Previous Article | Next Article 
Mol Cell Biol, January 1998, p. 598-607, Vol. 18, No. 1
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Numb-Associated Kinase Interacts with the Phosphotyrosine Binding
Domain of Numb and Antagonizes the Function of Numb In Vivo
Cheng-ting
Chien,
Shuwen
Wang,
Michael
Rothenberg,
Lily Y.
Jan, and
Yuh Nung
Jan*
Howard Hughes Medical Institute and
Department of Physiology and Biochemistry, University of California
at San Francisco, San Francisco, California 94143-0724
Received 23 June 1997/Returned for modification 14 August
1997/Accepted 11 October 1997
 |
ABSTRACT |
During asymmetric cell division, the membrane-associated Numb
protein localizes to a crescent in the mitotic progenitor and is
segregated predominantly to one of the two daughter cells. We have
identified a putative serine/threonine kinase, Numb-associated kinase
(Nak), which interacts physically with the phosphotyrosine binding
(PTB) domain of Numb. The PTB domains of Shc and insulin receptor
substrate bind to an NPXY motif which is not present in the region of
Nak that interacts with Numb PTB domain. We found that the Numb PTB
domain but not the Shc PTB domain interacts with Nak through a peptide
of 11 amino acids, implicating a novel and specific protein-protein
interaction. Overexpression of Nak in the sensory organs causes both
daughters of a normally asymmetric cell division to adopt the same cell
fate, a transformation similar to the loss of numb function
phenotype and opposite the cell fate transformation caused by
overexpression of Numb. The frequency of cell fate transformation is
sensitive to the numb gene dosage, as expected from the
physical interaction between Nak and Numb. These findings indicate that
Nak may play a role in cell fate determination during asymmetric cell
divisions.
| |
INTRODUCTION |
A multicellular organism originates
from a single cell via mitosis, leading to the generation of different
cell types. The asymmetric cell division which generates two daughter
cells of different fates is one of the ways to produce diversity of
cell types. Drosophila sensory organ development is well
suited for the study of asymmetric cell division (10, 32,
41). These sensory organs are generated through a few rounds of
asymmetric cell divisions from sensory organ precursor (SOP) cells. For
example, in the external sensory organ lineage (see Fig. 8H and I), a
single SOP cell divides to give rise to two different daughter cells, A
and B cells. The A cell divides to produce a hair cell and a socket
cell, whereas the B cell generates a neuron and a sheath cell. All
three divisions are asymmetric, and each generates two daughter cells
of different fates.
Asymmetric cell division may arise from unequal distribution of an
intracellular determinant, leading to its preferential segregation into
one of the two daughter cells (for a review, see reference
29). The membrane-associated Numb protein is such a
determinant for the asymmetric cell divisions of the sensory organ
lineage (42). The Numb protein localizes to a cortical crescent in SOP during prophase and segregates preferentally to one of
the two daughter cells in telophase (35). Genetic analysis reveals a role of numb in the asymmetric cell divisions
(42, 49). Loss of numb function can be induced at
different developmental stages and causes both daughter cells of a
normally asymmetric cell division to adopt the same fate; the SOP
divides to give rise to two A cells (B-cell-to-A-cell transformation;
see Fig. 8I for external sensory organ lineage). The A cell divides to produce two hair cells (socket-to-hair cell transformation), and the B
cell divides to generate two sheath cells (neuron-to-sheath cell
transformation). By contrast, overexpression of Numb results in a
transformation opposite that observed in loss-of-function mutants in
each of these three divisions, suggesting that the Numb protein level
influences cell fate specification. Numb is also localized during the
mitosis of neuroblasts in the central nervous system (35,
42), and it is required for the asymmetric division of the MP2
precursors in the central nervous system (47).
Besides intracellular determinants, extracellular cues, such as
signaling molecules or cell-cell interactions, can also affect cell
fate specification and lead to asymmetric cell division
(31). Cell-cell interaction mediated by the membrane Notch
receptor represents one mechanism for external cues to affect
asymmetric cell division. Notch may be activated by Delta or other
ligands to regulate downstream targets, such as Suppresser of Hairless [Su(H)] (2, 36) and Tramtrack (19), which are
required for cell fate specification in the sensory organ lineage. Loss or gain of Notch function during the formation of the
sensory organs and MP2 neurons causes cell fate transformations, which are opposite those caused by the loss or gain of numb
function, respectively (19, 25, 46). Epistasis analysis
indicates that Notch acts downstream of numb
(19, 46). Direct protein-protein interaction has been
observed between Numb and Notch as well as mammalian homologs of Numb
and Notch (19, 53). Moreover, Numb inhibits Delta-dependent
Notch signaling in cultured Drosophila melanogaster S2
cells, as indicated by the failure of Su(H) to translocate into the
nucleus, and ectopically expressed Numb inhibits Notch function during
wing development (17). Thus, both the cell-intrinsic factor
Numb and the cell-extrinsic influence mediated by Notch are important
for proper cell fate specification during asymmetric division, and Numb
functions at least in part by antagonizing Notch activity.
The Numb protein contains a phosphotyrosine binding (PTB) domain (also
called a phosphotyrosine-interacting domain) (7). The PTB
domains of a mouse homolog of Numb (mNumb) (53) and a rat
homolog of the mouse Numblike protein (rNbl) (54) show 71.5 and 75.2% amino acid identity with the PTB domain of
Drosophila Numb (dNumb). Like dNumb, mNumb is asymmetrically
localized during divisions of neural progenitors, and overexpression of
either mNumb or rNbl causes cell fate transformation in the
Drosophila sensory organ lineage, indicating that the
functions of the Numb proteins are conserved through evolution. The PTB
domain of dNumb is not necessary for its asymmetric localization but is
required for its function (17). Overexpression of PTB
domain-deleted dNumb does not cause cell fate transformation in vivo,
nor does it inhibit Su(H) nuclear translocation mediated by Notch
signaling in cultured S2 cells, even though it still forms a crescent
during mitosis.
The PTB domain is also found in signaling molecules such as Shc
(5, 34) and insulin receptor substrate (IRS) families (27). As adapter proteins, Shc binds to activated receptors such as epidermal growth factor, nerve growth factor, and antigen receptors, and IRS binds to activated insulin receptor, so as to
transduce signals to downstream effector proteins (reviewed in
reference 37). When activated, those receptors
autophosphorylate at the tyrosine residues, leading to the binding of
phosphotyrosines with the Src homology 2 (SH2) or PTB domains of the
adapter proteins. Despite the lack of strong sequence similarity
between the Shc and IRS PTB domains, interaction of both PTB domains
with the NPXpY motifs (in which tyrosine residue is phosphorylated) has been shown (22, 27, 33). However, recognition of the Shc PTB
domain and the IRS-1 PTB domain depends on the sequence context of the
NPXpY motif (28, 52), suggesting different binding specificity of different PTB domains. Structural analysis further indicates that the topology of the two PTB domains resembles that of
the pleckstrin homology (PH) domain (15, 37, 55), with a
-sandwich formed by two nearly antiparallel -sheets and capped on
one end by a C-terminal 
-helix. In the complex of the PTB domain and
the phosphopeptide-bearing NPXpY motif, the peptide forms a -strand
antiparallel to one of the two -sheets of the PTB domain and the
phosphotyrosine fits into a largely hydrophobic pocket. The Shc PTB
domain and the IRS PTB domain exhibit similar mechanisms of ligand
binding but use different residues for phosphotyrosine recognition.
Although the PH domain is similar in topology to the PTB domains, the
mechanism of ligand recognition by PH domain is different from that of
PTB domain; the surface of the PH domain of phospholipase C
recognized by its ligand, inositol 1,4,5-triphosphate (16),
is different from the surface of the PTB domain recognized by the NPXpY
motif.
The involvement of Numb protein in asymmetric cell division raises a
number of questions. How does Numb protein become asymmetrically localized during mitosis? How might the physical interaction between Numb and Notch lead to the inhibition of Notch signaling? What is the
function of the PTB domain of Numb? Does Numb have other functions in
addition to the inhibition of Notch signaling? Given the possibility
that the localization or the function of Numb may involve proteins that
bind to Numb, we have searched for Numb-interacting proteins by using
the yeast two-hybrid system (12) as a first step to answer
those questions. We have isolated a novel gene product, Numb-associated
kinase (Nak), which showed strong interaction with Numb in biochemical
assays. Deletion analysis indicated that the interaction involves the
PTB domain of Numb. Whereas the PTB domains of Shc and IRS bind to the
NPXpY sequence motif, the interaction of the Numb PTB domain with Nak
does not require this motif or a tyrosine. The specificity of Nak
interaction with Numb was examined by testing its binding with the PTB
domain of Shc. The possible activity of Nak in asymmetric cell division
was explored in transgenic flies overexpressing Nak.
| |
MATERIALS AND METHODS |
Yeast two-hybrid screening and assays.
