From the New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314
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ABSTRACT |
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Spectrin is a widely expressed protein with
specific isoforms found in erythroid and nonerythroid cells. Spectrin
contains an Src homology 3 (SH3) domain of unknown function. A cDNA
encoding a candidate spectrin SH3 domain-binding protein was identified by interaction screening of a human brain expression library using the
human erythroid spectrin (I) SH3 domain as a bait. Five isoforms of
the
I SH3 domain-binding protein mRNA were identified in human brain. Mapping of SH3 binding regions revealed the presence of two
I
SH3 domain binding regions and one Abl-SH3 domain binding region. The
gene encoding the candidate spectrin SH3 domain-binding protein has
been located to human chromosome 10p11.2
p12. The gene belongs to a
recently identified family of tyrosine kinase-binding proteins, and one
of its isoforms is identical to e3B1, an eps8-binding protein (Biesova,
Z., Piccoli, C., and Wong, W. T. (1997)Oncogene 14, 233-241). Overexpression of the green fluorescent protein fusion of
the SH3 domain-binding protein in NIH3T3 cells resulted in cytoplasmic
punctate fluorescence characteristic of the reticulovesicular system.
This fluorescence pattern was similar to that obtained with the
anti-human erythroid spectrin
I
I/
I
I antibody in
untransfected NIH3T3 cells; in addition, the anti-
I
I/
I
I
antibody also stained Golgi apparatus. Immunofluorescence obtained
using antibodies against
I
I/
I
I spectrin and Abl tyrosine
kinase but not against
II/
II spectrin colocalized with the
overexpressed green fluorescent protein-SH3-binding protein. Based on
the conservation of the spectrin SH3 binding site within members of
this protein family and published interactions, a general mechanism of
interactions of tyrosine kinases with the spectrin-based membrane
skeleton is proposed.
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INTRODUCTION |
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Erythroid spectrin is the predominant component of the
two-dimensional protein network called the membrane skeleton,
underlying the lipid bilayer of red cells (for recent reviews, see
Refs. 1-3). Formation of the membrane skeleton involves multiple
protein-protein interactions among integral membrane proteins.
Interactions of spectrin with other membrane proteins such as ankyrin,
protein 4.1, and adducin provide a linkage of spectrin either to the
plasma membrane or among spectrin tetramers. Many hereditary anemia
mutations affect interactions of these integral membrane proteins,
resulting in increased fragility and shortened lifespan of
erythrocytes. In hereditary elliptocytosis and pyropoikilocytosis, the
mutations have been localized in the - and
-subunits of spectrin
(reviewed in Refs. 4 and 5). Many of these proteins, including
spectrin, which were first identified in red cells, have isoforms
expressed in nonerythroid cells, but the structure and regulatory
processes of the nonerythroid membrane skeleton are less well
understood (reviewed in Refs. 1-3, 6, and 7). Functional differences between the membranes of erythroid and nonerythroid cells argue against
the simple erythrocyte model of the membrane skeleton. Major
differences between the erythroid model and other cells include
differences in the expression of spectrin (8-11) and ankyrin isoforms
(12-15) (reviewed in Ref. 16), interactions of spectrin and ankyrin
with additional proteins (17-21), localization of spectrin in the
cytoplasm as well as in the plasma membrane (10, 11, 22), and the
potential for dramatic rearrangements of spectrin's cellular location
(23, 24) (reviewed in Refs. 2 and 7).
Several studies have demonstrated that both erythroid and nonerythroid
spectrins are expressed in brain tissue (8-11, 25). Neuronal
compartmentalization of brain spectrin isoforms into axons and
presynaptic terminals (nonerythroid spectrin) and into cell bodies and
dendrites (erythroid spectrin) (10, 25) suggests that brain spectrin
isoforms may perform related but distinct functions in neuronal cells.
It has been suggested that nonerythroid spectrin performs a more
general, constitutive role, while erythroid spectrin takes part in more
specialized activities of differentiated cells (26). The -subunit of
erythroid spectrin,
I
(27),1 and the
-subunit of
nonerythroid spectrin,
II (28, 29), each contains a unique
SH32 domain. Distinct protein
interactions are likely to involve these domains, and they may be
important for specific distribution and specialized roles of brain
spectrin isoforms.
The SH3 domain was originally identified in the regulatory region of
Src and Src-like tyrosine kinases (Abl, Fps) and then identified in
other proteins, including Raf, phospholipase C, Ras GTPase-activating
protein, and phosphatidylinositol 3'-kinase, all involved in
transmitting signals within cells (reviewed in Refs. 30-32). The SH3
domains are involved in protein interactions thought to control
signaling pathways. In Src and Abl, oncogenic mutations have been
identified in the SH3 domain, indicating that this region might have a
negative regulatory effect on transformation (33). The SH3 domains of
several tyrosine kinases were found to bind to short proline-rich
sequences containing a PXXP motif (34, 35) and a general
model for the SH3-ligand complex has been proposed based on NMR studies
(36, 37). The list of SH3-containing and SH3-binding proteins is
rapidly growing (reviewed in Ref. 32). Diversity in SH3 domains and in
their ligand binding sites indicate that their binding specificities
are variable and thus mediate different protein-protein interactions.
