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1 Department of Experimental Oncology, European Institute of Oncology, Milan 20140, Italy; 2 Dipartimento di Biologia, University of Rome "Tor Vergata," Rome 00100, Italy; 3 Istituto di Patologia Speciale Medica, University of Parma, Parma 43100, Italy; 4 Instituto di Microbiologia, University of Bari, Bari 70100, Italy
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Abstract |
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 Top
Abstract
Introduction
Results
Discussion
Materials & Methods
References
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EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct screening of expression libraries with EH domains yielded a number of putative EH interactors, all of which possessed NPF motifs that were shown to be responsible for the interaction. Among these interactors were the human homolog of NUMB, a developmentally reguated gene of Drosophila, and RAB, the cellular cofactor of the HIV REV protein. We demonstrated coimmunoprecipitation of Eps15 with NUMB and RAB. Finally, in vitro binding of NPF-containing peptides to cellular proteins and EST database screening established the existence of a family of EH-containing proteins in mammals. Based on the characteristics of EH-containing and EH-binding proteins, we propose that EH domains are involved in processes connected with the transport and sorting of molecules within the cell.
[Key Words: EH domain; protein interaction; Eps15; internalization; trafficking]
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Introduction |
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Abstract
Introduction
Results
Discussion
Materials & Methods
References
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Many cellular functions, including proliferation,
differentiation, cytoskeleton organization, and apoptosis, are
regulated through a complex intracellular network of signal
transducers. Specialized protein domains, such as
SH2, SH3, PTB, or WW, mediate this vast array of interactions by
binding to specific sequences located in target proteins (for review,
see Musacchio et al. 1994
; Cohen et al. 1995; van der Geer and Pawson
1995 and references therein). Within individual groups, binding domains
have diverged to recognize specific consensus sequences in which some
positions are obligatory occupied by given amino acids and other can
vary. For example the SH2 and PTB domains both recognize
phosphotyrosine-containing motifs (Songyang et al. 1993, 1994; Blaikie
et al. 1994; Kavanaugh and Williams 1994; Zhou et al. 1995).
Specificity appears to be determined by variations in amino acids
distal or proximal to the phosphotyrosine residue for SH2 and PTB
domains, respectively (for review, see Cohen et al. 1995; van der Geer
and Pawson 1995). SH3 domains, on the other hand, interact with
proline-rich peptides in target proteins (Cichetti et al. 1992; Gout et
al. 1993; Ren et al. 1993; Yu et al. 1994). Screening of biased random
libraries allowed the identification of two classes of SH3 ligands,
conforming to the general structure RXLPPZP (class 1) and XPPLPXR
(class 2), whereas X is any amino acid other than C and
Z is either L or R (Feng et al. 1994; Schlessinger 1994).
SH2 and SH3 domains contribute to the propagation of signals by
affecting the subcellular localization of the domain-containing proteins or their targets, or by recruting these proteins to specific enzymes (for review, see Cohen et al. 1995). Recent evidence, however,
suggests that these domains might not serve solely as passive binding
interfaces. In the case of SH3s, in particular, it has been shown that
binding to the dynamin GTPase and to phosphatidylinositol-3
kinase (PI-3K) activates these enzymes in vitro (Gout et al. 1993; Herskovits et al. 1993; Pleiman et al. 1994).
Finally, a phylogenetic hierarchy of interactions might also exist, as
witnessed by the presence of some binding domains, such as SH3 and WW
in lower eukaryotes like yeast, and the appearance of others, such as
SH2 and PTB, concomitantly with the emergence of tyrosine
kinase-mediated signaling in multicellular eukaryotes (for review, see
Cohen et al. 1995).
We have recently described a novel protein-protein interaction domain,
called EH (for Eps15 homology), present in three copies in the
amino terminus of the tyrosine kinase substrates Eps15 and Eps15R and
shared with other proteins of yeast and nematode (Wong et al. 1995).
Except for its protein-binding ability, no other clues to the
function(s) of the EH domain are available, as EH-containing proteins
appear to serve different roles in cellular homeostasis. One
EH-containing protein, End3p, is involved in the internalization of the
yeast 
-mating factor (Benedetti et al. 1994); another, Pan1p, is
required for normal organization of the actin cytoskeleton in yeast
(Tang and Cai 1996). Other EH-containing proteins still have unknown
functions. However, mutagenesis studies in END3 suggested that the
presence of its EH domain is necessary for function (Benedetti et al.
