(Received for publication, October 19, 1995; and in revised form, December 20, 1995)
From the
Src homology 2 (SH2) domains are structural modules that
function in the assembly of multicomponent signaling complexes by
binding to specific phosphopeptides. The tetratricopeptide repeat (TPR)
is a distinct structural motif that has been suggested to mediate
protein-protein interactions. Among SH2-binding phosphoproteins
purified from the mouse B cell lymphoma A20, a 150-kDa species was
identified and the corresponding complementary DNA (cDNA) was
molecularly cloned. This protein encoded by this cDNA, which we have
termed p150 (for TPR-containing, SH2-binding
phosphoprotein), is located predominantly in the nucleus and is highly
conserved in evolution. The gene encoding p150
(Tsp) was mapped to chromosome 7 of the mouse with gene
order: centromere-Tyr-Wnt11-Tsp-Zp2. The amino-terminal two-thirds of p150
consist almost
entirely of tandemly arranged TPR units, which mediate specific,
homotypic protein interactions in transfected cells. The
carboxyl-terminal third of p150
, which is serine- and
glutamic acid-rich, is essential for SH2 binding; this interaction is
dependent on serine/threonine phosphorylation but independent of
tyrosine phosphorylation. The sequence and binding properties of
p150
suggest that it may mediate interactions between
TPR-containing and SH2-containing proteins.
Src homology 2 (SH2) ()domains are conserved
structural modules of about 100 amino acid residues that have been
identified in tyrosine kinases of the Src family and in more than 60
other proteins(1) . SH2 domains bind subsets of
phosphotyrosine-containing peptides with high affinity (K
10-000
nM)(2, 3, 4, 5) ; these
interactions mediate assembly of diverse multicomponent signaling
complexes. In addition to phosphotyrosine-dependent interactions,
phosphoserine/threonine-dependent binding to SH2 domains has also been
reported(6, 7, 8, 9) . In Bcr-Abl
chimeras that are implicated in the pathogenesis of chronic myelogenous
leukemia, the Bcr segment contains serine/threonine- and glutamic
acid-rich regions that bind SH2 domains in a phosphorylation-dependent
manner but independent of phosphotyrosine(6) .
Phosphotyrosine-independent binding of Raf-1 to the SH2 domains of Fyn
and Src has also been described(8) . More recently, we
described SH2 binding by the cyclin-dependent kinase homologue
p130
(9) . This interaction is mediated by a
serine- and glutamic acid-rich region of p130
and is
likely to involve the same site in the SH2 domain that binds
phosphotyrosine-containing peptides.
The tetratricopeptide repeat
(TPR) is a 34-amino acid motif found in proteins that function in
diverse processes, including cell cycle control, transcriptional
repression, protein transport, and protein
dephosphorylation(10) . TPRs contain eight consensus residues
whose size, hydrophobicity, and spacing are conserved. TPRs are
predicted to form a pair of amphipathic, -helical domains (A and
B) that have been proposed to mediate TPR-TPR
interactions(11, 12) . While there is as yet no
evidence that TPR motifs interact directly, they have been shown to
participate in interactions between TPR-containing proteins. For
example, the TPR-containing proteins CDC23 and CDC27 form part of a
complex that promotes anaphase(13, 14) ; a mutation in
the TPR region of CDC27 impairs its ability to interact with
CDC23(14) . There is also evidence that TPRs mediate
interactions with non-TPR-containing proteins: the transcriptional
repression protein SSN6 (Cyc8), for example, interacts with specific
DNA-binding proteins by means of its TPR region(15) .
In
work described here, SH2-binding phosphoproteins from the B-lymphoid
cell line A20 were isolated by affinity chromatography. Internal
peptide sequences from one of these proteins were used to molecularly
clone a complementary DNA that encodes a hitherto unidentified protein
of 150 kDa. This protein, which we have termed p150 (for
TPR-containing, SH2-binding phosphoprotein), contains 1173 amino acid
residues and is located predominantly in the nucleus. The
amino-terminal portion of p150
contains a tandem array of
15 TPRs; the TPR-containing region mediates p150
self-association in transfected cells. Specific binding of
p150
to SH2 domains is mediated by a serine- and glutamic
acid-rich region near the carboxyl terminus. This interaction requires
serine/threonine phosphorylation but is independent of tyrosine
phosphorylation. The sequence and binding properties of p150
suggest that it may mediate interactions between TPR-containing
and SH2-containing proteins.
