the most severe unc-51 mutant
phenotypically and molecularly (Ogura et
al., 1994
) (data not shown). e369, however, contains an
amber nonsense mutation predicted to eliminate the VAB-8-binding site in
UNC-51. The ability of vab-8 overexpression to suppress this
unc-51 mutation demonstrates that increased VAB-8 levels can bypass
the requirement for the VAB-8 and UNC-51 interaction, and suggests that
vab-8 functions downstream of unc-51. We could not determine
the effect of unc-51 overexpression on vab-8, because the
transgenic array containing unc-51 full-length cDNA did not express
UNC-51 stably.
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Discussion |
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During the course of these experiments, we discovered that a peptide predicted to disrupt interactions between UNC-51 and VAB-8 also disrupted the interaction between UNC-51 and UNC-14. We showed that UNC-51 could phosphorylate UNC-14, suggesting that UNC-51 regulates the activity of UNC-14. To our knowledge, VAB-8 and UNC-14 are the first proteins identified as targets of the UNC-51 kinase family. Based on our genetic and molecular results, we propose that VAB-8 and UNC-14 mediate the effects of UNC-51 to regulate posteriorly directed axon outgrowth.
The UNC-51 protein family appears to function in membrane trafficking
UNC-51 is a conserved kinase with homologs found in organisms as diverse as
yeast and humans. The yeast homolog of UNC-51, Apg1p, is required for the
cytoplasm-to-vacuole (Cvt) targeting pathway, which is involved in the
transport of the vacuolar hydrolase aminopeptidase I, and for the induction of
autophagy, a process that involves the delivery of organelles and cytoplasm to
the lysosome (for reviews, see Huang and
Klionsky, 2002; Noda et al.,
2002
; Reggiori and Klionsky,
2002
). Both the Cvt pathway and autophagy require the de novo
synthesis of cup-shaped membrane cisternae, which elongate and fuse to form
double-membraned Cvt vesicles and autophagosomes. Apg1p, complexed with
different binding partners, is proposed to regulate the formation of these
membrane structures (Noda et al.,
2002
).
While VAB-8 and UNC-14 are the first putative phosphorylation targets of
UNC-51 identified, several other proteins have been shown to physically
interact with UNC-51 homologs. Humans have two
UNC-51-like kinases, ULK1 and ULK2
(Kuroyanagi et al., 1998;
Yan et al., 1998
;
Yan et al., 1999
). In a yeast
two-hybrid screen for proteins that interact with human ULK1, Okazaki et al.
(Okazaki et al., 2000
)
identified both the GABAA receptor associated protein (GABARAP) and
the Golgi-associated ATPase enhancer of 16 kDa (GATE-16). GABARAP was
originally identified as a protein that could physically interact with the
2 subunit of the GABAA receptor and was found to co-localize
with this receptor in cultured cortical neurons
(Wang et al., 1999
). GABARAP
shares similarity with microtubule-associated proteins and can bind
microtubules (MTs), raising the possibility that it provides a link between
MTs and the GABAA receptor
(Wang et al., 1999
). GATE-16
is an essential component for intra-Golgi transport and regulates SNARE
function (Muller et al., 2002
;
Sagiv et al., 2000
). GABARAP
and GATE-16 are related to one another, and to the yeast autophagic factor
Aut7p. Taken together, these observations suggest that UNC-51 homologs and
their interacting proteins could function in membrane dynamics and vesicle
trafficking.
As with C. elegans UNC-51, its mammalian homologs also regulate
neuronal development. Mouse Ulk1 (a.k.a. Unc51.1) has been implicated in
neurite outgrowth of cerebellar granule cells, suggesting that the function of
UNC-51 homologs in axon outgrowth is conserved
(Tomoda et al., 1999). In a
yeast two-hybrid screen, Unc51.1 was found to interact with SynGAP, a Ras GAP,
and syntenin, a PDZ domain-containing protein
(Tomoda et al., 2004
). SynGAP
was also found to be a GAP for Rab5, and syntenin binds Rab5. Unc51.1 can
downregulate SynGAP, leading to Rab5 activation, suggesting that a complex of
Unc-51.1, SynGAP and syntenin controls axon outgrowth through its regulation
of Rab5 activity (Tomoda et al.,
2004
). Since axon outgrowth requires membrane synthesis and
vesicle trafficking to deliver cellular components necessary for the formation
and steering of the growth cone, UNC-51 and its homologs could regulate these
processes.
UNC-51 and UNC-14 also function in membrane trafficking
The requirement of Apg1p in autophagy and the interactions between UNC-51
homologs and proteins involved in receptor and membrane trafficking, suggest a
conserved role for UNC-51-like kinases. The Unc-51 phenotypes described in
C. elegans support this hypothesis. unc-51 mutant animals
have axons that form unusually large varicosities that accumulate internal
membrane structures, including abnormal vesicles and cisternae-like structures
(McIntire et al., 1992). The
same study found similar but less severe defects in unc-14 mutants.
Based on these observations, McIntire et al.
(McIntire et al., 1992
)
proposed that UNC-51 and UNC-14 functioned together in membrane trafficking.
The functional link between UNC-51 and UNC-14 was strengthened by the ability
of the two proteins to physically interact
(Ogura et al., 1997
). Our
results suggest that UNC-51 can regulate the activity of UNC-14.
UNC-14 contains a RUN domain (Callebaut
et al., 2001). While the function of this domain is unknown,
several RUN domain-containing proteins are linked to the functions of Rab and
Rap GTPases, and appear to function in vesicular trafficking
(Callebaut et al., 2001
;
Mari et al., 2001
;
Yang et al., 2002
), supporting
the hypothesis that UNC-14 regulates membrane trafficking.
The role of VAB-8 and UNC-51 in axon outgrowth
Our results suggest that VAB-8 mediates the function of UNC-51 in axon
guidance. The role of VAB-8 in posteriorly directed migrations, however,
remains enigmatic. Although VAB-8 may act within an UNC-51 pathway to mediate
vesicle transport, vab-8 mutants lack the prominent axon varicosities
displayed by unc-51 and unc-14 mutants. Alternatively,
UNC-51 could act with VAB-8 in a distinct process required for directed axon
outgrowth. VAB-8 has been placed into a subfamily of kinesin-like molecules
that includes Drosophila Costal2
(Miki et al., 2001). Although
this molecule appears to lack kinesin motor activity, it retains an ability to
bind to MTs and serves as a cytoplasmic tether for the ci transcription factor
in Hedgehog signaling (Sisson et al.,
1997
). Perhaps as proposed for GABARAP, VAB-8 could provide a link
between the cytoskeleton and guidance receptors. Given the specificity of
VAB-8 in regulating posteriorly directed migrations, one interesting
speculation is that VAB-8 regulates the activity of receptors involved in
posteriorly directed guidance.
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ACKNOWLEDGMENTS |
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