1994). Understanding of the fuctions of EH will ultimately rely on the
identification of its binding specificity and of the intracellular
targets recruited in a hypothetical EH-based network. Studies described
in this paper were undertaken to shed light on these issues.
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Results |
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Abstract
Introduction
Results
Discussion
Materials & Methods
References
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EH domains from Eps15 and Eps15R bind to NPF-containing peptides
In their amino termini, Eps15 and Eps15R contain three
copies of a novel protein-protein interaction domain, which we named EH (Fig. 1A). To understand the binding specificity
of EH domains, we engineered glutathione S-transferase (GST)
fusion proteins containing the three EH domains of Eps15 (GST-EH) or
Eps15R (GST-EHR). These fusion proteins were employed to screen a
random phage-displayed peptide library, and the selected phages were
sequenced in the region corresponding to the random inserts. In Figure
1B, the conceptually translated peptides are displayed. Of 48 selected peptides, 46 contained the motif NPF
(asparagine-proline-phenylalanine). Two additional phages containing
either NHF (asparagine-histidine-phenylalanine) or HPF
(histidine-proline-phenylalanine) were also selected by GST-EHR
(Fig. 1B). Position +1 (with respect to NPF) exhibited a strong
preference for basic and basic/hydrophobic residues in peptides selected by GST-EHR and GST-EH, respectively. Of note, negatively charged residues were never present in this position. Positions 
1 and 2 displayed a weaker preference for serine or threonine (Fig. 1B).
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Cellular proteins binding to EH in vitro invariably contain NPF motifs
We have shown previously that the GST-EH fusion protein is able
to specifically bind to a number of cellular proteins, in Far Western
assays (Wong et al. 1995). To clone the cDNAs encoding these proteins,
we employed the GST-EH fusion protein to screen a prokaryotic
expression library from M426 human fibroblasts. We identified several
positive plaques that specifically reacted with GST-EH but not with
control GST. By cross-hybridization experiments, the positive phages
could be assigned to seven groups, corresponding to distinct cDNAs (not
shown).
Nucleotide sequence of the longest phage inserts of these cDNAs
revealed that they represented partial cDNAs of the human homolog of
NUMB, a developmentally regulated gene of Drosophila (Uemura et al. 1989); a NUMB-related gene, which we named
NUMB-R; RAB, coding the cellular cofactor of the HIV REV
protein (Bogerd et al. 1995; Fritz et al. 1995); and a
RAB-related gene, which we named RAB-R (see next
section). We also identified three novel genes that we named ehb3,
ehb10, and ehb21 (for EH
binding, followed by the original plaque identifier; data
not shown).
There were no homologies among the seven partial cDNAs (apart from
those between NUMB and NUMB-R, and RAB, and RAB-R, respectively), except for the presence of NPF motifs, which frequently are present in
multiple copies (Fig. 1C). Alignment of all NPF-containing stretches
revealed preference for hydrophobic residues at position +1
(relative to NPF) and for S/T at positions 1 and
2 (Fig. 1C).
Cloning of cDNAs containing entire ORFs for human NUMB, NUMB-R, RAB, and RAB-R and characterization of their predicted products
The cDNAs obtained from the screening of the prokaryotic
expression library represented partial cDNAs that did not contain entire open reading frames (ORFs) (data not shown). We therefore screened a human fibroblast cDNA library to obtain the entire ORFs of
human RAB, RAV-R, NUMB, and NUMB-R. Several cDNAs
were isolated, and the longest ones, representative of each gene, were sequenced. A schematic of the cDNAs containing the entire ORFs of human
NUMB and NUMB-R and of human RAB and
RAB_R is presented in Figures 2A and
3A, respectively, and details are
given in the figure legends. The sequence of human RAB
corresponded to that already reported by Bogerd et al. (1995) and Fritz
et al. (1995). Conceptual translation of the cDNAs allowed for
comparison of human NUMB and NUMB-R (Fig. 2B) and RAB and RAB-R (Fig.
3B).
