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Molecular and Cellular Biology, July 2000, p. 4736-4744, Vol. 20, No. 13
Department of
Pharmacology1 and Department of Cell
Biology,2 Skirball Institute of Biomolecular
Medicine, New York University Medical Center, New York, New York
Received 29 November 1999/Returned for modification 20 February
2000/Accepted 27 March 2000
We have previously shown that the Ste20 kinase encoded by
misshapen (msn) functions upstream of the c-Jun
N-terminal kinase (JNK) mitogen-activated protein kinase module in
Drosophila. msn is required to activate the
Drosophila JNK, Basket (Bsk), to promote dorsal closure of
the embryo. A mammalian homolog of Msn, Nck interacting kinase,
interacts with the SH3 domains of the SH2-SH3 adapter protein Nck. We
now show that Msn likewise interacts with Dreadlocks (Dock), the
Drosophila homolog of Nck. dock is required for
the correct targeting of photoreceptor axons. We have performed a
structure-function analysis of Msn in vivo in Drosophila in order to elucidate the mechanism whereby Msn regulates JNK and to
determine whether msn, like dock, is required
for the correct targeting of photoreceptor axons. We show that Msn
requires both a functional kinase and a C-terminal regulatory domain to
activate JNK in vivo in Drosophila. A mutation in a PXXP
motif on Msn that prevents it from binding to the SH3 domains of Dock
does not affect its ability to rescue the dorsal closure defect in
msn embryos, suggesting that Dock is not an upstream
regulator of msn in dorsal closure. Larvae with only this
mutated form of Msn show a marked disruption in photoreceptor axon
targeting, implicating an SH3 domain protein in this process; however,
an activated form of Msn is not sufficient to rescue the
dock mutant phenotype. Mosaic analysis reveals that
msn expression is required in photoreceptors in order for
their axons to project correctly. The data presented here genetically
link msn to two distinct biological events, dorsal closure
and photoreceptor axon pathfinding, and thus provide the first evidence
that Ste20 kinases of the germinal center kinase family play a role in
axonal pathfinding. The ability of Msn to interact with distinct
classes of adapter molecules in dorsal closure and photoreceptor axon
pathfinding may provide the flexibility that allows it to link to
distinct upstream signaling systems.
Ste20 kinases play a critical role
in mediating activation of the c-Jun N-terminal kinase (JNK)
mitogen-activated protein (MAP) kinase pathway (23). Both
genetic and biochemical evidence has indicated that Ste20 kinases
mediate JNK activation by functioning as MAP kinase kinase kinase
kinases (MAP4Ks) (17, 23). Two families of Ste20 kinases
have been identified in mammalian cells and lower organisms, the
p21-activated protein kinase (Pak) and germinal center kinase (GCK)
families (16, 26, 28). In contrast to Paks, GCK family
kinases lack binding motifs for Rho family GTPases. In addition, unlike
PAKs, which have a C-terminal kinase and an N-terminal regulatory
domain, GCK family members contain an N-terminal kinase and a
C-terminal regulatory domain (5, 28).
We have recently placed the Ste20 kinase encoded by
misshapen (msn), a member of the GCK family of
Ste20 kinases, genetically upstream of the JNK MAP kinase pathway in
Drosophila (44). The failure to activate JNK in
Drosophila leads to embryonic lethality due to defects in
dorsal closure; in embryos with mutations in components of the JNK
pathway, the lateral epithelial sheets fail to elongate and migrate
dorsally (29). This coordinated movement of the lateral
epithelia functions to internalize the amnioserosa and connect the two
sides of the embryo (6, 48). A number of signaling molecules
that are critical for stimulating dorsal closure in
Drosophila can now be ordered on a signaling pathway. The
most proximal molecule identified on this pathway is the Ste20 kinase
encoded by msn (44). Msn likely functions as a
MAP4K and activates the JNK pathway by activating a yet-to-be-defined Drosophila MAP3K. A Drosophila MAP3K would likely
phosphorylate and activate the JNK kinase Hemipterous (Hep), which in
turn phosphorylates and activates Drosophila JNK (DJNK),
encoded by basket (bsk) (13, 35, 41).
DJNK phosphorylates and activates DJun, which in turn cooperates with
DFos to stimulate transcription of dpp, a member of the
transforming growth factor- The mammalian homolog of Msn, Nck interacting kinase (NIK), was
identified in a two-hybrid screen for proteins that interact with the
SH3 domains of the adapter protein Nck (43). Nck is a
ubiquitously expressed protein composed entirely of a single SH2 and
three SH3 domains and thus fits into the adapter class of signaling
molecules (32, 38). Nck and related adapter proteins, such
as Grb2 and Crk, are thought to regulate signaling pathways downstream
of tyrosine kinases by coupling catalytic subunits, bound to their SH3
domains, to phosphotyrosine-containing proteins that interact with
their SH2 domains (10, 32, 38).