The GAL4 activating
domain (GAD) library used in the yeast two-hybrid screen was a gift
from S. Elledge (Baylor College of Medicine). It was made from
third-instar larval cDNA. The methodology in the yeast two-hybrid
screening is as described in reference 3. Briefly,
the library cDNA was cotransformed with pBHA-Numb (encoding the
LexA-Numb fusion protein) into L40 strain (30), and 2 million colonies were selected for His+. The
His+ colonies were filter lifted for blue color assay,
using 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) as the substrate for LacZ activity. After a secondary screening
against LexA-Lamin (3) instead of LexA-Numb, five positive
clones were obtained; two of the positive clones encode Nak, as shown
in Fig. 1A.
View larger version (62K):
[in this window]
[in a new window]
|
FIG. 1.
(A) Schematic representation of the open reading frame
of nak, which encodes 1,490 aa. The hatched box indicates
the serine/threonine kinase catalytic domain. Also indicated are the
starting points of the two positive clones, 13-1 and 7-2, from the
two-hybrid screen. (B) Comparison of the kinase catalytic domains of
Nak, two proteins from yeast (GenBank accession no. p38080 and p40494),
and one from C. elegans (z46242). Only those residues that
are identical in these four proteins are boxed. Some of these residues
are characteristic of the serine/threonine kinase family and are marked
with black dots. The subdomains of the catalytic domain are marked by I
to XI. (C) Complete predicted amino acid sequence of Nak. The 11-aa
peptide near the C terminus that binds the Numb PTB domain is
highlighted.
|
|
The LacZ (
-galactosidase) activities given in Fig.
2 to
5 are the
averages and standard deviations of six independent samples.
-Galactosidase activity was tested for and activity as described
in
reference
4, with the following modifications.
Four-tenths
milliliter of log-phase yeast culture grown in standard
yeast
medium with 2% galactose, 2% glycerol, and 2% ethanol as
carbon
sources was mixed with 0.4 ml of Z buffer. After addition of 50
µl of chloroform and 50 µl of 0.1% sodium dodecyl sulfate, the
yeast cultures were vortexed for 30 s and preincubated at 30°C
for 5 min before the introduction of 160 µl of the substrate,
o-nitrophenyl-
-
D-galactopyranoside (4 mg/ml).
The tubes were
further incubated at 30°C until the yellow color
developed.
The pBHA vector was modified from pBTM116 (
4) with a
hemagglutinin tag (YPYDVPDYA) inserted at the beginning of the multiple
cloning sites and was used to clone the various fragments of Numb
and
Shc. The hemagglutinin tag was used to examine the expression
of the
fusion proteins in yeast (data not shown).
pGAD-GH (
23) was used to clone the C-terminal fragments of
Nak shown in Fig.
4B. The various deletions of Nak shown in Fig.
4A
were done in the original pACT (
14) vector used for
constructing
the library.
The DNA constructs for fusion proteins of LexA and PTB domains of mNumb
and rNbl were provided by W. Zhong, and the DNA fragments
for the PTB
domains of
Drosophila Shc (dShc) and mouse Shc (mShc)
were
obtained by PCR from a
Drosophila and a mouse cDNA library
(a gift from Y.-W. Chen, University of California, San Francisco),
respectively. The junctions for the PTB domains were as given
in
reference
55.
Molecular cloning.
The full-length cDNA encoding Nak was
obtained by screening both Drosophila 
-ZAP larval and
pupal cDNA libraries (gifts from C. S. Thummel, University of
Utah).
In vitro binding assays.
Various Nak C-terminal fragments
were cloned into in-frame pGEX vectors (Pharmacia Co.) for expression
in Escherichia coli DH5 as glutathione
S-transferase (GST) fusion proteins. The expression of
fusion proteins was induced for 2 to 3 h with 100 µM
isopropylthiogalactopyranoside added to log-phase bacteria. Following
sonication of the bacteria in phosphate-buffered saline, 0.1% Triton
X-100 was added before centrifugation to remove the insoluble pellet.
The supernatant were kept in 10% glycerol at 
80°C.
The DNA fragments containing coding sequences for PTB domains of Numb
and Shc were cloned into pNAC vector (a gift from J.
P. O'Connor,
University of Penn.), and the
35S-labeled proteins were
expressed in the TnT coupled lysate system
(Promega Co.). For in vitro
binding assay, 2 to 5 µg of GST fusion
proteins was incubated at
4°C for 30 min with 5 to 10 µl of lysate
containing
35S-labeled proteins. After addition of 20 µl of
GST-agarose beads
(Sigma Co.), the tubes were incubated at room
temperature for
2 min. These beads were washed four times with
phosphate-buffered
saline-100 mM NaCl-0.1% Triton X-100 and
then mixed with protein
loading buffer to elude the bound proteins.
To estimate the dissociation constant for binding of the GST-c1 protein
fusion to the Numb PTB domain, we used several concentrations
of GST-c1
protein to precipitate the in vitro-translated Numb
PTB. The
concentration of Gst-c1 required to precipitate 50% of
labeled Numb
PTB is about 1 µM.
Immunoprecipitation.
Embryos were collected overnight from a
cross of UAS (upstream activation
sequence)-myc-nak males and scabrous-GAL4
females (described below). Embryo extract was prepared according to
reference 13. Anti-c-Myc antibody-agarose conjugate
(Santa Cruz Biotechnology Co.) was used for coimmunoprecipitation, and
the precipitates were run on sodium dodecyl sulfate-polyacrylamide gels
for Western blotting with either anti-c-Myc or anti-Numb antibodies.
Fly genetics.
The full-length DNA fragments for
nak and numb genes were the
NotI-XbaI fragment from pBS-13-1F and
KpnI fragment from hs-numb#2 (42), respectively.
Each DNA fragment was subcloned into the respective restriction sites
of the pUAST vector. The pUAST-nak and pUAST-numb DNAs were injected
into w
flies to create transgenic flies
(43). These transgenic flies were crossed to
Sca-GAL4 (29) or 109-68 flies
(17); these GAL4 enhancer trap lines probably have GAL4
coding sequence inserted at the scabrous locus. The larval
progenies from these crosses were kept at either 25 or 30°C (normally
stronger phenotype is obtained at 30 than at 25°C), and their
overexpression phenotypes in the sensory organs were examined. About
one-third (10 of 33) of the UAS-nak and most (15 of 18) of
the UAS-numb independent transformants gave similar
phenotypes, though the strength of the phenotypes varied with the
transformant lines. Transformants 15 of UAS-nak and 14 of
UAS-numb gave strong and consistent phenotype and were used
for the quantitative analysis in this report.
pUAS-myc-nak was created by insertion of
nak open reading
frame sequence into pBS-
G vector (from I. Clark) which includes
5'
leader sequence of the
Xenopus globin gene and a
myc tag sequence.
The
myc-nak fusion sequence was
subcloned into pUAST vector for
embryo injection.
Phenotype examination.
For examining the embryonic
neuron/sheath cell transformation, homozygous UAS-nak and
UAS-numb transgenic flies were crossed to homozygous
Sca-GAL4 flies. Embryos were collected over a period of
4 h at room temperature and moved to 30°C for another 9.5 h. The fixation and staining procedures were as described in reference 16. Monoclonal antibody 22C10 (56) and
rabbit anti-Prospero (50) were used at 1:250 and 1:1,000
dilutions to stain neurons and sheath cells, respectively. The images
were analyzed with a Zeiss microscope and a Bio-Rad MRC-600 confocal
microscope.
Adult nota were dissected from flies in 80% isopropanol and mounted in
Hoyer's medium (
1).
| |
RESULTS |
Identification of the nak gene.
To identify genes
encoding proteins which interact physically with the Numb protein, we
used the yeast two-hybrid system to screen a library of third-instar
larval cDNA fused to the coding sequences of GAD (see Materials and
Methods). From 2 million colonies, we isolated two independent clones
(13-1 and 7-2 [Fig. 1A]) that correspond to a novel gene,
nak, with an open reading frame of 1,490 amino acids (aa)
(Fig. 1C). Nak protein includes a putative serine/threonine kinase
domain at the N terminus (Fig. 1A), which has about 40% amino acid
identity with the kinase domains encoded by a gene from
Caenorhabditis elegans and two genes from
Saccharomyces cerevisiae (Fig. 1B). The functions of these
genes are not known. Nak and the three related putative kinases contain
the characteristic residues that are conserved in the subdomains of
known kinases with the exception of domain I: instead of the GXGXXG
nucleotide binding motif present in domain I of most kinases, Nak and
related kinases contain a GGFA motif. The GGFA motif is also present in the polo kinase family, plo1+ of Schizosaccharomyces
pombe, CDC5 of S. cerevisiae, polo of D. melanogaster, and polo-like kinase of mammals (39),
suggesting that these proteins may represent a new class of kinases.
