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Ras target protein canoe is a substrate for Cdc2 and Cdk5 kinases.

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102
Takahashi et al.
Archives of Insect Biochemistry and Physiology 49:102–107 (2002)
Ras Target Protein Canoe Is a Substrate for Cdc2 and
Cdk5 Kinases
Kuniaki Takahashi,1,3 Noriko Hamada,2 and Daisuke Yamamoto1–3*
Mutations in the canoe locus of Drosophila lead to failure in the dorsal closure of the embryonic epidermis and pattern
formation defects in imaginal eyes and wings. In the wing, the canoe mutants develop extra veins when they are heterozygous
for shaggy, a mutation in the locus encoding the glycogen synthase kinase 3β (Gsk3β), which has been known to phosphorylate the Armadillo protein. Although Canoe has a putative target sequence for phosphorylation by Gsk3β similar to that
found in Armadillo, in vitro experiments indicate that Canoe is not phosphorylated by Gsk3β. Instead, Canoe is demonstrated
to be a good substrate of Cdc2 and Cdk5 kinases. Thus, Cdc2 and Cdk5 kinases are the potential regulators of the function of
Canoe in morphogenesis. Arch. Insect Biochem. Physiol. 49:102–107, 2002. © 2002 Wiley-Liss, Inc.
KEYWORDS: phosphorylation; Drosophila; adherens junctions; Cdc2; Cdk5; Canoe; AF-6
INTRODUCTION
The Canoe protein plays an essential role in
Drosophila morphogenesis. Complete loss-of-function mutations in the canoe (cno) locus cause developmental arrest in mid-embryogenesis as a
result of failure in dorsal closure of the bilateral
epidermis (Jürgens et al., 1984; Takahashi et al.,
1998). A hypomorphic allele canoemisty1 (cnomis1)
yields some adults, the compound eyes of which
are roughened due to perturbed cone cell fate determination (Matsuo et al., 1997; Yamamoto, 1996)
and incomplete extension of photoreceptor rhabdomeres (Matsuo et al., 1999). These observations
indicate that the Canoe protein has multiple functions in Drosophila development, including the
regulation of cell shape and motility, as well as
the fate induction of certain cell types (Peifer and
Tepass, 2000; Yamamoto, 1996).
In accordance with such functional pleiotropy,
the Canoe protein has several structural motifs
that mediate direct interactions with other proteins (Miyamoto et al., 1995). In the N-terminal
end are two repeats of the RA (Ral GDS/AF-6; Rasassociating) domain (Ponting and Benjamin,
1996), which serves as the binding site for Ras1
(Kuriyama et al., 1996; Matsuo et al., 1997). At
the center of the molecule, the Canoe protein has
the PDZ (PSD-95/Dlg/ ZO-1) domain, although
its binding target has not been identified for Canoe (see Songyang et al., 1997). Between the RA
and PDZ domains, there exist two additional conserved regions, i.e., the kinesin-like and myosinV-like domains (Ponting, 1995), the latter recently
renamed the AF-6/cno-homology region, which
mediates binding of the myosin Va tail to the conventional kinesin tail (Huang et al., 1999). In addition, Afadin, a splice variant of AF-6, was
identified based on the actin-binding ability of its
unique C-terminal sequence (Mandai et al., 1997).
1
ERATO Yamamoto Behavior Genes Project, JST, Mitsubishikagaku Institute of Life Sciences, Machida, Japan
2
School of Human Sciences, Waseda University, Tokorozawa, Japan
3
Advanced Research Institute for Science and Engineering, Waseda University, Tokorozawa, Japan
Grant sponsor: Ministry of Education, Culture, Sports, Science and Technology; Grant sponsor: Waseda University; Grant number: 2000B-029.
*Correspondence to: Prof. Daisuke Yamamoto, 2-579-15, Mikajima, Tokorozawa, 359-1192, Saitama, Japan. E-mail: daichan@mn.waseda.ac.jp
Received 13 March 2001; Accepted 10 September 2001
© 2002 Wiley-Liss, Inc.
DOI 10.1002/arch.10012
Archives of Insect Biochemistry and Physiology
Cdc2 and Cdk5 Phosphorylate Canoe
The functional significance of molecular interactions mediated by the Canoe protein has been
demonstrated by phenotypic interactions between
mutants of the canoe gene and other genes coding
for binding partners. For example, halving the canoe wild-type dosage significantly enhances the effects of the RasN17 or RasV12 transgene in reducing
or increasing the cone cell number in the developing compound eye (Matsuo et al., 1997). The Canoe protein binds to ZO-1 by its N- and C-termini,
and hypomorphic mutants of the genes encoding
these two proteins synergistically interact to block
embryonic dorsal closure (Takahashi et al., 1998;
see also Yamamoto et al., 1997, 1999). These observations suggest that a search for mutants genetically interacting with the canoe mutant can provide
a list of candidate proteins associated with Canoe.