In addition to I and
II spectrin, several unrelated cytoskeletal
or membrane proteins have been shown to contain the SH3 domain,
including a major palmitoylated erythrocyte membrane protein p55 (38), several isoforms of myosin Ib, a yeast actin-binding protein implicated in the regulation of cytoskeletal assembly (39), and yeast protein BEM1, which is involved in cell polarization (40).
Using interaction cloning, we identified a cDNA encoding a
candidate human I spectrin SH3 domain-binding protein. Five isoforms of the candidate mRNA were identified in human brain. Using the recombinant polypeptides, we located the
I SH3 domain and Abl-SH3 domain binding regions. Immunofluorescence studies suggest association of the
I spectrin SH3 domain-binding protein and Abl tyrosine kinase
with an erythroid-like spectrin in transfected NIH3T3 cells. The
candidate
I SH3-binding protein belongs to a recently identified family of tyrosine kinase-binding proteins (41-44), and one of its
isoforms is identical to e3B1, an eps8-binding protein (44). Based on
conservation of the spectrin SH3 binding site within members of this
protein family and published data (41-44), a general mechanism of
interactions of tyrosine kinases with the spectrin-based membrane
skeleton is proposed.
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EXPERIMENTAL PROCEDURES |
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Expression of the I and
II SH3 Domain in Yeast, and
Expression Library Screening--
The sequence encoding the
I
spectrin SH3 domain (nucleotides 3115-3291 of the
SpI cDNA;
Ref. 27) and the
II spectrin SH3 domain (nucleotides 2995-3177 of
the
SpII cDNA; Ref. 29) were amplified using specific primers
(see Table I) and subcloned into the pAS vector; the
I SH3 domain
plasmid is called pAS-Sp, and the
II SH3 domain plasmid is called
pAS-F. In both cases, the SH3 domain sequence was fused to the C
terminus of the GAL4 DNA binding domain. The human adult brain
expression library was obtained from CLONTECH
(catalog no. HL4004AB), and the library screening was performed using
the yeast strain Y190, with addition of 25 mM aminotriazole
into the medium. Yeast transformations and library screening were
performed as described in Ref. 45. For the liquid
-galactosidase
assays, chlorophenol red-
-D-galactopyranoside was used
as the substrate.
Rapid Amplification of cDNA Ends and PCR Cloning of hssh3bp1/e3B1 Isoforms-- Marathon-Ready human brain cDNA and Klentaq DNA polymerase mix (CLONTECH) were used to clone the 5' (primer A3': 5'-TATGAATTCGCTGGAGTACATTGTTG-3') and 3' (primer T25', Table I) ends of the hssh3bp1/e3B1 mRNA and to clone its isoforms. Although no additional sequence was obtained at the 5' end, the 3'-untranslated region was extended to 1044 base pairs; it contained a poly(A) addition signal, AATAAA, located 17 base pairs from the 3' poly(A) tail. At the 5' end of the hssh3bp1/e3B1 cDNA, the first ATG codon was located 81 base pairs downstream from the 5' EcoRI restriction site. No additional ATG codons were found following the TATA box identified in the genomic DNA fragment that hybridized to the 5' end sequence (data not shown); therefore, it is likely that the first ATG identified in the original cDNA clone represents the translation initiation site of the candidate mRNA. The amplified DNA fragments were cloned into M13mp18 or 19 or into pGEX2T vector (Amersham Pharmacia Biotech) and sequenced (46). Each hssh3bp1/e3B1 isoform was sequenced from two independent subclones. The variable alanine residue (see Fig. 1A) was present in five of eight subclones.
Expression of Glutathione S-Transferase (GST) Fusion Proteins:
I and
II SH3 Domain--
The desired regions of
I and
II
spectrin were obtained by amplification using specific primers and
subsequently cloned into the pGEX-2T vector using BamHI and
EcoRI restriction sites incorporated into amplification
products by primers. All primer sequences used for expression plasmids
are listed in Table I. The plasmid GST-E-SH3 encodes residues 977-1062
of the
I spectrin that includes the SH3 domain (residues 3115-3370
in the
I cDNA; Ref. 27). In this case, the termination codon was
present in the vector sequences, and this resulted in addition of
residues EFIVTD to the C terminus of the spectrin sequence. The
construct GST-F-SH3 encodes residues 965-1025 of
II spectrin
(nucleotides 2995-3177 of
SpII cDNA sequence; Ref. 29). The
nucleotide sequences of all plasmid constructs were confirmed by DNA
sequencing. pGEX-2T plasmids expressing SH3 domains of Abl, Crk, Src
and n-Src (34, 35) were obtained from Dr. Bruce J. Mayer (Howard
Hughes Medical Institute, Children's Hospital, Boston,
MA).