To determine the expression pattern of
nak, we performed
Northern analysis, which showed a 5.5-kb mRNA expressed in all stages
(embryo, larvae, pupae, and adult) of development, a 4.5-kb mRNA
expressed at least in 0- to 12-h embryos and a 3-kb mRNA expressed
in
0- to 3-h, but not 3- to 12-h, embryos. All three messages
hybridized
to a probe containing coding sequence of the kinase
domain, but only
the 5.5-kb mRNA hybridized to a probe corresponding
to the Nak C
terminus (aa 1088 to 1490). A ubiquitous expression
pattern in whole
mount embryos was observed (data not shown).
Binding of Nak to the PTB domain of Numb.
The Nak-interacting
domain of Numb was mapped by using the yeast two-hybrid system. Either
the N-terminal part (Fig. 2A, line 2) or
the PTB domain (Fig. 2A, line 5) of Numb interacted with the 13-1 clone, which includes the Nak C-terminal region (aa 1088 to 1490).
Fragments on either end of the Numb PTB domain did not show any
interaction with 13-1 in the two-hybrid assay (Fig. 2A, lines 3 and 4).
Thus, the PTB domain of Numb is necessary and sufficient for the
interaction with the C-terminal fragment of Nak.
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 2.
Nak binding to the PTB domain of Numb. (A) Mapping the
interaction domain of Numb, using the yeast two-hybrid system. Various
Numb fragments were fused with LexA and tested with GAD alone () or
the 13-1 clone encoding a fusion of GAD and aa 1088 to 1490 of Nak
(13-1). For the assay and calculation of -galactosidase (LacZ)
activity, see Materials and Methods. The means and standard deviations
calculated from six independent samples are given. (B) In vitro binding
of the Numb PTB domain to the Nak C terminus. GST-Nak(1385-1490) (lane
2), but not GST alone (lane 1) or GST-Nak(1385-1428) (lane 3), binds
to 35S-labeled Numb-PTB domain. Neither GST nor
GST-Nak(1385-1490) interacts with luciferase (lanes 4 and 5). The
loading controls for Numb-PTB and luciferase are shown in lanes 6 and
7.
|
|
The interaction between the Numb PTB domain and Nak C terminus was also
evident in an in vitro binding assay, namely, the
coprecipitation of
the bacterial fusion protein GST-Nak (aa 1385
to 1490), which includes
the Numb-interacting region of Nak (see
below), and in vitro-translated
PTB domain of Numb (Fig.
2B, lane
6) by glutathione-agarose beads (Fig.
2B, lane 2). This coprecipitation
was not observed with either GST or
GST-Nak (aa 1385 to 1428),
which lacks the Numb-interacting region of
Nak (see below). In
another control, both GST and GST-Nak (aa 1385 to
1490) failed
to coprecipitate with in vitro-translated luciferase
protein (Fig.
2B, lanes 4 and 5). Therefore, the PTB domain of Numb
interacts
specifically with the Nak C terminus.
Novel interactions of Nak with the PTB domain of Numb.
An
NPXpY motif containing a phosphotyrosine is recognized by the PTB
domains of Shc and IRS-1 (22, 27, 33) and possibly also the
Numb PTB domain (51). In contrast, the Numb-interacting region of Nak (aa 1088 to 1490) does not include any NPXY motif, suggesting that the binding of the PTB domain of Numb with Nak is
mechanistically different from interactions of PTB domains with the
NPXY motif. To test this possibility, we examined the effects of point
mutations in the Numb PTB domain which correspond to point mutations in
the Shc PTB domain known to disrupt the interaction between Shc and
tyrosine-phosphorylated p145 (55). The S148A mutation of the
Numb PTB domain corresponds to a mutation in the Shc PTB domain which
reduces the interaction between Shc and tyrosine-phosphorylated p145 by
60%, but it did not affect interaction between the Numb PTB domain and
the Nak C terminus (aa 1385 to 1490) (Fig.
3A, lane 8). Another point mutation of the Numb PTB domain, R171N, corresponds to a mutation in the Shc PTB
domain which eliminates the interaction between Shc and
tyrosine-phosphorylated p145 completely. Like S148A, the R171N mutation
did not abolish the interaction between Numb PTB domain and Nak C
terminus (Fig. 3A, lane 5). Moreover, in the two-hybrid assay, these
two mutants were indistinguishable from wild-type Numb in their
interaction with Nak C-terminal fragment encoded by the 13-1 clone
(Fig. 3B, lines 2, 4, and 6). Taken together, these observations
indicate that Nak interacts with the Numb PTB domain in a manner
different from interactions involving the Shc PTB domain and NPXY
motif.
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 3.
Numb PTB domain mutations predicted to disrupt the
interaction with the NPXpY motif do not affect the interaction with
Nak. (A) The mutation R171N (substitution of arginine with asparagine
at position 171) slightly reduces the interaction between Numb-PTB and
GST-Nak(1385-1490) (the middle three lanes). The mutation S148A
(substitution of serine with alanine at position 148) does not affect
the interaction at all (the last three lanes). The first three lanes
are wild-type controls as shown in Fig. 2B. (B) Two-hybrid assay for
the interactions between Numb-PTB (wild type and two mutants) and
Nak(1385-1490). Both mutants, R171N and S148A, yielded LacZ activities
similar to that of the wild-type control.
|
|
Identification of the Numb-binding sequence in Nak.
The
C-terminal fragment of Nak is sufficient for interaction with Numb, as
indicated by the isolation of the 13-1 clone (aa 1088 to 1490 of Nak)
and 7-2 clone (aa 1193 to 1490 of Nak) from the two-hybrid screen. Both
C-terminal fragments showed interaction with the PTB domain of Numb
(Fig. 4A, lines 2 and 3) and the entire Numb protein (Fig. 2A and data not shown). A large internal deletion of
the 13-1 clone that leaves the C-terminal 66 residues joined with its
N-terminal 26 residues (aa 1088 to 1113 and 1425 to 1490) did not
abolish the interaction with the Numb PTB domain (Fig. 4A, line 4). On
the other hand, deletion of the most C-terminal 66 aa (Fig. 4A, line 5)
eliminated the interaction completely. These data indicate that the
C-terminal 66 aa of Nak are responsible for most of the interaction
with Numb PTB domain.
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 4.
Mapping the interaction region of Nak. (A) Various
deletions of the 13-1 constructs affect the interaction with
LexA-Numb-PTB to different extent, as indicated by the LacZ activities.
(B) Various Nak C-terminal regions were tested for their interaction
with LexA-Numb-PTB as indicated by the growth of yeast colonies on a
His plate and by the appearance of blue color in filter
lift X-Gal assays. Growth after two days on His plates
was scored, and the number of +'s indicates relative size. , no
growth. For filter lift X-Gal assay, +++++ represents colonies turning
blue in 15 min and very dark blue at 300 min, ++++ represents colonies
turning blue in 30 min and dark blue at 300 min, +++ represents
colonies turning blue in 60 min and medium blue at 300 min, and represents colonies with no blue color in 300 min. (C) In vitro binding
assay to test the interaction between the Nak C-terminal fragments and
Numb-PTB. The results of this assay are summarized in the rightmost
column of panel B. (D) Amino acid sequence for the smallest region of
Nak that interacts with Numb-PTB.
|
|
To further define the Numb-binding region of Nak, we tested a series of
small fragments from the Nak C terminus for their
interaction with the
Numb PTB domain in the two-hybrid system
and in in vitro binding assays
(Fig.
4B). A peptide of 11 aa (aa
1439 to 1449 of Nak, c1.5CN) is
sufficient for the interaction
with the PTB domain of Numb. The Numb
PTB domain also recognized
three other Nak constructs that contain
these 11 aa (c1, c1.5,
c1.5C, and c1.5CN) but not those Nak fragments
without this sequence
(c2, c1.5N, and c1.5CC) (Fig.
4B and C). The
sequence of the 11
aa of c1.5CN, as shown in Fig.
4D, does not contain
any tyrosine
residues, though it is conceivable that the two serine
residues
in this peptide could be phosphorylated.
Specificity of Nak interaction with the PTB domain of Numb.
