From the Department of Biology, ** Howard Hughes
Medical Institute, and the Department of ¶ Internal Medicine and
Biological Chemistry, University of Michigan,
Ann Arbor, Michigan 48109 and the
Department of
Pharmacology, New York University Medical Center,
New York, New York 10016
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ABSTRACT |
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The Numb protein is involved in cell fate determination during Drosophila neural development. Numb has a protein domain homologous to the phosphotyrosine-binding domain (PTB) in the adaptor protein Shc. In Shc, this domain interacts with specific phosphotyrosine containing motifs on receptor tyrosine kinases and other signaling molecules. Residues N-terminal to the phosphotyrosine are also crucial for phosphopeptide binding to the Shc PTB domain. Several amino acid residues in Shc have been implicated by site-directed mutagenesis to be critical for Shc binding to receptor tyrosine kinases. We have generated homologous mutations in Numb to test whether, in vivo, these changes affect Numb function during Drosophila sensory organ development. Two independent amino acid changes that interfere with Shc binding to phosphotyrosine residues do not affect Numb activity in vivo. In contrast, a mutation shown to abrogate the ability of the Shc PTB domain to bind residues upstream of the phosphotyrosine virtually eliminates Numb function. Similar results were observed in vitro by examining the binding of the Numb PTB domain to proteins from Schneider S2 cells. Our data confirm the importance of the PTB domain for Numb function but strongly suggest that the Numb PTB domain is not involved in phosphotyrosine-dependent interactions.
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INTRODUCTION |
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During the development of an organism, different cell fates can be acquired through both intrinsic or extrinsic mechanisms. In an intrinsic mechanism, a certain factor in the cell is asymmetrically distributed within the mother cell, such that when the mother cell undergoes cytokinesis, the factor is distributed differentially to the two daughter cells. Conversely, in an extrinsic mechanism, the daughter cell receives signals from neighboring cells (or its own sibling cell) to acquire a certain cell fate. The Numb protein represents a key point in linking both intrinsic and extrinsic means of assigning cell fates during asymmetric divisions in the Drosophila peripheral nervous system (Refs. 1 and 2; for reviews see Refs. 3-5). In Drosophila, Numb is necessary for the asymmetric division of cells in neural lineages (1, 6-8). In the case of simple external sensory organ lineages, the sensory organ precursor undergoes two sets of divisions as follows: the first set produces two daughter cells (IIa and IIb), and the second set produces a bristle and socket cell from the IIa cell and a neuron and glial cell from the IIb cell (Fig. 1A). Numb protein has been shown to be localized to one side of the sensory organ precursor prior to division (1). After cell division it is then segregated asymmetrically to only the IIb cell. In numb mutants, the IIb daughter cell is transformed to a IIa cell fate resulting in two sets of bristle and socket cells (1). In numb mosaics, occasionally four socket cells are observed suggesting that Numb is also needed during the second division (Fig. 1B). Conversely, when Numb protein is overexpressed, the IIa cell is transformed into a IIb cell, resulting in two sets of neuron and glial cells (see Fig. 1C, balding; Ref. 1). When Numb is overexpressed after the first cell division, only the second stage of the lineage is affected, resulting in a transformation of a socket to a bristle cell (see Fig. 1D, twinning) or a glia into a neuron (1). Genetic studies have indicated that numb functions to antagonize signaling by the Notch receptor (2, 9). Lack of Notch function leads to an increase in neuronal cells in the Drosophila peripheral nervous system, whereas activated Notch leads to a decrease in cells adopting the neuronal fate (2, 10). A reduction in Notch signaling partially suppresses the numb mutant phenotype leading to the generation of some neuronal cells. These data suggest that numb functions to inhibit Notch signaling and thus promotes the neuronal phenotype. Furthermore, there is evidence that Notch binds directly to the Numb PTB1 domain (2).
The phosphotyrosine binding (PTB) domain of Numb is critical for Numb
function but not for the asymmetric localization of Numb (11). The PTB
domain was first described in the protein, Shc, and was subsequently
identified by sequence homology to be present in several other proteins
including Numb (12-16). The PTB domain of Numb and Shc are 20%
identical at the amino acid level (Fig. 2A, see Refs. 12 and
17), suggesting that the Numb PTB domain may act similarly to the Shc
PTB domain in mediating signal transduction events by binding to the
NPXpY motif (18). In Shc, the PTB domain binds a
XNPXpY motif on receptor tyrosine kinases, where
is a hydrophobic residue, X is any amino acid
residue, N is asparagine, P is proline, and pY is phosphotyrosine.