Recent studies with Drosophila have shed light on the likely
function of Nck in mammalian cells. The Drosophila homolog
of Nck, encoded by dreadlocks (dock), was
identified in a genetic screen for proteins that are critical for the
correct targeting of photoreceptor axons (11). The
Drosophila compound eye is composed of about 800 repeated
units called ommatidia, with each ommatidial unit being composed of
eight neurons, R1 to R8 (8). The axons from each ommatidium
form a single fascicle and project in a specific topographic pattern
into the optic ganglia. R1 to R6 axonal growth cones terminate in the
lamina, whereas R7 and R8 growth cones project through the lamina and
terminate in the second optic ganglion, the medulla. In dock
mutants, photoreceptor axons show abnormal clumping and crossing over,
and some axons of R1 to R6 fail to terminate in the lamina and project
abnormally into the medulla. In addition, the R7 and R8 axons in
dock mutants fail to form an even array in the medulla and
lack expanded growth cones (11, 33). It has been proposed
that Dock provides a link between a receptor tyrosine kinase located at
the axonal growth cone and a downstream signaling pathway by targeting
a catalytic molecule bound to its SH3 domain to tyrosine-phosphorylated proteins. The Ste20 kinase Pak has recently been shown to function downstream of Dock in Drosophila (18) and may be
such a catalytic molecule. In this study, we have performed a
structure-function analysis of Msn in vivo in Drosophila in
order to elucidate the mechanism whereby Msn regulates JNK and to
determine whether msn, like dock and
PAK, is required for the correct targeting of photoreceptor axons.
Constructs, mutagenesis, and JNK assays.
The
kinase-defective mutant Msn(KD) contains the substitution of aspartic
acid for asparagine at position 160 in the kinase domain and has been
described previously (44). The gene for a truncated protein
lacking its C-terminal regulatory domain, Msn( GST fusions, in vitro binding studies, and yeast two-hybrid
assays.
Glutathione S-transferase (GST) fusion proteins
were made using the PCR as previously described (43).
Briefly, oligonucleotides flanking the regions of interest and
containing appropriate restriction sites were synthesized and used to
amplify by PCR the region of interest. The products obtained were
subcloned into PGEX3X, and GST fusion proteins were isolated as
previously described (43). To assay binding of Msn to the
various GST fusions, myc epitope-tagged Msn was overexpressed in 293 cells. Five hundred micrograms of lysates containing myc epitope-tagged
Msn was incubated with 5 µg of the various GST fusions coupled to
glutathione-agarose beads at 4°C for 4 h. After four washes with
lysis buffer, bound proteins were separated by sodium dodecyl
sulfate-8% polyacrylamide gel electrophoresis and immunoblotted with
the anti-myc antibody 9E10.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Ste20 Kinase Misshapen Regulates Both
Photoreceptor Axon Targeting and Dorsal Closure, Acting Downstream
of Distinct Signals
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
family (12, 20, 22, 34, 36).
Dpp then acts on cells adjacent to the leading-edge cells to promote
their elongation, probably through alterations in the cytoskeleton
(1, 4, 24, 37a).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
CT), was amplified by
PCR using oligonucleotides corresponding to regions that flanked amino
acids (aa) 1 to 772 of full-length Msn and that contained appropriate
restriction sites. The genes for Msn(P656A, P659A), Msn(
332-667),
and Msn-F were made using overlapping PCR as previously described
(21). To generate the mutation for Msn(P656A, P659A), two
complementary primers in opposite orientations spanning the sites of
mutation were synthesized. The msn sequence 5' to the
mutation was then amplified using the mutant primer in the reverse
orientation together with a primer 5' to a convenient restriction site
in msn, and the msn sequence 3' to the mutation
was amplified using the complementary mutant primer in the forward
orientation together with a primer containing the 3' end of full-length
msn and a myc epitope tag. The two fragments were then mixed
and used as a template for a second round of PCR using only the
nonmutagenic flanking oligonucleotides. The same approach was used to
mutate the other PXXP motifs of Msn. A similar strategy was used to
generate the mutation for Msn(332-667), with the exception that the
complementary mutant primers synthesized for the first PCR contained
sequences flanking the regions deleted in the sequence corresponding to
Msn(332-667). To add the last 20 aa of Ras containing the Ras
farnesylation sequence to the C terminus of Msn, complementary primers
containing the 3' end of msn and the 5' end of the gene for
the Ras farnesylation sequence were synthesized (Msn-Ras). Msn was then
amplified using the msn-Ras primer in the reverse orientation together
with the same 5' nonmutant primer mentioned above, and the gene for the
Ras farnesylation sequence was amplified using the complementary
msn-Ras primer in the forward orientation with a primer containing the
3' end of the Ras gene. The two fragments were then mixed, and a second PCR was performed as described above to generate Msn-F. All
msn constructs were initially cloned into the mammalian
expression vector pRK5, and all PCR-generated fragments were subjected
to DNA sequencing to eliminate the possibility that errors were
introduced during the PCR. In addition, all constructs encoded proteins
of the correct molecular size when transfected into 293 cells (data not shown).
