Molecular cloning and characterization of a novel SH3 protein (SLY) preferentially expressed in lymphoid cells

A novel full-length cDNA was isolated from a murine T-cell lymphoma library that has an open reading frame encoding 381 amino acids. The predicted protein (termed SLY) contains a Src homology 3 domain and a sterile alpha motif, suggesting that it functions as a signaling adaptor protein in lymphocytes. Northern blot and in situ hybridization analysis showed a preferential expression in lymphoid tissues. The sly gene is located on the X-chromosome in close proximity to genes involved in various immune disorders. This is consistent with an additional role of SLY in immune pathology.     

Identification of novel nuclear localization signal within the ErbB-2 protein

ErbB2, a member of the receptor tyrosine kinase family, is frequently over-expressed in breast cancer. Proteolysis of the extracellular domain of ErbB2 results in constitutive activation of ErbB2 kinase. Recent study reported that ErbB2 is found in the nucleus. Here, we showed that ErbB2 is imported into the nucleus through a nuclear localization signal（NLS）-mediated mechanism. The NLS sequence KRRQQKIRKYTMRR （aa655-668） contains three clusters of basic amino acids and it is sufficient to target GFP into the nucleus. However, mutation in any basic amino acid cluster of this NLS sequence significantly affects its nuclear localization. Furthermore, it was found that this NLS is essential for the nuclear localization of ErbB2 since the intracellular domain of Erb2 lacking NLS completely abrogates its nuclear translocation. Taken together, our study identified a novel nuclear localization signal and reveals a novel mechanism underlying ErbB2 nuclear trafficking and localization.     

SH3GLB, a new endophilin-related protein family featuring an SH3 domain

A new cDNA encoding a protein of 362 amino acids designated SH3GLB1, for SH3 domain GRB2-like endophilin B1, was identified in a yeast two-hybrid screen devoted to the identification of new partners interacting with the apoptosis inducer Bax. SH3GLB1 shows strong similarities to the SH3 domain-containing proteins of the endophilin family and presumably represents the human homologue of the potential Caenorhabditis elegans SH3 containing-protein identified by systematic translation of the C. elegans genome (GenBank Accession No. U46675). Reversing prey to bait in the yeast screen, a second protein, SH3GLB2, of 395 amino acids showing 65% identity to SH3GLB1 was identified as an interacting partner of SH3GLB1. The discovery of SH3GLB1 itself in the screening with SH3GLB1 as a bait and further mapping experiments demonstrated that a core coiled-coil-type region is required for the formation of SH3GLB homo- and/or heterodimers, whereas the SH3 domain is not involved in these interactions. Interestingly, the similarities with the endophilin proteins cover the entire sequence of the SH3GLB family, suggesting a common fold and presumably a common mode of action. Furthermore, SH3GLB members colocalize to the cytoplasmic compartment of the cell together with Bax and are excluded from the nucleus. SH3GLB1 and SH3GLB2 do not significantly influence the onset and time course of Bax-mediated apoptosis in HeLa or 293T cells.     

Identification of a nuclear localization signal in mouse polycomb protein, M33

The mouse Polycomb group (PcG) protein M33 forms nuclear complexes with the products of other PcG members and maintains repressed states of developmentally important genes, including homeotic genes. In this context, nuclear localization is a prerequisite for M33 to exert its function. However, we previously found that M33 in mouse liver shuttles dynamically between the nucleus and the cytoplasm, depending on the proliferative states of cells, coupled with phosphorylation and dephosphorylation of M33 protein. To understand the mechanism and significance of this phenomenon, we identified the functional nuclear localization signal (NLS) of M33 protein. Deletion mutants that lack a particular one of three putative NLS motifs failed to localize in the nucleus. Green fluorescent protein (GFP) fused to this motif specifically localized in the nucleus. We conclude that this amino-acid stretch in M33 acts as the functional NLS for this protein.     

