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International Immunology, Vol. 11, No. 12, 1957-1964, December 1999
© 1999 Japanese Society for Immunology

Isolation and characterization of a novel HS1 SH3 domain binding protein, HS1BP3

Yoshihiro Takemoto1,3, Masaaki Furuta1,3, Mitsuru Sato1,3, Masato Kubo2 and Yasuhiro Hashimoto1,3

1 Institute of Immunology, Syntex-Roche, 2669 Yamazaki, Noda, Chiba 278, Japan.
2 Science University of Tokyo, 2669 Yamazaki Noda, Chiba 278, Japan

Correspondence to: Y. Takemoto, Tsukuba Research Laboratories, Glaxo Wellcome K.K., 43, Wadai, Tsukuba-shi, Ibaraki 300-4247, Japan


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
We have isolated a novel gene, HS1BP3, which encodes an HS1 binding protein. Analysis of HS1BP3 cDNA indicates several potentially important segments, including a PX domain, a leucine zipper, immunoreceptor tyrosine-based inhibitory motif-like motifs and proline-rich regions. HS1BP3 associates with HS1 proteins in vivo as confirmed by immunoprecipitation in B and T cell lines. HS1BP3 preferentially associates with the HS1 SH3 domains rather than with other SH3 molecules, suggesting a role of HS1BP3 as an HS1 signaling mediator. Overexpression of mutant HS1BP3 protein in T cell lines results in decreased IL-2 production. Our data suggest a novel role for HS1BP3 in lymphocyte activation.

Keywords: HS1, HS1BP3, IL-2, Lck, PX domain, tyrosine kinase


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The tyrosine kinase p56lck controls T cell development and activation. Lck-deficient mice display a dramatic reduction in double-positive (CD4+CD8+) thymocytes and nearly undetectable single-positive (CD4+CD8– or CD4–CD8+) thymocytes (1). In transgenic mice that overexpress the catalytically inactive version of Lck, T cell differentiation is blocked at the double-negative to double-positive stage (2,3). Lck mutant human cell lines with a defect in TCR-mediated signaling can be rescued by the introduction of an intact lck gene (4).

Several molecules have been described as Lck binding proteins, e.g. phospholipase C-{gamma}1 (5), tyrosine phosphorylated CD45 (6) and ZAP-70 (7), which are Lck SH2 binding proteins, and phosphatidylinositol 3-kinase (8,9) and p120 (10), which are Lck SH3 binding proteins. However, the detailed molecular mechanism of Lck signaling remains unclear. We previously isolated a Lck binding protein (11–13) that is identical to HS1 (14) and found that HS1 becomes tyrosine phosphorylated upon TCR stimulation (11,15). HS1 is also associated with Lyn and is rapidly tyrosine phosphorylated after stimulation with antibody to IgM (16). HS1 links Src family tyrosine kinase and Grb2 (13). These data suggest that HS1 mediates antigen receptor signaling through Lck in T cells and Lyn in B cells. HS1 appears to be involved in B and T cell activation and in apoptosis of lymphoid lineage cells (15,17,18). HS1 has also been shown to mediate signals from the IL-5 receptor (19) and Fc{varepsilon} receptor (20).

HS1 consists of several potentially important segments, i.e. a four tandem 37 amino acid repeat motif with a potential helix-turn-helix DNA binding motif, proline-rich regions and an SH3 domain (11,21). These segments are also found in Cortactin (11,21), which is known to be a Src/Syk substrate actin binding protein (22,23).