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The ORFs of human NUMB and NUMB-R have the capacity
of encoding peptides of 603 and 609 amino acids, respectively, with a predicted molecular mass of 66 and 65 kD. The two predicted proteins display an overall relatedness of 74% with 57% identity (Fig. 2B). As
already reported for murine and Drosophila (Verdi et al. 1996;
Zhong et al. 1996), both human NUMB and NUMB-R contained a
phosphotyrosine interaction domain (PID/PTB; van der Geer
and Pawson 1995) in their amino terminus (Fig. 2B), and putative
SH3-binding sites in their carboxyl terminus. In addition, they both
contained a single NPF motif, located a few amino acids before the end
of the proteins (Fig. 2B).
The ORFs of human RAB and RAB-R have the capacity of
encoding peptides of 562 and 481 amino acids, respectively, with a
predicted molecular mass of 58 and 49 kD. The two predicted proteins
display an overall relatedness of 71% with 46% identity (Fig. 3B).
Conserved features between RAB and RAB-R include a zinc finger region,
in the amino terminus of the proteins, and several FG
(phenylalanine-glycine) motifs, characteristic of nucleoporin-like
proteins (Bogerd et al. 1995, Fritz et al. 1995). In addition, they
both contained four NPF motifs, located in the carboxy-terminal half of
the molecule (Fig. 3B). It has not been established whether all of the
NPF motifs are endowed with EH-binding ability, and if so, whether they
bind to different EH-containing proteins.
Binding of various cellular proteins to EH domains in vitro and in vivo
To characterize the binding of the EH interactors to Eps15, we engineered GST fusion proteins encompassing fragments derived from the seven identified EH-binding proteins. In all cases, the original partial cDNAs were used (see Materials and Methods), as they should contain all of the determinants necessary and sufficient for EH binding. As shown in Figure 4A, all of the GST fusion proteins were able to specifically recover native Eps15 from cell lysates in vitro binding experiments.
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To address the issue of whether the NPF-containing peptides, present in the EH-binding proteins, were actually responsible for binding to Eps15, we engineered GST fusion proteins encompassing short NPF-containing peptides from the proteins of interest. The peptides engineered as GST fusions are underlined in Figure 1C. Also in this case (Fig. 4B), we evidenced specific binding of the GST peptide fusions to native Eps15 from total cellular lysates. Although the participation of other regions of the EH-interacting proteins to the binding cannot be excluded, the sum of the above results indicates that NPF-containing peptides are sufficient for binding to Eps15.
We then tested whether Eps15 can physically interact with some of the EH-binding proteins in vivo. To this end, we utilized the GST-NUMB and GST-RAB fusion proteins, described in Figure 4A, as immunogens to generate polyconal sera. The anti-RAB and anti-NUMB sera recognized specifically proteins of ~60 kD and a doublet of ~70-72 kD, respectively, in several human and mouse cell lines (data not shown). The two sera were then used to coimmunoprecipitate Eps15 from lysates of NIH-3T3 cells (Fig. 5A) or of U2OS cells (not shown). As shown in Figure 5A, Eps15 was recovered in immunoprecipitates obtained with anti-NUMB or anti-RAB sera but not with control sera.
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The amount of Eps15 coimmunoprecipitated by NUMB was higher than that coimmunoprecipitated by RAB. Although this might be attributable to a lower affinity of the RAB/Eps15 interaction, we favor the possibility that it simply reflects less efficient immunoprecipitation of RAB by the anti-RAB antibody. In another set of experiments, we engineered eukaryotic expression vectors encoding for either NUMB or RAB fused to a hemagglutinin (HA) epitope, at their amino termini. C33A cells were then transiently transfected with vectors coding HA-NUMB or HA-RAB. As shown in Figure 5B (top) HA-NUMB and HA-RAB were expressed at similar levels. Under these conditions, Eps15 was readily and comparably recoverable by immunoprecipitation with an anti-HA antibody but not by control antibodies, in both HA-NUMB or HA-RAB transfectants (Fig. 5B, bottom). The sum of the above results strongly argues that Eps15 can interact with NUMB and RAB in vivo.
To prove that the in vivo interaction of Eps15 with a representative EH-binding protein protein was attributable to a NPF-mediated interaction, we engineered a HA-tagged mutant of NUMB (HA-NUMB/NAA) in which its unique NPF motif was mutageneized to NAA (asparagine-alanine-alanine). Upon transfection in C33A cells, both the HA-NUMB/NAA and the wild-type HA-NUMB mutants were expressed at compatable levels (Fig. 5C, top). In HA-NUMB/NAA transfectants, however, little if any Eps15 was visible in anti-HA immunoprecipitates, whereas it was readily detectable in similar immunoprecipitates obtained from wild-type HA-NUMB transfectants (Fig. 5C, middle). These differences were not attributable to the levels of expression of endogenous Eps15, which appeared comparable in all transfectants (Fig. 5C, bottom). Thus, the in vivo binding of Eps15 to NUMB is most likely mediated by an EH-NPF interaction.