To
examine the ability of Nak to interact with PTB domains of different
proteins, we looked for its interaction with mammalian homologs of Numb
as well as the PTB domains of Shc from mouse and from fly proteins. The
PTB domains of fly and mammalian Numb proteins show about 70 to 75%
amino acid identity (54). PTB domains of both mNumb and rNbl
interacted with 13-1 clone (aa 1088 to 1490 of Nak) as strongly as the
dNumb PTB domain (Fig. 5A, lines 1 to 3),
suggesting that the interaction between Numb and Nak may also be
conserved in mammals. In contrast, the PTB domains of dShc and mShc did
not show any interaction with this Nak fragment in the two-hybrid assay
(Fig. 5A, lines 4 and 5), nor did we observe any binding of the PTB
domains of dShc and mShc to the c1 fragment of Nak in the in vitro
binding assays (Fig. 5B, lanes 8 and 9). These results indicate that
Nak interacts with PTB domain of Numb specifically. The dissociation
constant for GST-c1 binding to the dNumb PTB domain was estimated to be ~1 µM (see Materials and Methods).
View larger version (49K):
[in this window]
[in a new window]
|
FIG. 5.
Test for the specificity of interaction between Nak and
PTB domains. (A) The PTB domains of mNumb, rNbl, dShc, and mShc were
cotransformed with GAD or GAD-Nak (aa 1088 to 1490, the 13-1 clone)
into the yeast two-hybrid reporter strain for LacZ activity assay. (B)
The PTB domains of dShc and mShc showed no interaction with the c1
fragment (amino acids 1422 to 1490) of Nak in the in vitro binding
assay. The control is as for Fig. 2B.
|
|
Nak interacts with Numb in vivo.
To identify in vivo
interaction between Nak and Numb, we created transgenic flies
expressing Myc-tagged Nak protein (see Materials and Methods). Embryo
extracts were prepared from the strains with or without Myc-Nak. Using
anti-c-Myc antibody, Myc-Nak was detected as doublet of 200- and
180-kDa proteins (Fig. 6, lane 2),
similar to the size of in vitro-expressed Nak proteins (data not
shown), but not in the control lane 1. Immunoprecipitation was
performed with the anti-c-Myc antibody. The two extracts contained
similar amounts of Numb protein (Fig. 6, lanes 3 and 4). Numb protein was coprecipitated with Myc-Nak, detected with anti-Numb antibody (Fig.
6, lane 6), but not from embryo extract prepared from the strain
without expressing Myc-Nak (Fig. 6, lane 5), indicating an in vivo
interaction between Numb and Nak.
View larger version (65K):
[in this window]
[in a new window]
|
FIG. 6.
Western blot showing the coimmunoprecipitation of Numb
and Nak in embryo extract. Lanes 1, 3, and 5 are embryo extracts
prepared from scabrous-GAL4 strains; lanes 2, 4, and 6 are
extracts from the cross of scabrous-GAL4 and
UAS-myc-nak strains. After immunoprecipitation with
anti-c-Myc antibody, lanes 1 and 2 were probed with anti-c-Myc antibody
and lanes 5 and 6 were probed with anti-Numb antibody. Control lanes 3 and 4 are embryo extracts before immunoprecipitation and probed with
anti-Numb antibody.
|
|
The cell fate transformation due to Nak overexpression is opposite
to that caused by Numb overexpression.
By doing in situ
hybridization to third-instar larval salivary gland polytene
chromosomes, we mapped the nak gene to the cytological location 37B4-7 on the left arm of the second chromosome. The smallest
available chromosomal deficiency, Df(2L)TW3
(36F7-37A1;37B2-37B7), deletes a number of genes located in the 37A and
37B region, including nak. We found that the homozygous
Df(2L)TW3 embryos are quite disorganized, thus precluding us
from assessing whether loss of nak function would lead to
cell fate change in sensory organs. Another confounding problem to the
analysis of zygotic mutant embryos is the presence of a significant
maternal contribution of the nak gene activity in early
embryos. Thus, we were unable to infer the loss of function phenotype
by analyzing homozygous Df(2L)TW3 embryos. We therefore
turned to gain-of-function experiments.
We use the GAL4-UAS system (
8) to express the Nak protein
and to test its function in vivo. The
UAS-nak flies were
crossed
to
scabrous-GAL4 enhancer trap lines (
17,
29), which leads
to the expression of Nak protein in the neural
precursor cells.
Overexpression of Nak during sensory organ development
resulted
in several phenotypes suggestive of cell fate transformations
within the sensory organ lineage. In the wild-type embryos, five
chordotonal neurons and five sheath cells are aligned in the lateral
5 region (lch5 [Fig.
7A, D, G, and J]) of
each abdominal hemisegment.
In addition, there are one chordotonal
organ and three other sensory
organs (Fig.
7J) in the same region. When
Nak is overexpressed,
we observed cell fate transformation from neuron
to sheath cell
in the lineage of chordotonal organs (Fig.
7B, E, and
F), as shown
by the increased number of sheath cells and the reduced
number
of neurons. This is in contrast to the overexpression of Numb
protein, which increases the number of neurons and reduces the
number
of sheath cells in the same lineage (Fig.
7C, F, and I).
View larger version (100K):
[in this window]
[in a new window]
|
FIG. 7.
Overexpression of Nak or Numb causes opposite cell fate
transformation between neurons and sheath cells in the embryonic
lateral chordotonal organs. (A, D, and E) From a wild-type embryo; (B,
E, and H) from an embryo with Nak overexpression; (C, F, and I) from an
embryo with Numb overexpression. (A to C) Staining of neurons with
antibody 22C10; (D to F) staining of the sheath cells with
anti-Prospero antibody; (G to I) superimposition of the neuron and the
sheath cell staining patterns; (J) Schematic drawing of the lch5
chordotonal organs and their neighboring sensory organs; (K) lineage of
lch5 (9), with the solid arrow indicating the direction of
cell fate transformation due to overexpression of Nak and the lower,
empty arrow indicating the direction of transformation due to
overexpression of Numb. n, neuron; sh, sheath cell; cap, cap cell; ect,
ectodermal cell; lig, ligament cell; chIII, tertiary chordotonal
precursor cell.
|
|
Similar to the embryonic phenotypes, Nak overexpression in the adult
sensory organs caused cell fate transformations in the
bristles in
various parts of the flies, including the head, notum,
and wing margin.
Instead of the normal appearance of a hair and
a socket (with a neuron
and a sheath cell underneath), the mutant
bristles of the transgenic
flies sometimes contain two hairs and
two sockets (Fig.
8B and C, arrowhead), indicating that the
SOP
cell divides symmetrically to two A cells (i.e., a transformation
of B to A cell [Fig.
8I]), instead of one A and one B cell. In
some
cases, the sensory bristle contained two sockets but no hair
(Fig.
8D,
solid arrowhead), indicating that the SOP divided asymmetrically
to
produce an A and a B cell, but the A cell divided symmetrically
to give
rise to two sockets but no hair (i.e., a transformation
of hair to
socket [Fig.
8I]). Other phenotypes are characterized
by four sockets
(Fig.
8D, empty arrowhead) or one hair and three
sockets (Fig.
8C,
arrow), suggesting that the SOP cell divided
symmetrically and at least
one of the two A cells that it generated
also divided symmetrically.
Besides examination of the external
sensory structures, we carried out
immunocytochemical studies
using neuron-specific antibody and found
that neurons were missing
under these mutant bristles (data not shown).
The absence of neurons
could arise either from a transformation of B
cell to A cell during
SOP division or from neuron-to-sheath cell
transformation during
the B-cell division. All of the cell fate
transformations due
to overexpression of Nak are opposite those caused
by overexpression
of Numb, which resulted in twin hair (socket-to-hair
cell transformation
[Fig.
8E and F]) and balding (no hair and no
socket; A-to-B cell
transformation [Fig.
8E]). Thus, during sensory
organ development,
overexpression of Nak and overexpression of Numb
lead to opposite
cell fate transformation in every cell division of the
sensory
organ lineage examined (Fig.
8K and I).
View larger version (73K):
[in this window]
[in a new window]
|
FIG. 8.
Cell fate transformation in the external sensory organs
on the nota. (A) From a wild-type fly; (B) from a Nak overexpression
fly; (E) from a Numb overexpression fly; (G) from a fly with both Nak
and Numb overexpression; (C and F) from the inlets of panels B and E,
respectively. In panel C, the arrowhead indicates a 2-hair-2-socket
bristle and the arrow indicates a 1-hair-3-socket bristle. In panel D,
the filled arrowhead indicates a 2-socket phenotype and the empty
arrowhead indicates a 4-socket phenotype due to overexpression of Nak.