Overall, the evidence suggests the involvement of HS1 in the activation, differentiation and apoptosis of hematopoietic lineage cells. In the present study, we have identified an HS1 binding protein, HS1BP3 (HS1 binding protein 3), that preferentially associates with the HS1 SH3 domain in vitro and binds to HS1 in vivo, suggesting a role of HS1BP3 as an HS1 signaling mediator.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Isolation of HS1BP3
HS1BP3 was isolated by expression cloning from the murine pre-T cell line KKF cDNA library with the glutathione-S-transferase (GST)–HS1 SH3, HS1-#4 and -HS1-P3 fusion proteins as probes respectively (11). The cDNA of the HS1 SH3 binding protein, HS1BP3, derived from the KKF library was subcloned into a NotI site of pGEX-4T (Pharmacia, Uppsala, Sweden), and a BamHI fragment (nucleotides 197–646) of the HS1BP3 cDNA was radiolabeled and used to screen a thymus a {lambda}ZAPII cDNA library. The HS1BP3 cDNA was excised from {lambda}ZAPII phage using the ExAssist/SOLR system (Stratagene, La Jolla, CA), resulting in a pBluescript phagemid containing the HS1BP3 cDNA. The sequence was determined by the Sanger method (24). The HS1BP3 nucleotide sequence is available from the EMBL database under accession no. AJ132192.

Phage spot assay
Phage solutions of HS1BP3 and Lck cDNA were diluted and spotted onto agarose plates containing Escherichia coli. After 3 h incubation at 43°C, isoproply-ß-D-galactopyranoside (IPTG)-treated nitrocellulose filters were overlaid on the agarose plates to induce synthesis of the ß-galacotosidase (ß-gal) fusion proteins. The filters were blocked with Tris-buffered saline/Tween containing 5% skim milk and incubated with probe proteins. Binding was detected by sequential incubation of filters with anti-GST antibody (1/2000 dilution) (11) and alkaline phosphatase-conjugated anti-rabbit antibody (Dako, Carpinteria, CA; 1/2000 dilution), followed by a color development reagent.

Construction of GST fusion proteins and antibodies
The GST–Lck-SH3 and GST–HS1-SH3 fusion proteins have been described (11). The GST–Grb2 N-terminal SH3 and C-terminal SH3 fusion proteins have also been described (13). The cDNA of HS1BP3 (nucleotides 197–1082 ) derived from the KKF library was subcloned into a NotI site of pGEX-4T (Pharmacia) (GST–HS1BP3-C2). To construct GST–HS1BP3-C5, a BamHI fragment (nucleotides 197–646) of GST–HS1BP3-C2 was subcloned into a BamHI site of pGEX-4T. Expression and purification of the GST fusion proteins have been described (11). The HS1BP3-specific antibodies, L3 and L10-1, were generated in rabbits immunized with GST–HS1BP3-C2 (amino acids 57–351 of HS1BP3) and HS1BP3-C5 (amino acids 57–208 of HS1BP3) respectively.

Construction of expression vector
The pCAGGS vector has been described (25). To introduce a multi-cloning site, the XbaI and HindIII sites of the pCAGGS vector were eliminated by Klenow filling and ligation, and a linker containing a multi-cloning site (HindIII–NotI–XbaI–EcoRV–EcoRI: 5' linker, 5'-AATTAAGCTTGCGGCCGCTCTAGAGATATCG-3' and 3' linker, 5'-AATTCGATATCTCTAGAGCGGCCGCAAGCTT-3') was cloned into an EcoRI site of the pCAGGS, termed the pCAGGS-MCS vector. The NotI cDNA fragment of HS1BP3 was subcloned into a NotI site of pCAGGS-MCS. To construct the double-tagged expression vectors, the cDNAs of HS1BP3 and HS1BP3-C5 were subcloned into a NotI site of the double HA- or Myc-tagged expression vectors respectively (26). All PCR-derived sequences used in these studies were confirmed by the Sanger method (24).

RT-PCR
Total RNA was isolated by ISOGEN (Nippongene, Toyama, Japan) according to the manufacturer's instructions. cDNA was prepared from 5 µg of total RNA using MMLV reverse transcriptase (Life Technologies, Rockville, MD) to a final volume of 100 µl. After a 90 min incubation of the mixture at 37°C, the cDNA solution was ethanol-precipitated and resuspended in 100 µl of water. The cDNA was amplified by PCR with the HS1BP3-specific primers (sense primer, nucleotides 687–703, 5'-GCCCAGAAGTGGCCGTG-3'; reverse primer, nucleotides 1058–1074, 5'-GTTGGTTTCCTGGGCAA-3'), reverse primer, 5'-GCTTGTTGAGATGCTTTGACA-3') and the G3PDH-specific primers (Clonetech, Paolo Alto, CA; sense primer, 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3'; reverse primer, 5'-CATGTAGGCCATGAGGTCCACCAC-3'). Obtained PCR products (388 bp for HS1BP3, 451 bp for IL-2 and 983 bp for G3PDH) were size-fractionated onto a 1.8% agarose gel and stained with ethidium bromide.