Clone 4-1--
The 1.63-kb EcoRI fragment was
obtained from the clone pGAD4-1 (isolated from the human brain
expression library) and subcloned into the plasmid pGEX1 (Amersham
Pharmacia Biotech), plasmid C1. The other GST-hssh3bp1/e3B1 fusion
plasmids are pGEX-2T subclones. Plasmids C2 and C3 were obtained by
truncation of the 1.63-kb fragment at the BamHI site and
NcoI site, respectively. The plasmid C12 was obtained by
subcloning the HincII fragment of the 1.63-kb EcoRI fragment, clone pGAD4-1, into the SmaI site
of pGEX-2T. All subsequent plasmids are subclones of amplification
products obtained using specific primers listed in Table I and the
clone pGAD4-1 as a template; for the plasmid C7, the PCR fragment
encoding isoform 5 was used. The plasmid C4 was obtained by subcloning the amplification product digested with BamHI. The plasmid
C5 was obtained by ligating the BglII-BamHI
fragment obtained from the PCR amplification with the
BamHI-EcoRI fragment from the plasmid C12 into
the BamHI-EcoRI sites of pGEX2T and checking for
the correct orientation of inserts. Plasmids C6, C8-C10, and C13 were assembled in each case by subcloning simultaneously the
BamHI-BglII fragment obtained from the PCR
amplification and the BamHI-EcoRI fragment from
the plasmid C12 as described for C5. The plasmid C15 was assembled by
annealing two complementary primers. The rest of the plasmids
(C16-C19) are PCR amplification products subcloned into the
BamHI and EcoRI restriction sites of pGEX-2T.
Green Fluorescent Protein (GFP) Fusions--
The coding
sequence of hssh3bp1/e3B1 isoform 1 was obtained by PCR amplification
(primers M5' and NGFP413') and was subcloned into the plasmid pEGFP-N3
(BglII and EcoRI sites)
(CLONTECH) so that the GFP sequences were located
at the C terminus of hssh3bp1/e3B1 (plasmid N3-1). The I SH3 domain
containing the same region of
I spectrin as clone GST-E-SH3 was
obtained by PCR amplification (primers SH5-1 and 2E-SH3) and cloned
into the BglII and EcoRI sites of the plasmid
pEGFP-C1 (CLONTECH) (plasmid C1-2E1). In plasmid
NG-1, the GFP sequence was removed from plasmid N3-1 by digestion with
EcoRI and BsrGI restriction enzymes followed by the Klenow
fill-in reaction and ligation.
Expression Fusion Proteins and Filter Binding Assay-- The affinity purification of GST fusion proteins on glutathione-Sepharose was followed by gel filtration using Sephacryl S-100 (47). Protein concentrations were estimated using the bicinchoninic acid assay (Pierce). Biotinylation of recombinant polypeptides was performed using 6-[(6-{(biotinoyl)amino}hexanoyl)amino]hexanoic acid, succinimidyl ester (Biotin-XX, SE; Molecular Probes, Eugene, OR), and the filter binding assay was performed essentially as described (34, 35). All GST fusion proteins were expressed in BL21 strain of Escherichia coli. Inductions of GST recombinant polypeptides were performed as described (47). In each case, bacterial lysates from 50 µl of induced cell cultures were solubilized and separated on SDS-Tricine polyacrylamide gels and blotted onto polyvinylidene difluoride membranes. After the transfer, blots were blocked in TBST buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, v/v) containing 2% nonfat dried milk for 1 h at room temperature. The blots were then incubated in the same buffer with biotinylated recombinant fusion proteins (0.4 µg/ml). Following incubation for 1 h at 20 °C, the blots were extensively washed with TBST buffer and incubated with a streptavidin-alkaline phosphatase conjugate at 1:5,000 dilution (Boehringer Mannheim) in 2% milk TBST for 1 h. After washing, the blots were developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrates. The band located between the 19.8- and 33.5-kDa molecular mass markers, present on all blots developed with the streptavidin-alkaline phosphatase conjugate, most likely represents a naturally occurring biotinylated bacterial protein and has been observed previously (34, 35). To quantitate the intensity of bands, the blots were scanned and bands were quantified using Scan Analysis software (Biosoft, Ferguson, MO).
Cell Transfections and Immunofluorescence--
NIH3T3 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
glutamine, 10% bovine calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were transiently transfected with GFP
fusion plasmids or NG-1 using LipofectAMINE (Life Technologies, Inc.).
24-48 h after transfection, cells were fixed with 20 °C methanol
and 1 mM EGTA, and processed for imunofluorescence essentially as described in Ref. 63. Texas red-conjugated goat anti-rabbit secondary antibody (Molecular Probes) was used in all
experiments; no staining of GFP alone was observed with this antibody.
All images were obtained from the Texas Red channel first, and this
procedure prevented possible bleed-through of GFP immunofluorescence
into the Texas Red channel. For experiments with plasmid NG-1,
hssh3bp1/e3B1 was stained with mAb 4E2 raised to
GST-hssh3bp1/e3B1,3 followed
by FITC-conjugated goat anti-mouse secondary antibody (Molecular
Probes).
Spectrin Antibodies--
The polyclonal antibody 992 (anti-axonal brain spectrin 240/325 or anti-II/
II) was purchased
from Chemicon International Inc., Temecula, CA (10). The polyclonal
antibody raised against human red cell spectrin
I
I/
I
I
(anti-HS) was obtained from ICN Biomedicals, Costa Mesa, CA. The
polyclonal antibody raised against canine erythrocyte spectrin, which
recognizes Golgi spectrin
I
*, was a generous gift from Drs.