Several individual amino acid residues in Shc have been implicated by site-directed mutagenesis to be critical for Shc binding to the
XNPXpY motif (17, 19, 20). These include
residues involved in binding the phosphotyrosine as well as residues
necessary for contacting the amino acids upstream of the
phosphotyrosine in the
XNPXpY peptide. An
important question is whether all or only some of the PTB domains are
involved in phosphotyrosine-dependent interactions
(21-26). In an attempt to address whether or not the Numb PTB domain
is involved in phosphotyrosine binding, we generated point mutants in
the Numb PTB domain analogous to those generated in Shc (19, 21). Two
of these mutations affect conserved residues involved in the binding of
the Shc PTB domain to phosphotyrosine. These are serine 148 and
arginine 171 of Numb. Another mutation targets phenylalanine 195 of
Numb. In Shc, this phenylalanine binds to the hydrophobic residue
5
and the asparagine
3 to the phosphotyrosine without directly
interacting with phosphotyrosine (17). Finally, a fourth mutation,
phenylalanine to leucine 149 of Numb, which results in reduced binding
affinity of the Shc PTB domain, was also tested.
We have examined how these mutations affect Numb function in vivo both during Drosophila sensory organ development and mesodermal development. The phenylalanine to valine mutation (amino acid residue 195) is able to virtually eliminate the gain-of-Numb function when overexpressed in transgenic flies using the UAS-GAL4 system (27). Altering the residues in Numb homologous to those involved in Shc phosphotyrosine binding has little or no effect on the lineage transformation activity of Numb overexpression in vivo. Another mutation, phenylalanine to leucine (position 149), which causes Shc to bind with a reduced affinity in vitro, exhibits an intermediate phenotype in vivo. In binding studies using Drosophila cell culture, we detect several proteins that bind to the wild-type PTB domain and to the S148A and R171Q mutants but not to the F195V mutant of Numb. These data with site-directed mutagenesis confirm that the PTB domain is crucial for Numb function but strongly suggest that phosphotyrosine is not required for Numb PTB domain function in vivo.
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MATERIALS AND METHODS |
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DNA Constructs and Mutagenesis-- Wild-type Numb PTB domain GST fusion protein constructs were made by subcloning a polymerase chain reaction-generated fragment of numb DNA spanning the 966-1490-base pair region of numb cDNA into the pGSTag vector (28) in the EcoRI site. PTB domain point mutants were generated by polymerase chain reaction-based site-directed mutagenesis from the wild-type pGSTag-Numb PTB domain.
The full-length numb cDNA (760-2800 base pairs) was subcloned into the pUAST vector in the KpnI site. Site-directed mutagenesis of full-length numb was done by using Transformer Site-directed Mutagenesis kit (CLONTECH) or U.S.E. Mutagenesis kit (Amersham Pharmacia Biotech). A Myc epitope tag was attached to the C-terminal end of the Numb coding region. All constructs were sequenced using Sequenase version 2.0 (U. S. Biochemical Corp.).Western Blot of Embryo Lysates-- To assay for the expression of both wild-type and Numb protein, a Western blot was performed. The UAS-Numb flies (wild-type and mutant) were crossed to the daG32-GAL4 driver (29). Embryos from this cross were collected from 0 to 8 h at 25 °C or 0 to 16 h at 18 °C. They were then washed in NaCl/Triton X (0.7% NaCl, 0.04% Triton X-100), dechorionated in 50% bleach in NaCl/Triton X, and then washed again in NaCl/Triton X, followed by a wash in PBT (0.1 M NaPO4, 0.3% deoxycholate, 0.5% Triton X-100). The embryos were then lysed in RIPA buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2 with 1 mM EGTA, 1% sodium deoxycholate, 0.1% SDS, 10% glycerol, and 1% Triton X-100) with 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 100 mM sodium fluoride, 200 mM sodium vanadate, and 10 mM tetrasodium pyrophosphate by grinding using a Dounce homogenizer. Lysates were then subjected to electrophoresis and transferred to nitrocellulose membrane. The membrane was then blotted with anti-Numb antibody (1).