Immunoprecipitation and Western blot analysis
Cell lysates (2x107) were prepared by lysis with TNE buffer, and incubated with 50 µl of L10-1 antibody for immunoprecipitation of proteins from T cells and B cells. Immunoprecipitation was as described (11,12). The mAb used in the Western analysis was anti-HS1 (Sumitomo Denko, Kanagawa, Japan).

Cells and TCR stimulation
The T cell lines BgV, KKE, KKF, KgV and KKC were originally derived from Gross virus-infected BALB/k thymocytes (27,28). The T cell lines and the T cell hybridoma DO-11.10 (29), Jurkat, B cell line Ig6.3 and p815 were maintained in RPMI 1640 with 10% FCS. Murine NIH 3T3 and HeLa cells from the RIKEN cell bank (Ibaraki, Japan) were maintained in DMEM with 10% FCS. TCR on T hybridoma cells were stimulated by plating in anti-CD3{varepsilon}-coated plastic culture dishes (100 µg/ml) for 15 min. Non-stimulated cells were used as a negative control. After T cell stimulation, plates were washed with PBS 3 times and cells were directly lysed by the addition of TNE buffer (10 mM Tris–HCl, pH 7.8, 1% NP-40, 0.15 M NaCl, 1 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 10 µg/ml aprotinin and 10 µg/ml leupeptin).

DO-11.10 cell lines that stably expressed tagged HS1BP3 were obtained by electroporation in RPMI 1640 containing 20% FSC at a density of 5x106 cells/400 µF/400 µl cuvette with a BioRad gene pulser at 250 mV and 960 µF followed by selection in 500 µg/ml of geneticin.

IL-2 bioassay
The bioassay of IL-2 production has been described (30). Briefly, 1x 106 cells were stimulated by plating in plastic culture dishes coated with anti-CD3{varepsilon} at various concentrations for 24 h. The culture supernatant was harvested and the biological activity of IL-2 was tested based on the proliferative response of the murine indicator cell line CTLL-2.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Isolation of HS1 binding protein
To isolate HS1 binding proteins, we used expression cloning techniques with a bacterially synthesized GST fusion proteins containing the HS1-#4 (amino acids 215–335, containing a potential SH3 binding region, PSLPTR), HS1-P3 (amino acids 336–360, containing a potential SH3 binding region, PPALPPR) or HS1-SH3 region to screen the {lambda}gt11 cDNA expression library obtained from the murine pre-T cell line KKF (Fig. 1Go) (11,28). Three clones were obtained after three rounds of screening 1x106 independent phage clones with these probes. Two clones with GST–HS1-#4 and GST–HS1-P3 probes were isolated, and found to be identical to the Lck SH3-SH2 domains (amino acids 55–282, nucleotides 326-1010 ) (31), confirming the Lck SH3 domain binding to HS1 as reported previously (11,12). The third clone probed with GST–HS1-SH3 encodes a novel gene, termed HS1BP3. A cDNA of HS1BP3 containing further 5' and 3' regions was obtained by screening a murine thymus cDNA library using the 5' end of the cDNA derived from the KKF library (11,28) as a probe. Sequence analysis revealed that the thymus-derived cDNA was 1393 bp in size, with a 1188 bp open reading frame, a Kozak consensus-like sequence (32) and a termination codon (Fig. 1Go). The 5' end of the cDNA has no stop codon in front of the first methionine (nucleotides, 29–31). We obtained a similar site for the 5' end of HS1BP3 cDNA using the 5'RACE method (Gibco/BRL, Life Technologies, Rockville, MD) with the KKF mRNA (data not shown), suggesting a possibility that the first methionine is an initiation codon. The cDNA encodes 396 amino acids and the predicted molecular mass of the protein is 44 kDa. The deduced amino acid sequence shows an acidic region (amino acids 195–204), a basic region (amino acids 213–220), a potential leucine zipper (amino acids 246–267), proline-rich regions as potential SH3 domain binding regions (PPLPRK, amino acids 91–96; KPKPLVP, amino acids 333–339; PLVPAK, amino acids 336–341; PALPRK, amino acids 342–347), immunoreceptor tyrosine-based inhibitory motif (ITIM)-like motifs [T/SxxYxxL (33)] (YQIL, amino acids 41–44; SKKYSEI, amino acids 68–74; YQKL, amino acids 78–81) and a PX (phox) domain (Figs 1 and 3AGoGo). The PX domains were found in the NADPH oxidase (p47phox and p40phox), a sorting nexin (SNX1), phosphatidylinositol 3-kinases and several yeast proteins, such as Bem1 and Vam7p (34).