Characterization of the NPF-EH interaction
To assess the relevance of the NPF motif and of the surrounding positions for binding to EH, we selected the peptide SSSTNPFL, which is present in RAB and strongly binds to Eps15 and mutated individual amino acid positions in a GST fusion protein background. The various GST fusion proteins depicted in Figure 6 (left panels) were then tested for their ability to recover native Eps15 from cellular lysates in in vitro binding assays.
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Mutations in the NPF motif to NGF, DPF, and NPY abolish binding
completely (Fig. 6A, right). We then performed alanine scanning of the
positions surrounding the NPF motif. This unconvered a hierarchy of
contribution to binding, as A(1), A(2), and A(+1) mutants,
containing mutations to alanine at positions 1, 2, and +2
respective to the NPF motif, displayed binding reduced by 95%, 60%,
and 55%, respectively (Fig. 6B, right). Mutations of residues 3
and 4 to alanine did not appreciably affect binding (Fig. 6B,
right). Finally a GST peptide bearing five simultaneous mutations to
alanine (A5 mutant), leaving the NPF motif intact, displayed little if
any detectable binding (Fig. 6B, right). These results indicate that an
intact NPF is necessary but not sufficient for binding and that optimal
binding is conditioned by the presence of certain amino acids in the
surrounding positions. Whether this is attributable to impact on
conformation of directly on binding remains to be established.
A single EH is necessary and sufficient for interaction with an EH-binding protein
Many EH-containing proteins display repeated copies of the binding
module (Wong et al. 1995); this observation raises the question as to
whether a single EH is endowed with binding ability or whether multiple
copies are required. Our screenings of phage-displayed and bacterial
expression libraries were performed with GST fusion proteins containing
the three EH domains of Eps15 and Eps15R, to maximize the chance of
detecting protein-protein interactions. To design a strategy to
engineer GST fusions containing single EH domains, we had to take into
account the fact that the EH domains of Eps15 are contiguous. Thus,
definition of the exact boundaries and of the structural consequences
of extrapolating sequences from their natural context was difficult to
predict.
We thought, therefore, to take advantage of secondary structure predictions of the amino terminus of Eps15. As shown in Figure 7A, a Chous-Fasman-Rose algorithm predicted that the three EH domains of Eps15 are flanked by regions with high propensity for turns, possibly underlying the requirement for domain exposure, to achieve binding. Thus, we engineered a GST fusion protein containing residues 106-216 of Eps15, which encompass the second EH flanked by the predicted amino and carboxy-terminal turn (M2 protein, Fig. 7B). This protein displayed strong binding to RAB, obtained by in vitro transcription/translation (Fig. 7C). In addition, binding of GST-M2 to RAB was quantitative and of comparable intensity to that obtained with GST-EH (Fig. 7C), thus establishing that an individual EH domain is endowed with binding ability.
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A family of EH-containing proteins in mammals
Eps15 and Eps15R are the only EH-containing proteins known in mammals. The identification of peptide sequences that bind to EH allowed for the identification of putative EH-containing proteins in mammalian cells. GST fusion proteins, encompassing NPF-containing peptides from NUMB, RAB, and RAB-R were challenged with 35S-labeled lysates from NIH-3T3 cells, and several cellular proteins were specifically recovered (Fig. 8A). In the case of the RAB peptide, mutations of NPF to DPF, NGF, or NPY totally abolished recognition, arguing strongly that the identified proteins represent EH-containing species (Fig. 8A). In addition, individual NPF-containing peptides were able to bind to both common and different proteins, thus indicating a certain degree of specificity for EH-mediated interactions.
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The existence of a family of EH-containing protein in mammals was confirmed by screening the Expressed Sequence Tags Database (dbEST) with the EH domains of Eps15 and Eps15R. Four novel mammalian sequences, two from Homo sapiens and two from Mus musculus, were identified that contained EH domains (Fig. 8B).