Cell Culture-- Notch expressing Schneider S2 cells (30) were grown at room temperature in Schneider cell media (Life Technologies, Inc.), supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and expression maintained with 200 mg/ml hygromycin B. Notch expression was induced by heat shock at 37 °C for 30 min followed by recovery at room temperature for 2 h. The cells were then lysed in lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2 with 1 mM EGTA, 10% glycerol, 1% Triton X-100, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 µM phenylmethylsulfonyl fluoride). PC12 cells overexpressing TrkA receptor were stimulated with EGF or NGF for 5 min at 37 °C as described (12, 18). Cells were lysed as described above and incubated with GST fusion proteins.
Methionine Labeling of Schneider Cell Lysate-- Wild-type Schneider cells were first grown to 90% confluency. The cells were then placed in methionine-free Dulbecco's modified Eagle's medium supplemented with 5% heat-treated fetal bovine serum. Cells were labeled for 4 h in the same media with [35S]methionine (80 µCi/ml; Easytag Express Labeling Mix, Life Science Products). Cells were then rinsed with cold phosphate-buffered saline and lysed in lysis buffer with proteases and phosphatase inhibitors.
GST Fusion Protein Pull-down Assay-- GST (glutathione S-transferase) fusion proteins were expressed and bound to glutathione-agarose beads using standard protocols (13). GST fusion proteins of the various Numb constructs were quantitated by SDS-PAGE, and the equal amounts were incubated with radiolabeled Schneider cell lysates for 90 min at 4 °C. The beads were then washed three times with lysis buffer, boiled in 1× sample buffer, and separated by SDS-PAGE. The gel was then fixed and treated with Amplify (Amersham Pharmacia Biotech) prior to drying and subsequent autoradiography. For Western blotting, the proteins were transferred to nitrocellulose and blotted with antibody directed against the intracellular domain of Notch (C17.9C6; kindly provided by S. Artavanis-Tsakonas) or anti-phosphotyrosine (12).
Creation of UAS-Numb Transgenic Flies--
The UAS-Numb
constructs were purified on a CsCl gradient and then coprecipitated
with the p25.7wc
2-3 transposase helper plasmid (31) at a
concentration of 500 µg/ml UAS-Numb and 100 µg/ml p
25.7wc
2-3
in injection buffer (5 mM KCl, 0.1 mM
NaH2PO4, pH 6.8). The DNA was then injected in
w Oregon R flies. At least four independent lines were
generated for each construct (see Table I). The flies were grown on
standard yeast-glucose media at 25 °C, unless otherwise noted.
Immunohistochemical Staining-- To examine the effects of the UAS-Numb constructs (wild-type and mutant) during embryonic development, the UAS-Numb flies were crossed to a daG32-GAL4 line (29) for assaying neuronal development and twist-GAL4;24B-GAL4 for assaying mesodermal development. The embryos from this cross were then collected, dechorionated, and fixed as described previously (34). The neurons were then visualized using the 22C10 monoclonal antibody (35) at a concentration of 1:50. Cardiac and muscle cells were visualized with an anti-Evenskipped antibody (36) used at a concentration of 1:10,000. Horseradish peroxidase-conjugated goat anti-mouse IgG or anti-rabbit IgG (Bio-Rad) was used as the secondary antibody at a concentration of 1:200. Imaginal discs for immunohistochemical staining were prepared and stained as described by Patel (37). Anti-Myc antibody (9E10) was used at a 1:50 dilution to visualize transgenic Numb overexpression. Horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) was used as the secondary antibody at a concentration of 1:200.