Identification of a karyopherin alpha 2 recognition site in PLAG1, which functions as a nuclear localization signal

The activation of the pleomorphic adenoma gene 1 (PLAG1) is the most frequent gain-of-function mutation found in pleomorphic adenomas of the salivary glands. To gain more insight into the regulation of PLAG1 function, we searched for PLAG1-interacting proteins. Using the yeast two-hybrid system, we identified karyopherin alpha2 as a PLAG1-interacting protein. Physical interaction between PLAG1 and karyopherin alpha2 was confirmed by an in vitro glutathione S-transferase pull-down assay. Karyopherin alpha2 escorts proteins into the nucleus via interaction with a nuclear localization sequence (NLS) composed of short stretches of basic amino acids. Two putative NLSs were identified in PLAG1. The predicted NLS1 (KRKR) was essential for physical interaction with karyopherin alpha2 in glutathione S-transferase pull-down assay, and its mutation resulted in decreased nuclear import of PLAG1. Moreover, NLS1 was able to drive the nuclear import of the cytoplasmic protein beta-galactosidase. In contrast, predicted NLS2 of PLAG1 (KPRK) was not involved in karyopherin alpha2 binding nor in its nuclear import. The residual nuclear import of PLAG1 after mutation of the NLS1 was assigned to the zinc finger domain of PLAG1. These observations indicate that the nuclear import of PLAG1 is governed by its zinc finger domain and by NLS1, a karyopherin alpha2 recognition site.     

Molecular cloning and characterization of FHL2, a novel LIM domain protein preferentially expressed in human heart

A full-length cDNA clone encoding a novel LIM-only protein was isolated and sequenced from a human fetal heart cDNA library. This full-length clone consists of 1416 base pairs and has a predicted open reading frame (ORF) encoding 279 amino acids. The ORF of this polypeptide codes for the human heart-specific

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IM-only protein

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(FHL2). It possesses an extra zinc finger that is a half LIM domain and four repeats of LIM domain. When the human FHL2 cDNA probe was used to hybridize with poly-A RNA of various human tissues, a very strong signal could be seen in heart tissues, and only moderately low signals could be detected in placenta, skeletal muscle and ovary. Virtually no signal could be detected in brain, lung, liver, kidney, pancreas, spleen, thymus, prostate, testis, small intestine, colon or peripheral blood leukocyte. FHL2 was mapped to chromosome 2q12–q13 by fluorescent in-situ hybridization (FISH).     

Identification of a novel Mcl-1 protein binding motif

Recent characterization of Mcl-1 as the primary anti-apoptotic Bcl-2 family member expressed in solid tumors, coupled with its ability to enable therapeutic resistance, has provided the impetus for further study into how Mcl-1 is involved in apoptosis signaling. Here, we employ Sabutoclax, a potent and effective Mcl-1 antagonist, as a competing agent to screen a randomized 12-residue phage display library for peptides that bind strongly to the Bcl-2 homology 3 (BH3) binding groove of Mcl-1. Although the screen identified a number of α-helical peptides with canonical BH3 domain sequences, it also isolated a pair of unique peptide sequences. These sequences exhibit a reverse organization of conserved hydrophobic and acidic residues when compared with canonical BH3 sequences, and we therefore refer to them as reverse BH3 (rBH3) peptides. Furthermore, studies of the rBH3 peptides using NMR spectroscopy, fluorescence polarization displacement assays, and alanine scanning data all suggest that they bind to the BH3 binding groove of Mcl-1 selectively over Bcl-x(L). A search for proteins containing the rBH3 motif has identified a number of interesting Mcl-1 protein partners, some of which have previously been associated with apoptosis regulation involving Mcl-1. These findings provide insights into the development of more specific Mcl-1 antagonists and open the way to the identification of a previously unknown family of apoptosis-regulating and Mcl-1 interacting proteins.     

Identification of an unconventional nuclear localization signal in human ribosomal protein S2

Ribosomal proteins must be imported into the nucleus after being synthesized in the cytoplasm. Since the rpS2 amino acid sequence does not contain a typical nuclear localization signal, we used deletion mutant analysis and rpS2-beta-galactosidase chimeric proteins to identify the nuclear targeting domains in rpS2. Nuclear rpS2 is strictly localized in the nucleoplasm and is not targeted to the nucleoli. Subcellular localization analysis of deletion mutants of rpS2-beta-galactosidase chimeras identified a central domain comprising 72 amino acids which is necessary and sufficient to target the chimeric beta-galactosidase to the nucleus. The nuclear targeting domain shares no significant similarity to already characterized nuclear localization signals in ribosomal proteins or other nuclear proteins. Although a Nup153 fragment containing the importinbeta binding site fused to VP22 blocks nuclear import of rpS2-beta-galactosidase fusion proteins, nuclear uptake of rpS2 could be mediated by several import receptors since it binds to importinalpha/beta and transportin.     