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Discussion |
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Abstract
Introduction
Results
Discussion
Materials & Methods
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Data presented in this study establish the existence of an
expanding family of EH-containing proteins and of a network of EH-mediated interactions. Furthermore, they define the molecular basis
of EH-mediated interactions of two members of this family, Eps15 and
Eps15R, and their binding to NPF-containing peptides. We do not know
whether other EH domains will display similar binding preferences. By
analogy to other binding modules, it is conceivable that critical
binding residues will be invariant or conserved. However, a certain
degree of variation is expected, as EH domains are only ~40%
homologous. SH3 domains, by analogy, are 30% homologous, and their
binding preferences vary (Sparks et al. 1996).
With regard to Eps15, several proteins are known to date that interact
with it, including Crk, through its SH3 domain (Schumacher et al.
1995), the coated-pits adapter AP-2 (Benmerah et al. 1995, 1996;
Iannolo et al. 1997), and the EH-binding proteins described in this
paper. Thus, Eps15 is a multibinding protein that might function in the
assembling of macromolecular complexes. We do not know whether all of
the Eps15 targets interact simultaneously with it or whether a
hierarchy of interaction exists in vivo.
A wealth of evidence now supports the notion that EH domains are
involved in receptor-mediated internalization. The first indication
derived from the identification of EH domains in End3p, a yeast protein
essential for internalization of the -mating factor and of its
receptor Ste2p (Burkholder and Hartwell 1985; Nakayama et al. 1985;
Benedetti et al. 1994; Wong et al. 1995). Another EH-containing yeast
protein, Pan1p, also appears to regulate endocytosis (Wendland et al.
1996). Implication of eps15 in this pathway derived from the discovery
of its constitutive association with the clathrin adaptor protein
complex AP-2 (Benmerah et al. 1995). The related Eps15R protein is also
associated in vivo to AP-2 (Iannolo et al. 1997; A. Salcini, P.G.
Pelicci, and P. diFiore, unpubl.). Further characterization revealed
that the carboxyl termini of Eps15 and Eps15R and the -subunit of
AP-2 are involved in the interaction (Benmerah et al. 1996; Iannolo et
al. 1997). In addition, immunomorphological analysis demostrated
colocalization of Eps15 and Eps15R with markers of the plasma membrane
clathrin-coated pits and vesicles (Tebar et al. 1996; A. Salcini, P.G.
Pelicci, and P. diFiore, unpubl.).
Our present results might provide a molecular basis for the
understanding of the protein-protein interactions involved in receptor-mediated internalization, especially if viewed in the light of
recent findings by Tan et al. (1996). This group demonstrated that the
sequence NPFXD constitutes an internalization signal in
Saccharomyces cerevisiae. In particular, they showed that
NPF-based sequences in a Ste2p/Kex2p chimera and in
Ste3p, the receptor for the a-mating factor (Nakayama et al.
1985), are essential for endocytosis (Tan et al. 1996). Thus, the
possibility that receptor-mediated endocytosis is regulated by EH-NPF
interactions appears to be more than just a hypothesis.
The identification of an EH-mediated network of interactions sheds
additional light on the function of the EH domain, in that EH-containing and EH-binding proteins are involved in more functions than internalization. Pan1p, for instance, is required in yeast to
maintain proper organization of the actin cytoskeleton (Tang and Cai
1996). NUMB, identified in this study as an EH-binding protein, is
involved in cell fate determination through asymmetrical distribution
as mitosis (Uemura et al. 1989; Rhyu et al. 1994; Spana et al. 1995), a
function that requires precise sorting within the dividing cell. Thus,
the EH-based network appears to be involved in processes connected with
sorting and organization of molecules within the cell.
This contention is further strengthened by the identification of
additional candidate EH-binding proteins. We searched databases for
proteins containing multiple NPFs, as a characteristic of many of the
EH-binding proteins identified in this study is the presence of
multiple NPF-containing binding motifs, a feature that mirrors the
frequent presence of EH domains in multiple copies. This search yielded
proteins such as synaptojanin (three NPFs), which participates in
synaptic vesicle recycling (McPherson et al.1996); SCAMP 37 (three
NPFs), which is part of a family of molecules that are components of
membranes functioning in cell surface recycling (Brand and Castle
1993); and the Drosophila Dorsal protein (three NPFs), which
establishes a nuclear vemtral-to-dorsal gradient that is altered or
absent in dorsalized or ventralized embryos, thus probably determining
cell fate along the dorsal-ventral axis (Steward 1987). Other proteins
containing multiple NPFs include the Drosophila Suppressor of
sable protein (Voelker et al. 1991), the mammalian mitogen-responsive
proteins p97, p93, and p67 (Xu et al. 1995), and the Stn-b protein of
the Drosophila Stoned locus (GenBank accession no. U54982).