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RESULTS |
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Numb PTB Domain Function in Vivo-- Since we wanted to determine if the Numb PTB domain functions in vivo in a fashion analogous to the Shc PTB domain, we decided to use Numb overexpression within the Drosophila peripheral nervous system as our assay. The UAS-GAL4 system (27) was used so that Numb could be induced specifically in ectodermal tissues, either during embryonic or pupal development. The full-length wild-type Numb cDNA, tagged with a Myc epitope, was subcloned into the pUAST vector (27), and several transgenic lines were generated (see Fig. 2B; Table I). As expected, many different phenotypes, corresponding to those observed by Rhyu et al. (1), were observed when the wild-type Numb was overexpressed under the control of different GAL4 drivers, presumably because the different GAL4 drivers are turning on Numb expression at different points in the neural lineages (for details about some of the GAL4 drivers tested see "Materials and Methods"). When Numb is overexpressed before the first division of the sensory organ precursor, no bristles or sockets are formed, because the corresponding cells are transformed into neurons and glial cells in a simple external sensory organ lineage (Fig. 1C, balding). However, when Numb protein is overexpressed only after the first division in the sensory organ precursor lineage, a transformation of sockets into bristles is observed (Fig. 1D, twinning). An example of the twinning phenotype can be seen in Fig. 3B, where overexpression of wild-type UAS-Numb driven by the C96-GAL4 driver (32) resulted in twinning of bristles on the wing margin. One of the most striking phenotypes observed was seen in the progeny of 189-GAL4 crossed to UAS-Numb, in which most of the bristles on the body were missing compared with wild type (see Fig. 3, A and B, and Table I). We chose to use this GAL4 driver for our assay, since any reduction in Numb activity might be expected to produce a less severe phenotype, intermediate between the extensive baldness observed with wild-type Numb overexpression and the normal bristle pattern observed in wild-type flies.
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Numb PTB Domain Function in Vitro-- To confirm our results in vivo, we analyzed the in vitro binding ability of the Numb PTB domain. Wild-type and mutant Numb PTB domains were generated as a GST fusion protein in bacteria. These proteins were purified on glutathione-agarose and used as an affinity matrix to bind proteins from [35S]methionine-labeled Schneider S2 cell lysate. After binding, the beads were washed with lysis buffer containing 1% Triton X-100. The bound proteins were then separated by SDS-PAGE and exposed for autoradiography. Several proteins could be seen to bind to the Numb PTB domain but not to the beads containing GST alone. Similarly, specific proteins could be detected that bound to wild-type Numb PTB domain but not to the F195V mutant (see arrows Fig. 6A). However, the S148A and R171Q mutations of the Numb PTB domain did not affect this binding. The effect of the F195V mutation to abrogate binding of the Numb PTB domain correlated closely with the ability of this mutation to impair Numb gain-of-function in vivo. In contrast, the S148A and R171Q mutations had no effect on binding in vitro or Numb function in vivo. Although we identified the binding of the Numb PTB domain to methionine-labeled proteins, none of these proteins appear to contain phosphotyrosine. In the Schneider S2 lysate we used for these experiments, we found that many of the proteins contained phosphotyrosine as indicated by phosphotyrosine immunoblotting. However, none of these tyrosine-phosphorylated proteins bound to GST alone or to the GST Numb PTB domain (Fig. 6B). Furthermore we tested the ability of the Numb PTB domain to bind tyrosine-phosphorylated proteins from growth factor-stimulated PC12 cells that overexpress the TrkA receptor (Fig. 6C, NGF receptor). Cells were stimulated with EGF or NGF leading to the tyrosine phosphorylation of many proteins in the total lysates. Lysates were incubated with GST alone, GST-Numb PTB domain, or GST-Shc PTB and bound proteins detected by anti-phosphotyrosine immunoblotting. Although the activated growth factor receptors (EGF receptor and TrkA) bound to the Shc PTB domain, no tyrosine-phosphorylated proteins bound to the Numb PTB domain.
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DISCUSSION |
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The PTB domain of Shc has been shown to be critical for binding to a number of receptor tyrosine kinases. This domain is well conserved in the Drosophila Numb protein (12, 17), suggesting that there may also be some conservation of biochemical activity. A number of amino acid residues have been identified in Shc as being critical for its ability to bind to NPXpY motifs (17, 19, 20). Our data demonstrate that the mutation F195V severely impairs the function of the Numb PTB domain in vitro and in vivo. The phenylalanine mutated in this experiment is conserved in almost all members of the PTB domain family (19). These results confirm the utility of using this mutation to probe PTB domain function. Although our data are the first to use point mutants to disrupt Numb PTB domain function, the work of others has also pointed to a crucial role for this domain in Numb function. Frise et al. (11) showed that deletion of the PTB domain impaired Numb function in vivo, and Verdi et al. (23) showed that overexpression of rat Numb PTB domain alone affected neural development in neural crest derived MONC-1 cells.
Numb has been shown to be genetically upstream of Notch and is proposed to inhibit Notch signaling (2). In addition, yeast two-hybrid studies and protein binding assays show that Notch and Numb are capable of a direct physical interaction and that this interaction is probably mediated by the PTB domain of Numb (2). However, in our studies we were not able to detect high affinity interactions between Numb and Notch under conditions where we could detect the binding of several labeled proteins to the Numb PTB domain. Further work will be necessary to determine if the genetic interaction between Numb and Notch rely on direct interactions or occurs via secondary proteins.