Ectopic expression in Drosophila.
To express wild-type
msn [msn(wt)] and the msn
mutants in Drosophila, the GAL4/upstream activation sequence
(UAS) system was used (3). All constructs were subcloned
from pRK5 into the vector pUAST. Germ line transformations were then
performed using standard techniques, and transgenic lines containing
the various msn constructs on chromosomes 2 and 3 were
obtained (42). To induce expression of msn in the
ectoderm, brain, and eye disc, UAS-msn(wt);
msn102/SM6.TM6B flies were crossed with
69B-GAL4 msn102/TM6B flies
(44). A similar strategy was used for the other UAS
constructs. Two or three independent insertions of each UAS construct
were evaluated. To rescue the defect in targeting of photoreceptor
axons in dockP1 mutants,
UAS-msn(wt),
dockP1/Gla,Bc,Elp, UAS-msn-F,
dockP1/Gla, Bc,Elp, and
UAS-msn(332-667),
dockP1/Gla,Bc,Elp flies were crossed with
ELAV-GAL4 dockP1/Gla,Bc,Elp flies, and
photoreceptor axonal targeting of third-instar larvae was analyzed by
staining eye-brain complexes with monoclonal antibody (MAb) 24B10
(9).
Immunohistochemistry. Photoreceptor projection patterns in third-instar larval eye-brain complexes were visualized by MAb 24B10 (1:100) and a horseradish peroxidase-coupled goat anti-mouse secondary antibody.
Genetic mosaic analysis. To generate msn mutant clones in the eye disc, FRT80, msn/TM6B females were crossed with y, w, eyFLP1; FRT80, M(3)67C/TM6B males, and female larvae were analyzed. To generate dockP1 or bsk1 mutant clones, FRT40, dockP1 (or bsk1) males were crossed with y, w, eyFLP1; FRT40, M(2)24F/CyO females. To produce hep, lic mutant clones, FRT18, H6/FM6 females were crossed with FRT18, ormlacZ; hs FLP38 males and heat shocked at 37°C during the first and second instars, and female third-instar larvae were analyzed.
Cuticle preparations. Cuticle preparations were performed as previously described (44). Briefly, embryos were collected on yeast agar plates and dechorionated in 100% bleach, rinsed in water, and then fixed for 10 min at 65°C in a solution containing acetic acid and glycerol at a ratio of 3:1. Embryos were then mounted in Hoyer's medium and incubated for 24 h at 65°C.
ATF2 luciferase activity.
A fusion protein consisting of
ATF2 (aa 1 to 505) and the GAL4 DNA binding domain was expressed in 293 cells either alone or together with NIK (14).
Transcriptional activation of ATF2 was measured by cotransfecting a
luciferase reporter plasmid containing five GAL4 DNA binding sites. All
transfections were standardized by cotransfecting a control plasmid
expressing -galactosidase (Promega).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The kinase activity and C-terminal domain of Msn are required for
JNK activation in the Drosophila embryo.
The
requirement for msn to stimulate JNK activation and dorsal
closure in the Drosophila embryo afforded us a unique
opportunity to identify the domains of Msn that are essential for it to
couple to JNK activation in vivo (44). Msn can be divided
into three regions, an N-terminal kinase domain, a C-terminal domain
that is conserved in several GCK family members, and a region between the kinase and C-terminal domains that probably couples to upstream regulators (Fig. 1C). Previously, we have
demonstrated that the mammalian homolog of Msn, NIK, requires both a
functional kinase and a C-terminal regulatory domain to activate JNK in
293 cells (43). To determine whether these domains are also
critical for Msn to activate JNK in vivo in Drosophila, we
determined whether Msn(KD) or Msn(CT) could rescue the defect in
dorsal closure in msn mutant embryos. The
69B-GAL4 driver was used to express wild-type or mutant
forms of UAS-msn in the ectoderm of msn mutant embryos. Rescue was determined at the pupal stage by the absence of the
TM6B balancer chromosome marked with Tubby. We found that expression of wild-type msn was sufficient to rescue 100%
of msn mutant embryos to the pupal stage (Tables 1 and
2). In
contrast, none of the msn embryos rescued with Msn(KD)
survived to the pupal stage, and only 10% of msn embryos
rescued with Msn(CT) survived to the pupal stage (Tables 1 and 2).