Although the EH-binding properties of these proteins will have to be
validated experimentally, the hypothesis that the EH network is
implicated in processes connected with transport, protein sorting, and
organization of subcellular structures clearly appears worthy of
further investigation.
Binding of Eps15 to NPF-containing targets did not appear to require
tyrosine phosphorylation of the former, in that it was readily observed
in in vitro bindings performed on serum-starved cell lysates, in which
Eps15 is not phosphorylated. In addition, stoichiometry of binding was
not improved under conditions in which a sizable fraction of Eps15 was
tyrosine phosphorylated, as achieved in lysates from epidermal growth
factor (EGF)-stimulated cells (data not shown). None of the
Eps15-binding abilities known so far, including binding to Crk and
AP-2, appears to be phosphotyrosine-dependent (Benmerah et al. 1995,
1996; Schumacher et al. 1995; Iannolo et al. 1997). This raises the
question of how the functional coupling of Eps15 to receptor tyrosine
kinase (RTK)-activated pathways (Fazioli et al 1993; Wong et al. 1994)
influences its downstream function(s), with particular regard to its
EH-mediated interactions. One attractive possibility is that a
constitutive Eps15/target complex, such as
Eps15/NUMB or Eps15/RAB, can be directed to
a proper subcellular location by tyrosine phosphorylation. In this framework, tyrosine phosphorylation would act as a molecular switch through which RTKs can condition the targeting of components of the EH
network to their active site within the cell.
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Materials and methods |
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Abstract
Introduction
Results
Discussion
Materials & Methods
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Screening of phage-display libraries
The peptide library utilized and the panning conditions were
essentially as described by Felici et al. (1991), the main difference being that the target EH domains (3 µg) were immobilized by binding to 20 µl of GST-Sepharose matrix (Pharmacia). The peptide library was screened with either the GST-EH or the GST-EHR fusion protein, designed to encompass amino acid positions 2-330 and 15-368 of mouse
Eps15 and Eps15R, respectively. Two panning cycles were carried out for
each domain before the sequencing of the clones, which were enriched
during the selection procedure.
Isolation of cDNAs encoding EH-binding proteins
A GST fusion protein containing the three EH domains of Eps15
(GST-EH; Wong et al. 1995) was used to screen a pCEV-LAC-based prokaryotic expression library (a kind gift of T. Miki, National Institutes of Health, Bethesda, MD) from M426 human fibroblasts. Recombinant plaques (5 × 106) were screened, after
induction with IPTG, using a modification for the Far Western assay
developed to identify proteins interacting with GST fusions (Matoskova
et al. 1996a,b). Briefly, filters were blocked in 2% bovine serum
albumin (BSA) in TTBS (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 0.05% Tween 20) for at least 2 hr at room temperature and then in reduced glutathione (Sigma, 3 µM)
in TTBS with 0.5% (wt/vol) BSA for 1 hr at room
temperature. This latter step substantially reduced background (not
shown). Blots were then incubated with GST-EH (10 nM) in
TTBS in the presence of reduced glutathione (3 µM) and
BSA (0.5% wt/vol) for 1 hr at room temperature. After
extensive washing in TTBS, blots were detected with an
affinity-purified anti-GST antibody, as described (Matoskova et al.
1996a,b).
Positive phages that specifically reacted with GST-EH but not with control GST were subjected to cross-hybridization experiments and assigned to seven groups, corresponding to distinct cDNAs (not shown). For each group, phages containing the longest inserts were sequenced by the dideoxy-termination method on both strands of the cDNAs, using a commercial kit (Sequenase).
To obtain cDNA clones containing the entire ORFs of human NUMB, NUMB-R, RAB, and RAB-R, a pCEV-29-based eukaryotic expression library from M426 human fibroblasts (a kind gift of T. Miki) were screened using standard techniques. Screening of the GenBank, EMBL, and EST databases was performed by the BLAST program (Altschul et al. 1990). Alignments of peptide sequences were performed by a CLUSTAL4 algorithm on MacDNASIS software.