A central question that this study helps address is whether
phosphotyrosine is required for PTB domain interactions. Original studies of the Shc and insulin receptor substrate-1 PTB domains showed
that phosphotyrosine is crucial for the interactions of these PTB
domains and their binding partners. Affinity between these PTB domains
and their phosphotyrosine containing binding partners falls to
undetectable levels when phosphotyrosine is omitted (40). Residues
crucial for phosphotyrosine binding by the Shc PTB domain are conserved
in several members of the PTB domain family including Numb (17). This
has led to the suggestion that many members of this family might be
involved in phosphotyrosine-dependent interactions.
However, mutation of the conserved residues in Numb, which are involved
in phosphotyrosine recognition by Shc, had no significant effect on
Numb activity in vivo. Furthermore, we could detect binding
of the Numb PTB domain independent of phosphotyrosine in
vitro (Fig. 6). This finding is supported by earlier data with the
X11 and FE65 PTB domains that demonstrated binding to amyloid precursor
protein independently of tyrosine phosphorylation (21, 26). In the case
of X11 this binding occurs to a XNPXY motif on
amyloid precursor protein (21). In agreement, recent studies have
identified a
XNPXY containing protein that
binds to the Numb PTB domain independently of tyrosine phosphorylation
(41).3 This indicates that
the Numb PTB domain can also bind a non-phosphorylated NPXY
motif. This speculation is supported by our finding that the F195V
mutant of the Numb PTB domain eliminates Numb function. This
phenylalanine residue in the Shc PTB domain is crucial for interactions
with upstream elements of the
XNPXPY motif and
might serve a similar role in Numb. In contrast, however, Pawson and co-workers (25) have suggested that both phosphotyrosine- and non-phosphotyrosine-dependent interactions might occur
between the Numb PTB domain and target peptides. By using in
vitro peptide selection these authors identified the motif
YIGPY
as a target for the Numb PTB domain. This peptide can bind
with slightly higher affinity when the second tyrosine is
phosphorylated. These studies, however, did not identify a target
protein for Numb that might contain such a motif. Further evidence
identifying the exact protein involved in Numb function will clarify
these issues. The powerful genetic and biochemical studies now being
performed on Numb should allow the identification of the physiological
Numb PTB domain partner(s) and their mode of interaction. Our data
predict that this interaction will be crucial for Numb function, but
independent of phosphotyrosine binding.
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ACKNOWLEDGEMENTS |
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We thank Dr. Yuh Nung Jan for the Numb cDNA and Numb antibodies. We also thank the following people for providing us with GAL4 lines: Gabrielle Boulianne for providing the C96-GAL4 line; Elisabeth Knust for providing us with the daG32-GAL4 line; and Gerhard Technau for providing 60-GAL4, 160-GAL4, 189-GAL4, 281-GAL4, 605/6-GAL 4, 1407-GAL 4, 1445-GAL 4, and 1481-GAL4. Wendy Lockwood kindly donated the twist-GAL4;24B-GAL4 line. Other GAL4 lines were kindly provided by Kathy Matthews at the Bloomington Drosophila Stock Center. We also thank Toby Lieber and Mike Young for providing us with Notch expressing Schneider cells and Robert Mann and Spyros Artavanis-Tsakonas for providing the Notch C17.9C6 monoclonal antibody.
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FOOTNOTES |
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* This research was supported by Public Health Service Grant NS29119 (to R. B.), Public Health Service National Research Service award, and Chemical and Hearing Senses training grant (to L. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work and should be considered joint co-authors.
Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Dept. of Biology, University of
Michigan, Ann Arbor, MI 48109-1048. Tel.: 734-763-3182; Fax: 734-647-0884; E-mail: rolf{at}umich.edu.
1 The abbreviations used are: PTB, phosphotyrosine-binding domain; Eve, Evenskipped; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; Shc, Src homologous and collagen protein; EGF, epidermal growth factor; NGF, nerve growth factor; UAS, upstream activation sequence; w, white; pY, phosphotyrosine.
2 M. Park, L. Yaich, and R. Bodmer, unpublished observations.
3 J. McGlade, personal communication.
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REFERENCES |
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