Kenneth Beck and James Nelson (Stanford University, Stanford, CA) (22).
The polyclonal antibody raised against the 80-kDa domain of
I
I
erythrocyte spectrin was a generous gift from Dr. David W. Speicher (The Wistar Institute, Philadelphia, PA). The
polyclonal antibody against
I spectrin was obtained from
Affinity Bioreagents, Golden, CO (59). The polyclonal antibody against
Abl tyrosine kinase (Ab1) was obtained from Calbiochem-Novabiochem
International, La Jolla, CA. The monoclonal antibody against GST was
raised at the IBR Antibody Facility using standard techniques (49).
Chromosomal Localization of the hssh3bp1/e3B1 Gene-- A human P1 artificial chromosome (PAC) (50) library was screened using the EcoRI cDNA fragment from clone pGAD4-1 (BIOS Laboratories, New Haven, CT). The primers used for chromosomal mapping by PCR amplification were primer BS15' (see Table I) and primer Y3' (5'-CAA ATA TGC CTA TGT TTA TAA GTG GC 3'). The sequence of the primer BS15' was present within an exon identified in the PAC clone 102.J.10. The sequence of the primer Y3' was derived from an intron downstream from the BS15' sequence (data not shown). Each PCR amplification was performed in 25 µl using 50 ng of template DNA and Advantage PCR polymerase mix (CLONTECH) with 7 mM MgCl2; after an initial denaturation step (94 °C for 60 s), 32 cycles of PCR were performed: 92 °C for 30 s and 68 °C for 3 min. To localize the hssh3bp1/e3B1 gene by fluorescent in situ hybridization (FISH), 1-2 µg of PAC DNA from clones 102.J.10 and 305.I.23 were labeled separately by the random-primed method (46) using digoxygenin-11-dUTP (DIG; Boehringer Mannheim). Labeled DNA probes were solubilized at 5 ng/µl in Hybrisol 7 containing blocking DNA as indicated (Oncor, Gaithersburg, MD). Anonymous material remaining from clinical whole blood samples was cultured according to a previously described protocol (51). Metaphases were banded with a trypsin-Giemsa protocol and digitized using a PSI Genetiscan image analyzer. These pre-identified metaphases were then printed for retrospective chromosome identification (52) of FISH preparations. The slide preparations were destained by immersing them for 2 min each in three changes of 3:1 methanol-acetic acid. They were then treated according to a commercial protocol for in situ chromosome hybridization of unique sequence probes (Oncor catalog no. S5150), and the DIG-labeled probes 102.J.10 and 305.I.23 were hybridized to them. Subsequently, the preparations were counterstained with propidium iodide (orange) for FITC detection (yellow) of the probes on the hybridized chromosomes (see Fig. 2).
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Sequence Analyses-- were performed using the sequence analysis software package of the University of Wisconsin Genetics Computer Group (Madison, WI) (53) and the BLAST algorithm, National Center for Biotechnology Information (Bethesda, MD) (54).
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RESULTS |
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Yeast Two-hybrid Screening for Spectrin SH3 Domain-binding
Proteins--
The yeast two-hybrid system was used to screen the human
brain cDNA expression library for spectrin SH3 domain-binding
proteins. Approximately 1.9 × 106 yeast transformants
were screened with pAS-Sp (I SH3 domain construct) on drop-out
synthetic medium lacking tryptophan, histidine, and leucine. All yeast
colonies larger than 1 mm (over 550 colonies) were tested for
-galactosidase activity using the filter assay, and 17 clones were
positive as indicated by blue colonies. These clones were selected on
leucine and +cycloheximide (
leu, +cyh) medium
to retain only the pGAD-cDNA library fusion plasmids. After purification, each of the library fusion plasmids was retransformed separately with the appropriate pAS2-SH3 construct into yeast, and a
liquid
-galactosidase assay was performed. Clone pGAD4-1 showed over
33-fold above background
-galactosidase activity when transformed
with the construct containing the
I spectrin SH3 domain, indicating
an interaction (see Table II). The
II SH3 domain plasmid, pAS-F, showed only background values when cotransformed with the clone pGAD4-1. Relatively high
-galactosidase activity was observed when the pAS-F plasmid was transformed with the
unmodified library pGAD plasmid. This intrinsic transcriptional activity of pAS-F prevented the screening of two-hybrid system expression libraries using the
II SH3 domain. Control assays for
induction of
-galactosidase activity of the pGAD4-1 clone alone and
when cotransformed with the unmodified library plasmid pGAD or the
nonrelated plasmids pLAM and pVA3 were negative, indicating a specific
interaction with the
I SH3 domain. Therefore, we designated this
clone human spectrin SH3-binding protein 1, or hssh3bp1 (Fig. 1A).