In our attempt to isolate mutants interacting
with the canoe mutant, the shaggyD127 (sggD127) mutant heterozygote was found to develop extra wing
veins in the cnomis1 homozygous background (Miyamoto et al., 1995). Normally, sggD127 is recessive and
thus is not expressed phenotypically. The sgg gene
encodes a serine/threonine protein kinase homologous to glycogen synthase kinase 3β (Gsk3β), a human kinase involved in hyperphosphorylation of
the Tau protein. The hyperphosphorylated Tau is
known to be associated with Alzheimer’s disease
(Kobayashi et al., 1993; Ishiguro et al., 1994). In
Drosophila, the kinase is downregulated upon activation of the Wingless (Wg) pathway, leading to
dephosphorylation of Armadillo (Arm), which then
translocates to the nucleus for transcriptional regulation of target genes in this pathway (Peifer, 1999).
The fact that the canoe viable mutant allele exhibits lethality when heterozygous for arm (Miyamoto
et al., 1995) suggests that Canoe might interact molecularly with Sgg, Armadillo, or other components
in the Wg pathway. As the simplest model, one may
envisage that the Sgg kinase phosphorylates Canoe.
We examined this possibility in this study.
MATERIALS AND METHODS
A nucleotide sequence of cno corresponding to
amino acid residues 1552–1614 was amplified by
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103
polymerase chain reaction (PCR) and ligated into
the pGEX-5X vector (Amersham Pharmacia Biotech). The plasmid was introduced into XL-1 blue
cells, and a fusion protein with GST was induced
by 0.1 mM IPTG. After 5 h of culture, cells were
pelleted and resuspended in 50 mM Tris (pH 7.5),
25% sucrose, 0.5% NP-40, 5 mM MgCl 2, and 100
µM (p-amidinophenyl) methanesulfonyl fluoride
(p-APMSF). Cells were lysed by sonication for 5
min and the lysate was centrifuged. The supernatant obtained was mixed with glutathione-agarose
beads (Sigma) at 4°C for 30 min. The fusion protein was eluted with 5 mM glutathione and analyzed on a SDS-12.5% polyacrylamide gel.
Kinase assays were carried out at 30°C in TwG
buffer A [100 mM MES-NaOH (pH 6.5), 1 mM
Mg (CH3COO)2, 1 mM EGTA, 0.02% Tween 20,
and 10% glycerol], 10 µCi γ-32P-ATP, and either
GST-Canoe (80 µg/ml), or GST (80 µg/ml). Gsk3β
(TPKI), Cdk5 (TPKII), (TPKI+TPKII) or Cdc2 kinase (5 µg/ml) was added. Gsk3β and Cdk5 were
purified from the bovine brain (a gift from Dr. K.
Ishiguro). Cdc2 was purchased from Seikagaku Co.
After 10 min, the reactions were stopped by the
addition of Laemmli’s sample buffer. The samples
were boiled for 5 min, and then electrophoresed
on an SDS-polyacrylamide gel followed by image
analysis using a BAS 2000 system (Fuji Film).
RESULTS
Comparisons of amino acid sequences between
Arm and Canoe identify an eight-residue stretch
common to these two proteins, i.e., PSGAPS (or
T) SP (Fig. 1). This matches well with the consensus for Gsk3β phosphorylation sites, SXXXS (Kemp
and Pearson, 1990). This observation prompts us
to examine the possibility of phosphorylation of
Canoe by mammalian Gsk3β as an orthologue of
Drosophila Sgg. The Drosophila Sgg protein obtained
by bacterial expression or in vitro translation is
catalytically inactive, and purification of Sgg from
flies has never been employed. In contrast, the active form of mammalian Gsk3β has been obtained
from the bovine brain (Ishiguro et al., 1993;
Kobayashi et al., 1993).
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Takahashi et al.
Fig. 1. The putative phosphorylation sites in the Canoe
and Armadillo proteins. a: The RA domain, kinesin-like
domain, myosin 5-like domain, and PDZ domain of the
Canoe protein and the Armadillo repeat region in the Armadillo protein are indicated. The sequences that match
the consensus phosphorylation site for Cdc5 and Cdc2
kinases are found at a.a.1573–a.a.1593 of Canoe and a.a.
793–a.a. 809 of Armadillo. These segments are aligned
with the canonical sequence for the phosphorylation site
in b. The shaded region indicates the amino acids conserved between Armadillo and Canoe, and the open box
highlights residues common to the consensus sequence
of Cdc2/Cdk5 targets and Canoe.