The finding that some msn mutants are rescued with Msn(CT)
is consistent with our findings in 293 cells that C-terminally
truncated forms of NIK and Msn are able to partially activate JNK
(43, 44) (see Fig. 4). Similar results were obtained by
crossing the UAS-msn lines with a 69B-GAL4 driver
line containing a different inversion allele of msn
(msn172, as opposed to
msn102) (46), indicating that the
results obtained reflect a specific rescue of the loss of
msn function.
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/CT) were able to rescue
the defect in dorsal closure. The percentage of msn mutant
embryos expressing either of these constructs that were found to have a
defect in dorsal closure was about 14, similar to the number in a cross
lacking the GAL4 driver line (Tables 1 and 2). However, the finding
that a small percentage of msn mutant embryos rescued with
UAS Msn(CT), but not the control, survived to the pupal stage
suggests that Msn(CT) provides some signaling function that is
required for survival after hatching. It is not clear whether this
signaling function of Msn(CT) is related to the activation of JNK or
to JNK-independent pathways. No defect in dorsal closure was observed
when msn embryos were rescued with wild-type Msn. These
findings demonstrate that the defect in dorsal closure in
msn mutant embryos is due to a loss of msn
function and that both the kinase activity and the C-terminal regulatory domain of Msn are essential for it to activate JNK in vivo.
In evaluating msn mutant embryos rescued with Msn(KD), we
observed that many had ventral cuticular abnormalities, suggesting a
defect in gastrulation (data not shown). While embryos zygotically mutant for msn do not show such defects, the function of
maternal msn could not be evaluated because msn
is required for oogenesis (46). Although the level of
expression of maternal msn is sufficient to support
gastrulation of embryos lacking zygotic msn, Msn(KD) may
function as a dominant negative protein, inhibiting maternal Msn. In
the presence of wild-type levels of zygotic Msn we did not observe such
a dominant negative effect (data not shown). This role of
msn is likely to be independent of JNK activation, because
ventral defects are not seen in embryos lacking both
maternal and zygotic JNK (bsk) or the JNK
kinase encoded by hep (13, 35, 41).
A single PXXP motif in Msn mediates binding to the SH3 domains of
the SH2-SH3 adapter molecule Dock.
We isolated NIK in a two-hybrid
screen designed to identify proteins that interact with the mammalian
homolog of Drosophila Dock, Nck. The region between the
kinase and C-terminal regulatory domains of Msn, the
Drosophila homolog of NIK, contains several PXXP motifs that
match consensus SH3 binding motifs (49). To determine
whether Msn may be a physiological target of the SH3 domains of Dock,
we determined whether Msn bound the SH3 domains of Dock in vitro. We
found that Msn bound full-length Dock as well as a GST fusion protein
containing only the SH3 domains of Dock (Fig. 1A). Msn also bound the
SH3 domains of mammalian Nck, Grb2, and phospholipase C
. The
interaction of Msn with these fusion proteins was specific, because Msn
did not bind to GST alone or to GST fused to the SH3 domains of p85 or
interleukin 2-inducible T-cell kinase.
Interaction between Msn and Dock is not required for the function of Msn in dorsal closure. We used this mutant protein to test whether Msn required Dock binding to rescue the defect in dorsal closure in msn mutant embryos. While msn mutants die embryonically due to a defect in dorsal closure, dock mutants survive until the pupal stage (11), suggesting that dock may not function upstream of msn in dorsal closure. Loss of both maternal and zygotic dock causes embryonic lethality, but the embryos survive until after stage 16, when dorsal closure takes place (7), making a role for maternal dock in dorsal closure unlikely. We found that UAS-msn(P656A, P659A) driven by 69B-GAL4 completely rescued the defect in dorsal closure in msn mutant embryos (Tables 1 and 2), allowing 37% of rescued embryos to survive to the pupal stage. This finding is consistent with the idea that Dock does not function upstream of Msn in dorsal closure. Rather, we favor the idea that DTRAF1 acts to couple Msn to upstream signals important in mediating JNK activation and dorsal closure (25).
Formation of normal photoreceptor axonal projections requires the prolines on Msn that interact with SH3 domains. The finding that msn mutants could be rescued to the pupal stage by expression of either wild-type Msn or Msn(P656A, P659A) led us to test whether larval photoreceptor axonal projections are disrupted in msn mutants rescued with the msn transgenes. Recent evidence has indicated that Pak is one of the downstream signaling molecules activated by Dock and that activation of Pak by Dock is critical for correct targeting of photoreceptor axons (18). However, these studies cannot rule out the possibility that Dock interacts with multiple downstream targets, one of which is Msn.