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Characterization of the hssh3bp1/e3B1 Isoforms-- Northern blotting analysis using the EcoRI insert of the clone pGAD4-1 indicated the presence of alternative transcripts. The PCR amplification using human brain cDNA as a template and primers T15' and E3' (Table I) resulted in simultaneous amplification of five DNA fragments that differ in size (data not shown). Sequence analyses indicated that the fragments represent alternatively spliced forms of the candidate cDNA and were named isoforms 1 (the largest isoform) through 5 (the smallest isoform) (Fig. 1B). 5' and 3' rapid amplification of cDNA ends was used to clone the ends of the hssh3bp1 cDNA (see "Experimental Procedures"). Data base searches using BLAST revealed that hssh3bp1 (Fig. 1A) and e3B1 represent alternatively spliced isoforms of the same cDNA. e3B1 was recently reported as the eps8 SH3 domain-binding protein (44). The predicted amino acid sequence of e3B1 is identical to isoform 2 of hssh3bp1. Subsequently, hssh3bp1 will be referred to as hssh3bp1/e3B1.
The sizes of hssh3bp/e3B1 cDNAs, between 2394 base pairs for isoform 1 and 2049 base pairs for isoform 5, corresponded well to mRNA sizes represented by the lower bands observed on Northern blots, ranging from approximately 2.3 to 2.7 kb (data not shown; Ref. 44). The mRNA of 3233 bases reported for e3B1 corresponds to higher bands on Northern blots, which range between approximately 3.3 and 3.7 kb (44). Apparently, the hssh3bp1/e3B1 mRNA contains two functional polyadenylation consensus signals (data not shown), which result in alternative splicing of the 3'-untranslated region. Alternatively spliced forms correspond to the size of either of the bands observed on Northern blots. The hssh3bp1/e3B1 gene is closely related to a group of recently identified genes encoding tyrosine kinase-binding proteins (see Refs. 41-44). Sequence comparisons using the predicted amino acid sequence of isoform 1 showed 86% identity to Abi-1 (41), and 65% identity to Abi-2 (42). The above proteins were identified as Abl-binding proteins: Abi-1 as a protein binding to the Abl C-terminal proline-rich region, and Abi-2 as a protein binding to the Abl SH3 domain. Abi-1 and Abi-2 were shown to play a role in regulating the transforming ability of Abl; Abi-2 is also a substrate of Abl tyrosine kinase (42). Isoform 1 showed 76% identity to Arg protein-tyrosine kinase-binding protein, which is likely to be a splice isoform of Abi-2 (43), and 70% identity to a 35-kDa proline-rich protein from Xenopus Xlan 4 (55). Prominent features of all hssh3bp1/e3B1 isoforms are the presence of PEST sequences (56, 57) and numerous stretches of proline-rich sequences containing the SH3 binding consensus sequence, PXXP (Fig. 1A) (35-37). Overall, 13% of the residues in isoform 1 are prolines. The proline-rich region of hssh3bp1/e3B1 undergoes alternative splicing, suggesting that regulation of its binding specificities may be mediated by differential expression of proline containing sequences. All hssh3bp1/e3B1 isoforms contain an SH3 domain, residues 446-505.Chromosomal Localization of the hssh3bp1/e3B1 Gene--
The
chromosomal location of the hssh3bp1/e3B1 gene was
determined with PAC (50) clones 102.J.10 and 305.I.23, isolated using the hssh3bp1/e3B1 cDNA. Southern blotting and sequence analyses indicated that the 102.J.10 clone contains the entire
hssh3bp1/e3B1 gene while clone 305.I.23 contains only the
3'end portion of the gene (data not shown). Each of the PAC clones
contained at least 50 kb of human genomic DNA. In FISH analysis, the
probe 102.J.10 hybridized with the same intensity to chromosomes 10 and
18 at bands 10p11.2 p12 (gene symbol
SSH3BP1)4 and
18q11.2
q12.1 (gene symbol SSH3BP2),4
respectively (Fig. 2, upper
panel). This was observed in a total of 19 metaphases that were
pre-G-banded and digitized using the method described under
"Experimental Procedures." Probe 305.I.23, however, hybridized only
to chromosome 10p11.2
p12 in a total of 16 metaphases (Fig. 2,
lower panel). In both cases, 100% hybridization efficiency
was observed.
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Mapping of the Spectrin SH3 Domain Binding Region--
A number of
GST-hssh3bp1/e3B1 mutants (Fig. 3) were
created to map the spectrin SH3 domain binding region of hssh3bp1.
Mapping was performed using GST fusion polypeptides derived from
isoform 4 cDNA with the exception of clone C7 in which the desired
cDNA region was obtained from isoform 5. The first set of mutants
enabled us to localize the I SH3 domain binding to the proline-rich
region, residues 152-431 (Fig. 4). The
binding was stronger, as indicated by the intensity of bands, to the
I SH3 domain than to the
II SH3 domain (compare lanes
2, 3, 5, and 6 in the
GST-E-SH3 and GST-F-SH3 panels). It should be
noted that the expression of GST-hssh3bp1/e3B1 fusions was represented
by several bands in each lane (anti-GST panel, lanes
2-7), suggesting that GST-hssh3bp1/e3B1 fusions were rapidly
degraded in bacterial lysates.