In panel F, the 2-hair-no-socket phenotype caused by Numb
overexpression is marked with two arrows. (H) Schematic drawing
of an adult external sensory organ; (I) external sensory organ lineage,
with the solid arrow indicating the direction of cell fate
transformation due to overexpression of Nak and the lower, empty arrow
indicating the direction caused by overexpression of Numb. n, neuron;
sh, sheath cell; h, hair cell, so, socket cell.
|
|
Antagonistic actions of the nak transgene and the
endogenous numb gene.
We then investigated the genetic
interaction between nak and numb by examining the
effect of numb copy number on Nak overexpression phenotype.
The phenotype caused by overexpression of Nak is enhanced by reducing
the copy number of numb gene from two to one (Table 1; compare rows 1 and 2). Whereas eight
macrochaetes are present at the dorsocentral and scutellar positions on
the notum of a wild-type fly, overexpression of Nak caused 31.7% of
these macrochaetes to exhibit phenotypes indicative of cell fate
transformation when the transgenic flies contained two copies of the
wild-type numb gene. The fraction of mutant macrochaetes
with indication of cell fate transformation was increased to 67.1% in
transgenic flies with overexpression of Nak but only one copy of
wild-type numb gene (numb1/+); the
2/4-socket phenotype (see Table 1, footnotes b to d, for definitions of
phenotypes) was increased from 15.0 to 30.4%, and the 1-hair-3-socket
and 2-hair-2-socket phenotypes were increased from 16.7 to 36.7%. In
the absence of the nak transgene, the
numb1/+ flies have no bristle phenotype. Thus,
the effect of nak transgene in the sensory organ development
could be enhanced by reducing the gene dosage of numb.
Not only did a reduction of
numb gene enhance the cell fate
transformation phenotype caused by Nak overexpression, overexpression
of Numb as well as Nak suppressed most of the bristle phenotypes
due to
overexpression of either Numb or Nak alone, including the
2-socket (the
phenotype of Nak overexpression), the 2-hair (the
phenotype of Numb
overexpression) (Fig.
8G), and the 2-hair-2-socket
or 1-hair-3-socket
phenotypes due to overexpression of Nak. The
balding phenotype of
macrochaetes caused by overexpression of
Numb was also significantly
reduced; 44.3% of the bristles (or
3.6 bristles per fly on average)
were normal in flies overexpressing
Nak and Numb, compared to only
6.4% (or 0.5 bristle per fly) in
flies overexpressing Numb alone (Fig.
8E and G; Table
1, rows
3 and 4). Similarly, most of the balding
phenotype of microchaetes
was also suppressed in flies expressing Numb
as well as Nak (Fig.
8E and G). Thus, the phenotype of the opposite
cell fate transformation
due to the action of Nak is sensitive to both
increase and decrease
of the Numb protein level.
To further test the genetic interaction between
numb and
nak, we used the
Df(2L)TW3 deficiency, which
deletes
nak and several
other genes, and tested if
overexpression phenotype of Numb is
sensitive to the gene dosage of
nak. Indeed, heterozygous
Df(2L)TW3 embryos that
overexpressed Numb showed enhanced Numb overexpression
phenotype (Table
1; compare row 3 with row 5). This result suggests
that the Numb
overexpression phenotype is sensitive to the gene
dosage of a certain
gene(s) in the region deleted in
Df(2L)TW3. nak is likely to
be the gene that is responsible for this genetic
interaction.
| |
DISCUSSION |
In this study, we have used the yeast two-hybrid system to isolate
a novel Numb-interacting protein which includes a putative serine/threonine kinase domain at the N terminus and a Numb-binding region at the C terminus. Interaction between Numb and Nak requires the
PTB domain of Numb and an 11-aa peptide of Nak; this interaction is
specific for the Numb PTB domain and may represent a novel interaction
independent of tyrosine phosphorylation. Overexpression of Nak in
transgenic flies affects asymmetric cell division in a manner that is
antagonistic to the actions of Numb. The characteristics of Nak binding
to the PTB domain of Numb and the possible functions of Nak in vivo are
discussed below.
Nak binding to the PTB domain of dNumb, mNumb, and rNbl but not the
PTB domain of dShc and mShc.
Several observations suggest a novel
interaction of Nak with the PTB domain of Numb. First, unlike
interactions involving the PTB domains of proteins in the Shc family or
the IRS family (22, 27, 33), Nak interaction with the PTB
domain of Numb does not involve an NPXpY motif (Fig. 4D). In fact,
there are no tyrosine residues in the 11-aa peptide from Nak that is
sufficient for the binding to the Numb PTB domain. Second, Nak binding
was not affected by mutations of the Numb PTB domain that corresponds to those mutations of the Shc PTB domain known to disrupt Shc interaction with the tyrosine-phosphorylated p145 (55) (Fig. 3). Moreover, the Nak interaction is specific for the PTB domain of
Numb; no interaction with the Shc PTB domains can be detected (Fig. 5).
The requirement of a short peptide of 11 aa from Nak for binding to the
Numb PTB domain is suggestive of peptide-surface association,
similar
to the interactions between the SH2 domains and tyrosine-phosphorylated
peptides, the PDZ domain and a peptide containing the Ser/Thr-X-Val
motif, and the PTB domains of Shc or IRS families and a peptide
containing the NPXpY motif (for a review, see reference
24).
In another screen for Numb-interacting
proteins, we found that
the intracellular domain of the
Drosophila epidermal cell surface
receptor encoded by
stranded at second (
sas) (
44) binds
Numb
PTB domain. The 37 aa of the intracellular domain of Sas contains
the NPXY motif, and point mutations of either the Asn or Tyr residues
abolished the interaction between Sas and Numb in the two-hybrid
assay
(
11a). This result suggests that the Numb PTB domain is
capable of binding to the NPXY motif. Also, a mammalian homolog
of Numb
was shown to bind tyrosine-phosphorylated proteins (
51),
although the motif which mediates the specific interaction was
not
defined. The versatility of the PTB domain binding specificity
was
further shown by the binding of the dNumb PTB domain to GPpY
motifs
(
38) and the Shc PTB domain to NPLH motifs (
11).
In
addition, the PTB domains of two neuronal proteins, X11 and FE65,
are capable of binding to the YENPTY motif of the cytoplasmic
domain of
the amyloid precursor protein (
6). This binding is
not
tyrosine phosphorylation dependent even though this sequence
contains
an NPXY motif. It remains to be determined whether the
binding surfaces
of the PTB domain to various motifs are distinct
from each other and if
the PTB domain can utilize multiple binding
regions for its interaction
with different proteins simultaneously
or these binding events are
mutually exclusive.
Possible functions of Nak in asymmetric cell division.
Whereas
the specific physical interaction between Nak and Numb suggests that
Nak may be part of the Numb pathway in specifying daughter cell fate
during asymmetric division, it will be necessary to test this
possibility by examining both the loss-of-function phenotype and the
gain-of-function phenotype of the nak gene. No
loss-of-function mutations of the nak gene are currently
available. It is worth noting, however, that a number of genes known to
be involved in the Numb pathway for asymmetric division exhibit
overexpression phenotypes which correspond to cell fate transformations
opposite those caused by loss of gene function. Hence, overexpression
of the protein products of Delta, Notch,
tramtrack, Suppresser of Hairless, or
enhancer of split causes transformation of the B cell to the
A cell, the hair cell to the socket cell, and the neuron to the sheath
cell (18, 26, 40, 45, 48), opposite their respective
loss-of-function phenotypes. Phenotypes due to overexpression of these
genes are similar to the numb null mutant phenotype, whereas
overexpression of Numb causes the opposite cell fate transformation
(42). The overexpression phenotypes of nak are
very similar to those of Notch, tramtrack, and
other downstream genes of numb and are therefore highly
suggestive of the involvement of nak in asymmetric
divisions.
The in vivo interaction of Myc-Nak with Numb protein is also indicative
of the function of Nak in the asymmetric cell division
pathway. In
addition to the immunocoprecipitation of Numb and
Myc-Nak, we also
observed that the ectopically expressed Myc-Nak
localize to the
cortical membrane where Numb and Notch are distributed
(data not
shown), suggesting that Nak can localize to the site
for participation
in the asymmetric cell divisions. Due to the
overexpression of Myc-Nak,
it is difficult to analyze the segregation
of Myc-Nak during cell
division. Whether Nak is asymmetrically
localized during asymmetric
cell divisions awaits the availability
of an antibody that is suitable
for immunocytochemistry.