To begin to address whether msn plays a role in photoreceptor axon guidance, we tested whether msn mutant third-instar larvae rescued with UAS-msn(wt) or UAS-msn(P656A, P659A) displayed a defect in pathfinding of photoreceptor axons. We have observed that 69B-GAL4 is expressed in the photoreceptors and the brain as well as in the embryonic ectoderm (data not shown). We reasoned that if binding of Msn to Dock was necessary for Dock to function, photoreceptor axon projections would be rescued by UAS-msn(wt) driven by 69B-GAL4 but would not be rescued by UAS-msn(P656A, P659A). To assess photoreceptor projections, eye-brain complexes were stained with the MAb 24B10, which recognizes the membrane protein chaoptin present on photoreceptors and their axons (9). msn mutants rescued to the third-instar larval stage with UAS-msn(P656A, P659A) exhibited a severe defect in axonal targeting, confirming a requirement for msn in this process. Whereas axons from wild-type photoreceptors or photoreceptors from msn third-instar larvae rescued with UAS-msn(wt) fanned out evenly upon leaving the optic stalk and formed a smooth retinotopic array in both the lamina and the medulla (Fig. 2A and C), photoreceptor axons from msn third-instar larvae rescued with UAS-msn(P656A, P659A) formed very large axonal bundles and failed to defasciculate in the region of either the lamina or the medulla (Fig. 2D). This defect in axonal targeting is more severe than the defect reported for dock mutants (Fig. 2B). It is not due to a nonspecific effect of overexpression of Msn protein (for example, by titrating out an important signaling molecule that functions on this pathway), because these effects were not seen with Msn(wt) (Fig. 2C). In addition, overexpression of msn(P656A, P659A) in wild-type flies did not produce a defect in axonal targeting, indicating that msn(P656A, P659A) does not function as a dominant negative protein in a wild-type background (data not shown).
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msn is required in the eye for axonal pathfinding. To determine whether, like dock, msn acts autonomously in the photoreceptor growth cones, we made use of the FLP-FLP recognition target (FRT) recombinase system, which enables the generation of clones of cells with mutations in msn in a heterozygous animal (46, 47). To generate msn mutant clones in the eye disc, FRT-msn males were crossed with females carrying a Minute mutation, causing dominant slow growth and recessive lethality, on the same chromosome arm as the FRT site. The females also carried a construct expressing FLP recombinase under the control of the eye-specific eyeless promoter (28a). Using this approach, we were able to obtain third-instar larval eye discs in which >90% of the eye disc was mutant for msn. While photoreceptor axons derived from these msn mutant clones extended normally into the brain, the R1 to R6 axons failed to elaborate a smooth retinotopic array in the lamina (Fig. 2F). Whereas R1 to R6 axons from wild-type eye discs terminate at a precise depth in the lamina and therefore form a continuous line of chaoptin immunoreactivity, many R1 to R6 axons in msn mutant clones terminated prematurely, giving rise to an uneven lamina neuropil. Surprisingly, the growth cones of axons that innervate the medulla appeared to expand normally, although their projections were somewhat disorganized. This is in contrast to the R7 and R8 axons in dock mutants, which lack expanded growth cones and fail to form an even array in the medulla. Moreover, the axon bundles projecting between the lamina and medulla appeared to have the same thickness as those in the wild type, whereas in dock mutants these axons form larger bundles. Although a lack of msn in the eye causes defects in the shape of photoreceptor rhabdomeres (46), the relatively normal projection observed for R7 and R8 suggested that the axon guidance defects were not a consequence of these changes. In addition, the differences between the msn and dock phenotypes were not an experimental artifact arising from the generation of msn clones in the eye; the phenotype of dockP1 mutant clones in the eye made in a Minute background was identical to that of dockP1 homozygous mutants (compare Fig. 2G and B). These results confirm that msn is required in photoreceptors for their axons to project normally. However, some of the defects observed are different from those reported for dock mutants, suggesting that Msn performs functions in photoreceptor cells that are independent of those of Dock.
Msn is not the only downstream target of Dock.
The
above-described studies demonstrate that Msn and Dock interact in vitro
and that msn plays an essential role in axonal pathfinding.
However, these studies do not demonstrate that msn and
dock act in the same pathway. In fact, the finding that
photoreceptor axonal pathfinding is more severely disrupted in
msn mutants rescued with
UAS-msn(P656A, P659A) than in
dock mutants indicates that Msn must interact with at least
one protein other than Dock. To further study the relationship between
msn and dock in vivo, we looked for genetic
interactions between them. We found that defects in the photoreceptor
projection pattern were consistently more severe in
dockP1 third-instar larvae that were also
heterozygous for msn than in third-instar larvae that were
homozygous for the dockP1 mutation alone
(compare Fig. 3A and B). Because
dockP1 is likely to be a null mutation, its
enhancement by msn would be most consistent with
msn functioning on a pathway parallel to that of
dock.