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Spectrin SH3-binding Protein Colocalizes with the Erythroid-like
Spectrin in Transfected NIH3T3 Cells--
No relevant bands
representing hssh3bp1/e3B1 were detected by Western blotting in human
erythrocyte ghosts using several anti-hssh3bp1/e3B1 polyclonal and
monoclonal antibodies (data not shown). To study whether hssh3bp1/e3B1
interacts with erythroid spectrin in vivo, we tested several
cell lines for expression of erythroid isoforms of spectrin.
Immunostaining obtained with antibodies to erythroid spectrin indicated
expression of an erythroid-like spectrin in NIH3T3 cells. Staining with
the anti-HS antibody to I
I/
I
I spectrin (Fig.
7) resulted in perinuclear staining
characteristic of Golgi apparatus, and in cytoplasmic punctate
fluorescence, suggesting vesicular staining (Fig.
8a). The Golgi staining was very similar to that observed with anti-Golgi spectrin
I
*
antibody (Fig. 8b). No cytoplasmic punctate staining was
observed with the anti-
I
* antibody. Anti-
II/
II-specific
antibody 992 strongly stained cell edges, suggesting association of
nonerythroid spectrin with the plasma membrane (Fig. 8f).
These data indicated specific distribution of erythroid and
nonerythroid isoforms of spectrin in NIH3T3 cells and enabled us to
study the interaction of hssh3bp1/e3B1 with spectrin in transfected
NIH3T3 cells. Expression of the green fluorescence protein fusion of
hssh3bp1/e3B1 (GFP-hssh3bp1/e3B1) in NIH3T3 cells resulted in
cytoplasmic punctate fluorescence. This pattern was similar to that
observed with the anti-HS antibody in untransfected cells, but a lower
number of vesicular structures per cell and no Golgi staining were
observed (Fig. 8e). In many transfected cells, vesicular
structures were fused and produced tubulovesicular structures (Fig.
8g) or larger vesicles (Fig. 9a) or resulted in a
reticular-like pattern very similar to that observed in cells
transfected with GFP fusion protein containing the
I spectrin SH3
domain (Fig. 8d). In cells transfected with GFP-hssh3bp1/e3B1, regardless of the observed expression pattern, immunofluorescence of the fusion protein was coincident with the staining obtained with the anti-HS antibody (Fig. 8, g and
h), but not with the staining obtained with the 992 antibody
(Fig. 8, e and f). In addition, the
characteristic perinuclear Golgi staining was partially disrupted (Fig.
8, compare a with h). The pattern of
hssh3bp1/e3B1 expression was also coincident with that of the antibody
against Abl tyrosine kinase (Fig. 9, a and b, respectively). These data indicate colocalization of hssh3bp1/e3B1 with
the erythroid-like spectrin and Abl tyrosine kinase in vivo in NIH3T3 cells.
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DISCUSSION |
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Using interaction cloning, we identified a cDNA clone encoding a candidate human spectrin SH3 domain-binding protein. We have characterized five isoforms of its mRNA in human brain; each isoform contains: 1) an alternatively spliced proline-rich region in the middle of the molecule, and 2) an SH3 domain at the C terminus.
The I SH3 domain spectrin binding site was mapped to two regions of
hssh3bp1/e3B1, one spanning residues 360-372 (BS2) and the other
spanning residues 390-431 (BS3). Both regions contain a
PXXP SH3 binding consensus sequence (34-36). All five
isoforms of hssh3bp1/e3B1 contain BS3; isoforms 1, 2 and 4 contain both spectrin binding sites. Our data indicate that isoforms 4 and 5 can
interact with the
I spectrin SH3 domain. Isoform 5 lacks BS2, yet it
binds to
I SH3 domain, indicating that BS3 alone is sufficient for
the interaction; therefore, all isoforms of hssh3bp1/e3B1 are likely to
bind to the
I SH3 domain. BS2 may provide an additional
I SH3
binding site in isoforms 1, 2, and 4. The fact that BS2 is a part of an
alternatively spliced region in hssh3bp1/e3B1 and the fact that the
sequence N-terminal to BS2 is required for its activity suggest that
different isoforms of hssh3bp1/e3B1 bind to the
I SH3 domain with
different affinities. On the other hand, the alternatively spliced
proline-rich region of hssh3bp1/e3B1 may provide additional binding
sites for different hssh3bp1/e3B1 ligands.
Mapping of the I spectrin SH3 binding region was performed using the
GST fusion peptide containing residues 977-1062 of
I spectrin that
included the
I SH3 domain and several residues C-terminal to the SH3
domain. In addition, this peptide included at its C terminus six
nonsense residues, EFIVTD, which resulted from the translation of the
pGEX-2T sequences. GST fusions lacking the EFIVTD sequence showed
similar binding specificities to hssh3bp1/e3B1 as GST-E-SH3 (data not
shown). In a separate experiment, we established that the
I SH3
domain and the spectrin sequence located immediately C-terminal to the
I SH3 domain, residues 1036-1062, was required for optimal binding
of GST spectrin fusions to hssh3bp1/e3B1 in filter binding assays (data
not shown). Based on the Speicher model of the spectrin structure (60),
the crystal structure of a spectrin repeat (61), and the crystal
structure of the spectrin SH3 domain (62), the region C-terminal to the
I SH3 domain is a part of helix c of the spectrin repeat unit 9, which is adjacent to the SH3 domain.