Recombinant plasmid DNA was carried through a
second round of hybridization screening. Based on the sequences of
peptide 2 (QXSDLLSQAQYHVA) and peptide 3 (DKGNFYEASDVFK), degenerate
oligonucleotide probes SD945 (5` CA(A/G) GC(A/C/T/G) CA(A/G) TA(C/T)
CA(C/T) GT 3`) and SD944 (5` GA(T/C) AA(A/G) GG(A/T/G/C) AA(T/C)
TT(T/C) TA(T/C) GA 3`), corresponding to the underlined portions of
peptides 2 and 3, respectively, were synthesized. These were labeled
with P and hybridized sequentially to plasmid DNA that had
been digested with SalI and NotI, fractionated by
agarose gel electrophoresis, and transferred to nitrocellulose.
Hybridization was carried out overnight in 6
SSCPE, 20%
formamide, 5
Denhardt's solution, 10% dextran sulfate
(Pharmacia), 0.1% SDS, and 100 µg/ml salmon sperm DNA at an
oligonucleotide concentration of 33 µg/liter; hybridization was
performed at 42 °C for SD945 and at 44 °C for SD944. Membranes
were washed in 2
SSC, 0.1% SDS twice for 6 min at room
temperature and then once for 8 min at 42 °C (for SD945) or once
for 10 min at 44 °C (for SD944). Between hybridizations,the
membrane was stripped of probe by heating for 30 min at 68 °C in
0.1
SSC, 0.1% SDS.
For immunofluorescence,
affinity-purified Ab1543 and Ab1544 or total IgG from the corresponding
preimmune sera were used at 0.3 µg/ml in KB + Nonidet P-40.
Mouse anti-p150 antibody Ab635 and mouse preimmune serum
were used at 1:500 dilutions in KB + Nonidet P-40. For double
immunofluorescence experiments, microtubules were stained with the
mouse anti-tubulin antibody TU27B (19) at 1:75 dilution or with
rabbit anti-tubulin antibody Ra53 (provided by Dr. D. Murphy, Johns
Hopkins University School of Medicine) at 1:50 dilution.
Secondary
reagents used in these experiments included fluorescein
isothiocyanate-conjugated horse anti-mouse IgG (Vector Laboratories,
Burlington, CA) and fluorescein isothiocyanate-conjugated swine
anti-rabbit IgG (Accurate Chemicals, Westbury, NY). Biotinylated goat
anti-rabbit IgG or biotinylated horse anti-mouse IgG (Vector
Laboratories) were also used, in conjunction with Texas
Red-streptavidin (Life Technologies, Inc.). For competition
experiments, 150 µl of diluted, primary antibodies were
preincubated for 30 min with 15 µg of a purified GST fusion protein
containing residues 1059-1173 of p150.
Transfection and
metabolic labeling of 293 cells were carried out as described
previously(9) . Transfected cells were lysed in 2 ml of B
buffer (100 mM NaCl, 25 mM Tris-Cl (pH 7.6), 25
mM NaF, 1 mM EDTA, 2 mM NaVO
, 100 µM Na
MoO
, 1 mM phenylmethylsulfonyl
fluoride, 1% Nonidet P-40, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, and 5 µg/ml pepstatin) and incubated on ice for 10 min.
Lysates were clarified by centrifugation at 12,000
g for 12 min and assayed for the presence of SH2-binding proteins in
reactions containing 0.5 ml (2.5
10
cell
equivalents) of clarified lysate and 20 µg of GST or GST-BlkSH2
fusion protein, immobilized on glutathione-agarose as
described(9) .
To assay SH2 binding by endogenously
expressed p150, A20 cells (2
10
) were
washed twice in phosphate-buffered saline and lysed in D buffer (100
mM NaCl, 25 mM Tris-Cl (pH 8.0), 1 mM EDTA,
1 mM Na
VO
, 1 mM Na
MoO
, 1 mM phenylmethylsulfonyl
fluoride, 1% Nonidet P-40, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 5 µg/ml pepstatin). GST-BlkSH2 binding reactions were
carried out as above. In some experiments, the peptide SD12
(TWPAKSEQQRVKRGTSPRPPEGGLG) was used as a nonspecific competitor.