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  		Fig. 1. (A) Nucleotide sequence and predicted amino acid sequence of HS1BP3. Note the proposed first methionine (circle), a PX domain (boxes), ITIM-like sequences (double underlined), an acidic region (dotted lines), a basic region (dashed lines), typical proline-rich regions that are potential SH3 binding domains (underlined) and a leucine zipper (barred line). Arrows indicate the cDNA derived from KKF library. Numbers on the left indicate positions of the nucleotides. (B) Sequence homology of the PX domain of HS1BP3 (accession no. AJ132192) with yeast VPS5 (accession no. SCU84735), human SNX1 (accession no. U53225), yeast BEM1 (accession no. Z36069) and human p40phox (accession no. X77094). Dots indicate insertion. Conserved amino acid of the PX domain are in bold. ITIM-like sequences are underlined. The brackets show typical proline-rich regions of a potential SH3 binding domain. Numbers indicate positions of the amino acids.

 


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  		Fig. 3. Biochemical analysis of HS1BP3. (A) Schematic representation of HS1BP3 and antigen used to generate anti-HS1BP3 antibody. Note coding regions (open boxes), PX domain (closed box), proline-rich regions (vertical thin lines), acidic region (diagonal striped box), basic region (horizontal striped box) and leucine zipper (dotted box). (B) Immunoblot of anti-GST (lane 2) or L10-1 immunoprecipitates (lane 3) from T cell lysates using L3 antibody. Immunoprecipitates were prepared from lysates of 1x 106 cells. Approximately 20 µg of lysate of T cell hybridoma DO-11.10 was used as a positive control (T: DO-11.10). Numbers on the right indicate molecular size. (C) Expression of HS1BP3 cDNA in COS cells. COS cells were transfected with CAG-MCS vector alone (COS/Mock; lane 2) or the vector with HS1BP3 (COS/HS1BP3; lane 3). Lysate of T cell hybridoma DO-11.10 was used as a positive control (T: DO-11.10; lane 1). (D) Immunoblotting of various cell lines with L3 antibody. Murine T cell lines BgV (lane 1), KKE (lane 2), KKF (lane 3), KgV (lane 4), KKC (lane 5), DO-11.10 (lanes 6 and 11), murine B cell line Ig6.2 (lane 7), WEHI231 (lane 8), murine mast cell line p815 (lane 9), murine fibroblast NIH 3T3 (lane 10), human Jurkat (lane 12) and HeLa cells (lane 13). Approximately 20 µg of cell lysate for each line was blotted with L3 antibody.