The potential involvement of Nak in asymmetric divisions in
Drosophila is reminiscent of the involvement of the
par-1 gene
in asymmetric divisions during early embryonic
development of
C. elegans. Par-1 also contains a
serine/threonine kinase domain
and a C-terminal region that binds other
proteins; whereas the
C terminus of Nak binds Numb, the C terminus of
Par-1 binds a
nonmuscle myosin (
20,
21). A priori, a
Numb-binding protein
could be involved in asymmetric localization of
Numb during asymmetric
division or in executing the actions of
asymmetrically segregated
Numb in specifying daughter cell fate. It
appears unlikely that
Nak is involved in asymmetric localization of
Numb, for the following
reasons. First, the Nak overexpression
phenotypes could be suppressed
by Numb overexpression. This restoration
of the proper asymmetric
divisions could not have been achieved if
overexpression of Nak
had abolished asymmetric Numb localization.
Second, Nak binds
to the PTB domain but not to the rest of the Numb
protein. The
PTB domain is not necessary for asymmetric localization of
Numb
in dividing neural precursor cells but is necessary for the
ability
of Numb to inhibit Notch signaling (
17). Third, both
mNumb and
mouse Numblike (mNbl; homolog of rNbl) contain PTB domains
which
are 70 to 75% identical to the PTB domain of dNumb at the amino
acid level, and when overexpressed in
Drosophila, mNumb and
mNbl
can transform cell fate in the sensory organ lineages. But only
mNumb, not mNbl, is asymmetrically localized in transgenic flies
(
54). It thus appears unlikely that Nak plays a role in
asymmetric
Numb localization.
The observation of Nak activity thus far is consistent with the
possibility that Nak mediates or modulates the action of asymmetrically
distributed Numb. For example, Nak may phosphorylate Numb and
negatively regulate Numb function. Alternatively, the interaction
of
Nak and Numb may prevent the binding of Notch to Numb (
19,
53), thus relieving the inhibition of Notch from Numb. It is
also
conceivable that Nak may be recruited to the vicinity of
Notch due to
physical interactions of Numb with Notch and Nak,
so that it could
phosphorylate Notch or its downstream effectors,
thereby inhibiting
Notch signaling. These and other possible scenarios
may be tested by
future genetic and biochemical studies.
| |
ACKNOWLEDGMENTS |
We thank Weimin Zhong for mammalian Numb and Numblike DNA
constructs, Yenwen Chen (UCSF) for mammalian cDNA libraries, Steve Elledge (Baylor College of Medicine) for a Drosophila
two-hybrid library, Carl S. Thummel (University of Utah) for larval and
pupal cDNA libraries, Jue Wang for helping with some of the biochemical assays, and Susan Younger and Alice Turner for chromosomal in situ
hybridizations. We also thank Ming Guo for suggestions on the
manuscript.
Cheng-ting Chien was supported by the Jane Coffin Childs Memorial Fund
for Cancer Research. Shuwen Wang is a research associate and Lily Y. Jan and Yuh Nung Jan are investigators of the Howard Hughes Medical
Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Howard Hughes
Medical Institute and Department of Physiology and Biochemistry,
University of California at San Francisco, San Francisco, CA
94143-0724. Phone: (415) 476-8752. Fax: (415) 476-5774. E-mail:
ynjan{at}itsa.ucsf.edu.
Present address: Institute of Molecular Biology, Academia Sinica,
Nankang, Taipei 11529, Taiwan.
| |
REFERENCES |
| 1.
|
Ashburner, M.
1989.
.
Drosophila: a laboratory handbook.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 2.
|
Bailey, A. M., and J. W. Posakony.
1995.
Suppressor of Hairless directly activates transcription of Enhancer of split complex genes in response to Notch receptor activity.
Genes Dev.
9:2609-2622[Abstract/Free Full Text].
|
| 3.
|
Bartel, P. L.,
C.-T. Chien,
R. Sternglanz, and S. Fields.
1993.
Elimination of false positives that arise in using the two-hybrid system.
BioTechniques
14:920-924.
[Medline] |
| 4.
|
Bartel, P. L.,
C.-T. Chien,
R. Sternglanz, and S. Fields.
1993.
Using the two-hybrid system to detect protein-protein interactions, p. 153-179. In
D. A. Hartley (ed.), Cellular interaction in development: a practical approach.
Oxford University Press, Oxford, England.
|
| 5.
|
Blaikie, P.,
D. Immanuel,
J. Wu,
N. Li,
V. Yajnik, and B. Margolis.
1994.
A region in Shc distinct from the SH2 domain can bind tyrosine-phosphorylated growth factor receptors.
J. Biol. Chem.
269:32031-32034[Abstract/Free Full Text].
|
| 6.
|
Borg, J. P.,
J. Ooi,
E. Levy, and B. Margolis.
1996.
The phosphotyrosine interaction domains of X11 and FE65 bind to distinct sites on the YENPTY motif of amyloid precursor protein.
Mol. Cell. Biol.
16:6229-6241[Abstract].
|
| 7.
|
Bork, P., and B. Margolis.
1995.
A phosphotyrosine interaction domain.
Cell
80:693-694[Medline].
|
| 8.
|
Brand, A. H., and N. Perrimon.
1993.
Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.
Development
118:401-415[Abstract].
|
| 9.
|
Brewster, R., and R. Bodmer.
1995.
Origin and specification of type II sensory neurons in Drosophila.
Development
121:2923-2936[Abstract].
|
| 10.
|
Campos-Ortega, J. A.
1996.
Numb diverts Notch pathway off the Tramtrack.
Neuron
17:1-4[Medline].
|
| 11.
|
Charest, A.,
J. Wagner,
S. Jacob,
C. J. McGlade, and M. L. Tremblay.
1996.
Phosphotyrosine-independent binding of SHC to the NPLH sequence of murine protein-tyrosine phosphatase-PEST. Evidence for extended phosphotyrosine binding/phosphotyrosine interaction domain recognition specificity.
J. Biol. Chem.
271:8424-8429[Abstract/Free Full Text].
|
| 11a.
| Chien, C.-T., M. Rothernberg, L. Y. Jan,
and Y. N. Jan. Unpublished data.
|
| 12.
|
Chien, C.-T.,
P. Bartel,
R. Sternglanz, and S. Fields.
1991.
The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest.
Proc. Natl. Acad. Sci. USA
88:9578-9582[Abstract/Free Full Text].
|
| 13.
|
Cleghon, V.,
U. Gayko,
T. D. Copeland,
L. A. Perkins,
N. Perrimon, and D. K. Morrison.
1996.
Drosophila terminal structure development is regulated by the compensatory activities of positive and negative phosphotyrosine signaling sites on the Torso RTK.
Genes Dev.
10:566-577[Abstract/Free Full Text].
|
| 14.
|
Durfee, T.,
K. Becherer,
P. L. Chen,
S. H. Yeh,
Y. Yang,
A. E. Kilburn,
W. H. Lee, and S. J. Elledge.
1993.
The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit.
Genes Dev.
7:555-569[Abstract/Free Full Text].
|
| 15.
|
Eck, M. J.,
S. Dhe-Paganon,
T. Trub,
R. T. Nolte, and S. E. Shoelson.
1996.
Structure of the IRS-1 PTB domain bound to the juxtamembrane region of the insulin receptor.
Cell
85:695-705[Medline].
|
| 16.
|
Ferguson, K. M.,
M. A. Lemmon,
J. Schlessinger, and P. B. Sigler.
1995.
Structure of the high affinity complex of inositol triphosphate with a phospholipase C pleckstrin homology domain.
Cell
83:1037-1046[Medline].
|
| 17.
|
Frise, E.,
J. A. Knoblich,
S. Younger-Shepherd,
L. Y. Jan, and Y. N. Jan.
1996.
The Drosophila Numb protein inhibits signaling of the Notch receptor during cell-cell interaction in sensory organ lineage.
Proc. Natl. Acad. Sci. USA
93:11925-11932[Abstract/Free Full Text].
|
| 18.
|
Guo, M.,
E. Bier,
L. Y. Jan, and Y. N. Jan.
1995.
tramtrack acts downstream of numb to specify distinct daughter cell fates during asymmetric cell divisions in the Drosophila PNS.
Neuron
14:913-925[Medline].
|
| 19.
|
Guo, M.,
L. Y. Jan, and Y. N. Jan.
1996.
Control of daughter cell fate during asymmetric division: interaction of Numb and Notch.
Neuron
17:27-41[Medline].
|
| 20.
|
Guo, S., and K. J. Kemphues.
1995.
par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative ser/thr kinase that is asymmetrically distributed.