Figure 1:
Sequence
of p150 cDNA. A, the nucleotide sequence of
p150
cDNA, derived from the overlapping clones 19-4,
17-3, and 13-1, is shown on the upper line; the conceptual
translation of the p150
open reading frame is shown on
the lower line. Matches to peptide sequences derived from
affinity-purified p150
are underlined. B,
organization of TPRs in p150
. TPR consensus residues are
indicated in bold type. Individual TPR motifs are numbered at left. The canonical TPR consensus sequence as defined in (10) is shown at the bottom.
Interrogation of nucleotide and protein sequence data bases using
the TBLASTN algorithm (22) revealed 98.6% amino acid sequence
identity between p150 and a hypothetical, 1173-codon open
reading frame in the human genome (GenBank
accession
number D63875). In addition, p150
was found to share 31%
amino acid sequence identity with a hypothetical, 1245-amino acid
protein encoded at locus B0464.2 of Caenorhabditis
elegans(23) . The homology between p150
and
the putative B0464.2 product extends from near the amino terminus
(residue 14 of p150
) through the TPR-rich region and
includes most of the carboxyl-terminal domain (to residue 1111 of
p150
) (Fig. 2). Thus, B0464.2 is likely to encode
a C. elegans homologue of p150
. Remarkably,
interrogation of the dbEST data base of expressed sequence tags (24) revealed homology between p150
and the
conceptual translation product of an expressed sequence tag from the
higher plant Arabidopsis thaliana (T46289; 47% identity over
204 residues). The similarity between p150
and the
arabidopsis expressed sequence tag includes a TPR unit but extends
beyond it (Fig. 2). This suggested that a progenitor of
p150
first appeared before the animal and plant kingdoms
diverged. Consistent with this suggestion, a TBLASTN search also
detected a hypothetical, 1045-amino acid open reading frame in Saccharomyces cerevisiae (AOE1045) that exhibits significant
(smallest sum probability P(N) = 1.4
10
, N = 12) homology with
p150
(Fig. 2). The existence of p150
homologues in nematodes, plants, and yeast indicates an
extraordinary degree of evolutionary conservation.
Figure 2:
Comparison of mouse p150 and putative p150
homologues. Conceptual
translations of the S. cerevisiae AOE1045 coding sequence, the C. elegans B0464.2 coding sequence, the human open reading
frame homologous to Tsp, mouse Tsp, and the A.
thaliana expressed sequence tag T46289 are aligned and displayed
in single-letter code. TPR motifs are underlined; boundaries
between contiguous TPR motifs are indicated by vertical
arrows. Sequences are identified at left; amino acid
residues are numbered at right. Hyphens indicate gaps
introduced to maximize sequence identity. Deletions of the following
amino acid residues have been introduced to optimize alignment: AOE1045, 218, 226, 250, 338-339, 343-344, 361,
451, 463, 474, 576-580, 614-615, 670, 673-676, 772
and 803; B0464.2, 290-293, 619-624, 628,
636-637, 749-750, and 958-963; Tsp (human),
1051-1053.
Figure 3:
A single Tsp transcript is
expressed broadly among mouse tissues. Polyadenylated RNA from mouse
heart (He), brain (Br), spleen (Sp), lung (Lu), liver (Li), muscle (Mu), kidney (Ki), and testis (Te), fractionated by
electrophoresis and transferred to nylon, was assayed for hybridization
to a P-labeled, 2.0-kb SalI-NotI cDNA
insert from Tsp clone 17-3. The positions and sizes of RNA
markers, in kilobases, are indicated.
Figure 4:
Comparison of endogenous
p150 and the protein encoded by Tsp cDNA.
Lysates of 293 cells transfected with an expression vector containing
wild-type Tsp coding sequences (lane 1), or with
vector alone (lane 2), were fractionated by electrophoresis
through a 7.5% SDS-polyacrylamide gel. Total lysates of the B-lymphoid
cell lines A20/2J (2
10
cell eq; lane 3)
and WEHI 231 (2
10
cell eq; lane 4) were
fractionated in parallel. Lysates from A20 cells (2
10
cell eq) were incubated with bead-immobilized GST (lane
5) or GST-BlkSH2 fusion protein (lane 6); beads were
subsequently washed, and bound protein was fractionated. Protein was
transferred to a PVDF membrane, and protein was detected by
immunoblotting with antibody Ab1544. Bound primary antibody was
detected using a horseradish peroxidase-conjugated anti-rabbit antibody
and an enhanced chemiluminescence assay. The apparent sizes (in
kilodaltons) and positions of molecular mass standards are indicated at left.