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332-667)]. The region deleted in this mutant includes both
the region that we have shown binds DTRAF1 and prolines 656 and 659, which mediate binding to the SH3 domains of Dock, as well as all the
other PXXP motifs. The failure of Msn(332-667) to bind both Dock
and DTRAF was confirmed by two-hybrid analysis (data not shown). We
initially predicted that if DTRAF1 functioned upstream of Msn and JNK
activation in dorsal closure, ectopic expression of
UAS-msn(332-667) with 69B-GAL4
would fail to rescue the defect in dorsal closure in msn
mutant embryos. However, we found that
UAS-msn(332-667) rescued the defect in dorsal
closure in msn mutants, allowing them to survive to the
pupal stage (Table 1). Surprisingly, and in contrast to the results
shown for UAS-msn(P656A, P659A), the
photoreceptor projection pattern in larvae rescued with
UAS-msn(332-667) was essentially normal (Fig.
3C). Because both Msn(P656A, P659A) and Msn(332-667) are unable to
bind Dock, yet Msn(332-667) is able to rescue photoreceptor axonal
targeting, Msn(332-667) must be able to function without binding
Dock and is probably a constitutively active form of Msn.
To confirm biochemically that Msn(332-667) is an activated version
of Msn, we compared the abilities of Msn(wt) and Msn(332-667) to
activate JNK in 293 cells. The activation of JNK in 293 cells by NIK
and Msn is independent of upstream signals, and previous studies have
demonstrated that overexpression of either NIK or Msn is sufficient to
activate JNK in these cells (43, 44). To assess JNK
activation, Msn(wt) and various Msn mutants were transfected into 293 cells and activation of an ATF2 luciferase reporter was measured; JNK
has been shown to phosphorylate and activate ATF2. While overexpression
of Msn(wt) led to about a 10-fold increase in luciferase activity
compared to that for cells transfected with vector control,
overexpression of Msn(332-667) led to a >30-fold increase in
luciferase activity (Fig. 4). The increase in JNK activation by Msn(332-667) is not due to an
increase in protein expression, because Msn(wt) and Msn(332-667)
are expressed at equal levels (data not shown). Thus, these findings
are consistent with the idea that Msn(332-667) is an activated form
of Msn.
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332-667)
might partially rescue the defect in dock mutant
photoreceptors. To test this possibility, we expressed
UAS-msn(332-667) in a
dockP1 mutant background, using an
elav-GAL4 driver. However, the dock mutant
phenotype was still observed in these larvae (Fig. 3D). Thus, our
studies do not provide conclusive evidence that Msn and Dock act in the
same pathway in flies. Since a mutation in Msn sufficient to activate
it does not compensate for the lack of dock, either Msn must
function on a parallel pathway required for photoreceptor axon guidance
or Dock must act through both Msn and additional proteins, such as PAK.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Msn couples to distinct pathways to regulate dorsal closure and axon guidance. While a role for Ste20 kinases in promoting JNK activation has been previously identified, little is known about their regulation or about the specific in vivo function of these kinases. Members of our group have previously shown that the Ste20 kinase Msn functions upstream of the Drosophila JNK, Bsk, to stimulate dorsal closure of the Drosophila embryo (44). We now show that Msn requires both intact kinase activity and a C-terminal regulatory domain conserved in a number of Ste20 kinases of the GCK family in order to activate JNK in vivo in flies. The previous finding that the C-terminal regulatory domain of NIK bound the N-terminal regulatory domain of the Ste11 kinase MEKK1 led us to propose that the interaction of the C-terminal domain of NIK with downstream Ste11 kinases (DMKKK) was critical for NIK and other GCK family members to activate the JNK MAP kinase module (43). However, studies on NIK were performed in assays in which NIK protein was expressed at high levels, and under these circumstances, NIK is able to mediate JNK activation independent of an upstream activating signal. The requirement for both the C-terminal domain and the kinase activity of Msn to promote dorsal closure indicates that these domains are required in order for GCK family members to activate JNK in a physiologically relevant setting and suggests that a Drosophila Ste11 kinase also couples Msn to JNK activation and dorsal closure.