Cell
81:611-620[Medline].
|
| 21.
|
Guo, S., and K. J. Kemphues.
1996.
A non-muscle myosin required for embryonic polarity in Caenorhabditis elegans.
Nature
382:455-458[Medline].
|
| 22.
|
Gustafson, T. A.,
W. He,
A. Craparo,
C. D. Schaub, and T. J. O'Neill.
1995.
Phosphotyrosine-dependent interaction of Shc and IRS-1 with the NPXY motif of the insulin receptor via a novel non-SH2 domain.
Mol. Cell. Biol.
15:2500-2508[Abstract].
|
| 23.
|
Hannon, G. J.,
D. Demetrick, and D. Beach.
1993.
Isolation of the Rb-related p130 through its interaction with CDK2 and cyclins.
Genes Dev.
7:2378-2391[Abstract/Free Full Text].
|
| 24.
|
Harrison, S. C.
1996.
Peptide-surface association: the case of PDZ and PTB domains.
Cell
86:341-343[Medline].
|
| 25.
|
Hartenstein, V., and J. W. Posakony.
1989.
Development of adult sensilla on the wing and notum of Drosophila melanogaster.
Development
107:389-405[Abstract].
|
| 26.
|
Hartenstein, V., and J. W. Posakony.
1990.
A dual function of the Notch gene in Drosophila sensillum development.
Dev. Biol.
142:13-30[Medline].
|
| 27.
|
He, W.,
A. Craparo,
Y. Zhu,
T. J. O'Neill,
L. M. Wang,
J. H. Pierce, and T. A. Gustafson.
1996.
Interaction of insulin receptor substrate-2 with the insulin and IGF-1 receptors: evidence for two-distinct phosphotyrosine-dependent interaction domains within IRS-2.
J. Biol. Chem.
271:11641-11645[Abstract/Free Full Text].
|
| 28.
|
He, W.,
T. J. O'Neill, and T. A. Gustafson.
1995.
Distinct modes of interaction of SHC and insulin receptor substrate-1 with insulin receptor NPXY region via non-SH2 domains.
J. Biol. Chem.
270:23258-23262[Abstract/Free Full Text].
|
| 29.
|
Hinz, U.,
B. Giebel, and J. A. Campos-Ortega.
1994.
The basic-helix-loop-helix domain of Drosophila lethal of scute protein is sufficient for proneural function and activates neurogenic genes.
Cell
76:77-88[Medline].
|
| 30.
|
Hollenberg, S. M.,
R. Sternglanz,
P. F. Cheng, and H. Weintraub.
1995.
Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system.
Mol. Cell. Biol.
15:3813-3822[Abstract].
|
| 31.
|
Horvitz, H. R., and I. Herskowitz.
1992.
Mechanisms of asymmetric cell division: two Bs or not two Bs, that is the question.
Cell
68:237-255[Medline].
|
| 32.
|
Jan, Y. N., and L. Y. Jan.
1995.
Maggot's hair and bug's eye: role of cell interactions and intrinsic factors in cell fate specification.
Neuron
14:1-5[Medline].
|
| 33.
|
Kavanaugh, W. M.,
C. W. Turck, and L. T. William.
1995.
PTB domain binding to signaling proteins through a sequence motif containing phosphotyrosine.
Science
268:1177-1179[Abstract/Free Full Text].
|
| 34.
|
Kavanaugh, W. M., and L. T. William.
1994.
An alternative to SH2 domains for binding tyrosine-phosphorylated proteins.
Science
266:1862-1865[Abstract/Free Full Text].
|
| 35.
|
Knoblich, J. A.,
L. Y. Jan, and Y. N. Jan.
1995.
Asymmetric segregation of Numb and Prospero during cell division.
Nature
377:624-627[Medline].
|
| 36.
|
Lecourtois, M., and F. Schweisguth.
1995.
The neurogenic Suppressor of Hairless DNA-binding protein mediates the transcriptional activation of the Enhancer of split complex genes triggered by Notch signaling.
Genes Dev.
9:2598-2608[Abstract/Free Full Text].
|
| 37.
|
Lemmon, M. A.,
K. M. Ferguson, and J. Schlessinger.
1996.
PH domains: diverse sequences with a common fold recruit signaling molecules to the cell surface.
Cell
85:621-624[Medline].
|
| 38.
|
Li, S.-C.,
S. Zhou,
S. J. F. Vincent,
C. Zwahlen,
S. Wiley,
L. Cantley,
L. E. Kay,
J. Forman-Kay, and T. Pawson.
1997.
High-affinity binding of the Drosophila Numb phosphotyrosine-binding domain to peptides containing a Gly-Pro-(p)Tyr motif.
Proc. Natl. Acad. Sci. USA
94:7204-7209[Abstract/Free Full Text].
|
| 39.
|
Ohkura, H.,
I. M. Hagan, and D. M. Glover.
1995.
The conserved Schizosaccharomyces pombe kinase plo1, required to form a biopolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells.
Genes Dev.
9:1059-1073[Abstract/Free Full Text].
|
| 40.
|
Parks, A., and M. A. Muskavitch.
1993.
Delta function is required for bristle organ determination and morphogenesis in Drosophila.
Dev. Biol.
157:484-496[Medline].
|
| 41.
|
Posakony, J. W.
1994.
Nature versus nurture: asymmetric cell divisions in Drosophila bristle development.
Cell
76:415-418[Medline].
|
| 42.
|
Rhyu, M.,
L. Jan, and Y. N. Jan.
1994.
Asymmetric distribution of numb protein during division of the sensory organ precursor confers distinct fates to daughter cells.
Cell
76:477-491[Medline].
|
| 43.
|
Rubin, G. M., and A. C. Spradling.
1982.
Genetic transformation of Drosophila with transposable element vectors.
Science
218:348-353[Abstract/Free Full Text].
|
| 44.
|
Schonbaum, C. P.,
E. L. Organ,
S. Qu, and D. R. Cavener.
1992.
The Drosophila melanogaster stranded at second (sas) gene encodes a putative epidermal cell surface receptor required for larval development.
Dev. Biol.
151:431-445[Medline].
|
| 45.
|
Schweisguth, F., and J. W. Posakony.
1994.
Antagonistic activities of Suppressor-of-Hairless and Hairless control alternative cell fates in the Drosophila adult epidermis.
Development
120:1433-1441[Abstract].
|
| 46.
|
Spana, E. P., and C. Q. Doe.
1996.
Numb antagonizes Notch signaling to specify sibling neuron cell fates.
Neuron
17:21-26[Medline].
|
| 47.
|
Spana, E. P.,
C. Kopczynski,
C. S. Goodman, and C. Q. Doe.
1995.
Asymmetric localization of numb autonomously determines sibling neuron identity in the Drosophila CNS.
Development
121:3489-3494[Abstract].
|
| 48.
|
Tata, F., and D. A. Hartley.
1995.
Inhibition of cell fate in Drosophila by Enhancer of split genes.
Mech. Dev.
51:305-315[Medline].
|
| 49.
|
Uemura, T.,
S. Shepherd,
L. Ackerman,
L. Y. Jan, and Y. N. Jan.
1989.
numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos.
Cell
58:349-360[Medline].
|
| 50.
|
Vaessin, H.,
E. Grell,
E. Wolff,
E. Bier,
L. Y. Jan, and Y. N. Jan.
1991.
prospero is expressed in neuronal precursors and encodes a nuclear protein that is involved in the control of axonal outgrowth in Drosophila.
Cell
67:941-953[Medline].
|
| 51.
|
Verdi, J. M.,
R. Schmandt,
A. Bashirullah,
S. Jacob,
R. Salvino,
C. G. Craig,
A. E. Program,
H. D. Lipshitz, and C. J. McGlade.
1996.
Mammalian NUMB is an evolutionarily conserved signaling adapter protein that specifies cell fate.
Curr. Biol.
6:1134-1145[Medline].
|
| 52.
|
Wolf, G.,
T. Trub,
E. Ottinger,
L. Groninga,
A. Lynch,
M. F. White,
M. Miyazaki,
J. Lee, and S. E. Shoelson.
1995.
PTB domains of IRS-1 and Shc have distinct but overlapping binding specificities.
J. Biol. Chem.
270:27407-27410[Abstract/Free Full Text].
|
| 53.
|
Zhong, W.,
J. N. Feder,
M.-M. Jiang,
L. Y. Jan, and Y. N. Jan.
1996.
Asymmetric localization of a mammalian Numb homolog during mouse cortical neurogenesis.
Neuron
17:43-53[Medline].
|
| 54.
|
Zhong, W.,
M.-M. Jiang,
G. Weinmaster,
L. Y. Jan, and Y. N. Jan.
1997.