Figure 8:
Binding of p150 to SH2 is
direct and requires phosphorylation. The 293 cell line was transfected
with an expression construct encoding p150
and labeled
metabolically with [
P]orthophosphate. Cells were
lysed and p150
was immunoprecipitated with a mixture of
antibodies Ab1544 and Ab1543. Immunoprecipitates were treated with calf
intestinal alkaline phosphatase (lanes 1, 3, 5, 7, 9, and 11) or left untreated (lanes 2, 4, 6, 8, 10, and 12). Treated and untreated samples were each split into three
portions which were fractionated by electrophoresis through a 7.5%
SDS-polyacrylamide gel; protein was transferred to a PVDF membrane.
Pairs of treated and untreated samples were assayed for binding to a
biotinylated GST-BlkSH2 fusion protein (lanes 1-4) or
biotinylated GST (lanes 5-8); another pair of samples
was assayed for p150
by immunoblotting with a mixture of
antibodies Ab1544 and Ab1543 (lanes 9-12).
Membrane-bound biotinylated proteins or antibodies were detected by
enhanced chemiluminescence (ECL, lanes 1, 2, 5, 6, 9, and 10).
P-Labeled proteins were detected by autoradiography after
quenching of chemiluminescence (
P, lanes
3, 4, 7, 8, 11, and 12). The apparent sizes (in kilodaltons) and positions of
prestained molecular mass standards are indicated at right.
Figure 5:
p150 is localized
predominantly to the cell nucleus. NIH3T3 cells were grown to
subconfluence, fixed with methanol, and stained (red fluorescence) with
the affinity-purified, rabbit anti-p150
antibody Ab1544
in the absence (A) or presence (B) of 15 µg of a
purified, GST-p150
fusion protein containing amino acid
residues 1059-173 of p150
. Cells were similarly
stained with a 1:500 dilution of mouse anti-p150
antiserum Ab635 (C) or a 1:500 dilution of the
corresponding preimmune serum (D). Binding of biotinylated
secondary antibodies was detected with Texas Red-streptavidin.
Microtubules were stained with the mouse anti-tubulin antibody TU27B or
with rabbit anti-tubulin serum Ra53 (green fluorescence). DNA was
visualized by staining with 4,6-diamidino-2-phenylindole (blue
fluorescence).
Figure 6:
Specific binding of Tsp products
to the BlkSH2 domain in vitro. The 293 cell line was
transfected with a plasmid encoding p150 and labeled
metabolically with
P. Lysate was adsorbed to beads coated
with the following proteins: GST-BlkSH2 (lanes 1-4),
GST-BlkSH2 S147C (lane 5), or GST alone (lane 6).
Binding was carried out in the absence of competitor (lanes 1, 5, and 6), in the presence of EPQ(pY)EEIQYIL at 10
µM (lane 3) or 50 µM (lane
4), or in the presence of an irrelevant peptide (SD12) at 50
µM (lane 2). Protein retained by beads was
fractionated by electrophoresis through a 7.5% SDS-polyacrylamide gel
and detected by autoradiography for 2 h at -80 °C. Proteins
immunoprecipitated by anti-p150
antibody Ab1544 (lane
7) or Ab1543 (lane 8) were analyzed in parallel. The
apparent sizes (in kilodaltons) and positions of prestained molecular
mass standards are indicated at left.
Figure 7:
Localization of the SH2 binding region in
p150. Wild-type p150
or individual
p150
fragments were expressed in 293 cells by transient
transfection. Cells were labeled metabolically with
[
S]methionine/cysteine, and lysates were
incubated with GST-BlkSH2 beads. Beads were washed, and bound protein
was fractionated by electrophoresis through a 7.5% SDS-polyacrylamide
gel.
S was visualized by fluorography for 2 h. Lane
1, lysate of 293 cells transfected with vector alone; lane
2, lysate of cells expressing wild-type p150
; lanes 3-6, lysates of cells expressing individual
p150
mutant proteins, as indicated at the top.
Electrophoretic positions of p150
fragments recovered
from the SH2 beads are indicated at right. The apparent sizes
(in kilodaltons) and positions of molecular mass standards are
indicated at left.
Figure 9:
Self-association of p150.
p150
was tagged at its carboxyl terminus with a
nonapeptide influenza HA epitope. The HA-tagged p150
derivative was coexpressed with each of the following
p150
fragments in 293 cells by transient transfection:
p150(1-821) (lanes 1, 2, and 9);
p150(497-821) (lanes 3, 4, and 10);
p150 (3) (lanes 5, 6, and 11); and
p150 (3) (lanes 7, 8, and 12).