In addition to its role in JNK activation and dorsal closure, Msn is critical for the correct targeting of photoreceptor axons in Drosophila. Thus, our data indicate that msn is important in vivo for regulating at least two distinct biological events: dorsal closure and photoreceptor axon pathfinding. Interestingly, the upstream molecules that regulate msn in these two pathways are distinct, since a mutation eliminating the function of Msn in axon guidance does not affect its activity in dorsal closure. One molecule that may act upstream of Msn in the pathway leading to JNK activation and dorsal closure is a DTRAF; members of our group have recently shown that DTRAF1 can interact with Msn to activate the JNK pathway in cell lines (25). Mutation of a PXXP motif in Msn prevents it from binding to Dock and from rescuing photoreceptor axon pathfinding, indicating that Dock and/or related SH3 domain-containing molecules may act in concert with Msn in this process. We do not yet know the mechanism by which upstream factors regulate Msn. A common requirement for Msn activation may involve its increased local concentration. This could occur either by the recruitment of Msn to phosphotyrosine-containing proteins or by DTRAF1-induced aggregation of Msn, thereby allowing juxtaposed Msn molecules present in the complex to transphosphorylate and activate each other. Alternatively, the finding that deletion of the region between the kinase and C-terminal domains of Msn leads to its constitutive activation raises the possibility that upstream signals activate Msn by inducing a conformational change and/or displacing a negative regulator bound to this region.Msn does not behave as a simple downstream effector of Dock. The ability of axons to make precise connections during development requires the axonal growth cone, localized to the leading edge of projecting axons, to interpret multiple guidance cues that ultimately navigate axons to their destinations (45). Changes in the growth cone's actin cytoskeleton and/or the affinity for binding of the integrins to the matrix are thought to be the key elements whereby guidance cues regulate the path taken by developing axons (45). The finding that dock is required for Drosophila photoreceptor axon guidance and targeting provided a starting point for beginning to dissect the intracellular signaling pathways that are activated at the growth cone to mediate these guidance cues. Dock is a member of a large family of adapter proteins consisting essentially of SH2 and SH3 domains, of which the prototypic member is Grb2. SH2-containing adapter molecules regulate signaling pathways by coupling catalytic molecules bound to their SH3 domains to phosphotyrosine-containing proteins.
While a number of proteins that bind the SH3 domains of Nck and Dock have been identified, which of these serve as targets in vivo has been difficult to resolve. In contrast to the SH2-SH3 adapter molecule Grb2, for which interaction with the downstream SH3 binding partner Sos was demonstrated using genetic evidence, the physiologically relevant binding partners for Nck and Dock and the downstream signaling pathways have only recently begun to be defined (38). In this regard, the Ste20 kinase Pak has been shown to interact with Dock, and expression of a myristylated form of Pak can partially rescue the dock mutant phenotype (18). We show here that Msn also binds to the SH3 domains of Dock and that the amino acids that mediate this binding are required for the correct targeting of photoreceptor axons. However, our findings do not provide conclusive evidence that msn functions downstream of dock in photoreceptor targeting. Rather, our studies highlight the complex role of msn in photoreceptor targeting and suggest that unraveling the exact functions of msn in this process is unlikely to be simple. For example, it is likely that Msn functions in both photoreceptor cells and the brain. The severe photoreceptor axon guidance defects observed when msn mutants are rescued with UAS-msn(P656A, P659A) are stronger than those caused by either the absence of msn in the eye or the complete loss of function of dock. Interaction between Msn and an SH3 domain-containing protein or proteins other than Dock in nonphotoreceptor cells, such as those in the brain, is a likely explanation. Although we have shown that photoreceptor development in most of the eye disc is normal when rescue is carried out with UAS-msn(P656A, P659A), defects in brain development in these larvae may contribute to the axon guidance phenotype; an enhancer trap insertion in msn shows expression in the optic lobes as well as in the eye (data not shown). This hypothesis is difficult to test directly, as many aspects of optic lobe development are directly dependent on retinal innervation. Because the defects in photoreceptor axonal targeting are specific to a mutation in a proline motif that matches consensus SH3 binding motifs (49), this phenotype is probably due, at least in part, to the loss of interaction of Msn with an SH3 domain-containing protein. Our finding that the dock phenotype is enhanced by the presence of msn suggests that the signaling pathways regulated by msn, which are critical for the correct targeting of R-cell axons, intersect with the signaling pathways regulated by dock. However, this interaction does not clarify whether msn functions on the same pathway as dock or on a parallel pathway. In addition, expression of a form of Msn that we have shown to be constitutively active is not sufficient to rescue the dock phenotype. One possible explanation for these data is that msn acts downstream of dock but is not the only downstream mediator of its function. As discussed above, the Ste20 kinase Pak has been shown to interact with Dock, and expression of a myristylated form of Pak can partially rescue the dock mutant phenotype. Interestingly, this form of Pak predominantly rescues the expansion of growth cones in the medulla, a process that does not appear to require msn function in the photoreceptor axons. It is possible that msn and Pak mediate separable functions of dock in photoreceptor