Differential expression of mammalian Numb homologues and Notch1 suggests distinct roles during mouse cortical neurogenesis.
Development
124:1887-1897[Abstract].
|
| 55.
|
Zhou, M.-M.,
K. S. Ravichandran,
E. T. Olejniczak,
A. M. Petros,
R. P. Meadows,
M. Sattler,
J. E. Harlan,
W. S. Wade,
S. J. Burakoff, and S. W. Fesik.
1995.
Structure and ligand recognition of the phosphotyrosine binding domain of Shc.
Nature
378:584-592[Medline].
|
| 56.
|
Zipursky, S. L.,
T. R. Venkatesh,
D. B. Teplow, and S. Benzer.
1984.
Neuronal development in the Drosophila retina: Monoclonal antibodies as molecular probes.
Cell
36:15-26[Medline].
|
Mol Cell Biol, January 1998, p. 598-607, Vol. 18, No. 1
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Zhang, Y., Wang, Y.-G., Zhang, Q., Liu, X.-J., Liu, X., Jiao, L., Zhu, W., Zhang, Z.-H., Zhao, X.-L., He, C.
(2009). Interaction of Mint2 with TrkA Is Involved in Regulation of Nerve Growth Factor-induced Neurite Outgrowth. J. Biol. Chem.
284: 12469-12479
[Abstract]
[Full Text]
-
Lau, K.-F., Chan, W.-M., Perkinton, M. S., Tudor, E. L., Chang, R. C. C., Chan, H.-Y. E., McLoughlin, D. M., Miller, C. C. J.
(2008). Dexras1 Interacts with FE65 to Regulate FE65-Amyloid Precursor Protein-dependent Transcription. J. Biol. Chem.
283: 34728-34737
[Abstract]
[Full Text]
-
Smith, M. J., Hardy, W. R., Murphy, J. M., Jones, N., Pawson, T.
(2006). Screening for PTB Domain Binding Partners and Ligand Specificity Using Proteome-Derived NPXY Peptide Arrays. Mol. Cell. Biol.
26: 8461-8474
[Abstract]
[Full Text]
-
Tang, H., Rompani, S. B., Atkins, J. B., Zhou, Y., Osterwalder, T., Zhong, W.
(2005). Numb Proteins Specify Asymmetric Cell Fates via an Endocytosis- and Proteasome-Independent Pathway. Mol. Cell. Biol.
25: 2899-2909
[Abstract]
[Full Text]
-
Pece, S., Serresi, M., Santolini, E., Capra, M., Hulleman, E., Galimberti, V., Zurrida, S., Maisonneuve, P., Viale, G., Di Fiore, P. P.
(2004). Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis. JCB
167: 215-221
[Abstract]
[Full Text]
-
Nie, J., Li, S. S.-C., McGlade, C. J.
(2004). A Novel PTB-PDZ Domain Interaction Mediates Isoform-specific Ubiquitylation of Mammalian Numb. J. Biol. Chem.
279: 20807-20815
[Abstract]
[Full Text]
-
Bedirian, A., Baldwin, C., Abe, J.-i., Takano, T., Lemay, S.
(2004). Pleckstrin Homology and Phosphotyrosine-binding Domain-dependent Membrane Association and Tyrosine Phosphorylation of Dok-4, an Inhibitory Adapter Molecule Expressed in Epithelial Cells. J. Biol. Chem.
279: 19335-19349
[Abstract]
[Full Text]
-
Qin, H., Percival-Smith, A., Li, C., Jia, C. Y. H., Gloor, G., Li, S. S.-C.
(2004). A Novel Transmembrane Protein Recruits Numb to the Plasma Membrane during Asymmetric Cell Division. J. Biol. Chem.
279: 11304-11312
[Abstract]
[Full Text]
-
Zhou, Y., Zhang, J., King, M. L.
(2003). Xenopus Autosomal Recessive Hypercholesterolemia Protein Couples Lipoprotein Receptors with the AP-2 Complex in Oocytes and Embryos and Is Required for Vitellogenesis. J. Biol. Chem.
278: 44584-44592
[Abstract]
[Full Text]
-
Jones, N., Chen, S. H., Sturk, C., Master, Z., Tran, J., Kerbel, R. S., Dumont, D. J.
(2003). A Unique Autophosphorylation Site on Tie2/Tek Mediates Dok-R Phosphotyrosine Binding Domain Binding and Function. Mol. Cell. Biol.
23: 2658-2668
[Abstract]
[Full Text]
-
Yan, K. S., Kuti, M., Yan, S., Mujtaba, S., Farooq, A., Goldfarb, M. P., Zhou, M.-M.
(2002). FRS2 PTB Domain Conformation Regulates Interactions with Divergent Neurotrophic Receptors. J. Biol. Chem.
277: 17088-17094
[Abstract]
[Full Text]
-
Jaffrey, S. R., Benfenati, F., Snowman, A. M., Czernik, A. J., Snyder, S. H.
(2002). Neuronal nitric-oxide synthase localization mediated by a ternary complex with synapsin and CAPON. Proc. Natl. Acad. Sci. USA
10.1073/pnas.261705799v1
[Abstract]
[Full Text]
-
Pi, H., Wu, H.-J., Chien, C.-T.
(2001). A dual function of phyllopod in Drosophila external sensory organ development: cell fate specification of sensory organ precursor and its progeny. Development
128: 2699-2710
[Abstract]
[Full Text]
-
Rusten, T., Cantera, R, Urban, J, Technau, G, Kafatos, F., Barrio, R
(2001). Spalt modifies EGFR-mediated induction of chordotonal precursors in the embryonic PNS of Drosophila promoting the development of oenocytes. Development
128: 711-722
[Abstract]
-
Ong, S. H., Guy, G. R., Hadari, Y. R., Laks, S., Gotoh, N., Schlessinger, J., Lax, I.
(2000). FRS2 Proteins Recruit Intracellular Signaling Pathways by Binding to Diverse Targets on Fibroblast Growth Factor and Nerve Growth Factor Receptors. Mol. Cell. Biol.
20: 979-989
[Abstract]
[Full Text]
-
Meyer, D., Liu, A., Margolis, B.
(1999). Interaction of c-Jun Amino-terminal Kinase Interacting Protein-1 with p190 rhoGEF and Its Localization in Differentiated Neurons. J. Biol. Chem.
274: 35113-35118
[Abstract]
[Full Text]
-
Dho, S. E., French, M. B., Woods, S. A., McGlade, C. J.
(1999). Characterization of Four Mammalian Numb Protein Isoforms. IDENTIFICATION OF CYTOPLASMIC AND MEMBRANE-ASSOCIATED VARIANTS OF THE PHOSPHOTYROSINE BINDING DOMAIN. J. Biol. Chem.
274: 33097-33104
[Abstract]
[Full Text]
-
Homayouni, R., Rice, D. S., Sheldon, M., Curran, T.
(1999). Disabled-1 Binds to the Cytoplasmic Domain of Amyloid Precursor-Like Protein 1. J. Neurosci.
19: 7507-7515
[Abstract]
[Full Text]
-
Hawkins, N., Garriga, G.
(1998). Asymmetric cell division: from A to Z. Genes Dev.
12: 3625-3638
[Full Text]
-
Borg, J.-P., Straight, S. W., Kaech, S. M., de Taddeo-Borg, M., Kroon, D. E., Karnak, D., Turner, R. S., Kim, S. K., Margolis, B.
(1998). Identification of an Evolutionarily Conserved Heterotrimeric Protein Complex Involved in Protein Targeting. J. Biol. Chem.
273: 31633-31636
[Abstract]
[Full Text]
-
Roos, J., Kelly, R. B.
(1998). Dap160, a Neural-specific Eps15 Homology and Multiple SH3 Domain-containing Protein That Interacts with Drosophila Dynamin. J. Biol. Chem.
273: 19108-19119
[Abstract]
[Full Text]
-
Borg, J.-P., Yang, Y., De Taddeo-Borg, M., Margolis, B., Turner, R. S.
(1998). The X11alpha Protein Slows Cellular Amyloid Precursor Protein Processing and Reduces Abeta 40 and Abeta 42 Secretion. J. Biol. Chem.
273: 14761-14766
[Abstract]
[Full Text]
-
Jaffrey, S. R., Benfenati, F., Snowman, A. M., Czernik, A. J., Snyder, S. H.
(2002). Neuronal nitric-oxide synthase localization mediated by a ternary complex with synapsin and CAPON. Proc. Natl. Acad. Sci. USA
99: 3199-3204
[Abstract]
[Full Text]
Top
Abstract
Introduction