Identification of a single nuclear localization signal in the C-terminal domain of an Aspergillus DNA topoisomerase II

DNA topoisomerase II (topo II) is a major nuclear protein that plays an important role in DNA metabolism. We have isolated the gene for topo II ( TOP2) from the filamentous fungus Aspergillus terreus. The deduced amino acid sequence revealed that topo II consists of 1,587 amino acids and has a calculated molecular weight of 180 kDa; the protein expressed in Escherichia coli has an estimated molecular weight of 185 kDa. Expression of topo II polypeptides tagged with yellow fluorescent protein (YFP) in budding yeast suggests that the C-terminal region of the topo II is essential for transport of the fusion protein into the nucleus. The nuclear localization signal (NLS) sequence of topo II is a non-classical bipartite type containing two interdependent, positively charged clusters separated by 15 amino acids. Alanine scanning mutagenesis and deletion analyses showed further that a stretch of 23 amino acid residues (positions 1,234-1,256) is necessary for nuclear import. In addition, we confirmed, using co-immunoprecipitation and two-hybrid analysis, that this non-classical NLS interacts with importin alpha in budding yeast. These results suggest that the fungal topo II NLS is functional in yeast cells.     

Identification of a novel high molecular weight protein preferentially expressed by sinusoidal endothelial cells in normal human tissues

Mouse mAb MS-1, raised against human spleen, detects an endothelial cell antigen abundantly expressed by the sinusoidal endothelia of spleen, lymph node, liver, and adrenal cortex, but absent from nonsinusoidal continuous endothelia in these organs. Immunoelectron microscopy of splenic tissue demonstrates that the MS-1 antigen is predominantly deposited at zones of intercellular contact between adjacent sinusoidal endothelial cells. mAb MS-1 also reacts with a variable proportion of high endothelial venules in tonsil, but not in other lymphoid tissues, and with an interstitial dendritic cell population most abundant in placenta. mAb MS-1 does not react with cultured resting or mediator- activated human umbilical vein endothelial cells, dermal fibroblasts, peripheral blood mononuclear cells, or the cell lines U937, HL-60, K562 or Mo7E; it does react with the primitive myeloid cell line KG-1. mAb MS-1 immunoprecipitates a major protein of 215 kD and minor proteins of 320 and 120 kD from splenic extracts as analyzed by SDS-PAGE with reduction. These proteins are soluble in aqueous buffers. Immunoprecipitation from KG-1 cell lysates detects four proteins of 280, 300, 205, and 120 kD; the 300-, 205-, and 120-kD species, presumably corresponding to the 320-, 215-, and 120-kD species in spleen, respectively, are secreted into the media. Under nonreducing conditions, immunoprecipitates from KG-1 cell lysates or conditioned media contain one predominant 300-kD species; upon isolation and reduction, this 300-kD species separates into the previously observed 300-, 205-, and 120-kD species. Pulse-chase experiments and limited proteolysis peptide mapping suggest that the 280-kD species is a precursor of the mature 300-kD species which may be subsequently cleaved to yield the 205- and 120-kD species. Because of its size, solubility and expression pattern, the antigen recognized by mAb MS-1 is likely to be an extracellular matrix protein utilized by endothelial cells of contorted, large caliber, or leaky microvessels that lack a well-formed basement membrane.     

Identification of AMSH-LP containing a Jab1/MPN domain metalloenzyme motif

We have isolated a cDNA clone encoding a new AMSH (associated molecule with the SH3 domain of STAM) family protein, termed AMSH-like protein (AMSH-LP). AMSH-LP has similar characteristics to AMSH; both AMSH-LP and AMSH are expressed ubiquitously in various human tissues, contain a putative nuclear localization signal (NLS), an Mpr/Pad1/N-terminal (MPN) domain, and a Jab1/MPN domain metalloenzyme (JAMM) motif in their structures, and are excluded from the nucleus when lacking either the NLS or MPN domain. Moreover, we observed an enhancement of interleukin 2 (IL-2)-mediated c-myc induction in AMSH-LP-transfected cells similar to that seen in AMSH-transfected cells, suggesting a functional similarity between AMSH-LP and AMSH. However, the present study demonstrated that AMSH-LP, unlike AMSH, fails to bind to the SH3 domains of STAM1 (signal transducing adaptor molecule 1) and Grb2. These results suggest that AMSH-LP and AMSH may have different functions.     