Transfected cells were labeled metabolically with
[
S]methionine/cysteine and protein was
immunoprecipitated from cell lysates with the anti-HA monoclonal
antibody 12CA5 (lanes 1-8) in the absence (lanes
1, 3, 5, and 7) or presence (lanes
2, 4, 6, and 8) of an HA competitor
peptide. Alternatively, protein was immunoprecipitated with the
anti-p150
antibody Ab1544 (lanes 9-12).
Immunoprecipitated protein was fractionated by electrophoresis through
a 10% SDS-polyacrylamide gel.
S was visualized by
fluorography for 2 h. The electrophoretic positions of wild-type
p150
and p150
fragments are indicated by arrows at right. The apparent sizes (in kilodaltons)
and positions of molecular mass standards are indicated at left.
We have used SH2 affinity chromatography to isolate
SH2-binding proteins from the B-lymphoid cell line A20. By partial
peptide sequence determination and molecular cloning, one of these SH2
ligands was identified as a hitherto undescribed, ubiquitously
expressed protein of 1173 amino acid residues, which we have termed
p150. p150
has a predicted molecular mass
of 129 kDa but migrates as a protein of 150 kDa in SDS-polyacrylamide
gels; anomalous mobility may be conferred by the acidic,
carboxyl-terminal portion of the protein. Residues 74 through 815 of
p150
comprise a tandem array of 15 TPRs. Two of these
repeats (TPRs 4 and 8; residues 232-268 and 375-411) appear
to contain the amino-terminal helical domain (domain A) but not the
carboxyl-terminal domain (domain B). The TPR repeat region is
interrupted in four places by non-TPR-containing inserts (residues
75-162, between TPRs 1 and 2; residues 445-496, between
TPRs 9 and 10; residues 599-646, between TPRs 12 and 13; and
residues 715-781, between TPRs 14 and 15).
Comparison of the
individual TPR motifs of p150 provides the consensus
(I/L/V)xxx(I/L/V)xL(A/G)xx(Y/F)xxxx(D/E)xxxAxxx(F/Y)xxAL(R/K)xxxxx.
This is in close agreement with the canonical TPR motif, xxxWxxLGxxYxxxxxxxxAxxxFxxAxxxxPxx(11, 12) . The p150
TPR consensus
differs from the canonical sequence in that tryptophan is not well
conserved at position 4; nonetheless, in 10 out of the 15 TPR motifs in
p150
, hydrophobic residues are found at that position.
Another difference from the canonical TPR motif is the poor
conservation of proline at position 32. This difference, however, is
not unique to p150
; for example, in the human
serine/threonine phosphatase PP5 only one of four TPRs contains proline
at that position (26) . Structural, genetic, and biochemical
observations have suggested that TPRs mediate formation of specific
protein complexes(10) . Consistent with these data, we have
shown that p150
undergoes self-association and that this
interaction is mediated by the amino-terminal, TPR-containing region.
Whether this association is mediated by direct interactions between TPR
motifs has yet to be demonstrated.
The carboxyl-terminal acidic
region of p150 mediates binding to SH2 domains. While
binding of GST-BlkSH2 to filter-immobilized p150
was
observed at a fusion protein concentration of 100 nM,
estimation of the affinity of SH2 binding by p150
is
complicated by several factors, including the possible existence of
multiple binding sites in the acidic region of p150
,
multimerization of p150
through interactions between
TPR-containing regions, and the ability of GST-SH2 fusion proteins to
dimerize. Two lines of evidence indicate that SH2 binding by
p150
is dependent on phosphorylation but independent of
phosphotyrosine. First, a 287-amino acid fragment of p150
which lacks tyrosine residues retains the ability to bind SH2.
Second, SH2 binding was greatly reduced when p150
was
dephosphorylated by treatment with an alkaline phosphatase. Despite the
lack of a requirement for phosphotyrosine, p150
appears
to interact with the same site on the SH2 domain that binds
phosphotyrosine-containing peptides. Binding was abolished by excess
free phosphotyrosine and by the phosphotyrosine analogue
phenylphosphate(21) ; furthermore, a phosphotyrosine-containing
peptide that binds Src-type SH2 domains with high affinity was able to
compete specifically with p150
for SH2 binding.