cells. An alternative possibility is that the function of Msn expressed in photoreceptor cells is mediated by the binding of Msn to an SH3 domain-containing protein other than Dock. The difference in the phenotypes caused by loss of msn and loss of dock in the photoreceptor axons would support this hypothesis. Mutations in the gene encoding such a hypothetical protein, which would function on a pathway parallel to the dock pathway, have yet to be identified. While this report was under review, Ruan et al. (37) reported a role for msn in photoreceptor axonal targeting and Dock signaling. However, in contrast to our findings that msn mutant R1 to R6 axons terminate prematurely, Ruan et al. reported that the R1 to R6 axons overshoot the lamina and terminate in the medulla. In addition, they found that overexpression of Msn in photoreceptor cells in dock mutants reversed the overshoot of the R1 to R6 axons. These findings and other data led them to conclude that dock and msn act in the same pathway. The reason for the discrepancy between the findings reported here and their results is not clear at present. One possibility is that the expression of Msn was much higher in the studies by Ruan et al., enabling them to see rescue of the dock mutant phenotype; they used an enhancer promoter line containing a UAS element inserted in the 5' promoter region of msn to overexpress msn. However, Ruan et al. also found that overexpression of msn in a wild-type background led to the premature termination of many R-cell growth cones, essentially the same phenotype as they observed when msn was overexpressed in dock mutants; thus, it is not clear that this in fact constitutes rescue of the dock phenotype. In contrast, expression of a myristylated form of Pak largely rescues the dock mutant phenotype without inducing additional defects (18).Possible regulators and effectors for Msn in axon guidance. An attractive hypothesis is that Dock and/or related SH3 domain-containing molecules function as adapters to couple Msn to tyrosine-phosphorylated proteins in response to signaling by a receptor tyrosine kinase localized at the axonal growth cone. Eph receptors, which constitute the largest family of receptor tyrosine kinases, are good candidates for receptors that may function at the axonal growth cone to regulate changes in the actin cytoskeleton and/or adhesion of integrins to the matrix that ultimately facilitate the correct targeting of retinal axons. In this regard, we have recently found that NIK kinase activity is activated in mammalian cells by the EphB1 and EphB2 receptors and that NIK couples EphB1 to both JNK and integrin activation (2a). However, although a Drosophila Eph receptor kinase (DEK) is expressed on retinal axons, misexpression and overexpression of wild-type DEK or a kinase-defective form of DEK do not affect axonal pathfinding in Drosophila (39).
The intracellular signals activated downstream of Msn that mediate the correct pathfinding of photoreceptor axons are not yet known. The finding that regulation of the actin cytoskeleton is critical for growth cones to navigate correctly suggests that Msn may control the targeting of photoreceptor axons by regulating the actin cytoskeleton (45). Our studies have indicated that the downstream pathways regulated by Msn are likely to be diverse and will not be limited to the activation of JNK. This is suggested by the finding that msn is required for oogenesis, while bsk and hep are not, and that ventral defects can be induced by a kinase-defective form of Msn, although maternal and zygotic bsk mutants do not show such a phenotype. We do not think that msn directs axonal guidance via activation of the JNK MAP kinase pathway, because photoreceptor axonal targeting showed only minor defects, including occasional overshooting of R1 to R6 axons, in bsk1 mutant clones made in a Minute background in the eye disc (data not shown). However, because bsk1 is not a complete loss-of-function mutant, these studies cannot definitively rule out a role for JNK. Small clones with mutations in both hep and the other Drosophila p38 MAPK kinase encoded by licorne (44a) also showed an apparent overshoot of R1 to R6 axons, resembling the dock phenotype but not the msn phenotype (data not shown). However, we were unable to rescue the dock phenotype with an activated allele of hep, indicating that activation of the JNK pathway is not sufficient to rescue the dock phenotype (data not shown). While a direct link between Pak family Ste20 kinases and the actin cytoskeleton has been shown, a direct link between GCK family Ste20 kinases and the actin cytoskeleton has not yet been demonstrated (27, 30, 40). Thus, the ability to use genetics to identify and validate potential targets of Msn should provide a valuable tool to uncover not only the relevant biological functions regulated by Ste20 kinases but also their physiological downstream targets.| |
ACKNOWLEDGMENTS |
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We thank Barry Dickson, Paul Garrity, Naoto Ito, S. Noselli, M. Mlodzik, and Larry Zipursky for fly stocks and reagents and Zhai Li for technical help.
This work is supported by grants from the NIDDK and American Diabetes Association to E.Y.S. and a grant from the Edward Mallinckrodt, Jr., Foundation to J.E.T.
Y.-C.S. and C.M.-Z. contributed equally to this work.
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FOOTNOTES |
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* Corresponding author. Mailing address for Edward Skolnik: New York University Medical Center, Department of Pharmacology, Skirball Institute of Biomolecular Medicine, 540 First Ave., New York, NY 10016. Phone: (212) 263-7458. Fax: (212) 263-5711. E-mail: Skolnik{at}saturn.med.nyu.edu. Mailing address for Jessica Treisman: New York University Medical Center, Department of Cell Biology, Skirball Institute of Biomolecular Medicine, 540 First Ave., New York, NY 10016. Phone: (212) 263-1031. Fax: (212) 263-7760. E-mail: Treisman{at}saturn.med.nyu.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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