 
HS1BP3 selectively associates with the HS1 SH3 domain
The ß-gal–HS1BP3 expression phage was diluted and spotted onto agarose plates containing E. coli. As a control, a ß-gal fusion expression phage containing Lck SH3 and SH2 domain (amino acids 55–282) was used. After 3 h incubation at 43°C, the IPTG-treated nitrocellulose filters were overlaid on the agarose plates to induce the ß-gal fusion proteins. Filters containing the ß-gal fusion proteins were probed with 100 µg/ml of the GST, GST–HS1 SH3 (GST–HS1SH3), GST–Lck SH3 (GST–LckSH3), GST–Grb2 N-terminal SH3 (GST–Grb2NTSH3) and GST–Grb2 C-terminal SH3 fusion protein (GST–Grb2CTSH3) (Fig. 2Go). The complex of ß-gal fusion proteins and GST fusion proteins was detected by an anti-GST polyclonal antibody (11). GST–HS1SH3 specifically associates with ß-gal–HS1BP3; however, a very weak association of the GST, GST–LckSH3, GST–Grb2NTSH->GST–Grb2NTSH3 and GST–Grb2-CTSH3 probes to the ß-gal-HS1BP3 was observed (Fig. 2Go). Thus, significant binding of these probes to the control fusion protein was not observed. The Lck and Grb2 SH3 domain fusion proteins used in this experiment were capable of associating with other proteins (11–13). These results indicate that the HS1 SH3 domain specifically associate with HS1BP3 in vitro.



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  		Fig. 2. HS1BP3 binding pattern to various SH3 molecules. One microliter of phage solution of HS1BP3 and Lck was spotted onto nitrocellulose filters (lane 1, undiluted solution; lane 2, 1/5 dilution; lane 3, 1/52 dilution; lane 4, 1/53 dilution; lane 5, 1/54 dilution; lane 6, 1/55 dilution; lane 7, 1/56 dilution; lane 8, 1/57 dilution). After ß-gal fusion protein induction, the filters were probed with GST, GST–HS1-SH3, GST–Lck-SH3, GST–Grb2NTSH3 and GST–Grb2CTSH3 respectively. Binding was detected by sequential incubation of filters with anti-GST antibody and alkaline phosphatase-conjugated anti-rabbit antibody, followed by a color development reagent.

 
Biochemical analysis of HS1BP3
Anti-HS1BP3 polyclonal antibodies were derived from rabbits immunized with the GST–HS1BP3-C2 and GST–HS1BP3-C5 fusion proteins (Fig. 3AGo). To analyze the HS1BP3 molecule biochemically, proteins were immunoprecipitated from T cell lysates with an HS1BP3 antibody specific for HS1BP3-C5 (L10-1) and the immunoprecipitates were Western blotted with an HS1BP3 antibody specific for HS1BP3-C2 (L3) (Fig. 3BGo). The L3 antibody detected a 55 kDa molecule in the L10-1 immunoprecipitates and whole T cell lysates (Fig. 3BGo, lanes 1 and 3). HS1BP3 was detected as a 55 kDa molecule in SDS–PAGE. To further test whether the cDNA encodes a 55 kDa protein rather than the predicted 44 kDa, we transfected the HS1BP3 gene carried in an expression vector into COS cells and Western blotted the cell lysates with L3 antibody. A 55 kDa protein was abundantly expressed in the transfected cells (Fig. 3CGo, lane 3), identical in size to the endogenous HS1BP3 from the murine T cell lines (Fig. 3CGo, lane 1). The L3 antibody also detects a 55 kDa protein in monkey kidney COS cells. These data suggest that the thymus-derived cDNA encodes the full-length HS1BP3 coding region.

The expression pattern of HS1BP3 protein in the cell lines was analyzed by Western blot analysis (Fig. 3DGo). Cell lysates obtained from several cell lines were separated by SDS–PAGE and probed with L3 antibody. Expression of the 55 kDa molecule was observed in all murine cell lines examined. The L3 antibody also detected the 55 kDa molecule in the human Jurkat and HeLa cell lines (Fig. 3DGo, lanes 12 and 13).

The RNA expression of HS1BP3 was examined by RT-PCR. The HS1BP3 RNA was measured from various organs as indicated (Fig. 4Go). G3PDH was used as an internal control. The HS1BP3 RNA expression was observed in all murine organs examined. Thus, HS1BP3 appears to be widely or ubiquitously expressed.