Data from the filter binding assay and results of a -galactosidase
assay in which hssh3bp1/e3B1 bound to the
I SH3 domain but not to
the
II SH3 domain suggested that in fact
I spectrin may be the
preferred partner for hssh3bp1/e3B1 in vivo. Although we
were able to precipitate
I spectrin from human erythrocyte ghosts using GST-hssh3bp1/e3B1, hssh3bp1/e3B1 was not detected in
erythrocyte ghosts. These data suggest that the protein does not have a
role in mature red cells. More comprehensive biophysical studies using
GST-hssh3bp1/e3B1 were not possible owing to limited stability of the
fusion polypeptides in solution (see anti-GST panels in
Figs. 4 and 5). This is likely to be a result of several PEST sequences
present within hssh3bp1/e3B1; PEST sequences have been reported to
function as signals for rapid intracellular proteolysis and are usually
present in proteins with short half-lives (56, 57).
Several laboratories have demonstrated expression of isoforms of
erythroid membrane proteins in nonerythroid cells. Erythroid-like Golgi
spectrin (22) and cytoplasmic forms of ankyrins (13, 14, 64) are likely
to play a structural role in the membrane skeletons of the internal
membranes of endoplasmic reticulum, Golgi, and Golgi-associated
vesicles (reviewed in Refs. 2 and 7). In this report, we provide
immunostaining evidence for expression of the erythroid-like spectrin
in the cytoplasm of NIH3T3 cells. The antibody against human
I
I/
I
I spectrin stained Golgi membranes and a large number
of cytoplasmic vesicular structures. In cells overexpressing the
GFP-hssh3bp1/e3B1 fusion protein, the erythroid-like spectrin
colocalized with the spectrin SH3-binding protein, as indicated by
coincident pattern of immunofluorescence. These data suggest
interaction of hssh3bp1/e3B1 with the erythroid spectrin in NIH3T3
cells. The perinuclear Golgi staining observed with the
anti-
I
I/
I
I antibody in untransfected cells was partially disrupted in cells transfected with the
I SH3 domain-binding protein. In some cases, only cytoplasmic vesicular structures were
observed with the anti-
I
I/
I
I antibody in transfected cells.5 Disruption of Golgi
staining was observed in cells overexpressing centractin; however, a
different change in Golgi morphology was noted (63). Alternatively, an
increased expression of hssh3bp1/e3B1 cause redistribution of the
erythroid-like spectrin from Golgi membranes into the cytoplasmic
vesicles. Further work is necessary to identify the role of
hssh3bp1/e3B1 in Golgi function. Different patterns of hssh3bp1/e3B1
fluorescence observed in cells may reflect different expression levels
of the protein in transiently transfected cells. It should be noted
that the shape of cells did not change, suggesting that the plasma
membrane skeleton is not affected by the overexpression of
hssh3bp1/e3B1.
The staining of Golgi membranes with anti-I
I/
I
I antibody
indicated that this antibody recognizes Golgi spectrin. The sequence of
Golgi spectrin has not yet been reported as of date of this publication, but it is likely to be a homolog of erythroid
spectrin,
I
* (22). Our immunocolocalization data suggest that
either Golgi spectrin contains an SH3 domain similar to the
I
spectrin SH3 domain and/or that there is an erythroid
-like spectrin
associated with Golgi
I
* spectrin. A protein immunoreactive to
the anti-
I spectrin SH3 domain and the anti-HS antibodies can be
copurified with hssh3bp1/e3B1 from untransfected NIH3T3
cells.5 In addition, immunofluorescence patterns of the
I SH3 domain and of hssh3bp1/e3B1 expression in these cells were
very similar (Fig. 8, compare d with e and
g), indicating similar intracellular localization of
hssh3bp1/e3B1 and a protein containing the
I SH3 domain.
Hssh3bp1/e3B1 binds to the Abl-SH3 domain in vitro (this
report and Ref. 44). The Abl-SH3 domain binding site of hssh3bp1/e3B1 is separate from the ISH3 domain binding sites of hssh3bp1/e3B1 (see
Fig. 1A), suggesting the possibility of simultaneous
interactions of spectrin and Abl with hssh3bp1/e3B1. Another possible
interaction site of hssh3bp1/e3B1 with Abl tyrosine kinase would be the
SH3 domain of hssh3bp1/e3B1. It is closely related to the SH3 domain of
Abi-1, which has been demonstrated to bind to the proline-rich C-terminal region of Abl tyrosine kinase (41). A GST fusion protein of
Abl SH3 interacts with isoform 2 of hssh3bp1/e3B1, as indicated by
precipitation experiments (44). Our immunostaining data suggest an
association of Abl tyrosine kinase with hssh3bp1/e3B1. Abi-1, Abi-2,
ArgBP1, XLAN4, and hssh3bp1/e3B1 represent a family of nonreceptor
tyrosine kinase-binding proteins. The spectrin SH3 binding sites, BS2
and BS3, are well conserved among these proteins (see sequence
comparison in Fig. 10); therefore, it
is possible that other members of the family also bind to the
I spectrin SH3 domain. Spectrin molecules provide a number of SH3-ligand binding sites for hssh3bp1/e3B1 and possibly for other members of this
family of tyrosine kinase-binding proteins. These proteins in turn
could target various tyrosine kinases to spectrin-containing membranes,
as we observed for hssh3bp1/e3B1 and Abl tyrosine kinase.