The partial Canoe peptide of 62 amino acids
containing the potential phosphorylation site is
obtained as a fusion protein with glutathion-Stransferase (GST) using an Escherichia coli expression system. The GST-Cno fusion protein,
incubated with active Gsk3β purified from the bovine brain (a gift from Dr. K. Ishiguro) under the
standard reaction conditions, does not show any
significant γ32-ATP incorporation. In the case of Tau
protein phosphorylation by Gsk3β, the T231 residue can be phosphorylated only after phosphorylation of S235 by cyclin-dependent kinase-5 (Cdk5)
or mitogen-activated protein kinase (Ishiguro et al.,
1993; Goedert et al., 1994). We, therefore, examine the effect of consecutive incubation of GST-Canoe with active Cdk5 (a gift from Dr. K. Ishiguro)
and Gsk3β. Cdk5 successfully phosphorylates the
GST-Canoe protein, but subsequent incubation of
Cdk5-phosphorylated GST-Canoe with Gsk3β does
not result in additional phosphorylation by the latter kinase (Fig. 2).
Since the substrates of Cdk5 (including Tau) are
also often phosphorylated by Cdc2, we examine
this possibility. The experiment demonstrates that
Canoe is a good substrate of Cdc2 (Fig. 2). These
results indicate that Canoe is phosphorylated in
vitro by Cdk5 and Cdc2 but not by Gsk3β even
after its phosphorylation by Cdk5.
DISCUSSION
Contrary to the expectations from the phenotypic
interactions between cno and sgg mutants, Sgg kinase does not phosphorylate Canoe. This finding is
not surprising in light of the fact that both Sgg and
Canoe play multiple roles in different transduction
pathways (Matsuo et al., 1999; Martinez Arias et
al., 1999); therefore, the dominant enhancement
of the cno phenotype by sgg may reflect an indirect
effect of the reduced Sgg kinase activity on some
other element in an interacting signal network. It
is interesting to note that the Wg receptor Frizzed
Archives of Insect Biochemistry and Physiology
Cdc2 and Cdk5 Phosphorylate Canoe
105
Fig. 2. Phosphorylation of the Canoe protein in vitro.
Left panels: The GST-Canoe fusion protein (lane 1) and
GST (lane 2) expressed in E. coli were analyzed by SDS
PAGE gel electrophoresis. Right panels: In vitro phospho-
rylation of the GST-Canoe fusion protein and GST by
Gsk3β (a), Cdk5 (b), or Cdc2 (c). The GST-Canoe fusion
protein was phosphorylated by Cdk5 and Cdk2 but not
by Gsk3β.
(Fz) activates the c-Jun N-terminal kinase (Jnk)
pathway in addition to its “main route” toward
Arm (Wodarz and Nusse, 1998). Canoe is known
to contribute to the Jnk pathway in embryonic dorsal closure (Takahashi et al., 1998). Jnk signaling
is stimulated by Dishevelled (Dsh) (Boutros et al.,
1998), which, on the other hand, represses Sgg/
Gsk3β activity in the parallel pathway. It is worth
mentioning that the dsh mutant genetically interacts with the cno mutant, leading to the formation
of extra wing veins (Yamamoto, unpublished observations). Thus it appears possible that the observed phenotypic interaction of the cno mutant
with the sgg mutant is a result of crosstalk between
the Wg and Jnk pathways.
In contrast to Sgg/Gsk3β two other related kinases, i.e., Cdc2 and Cdk5, phosphorylate Canoe.
No evidence is available at present to indicate that
these two kinases modulate the Canoe functions
in vivo. Null alleles of the Drosophila cdc2 (Dm
cdc2) locus are lethal at the larval-pupal interphase
due to the absence of imaginal tissues resulting
from failure in mitosis (Stern et al., 1993; Hayashi,
1996). It is necessary to examine whether the severity of canoe-mutant phenotypes is affected by
the level of Cdc2 activity as altered by the use of a
temperature-sensitive cdc2ts mutant (Stern et al.,
1993; Hayashi, 1996). On the other hand, little is
known about the function of Drosophila Cdk5 due
to the lack of mutants in this locus (Hellmich et
al., 1994). However, the cdk5 mRNA expression is
developmentally controlled: it has the highest peak
during the late-larval stage to the pupal stage, while
it remains low throughout the embryonic stage
(Sauer et al., 1996). This pattern of expression implies the role of cdk5 in differentiation and morphogenesis of imaginal tissues. It is crucial to
isolate cdk5 mutants to examine their possible ge-
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106
Takahashi et al.
netic interactions with the canoe mutant, which are
inferred to occur by the in vitro experiment reported here.
ACKNOWLEDGMENTS
We thank K. Ishiguro for providing us with
the active Gsk3β and Cdk5, and Y. Kai for secretarial assistance. This study was supported in part
by Special Cooperation Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology
to D.Y., and by Waseda University grant no.
2000B-029 to O.Y.
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February 2002
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