LckBP1, a proline-rich protein expressed in haematopoietic lineage cells, directly associates with the SH3 domain of protein tyrosine kinase p56lck.

The Lck tyrosine kinase molecule plays an essential role in T cell activation and T cell development. Using the expression cloning technique, we have isolated a gene that encodes a molecule, LckBP1, able to associate with murine Lck. Analysis of full-length LckBP1 cDNA indicates at least four potentially important segments: a four tandem 37 amino acid repeat motif with a potential helix-turn-helix DNA binding motif; a proline-rich region; a proline-glutamate repeat; and an SH3 domain. These four regions are very similar to the human haematopoietic-specific protein 1 (HS1). Deletion mutant analysis of LckBP1 revealed two proline-rich regions that permit association with Lck SH3. One region contains prolines conserved among HS1 and cortactin, and the other region contains a potential MAP kinase recognition site. In vivo association between Lck and LckBP1 was confirmed by immunoprecipitation of lysates from a pre-T cell line and adult thymocytes using antibodies specific for Lck and LckBP1. LckBP1 is tyrosine phosphorylated after T-cell receptor stimulation. The SH3 domain and the potential helix-turn-helix motif in LckBP1 suggest that this molecule may associate with various molecules and function as a DNA binding molecule. The data also suggest that LckBP1 mediates intracellular signalling through Lck in T cells.     

Identification of a novel FGF, FGF-21, preferentially expressed in the liver

We isolated cDNA encoding a novel FGF (210 amino acids) from mouse embryos. As this is the 21st documented FGF, we tentatively term it FGF-21. FGF-21 has a hydrophobic amino terminus ( approximately 30 amino acids), which is a typical signal sequence, and appears to be a secreted protein. The expression of FGF-21 mRNA in mouse adult tissues was examined by Northern blotting analysis. FGF-21 mRNA was most abundantly expressed in the liver, and also expressed in the thymus at lower levels. We also isolated human cDNA encoding FGF-21 (209 amino acids). Human FGF-21 is highly identical ( approximately 75% amino acid identity) to mouse FGF-21. Among human FGF family members, FGF-21 is most similar ( approximately 35% amino acid identity) to FGF-19.     

Identification of a nuclear localization signal in the retinitis pigmentosa-mutated RP26 protein, ceramide kinase-like protein

Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new ceramide kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide the first evidence of the active nuclear import of CERKL and suggest that the identified NLS might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus.     

Identification of genes preferentially expressed in periodontal ligament: specific expression of a novel secreted protein, FDC-SP

Gene expression in human periodontal ligament (PDL) was examined by suppression subtractive hybridization to identify genes that are preferentially expressed in tissue compared to cultured PDL fibroblasts. The most enriched genes in a subtracted cDNA library are primarily genes for extracellular matrix components, types I and III collagen, lumican, periostin, and asporin, among others, whose expression conveys unique mechanical properties to the PDL. Also within this group is the gene for follicular dendritic cell secreted protein (FDC-SP), a small protein like statherin in saliva, not previously found in PDL. FDC-SP's presence in PDL was confirmed by in situ hybridization in mouse which also showed that it was definitely present in the parotid gland, but, surprisingly, not in the other salivary glands: submandibular and sublingual. Since only normal tissue was examined, these findings suggest that FDC-SP plays an important but previously unsuspected role within oral connective tissue.     

Identification of hTLR10: a novel human Toll-like receptor preferentially expressed in immune cells

Herein we describe the isolation of a cDNA encoding a novel human Toll-like receptor (hTLR) that we term hTLR10. Human TLR10 contains 811 amino acid residues. Deduced amino acid sequence analysis reveals that like the other known hTLRs (hTLR1-9) it is characterized by a signal peptide followed by multiple leucine-rich repeats (LRRs), a cysteine-rich domain, a transmembrane sequence and a cytoplasmic domain homologous to that of the human interleukin-1 receptor. Phylogenetic analysis indicates that among all the hTLRs, hTLR10 is most closely related to hTLR1 and hTLR6; the overall amino acid identity is 50% and 49%, respectively. hTLR10 mRNA is most highly expressed in lymphoid tissues such as spleen, lymph node, thymus, and tonsil. Expression analysis of cell lines indicates a predominance in a variety of immune cell types. Thus, hTLR10 is preferentially expressed in tissues and cells involved in immune responses.