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  		Fig. 4. HS1BP3 expression in murine tissues and cell line. (A) Schematic representation of the location of PCR primers of HS1BP3 and G3PDH. The locations of the primers of each genes are shown by arrows. Numbers indicate the nucleotide number of the starting point of the PCR primers. Note coding regions (open boxes) and non-coding regions (bar). (B) Expression of HS1BP3 RNA. Total RNA was purified from various organs as indicated and subjected to RT-PCR analysis with oligonucleotide primers specific for HS1BP3. PCR amplification of G3PDH was used as an internal control. After 35 cycles, the PCR products were electrophoresed onto a 1.8% agarose gel and stained with ethidium bromide.

 
HS1BP3 association with HS1 in vivo
To examine the in vivo association of HS1BP3 and HS1, cell lysates obtained from T cell hybridoma DO.11-10, with or without TCR stimulation, were immunoprecipitated with L10-1 antibody or with anti-GST antibody as a control and immunoblotted with HS1 antibody (Fig. 5Go). Anti-HS1 antibody detected the HS1 bands in the anti-HS1BP3 (L10-1) immunoprecipitate regardless of TCR stimulation in DO.11-10 cells (Fig. 5Go, lanes 1–6), whereas the GST antibody did not detect the HS1 band. The association of HS1 and HS1BP3 was also confirmed in the B cell line, Ig6.3 (Fig. 5Go, lanes 7 and 8). Thus, HS1BP3 associates with HS1 in vivo in the B and T lineage cells.



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  		Fig. 5. In vivo association of HS1 and HS1BP3. GST (lanes 3, 4 and 7) and L10-1 immunoprecipitates (lanes 5, 6 and 8) from murine T cell hybridoma DO-11.10 with TCR stimulation (lanes 4 and 6) or without stimulation (lanes 3 and 5), and murine B cell line Ig6.3 (lanes 7 and 8) were immunoblotted with HS1 antibody. Immunoprecipitates were prepared from lysates of 5x 107 cells. Lysates of T cell hybridoma DO.11-10 were used as a positive control (lanes 1 and 2). Numbers on the left indicate molecular size.

 
Role of HS1BP3 in IL-2 production
Our preliminary analysis of several cellular functions in the HS1BP3 overexpressing T cell lines revealed a change in IL-2 production in the transfectants. We therefore generated mutant HS1BP3 constructs and analyzed the effect on IL-2 production in greater detail by overexpression of the mutant HS1BP3. Double HA- or Myc-tagged HS1BP3 and HS1BP3-C2 (Figs 3A and 6AGoGo) were constructed in expression vectors and introduced into a T cell hybridoma DO.11-10. To examine the expression level of HS1BP3 or HS1BP3-C2, the cell lysates of antibiotic-resistant cell lines were Western blotted with anti-HS1BP3 antibody. The proteins derived from the exogenous genes were greater than the endogenous HS1BP3 (Fig. 6BGo). Next, the stable cell lines were stimulated with anti-CD3{varepsilon} antibody at several different concentrations and IL-2 production by these cells was quantitated. Cell lines containing double HA- (clones 9-3, 9-13 and 9-15) or Myc- (clone 9-36) tagged HS1BP3 produced IL-2 at levels similar to those by control cells (clones 3-1) (Fig. 6Go, open symbols), whereas IL-2 production by cell lines transfected with the truncated HS1BP3 (double HA- and Myc-tagged HS1BP3-C2; clones 3-26 and 3-45 respectively) was significantly lower than in control cells (Fig. 6Go, closed symbols). The expression level of surface TCR in these transfected or non-transfected cells was very similar (data not shown), excluding the artifact of reduced amount of surface TCR by transfection. The reproducibility of the results obtained with different transfectants and different DNA constructs excludes the possibility of artifacts due to DNA construction or DNA integration sites in the cell lines. Thus, these results indicate the involvement of HS1BP3 in IL-2 production.