|
Overexpression of isoform 2 of hssh3bp1/e3B1 in NIH/EGFR fibroblasts inhibits cell growth (44). Previous studies by the same group showed enhanced mitogenic response and enhanced cell growth in fibroblastic and hematopoietic cells that overexpressed eps8 (65). Hssh3bp1/e3B1 and eps8 can therefore be considered negative regulators of each other's functions, both playing a regulatory role in cell growth. Our data suggest that hssh3bp1/e3B1 may be important for the intracellular distribution of spectrin molecules in NIH3T3 cells and in this way plays a role in assembly of the erythroid spectrin-based membrane skeleton. Although the role of hssh3bp1/e3B1 and its isoforms in formation of the membrane skeleton has yet to be established, inhibition of cell growth following the membrane skeleton assembly would be a logical possibility.
The hssh3bp1/e3B1 gene has been located to the human
chromosome 10p11.2 p12 by PCR amplification and FISH. These data
indicate that hssh3bp1/e3B1 is not a human homolog of mouse
Abi-1 (in mouse, the gene for Abi-1 has been localized to the region of
chromosome 2, which, based on synteny, corresponds to human chromosome
9q32
q34; Ref. 41). The signal on human chromosome 18 (18q11.2
q12.1) observed in FISH raises a possibility that an
hssh3bp1/e3B1-related gene is present on chromosome 18.
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ACKNOWLEDGEMENTS |
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We thank Drs. Peter J. Curtis (Wistar
Institute, Philadelphia, PA) and Marshall Elzinga and Henry M. Wisniewski (New York State Institute for Basic Research in
Developmental Disabilities (NYS IBR), Staten Island, NY) for helpful
discussions. We thank Drs. Bernard G. Forget (Yale University, New
Haven, CT) and Randall T. Moon (University of Washington, Seattle, WA)
for providing the cDNA clones of I and
II spectrin and Dr.
Bruce J. Mayer (Howard Hughes Medical Institute, Children's Hospital,
Boston, MA) for providing pGEX-2T plasmids expressing SH3 domains of
Abl, Crk, Src, and n-Src. We are grateful to Dr. David W. Speicher (The
Wistar Institute, Philadelphia, PA) for providing antibodies against
I spectrin and to Dr. Steven R. Goodman (University of Alabama,
Mobile, AL) for providing some of the anti-spectrin antibodies used in
the preliminary studies. Dr. Michal Tarnawski's (NYS IBR, Staten
Island, NY) help in statistical analysis is acknowledged. We thank Dr.
Carl Dobkin (NYS IBR, Staten Island, NY) for providing Southern blots
containing human DNA and for discussions during this study and Dr.
George S. Merz (NYS IBR, Staten Island, NY) for help in
immunochemistry. We thank Dr. Patrick G. Gallagher (Yale University,
New Haven, CT) for critical review of the manuscript.
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Addendum |
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While this manuscript was in preparation, another group reported cloning of e3B1, an eps8 SH3 domain-binding protein (44). The predicted amino acid sequence of e3B1 is identical to isoform 2 of the candidate spectrin SH3-binding protein reported here. Since this is the first report of a spectrin SH3 domain-binding protein, we propose to use the name human spectrin SH3 domain-binding protein 1/e3B1 or the abbreviated form, hssh3bp1/e3B1. This name recognizes both functions identified by the two laboratories independently.
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FOOTNOTES |
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* This work was supported by National Institutes of Health NINDS Grant R29 NS32874 (to L. K.) and the New York State Office of Mental Retardation.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U87166.
To whom correspondence should be addressed: Laboratory of
Molecular Neurobiology, New York State Institute for Basic Research in
Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, NY
10314. Tel.: 718-494-5160; Fax: 718-698-3803; E-mail:
kotulal{at}interport.net.
1 Nomenclature for spectrin isoforms used in this paper is according to Winkelmann and Forget (6).
2 The abbreviations used are: SH3, Src homology 3; GST, glutathione S-transferase; FISH, fluorescence in situ hybridization; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PCR, polymerase chain reaction; DIG, digoxygenin; PAC, P1 artificial chromosome; mAb, monoclonal antibody; BS, binding site; kb, kilobase pair(s); GFP, green fluorescent protein; FITC, fluorescein isothiocyanate; TBS, Tris-buffered saline with Tween 20.
3 L. Kotula and K. S. Kim, unpublished data.
4 Gene symbols SSH3BP1 and SSH3BP2 have been approved by the Human Genome Organization (HUGO) Nomenclature Committee, London, United Kingdom.
5 L. Kotula, unpublished data.
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REFERENCES |
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