Consistent with the results of specific competition experiments,
binding of p150
was greatly reduced by mutation of a
single residue in the Blk FLI/VRES motif, Ser
, which is
predicted on the basis of structural data to interact with
phosphotyrosine(27, 28) . While it is possible that
impairment of p150
binding by free phosphotyrosine or the
phosphotyrosine-containing peptide reflects an allosteric interaction
between separate binding sites, the observation that the Ser
mutation also impairs binding makes this interpretation less
likely.
We recently showed that another protein,
p130, also binds SH2 domains in a
phosphorylation-dependent, phosphotyrosine-independent
fashion(9) . SH2 binding by both p150
and
p130
is mediated by an acidic region that contains
multiple casein kinase II phosphorylation sites; in the case of
p130
, phosphorylation of bacterially expressed
protein by casein kinase II was sufficient to confer SH2 binding
ability. While the structural basis of SH2 binding by p150
and p130
remains to be determined, we note that
several potential casein kinase II sites in the acidic regions of these
proteins exhibit the amino acid sequence SEEE. Three-dimensional
structures of Src and Lck SH2 domains in complex with the high-affinity
peptide EPQ(pY)EEIOIYL have been determined(25, 29) .
In these complexes, the SH2 domain makes critical contacts with
glutamic acid residues at Tyr(P)
and
Tyr(P)
. It is plausible that the SEEE sites in
p150
and p130
, when phosphorylated,
mimic the high-affinity SH2-binding site (pY)EEI.
The biological
significance of phosphotyrosine-independent SH2 interactions has yet to
be established, and physiologic ligands of p150 and
p130
have not yet been identified. We have been
unable to co-immunoprecipitate Blk and p150
, and
p150
does not appear to be a substrate for sIgG-activated
tyrosine kinases. Nonetheless, the ability of p150
and
p130
to bind SH2 domains in a
phosphorylation-dependent, phosphotyrosine-independent fashion suggests
that the number of proteins that interact with the classical
phosphopeptide binding sites of SH2 domains may be substantially larger
than appreciated.
Proteins homologous to p150 can be
found in other species. A search of nucleic acid and protein sequence
data bases identified a putative C. elegans coding sequence
specifying a protein 31% identical with p150
. In its
overall structure, including the arrangement of the TPR motifs and the
sequence of the acidic region, the hypothetical C. elegans homologue resembles p150
. In general, the homology
between the nematode and mouse TPR motifs extends beyond the consensus
residues; an exception is the seventh repeat, which is apparently not
conserved in the nematode protein. The gene that encodes p150
in the mouse was mapped to chromosome 7 between Wnt11 and Zp2; the putative C. elegans coding sequence
is located on chromosome 3 at locus B0464.2(23) . No mutations
in the mouse or in C. elegans have yet been mapped to those
loci. A search of the dbEST data base identified a partial cDNA from
the flowering plant A. thaliana which, when translated,
specifies a 68-amino acid sequence with 47% identity to
p150
. Strikingly, an anonymous, 1045-amino acid open
reading frame in the genome of S. cerevisiae(30) also
exhibits significant homology to p150
. The TPR-containing
region of the hypothetical yeast protein is most similar to that of
mouse p150
in regions corresponding to the second, tenth,
thirteenth, and fourteenth repeats of the mouse protein. Homology
between the yeast and mouse proteins is not restricted to TPR consensus
residues or to the TPR-containing region, suggesting that the yeast
protein is a homologue of mouse p150
and indicating an
extraordinary degree of evolutionary conservation. While the function
of p150
in higher eukaryotes is unknown, we have found
that homologous disruption of the yeast homologue is associated with
mitotic chromosomal instability and temperature-sensitive defect in
cell growth. (
)
In recent years, it has become apparent
that assembly of a diverse group of multicomponent protein complexes is
mediated by a relatively small number of conserved structural modules,
such as SH2 and SH3 domains, that bind specific target sites with high
specificity (31) . Some proteins contain multiple
ligand-binding modules and apparently function as linking molecules.
GRB-2, for example, which contains two SH3 domains and a single SH2
domain, functions as a bridge between transmembrane signaling complexes
and SOS, a guanine nucleotide exchange factor for
p21(32, 33, 34, 35, 36) .
The presence of TPR motifs and an SH2-binding region within
p150
suggests that this protein may be able to mediate
interactions between TPR-containing and SH2-containing proteins.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L49502[GenBank].