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  		Fig. 6. IL-2 production by stable HS1BP3 transfectants upon TCR stimulation. (A) Schematic representation of the double-tagged HS1BP3 and HS1BP3-C2. Note coding regions (open boxes), PX domain (closed box), proline-rich regions (vertical thin lines), acidic region (diagonal striped box), basic region (horizontal striped box) and leucine zipper (dotted box). Each clone represents stable transfectants of the following DNA constructs: 3-1, control DNA; 9-3, 9-13 and 9-15, double HA-tagged HS1BP3; 9-36, double Myc-tagged HS1BP3; 3-26, double HA-tagged HS1BP3-C2; and 3-45, double Myc-tagged HS1BP3-C2. (B) Western blot analysis of the stable transformant cell lines. Approximately 20 µg of cell lysate for each line was blotted with L3 antibody. (C) T cell transfectants were stimulated with anti-CD3{varepsilon} antibody for 24 h and IL-2 in the supernatant was quantitated based on the proliferative response of the murine IL-2 indicator cell line, CTLL-2. Concentrations of anti-CD3{varepsilon} antibody for stimulation are indicated on the x-axis. Proliferative response was converted to IL-2 international units.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We have isolated a novel SH3 domain binding protein, HS1BP3, by expression cloning. We have further demonstrated that HS1BP3 selectively associates with the HS1 SH3 domain in vitro. In addition, intracellular association of HS1BP3 and HS1 was confirmed by co-precipitation of HS1 in the HS1BP3 immunoprecipitates obtained from both T and B cell lysates. Overexpression of mutant HS1BP3 protein in T cell lines resulted in decreased IL-2 production, suggesting the possibility that HS1BP3 and HS1 complex is involved in IL-2 signaling.

HS1BP3 consists of the PX domain, proline-rich regions, ITIM-like regions and leucine zipper domains. The PX domain was first found in the NADPH oxidase, p47phox and p40phox, using a computer homology analysis (35). PX domains were further found in CPK-like phosphotidylinositol 3-kinases, proteins involved in vesicular trafficking including the SNX-1 family members, and the yeast proteins Mvp1p, Vps5p and Vps17p (34–37). Several PX domains have been reported to bind other proteins (38–40). The function of PX domains are not well understood; however, it has been reported that a potential role of the PX domain in Vam7p is the regulation of Saccharomyces cerevisiae vacuolar assembly (41). Many PX domains, including one in HS1BP3, contain a potential type II SH3 binding proline-rich region (34,42,43). In addition, two SH3 binding proline-rich regions are found in HS1BP3. Through the proline-rich regions, including one in the PX domain, HS1BP3 might bind to the SH3 and WW domains in other proteins.

In the PX domain, three potential ITIM-like sequences were found. ITIM has been shown to be a protein binding motif to the SH2 domain of several phosphatases (33), suggesting the possibility that HS1BP3 can associate with phosphatase molecules.

A leucine zipper motif, which is considered to be in the protein–protein interaction domain, and found in several proteins such as Fos, Jun and Myc (reviewed in 44), is located in the middle region of HS1BP3. Thus HS1BP3 might bind to several intracellular proteins through the PX domain, proline-rich regions, ITIM-like regions and a potential leucine zipper to assemble and localize associating proteins to certain areas of the cytoplasmic region.

We cannot formally exclude the possibility that the 5' end of the cDNA does not cover the complete N-terminal coding region, because the 5' region of HS1BP3 cDNAs in several DNA clones obtained by different methods does not have any stop codon in front of the first methionine. However, the HS1BP3 protein in the COS cell lines, in which our obtained HS1BP3 cDNA clones were overexpressed, was detected as a similar size molecule as seen in the other various cell lines. Thus it is likely that our obtained HS1BP3 cDNA encodes a full-length open reading flame.

The predicted mol. wt of HS1BP3 is 45 kDa based on the deduced amino acid sequence of the HS1BP3 cDNA, while the HS1BP3 is found as a 55 kDa protein in SDS–PAGE. The discrepancy between the predicted and observed mol. wt in SDS–PAGE may be due to phosphorylation, glycosylation or the protein folding of HS1BP3.