Production of recombinant proteins
GST fusion proteins containing large fragments of the proteins of interest were obtained by recombinant PCR of the appropriate fragment from the murine Eps15 and Eps15R cDNAs or from the cDNAs of the EH-binding proteins, followed by cloning in the pGEX expression vector, in-frame with the GST moiety.
GST fusions containing short peptides from the proteins of interest
were obtained by annealing in vitro complementary oligonucleotides with
the appropriate sequence followed by cloning in-frame with the GST
moiety in a gGEX-KT vector. Sequences of the oligonucleotides and
primers used are available on request. Purification of the GST fusion
protein onto agarose-glutathione and in vitro binding experiments were
performed as described (Wong et al. 1995; Matoskova et al. 1996a,b).
Protein studies
Expression vectors for HA-tagged RAB and NUMB proteins were
engineered in the pMT2 eukaryotic expression vector by inserting (by
insertional overlapping PCR) the sequence encoding the HA epitope
(YDVPDYASLP) between codons 1 and 2 of the ORF of the RAB and
NUMB cDNAs to obtain pMT2-HA-RAB and pMT2-HA-NUMB,
respectively. The HA-NUMB/NAA mutant was generated by
changing the sequence coding for the unique NPF motif to a sequence
coding for NAA by PCR-based oligonucleotide-directed mutagenesis (Ho et
al. 1989). Transient transfection of C33A cells by calcium phosphate
was performed as described previously for NIH-3T3 cells (Fazioli et al. 1993). Immunoprecipitation and immunoblotting experiments were
performed as described previously (Fazioli et al. 1993; Matoskova et
al. 1996a). Typically, we employed 50-100 µg of total cellular proteins for direct immunoblot analysis and 3-5 mg of total cellular proteins for immunoprecipitation/immunoblotting
experiments. For coimmunopreciptiation experiments total cellular
proteins were obtained in mild lysis conditions, in the absence of
ionic detergents, to preserve protein-protein interactions, as
described (Fazioli et al. 1993).
[35S]methionine-labeled RAB protein, employed in the
experiments in Figure 7, was synthesized by in vitro
transcription-translation using a commercial kit (Promega) and the
full-length RAB cDNA. Metabolic labeling with
[35S]methionine of NIH-3T3 cells was performed as
described previously (Fazioli et al. 1993).
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Acknowledgments |
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This work was supported in part by grants from the Associazione Italiana Ricerca sul Cancro to P.P.D.F., P.G.P, and G.C. and from the Consiglio Nazionale delle Ricerche to P.P.D.F. and P.G.P. The support of the European Community (BIOMED-2 Programme) to P.P.D.F. is also acknowledged. A.E.S. and E.S. are recipients of fellowships from the Fondazione Italiana Ricerca sul Cancro. M.D. is the recipient of a fellowship from the Fondazione Vollaro.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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Received May 21, 1997; revised version accepted July 2, 1997.
5 Corresponding author.
E-MAIL pdifiore{at}ieo.cilea.it; FAX 39-2-57489851.
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Top
Abstract
Introduction
Results
Discussion
Materials & Methods
References
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-Adaptin interacts with the COOH-terminal domain of the Eps15 protein.
J. Biol. Chem.
271:
12111-12116 kinase by Src-family kinase SH3 binding to the p85 subunit.
Science
263:
1609-1612
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-sheets; (thinner solid bars)
coils; (small solid boxes) turn. Amino acid positions are also
indicated. (B) Schematic representation of the Eps15
amino-terminal domain and of the GST fusion proteins engineered, with
predicted turns indicated by solid boxes. The indicated fragments of
Eps15 were engineered into GST fusion proteins and used for in vitro
binding experiments. The EH construct contains all three EH domains.
The M2 construct contains the region encompassing the second EH domain
flanked by the natural regions pedicting the turns shown in A. Amino acid positions are also indicated in parentheses. (C) In
vitro bindings. The GST fusions shown in B were used to bind
the 35S-labeled RAB protein, obtained by in vitro
transcription/translation of the RAB cDNA.
Detection was by autoradiography. The lane marked T/T was
loaded with the primary product of the in vitro
transcription/translation to serve as a reference. The
positions of RAB are also indicated.
5 were
considered for further analysis, and they are indicated with their
database accession numbers. The alignment of these sequences to the
second EH domain of Eps15 is shown, as obtained by a CLUSTAL4 algorithm
modified by visual inspection. The