HS1BP3 expression was observed in various tissues by RT-PCR and in the different tissue-derived cell lines by Western blot analysis, while expression of HS1 is found exclusively in hematopoietic lineage cells. Strong similarities in amino acid sequences between HS1 and Cortactin were found, especially the SH3 domain of HS1 which showed stronger similarity to the SH3 domain in the Cortactin and murine Drebrin-like molecule SH3P7 (45) than the SH3 domains in the Src family tyrosine kinases and Grb2. Coatactin is reported to be a Src/Syk substrate and expressed ubiquitously (22,23). Thus, HS1BP3 might associate with the SH3 domain of HS1 in hematopoietic lineage cells, while in non-hematopoietic lineage cells HS1BP3 might bind to Cortactin or murine Drebrin-like molecules through their SH3 regions.

The N- and C-terminal truncation of HS1BP3 affected IL-2 expression, whereas an expression of the intact protein makes no difference. Such an observation could be explained by the fact that HS1BP3-C2 lacks the N-terminal region and the C-terminal region of HS1BP3. In particular, the N-terminal region of HS1BP-C2 lacks a part of the PX domain, which has been proposed as functioning as the binding domain of some signaling proteins. Under overexpression of the mutant HS1BP3 molecules, a majority of HS1 molecules likely binds to the mutant HS1BP3, so that majority of the HS1 and mutant HS1BP3 complex does not transmit signals downstream because of a lack of association with other signaling molecules through the deleted regions of the HS1BP3 molecules.

We did not find reduced or increased levels of signal with overexpression of wild type HS1BP3. In cases of overexpression of enzymes, enhanced molecular activity is often found. However, the overexpression of a protein, such as an adapter molecule, reduces the number of proper molecular complex transmitting signals downstream due to a relative shortage of association molecules compared with the adapter proteins. The reduced number of molecular complexes might affect the downstream signals. On the other hand, if sufficient signals can be generated with a small number of molecular complexes, a reduced number of molecular complexes would not affect the transmission of signal downstream. Although we do not have molecular evidence, we believe that this might occur in the HS1BP3 overexpressed cell line, based on our previous experiments.

Immunoprecipitation using antiserum specific for HS1 and HS1BP3 showed the intracellular association of HS1 and HS1BP3 molecules. When the double T7tag molecule was attached to the full-length HS1BP3, we could not find an intracellular association of tag-HS1BP3 and HS1 using an antibody for the tag region. Therefore, The tag region might disturb the tertiary structure of the HS1BP3 molecule. To show the intracellular association of HS1 and HS1BP3, we need to use double T7 tagged-truncated HS1BP3 to make the distinction from endogenous HS1BP3 molecules. Thus, with the currently available reagents, we were not able to assess their association directly. However, we did show in vitro association of HS1 and truncated HS1BP3, and thus it is likely that the truncated HS1BP3 molecule associates with HS1 in vivo. Note that several of our past experiments have shown a very high probability of correlation between in vitro molecular association and in vivo association. Thus, these results might suggest that HS1BP3 affects the IL-2 signaling pathway via HS1 in hematopoietic lineage cells.


   Acknowledgments
 
We thank Dr J.-i. Miyazaki for the pCAGGS vector and Ms M. Hoffman for editorial preparation.


   Abbreviations
 
ß-gal ß-galactosidase
GST glutathione-S-transferase
HS1 hematopoietic-specific protein 1
HS1BP3 HS1 binding protein 3
ITIM immunoreceptor tyrosine-based inhibitory motif
IPTG isoproply-ß-D-galactopyranoside

   Notes
 
3 Present address: Tsukuba Research Laboratories, Glaxo Wellcome K.K., 43, Wadai, Tsukuba-shi, Ibaraki 300-4247, Japan Back

Transmitting editor: M. Taniguchi

Received 10 March 1999, accepted 19 August 1999.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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