Title: Distinct requirements for the naturally occurring splice forms Stat4 and Stat4 in IL-12 responses
Abstract: Article15 August 2003free access Distinct requirements for the naturally occurring splice forms Stat4α and Stat4β in IL-12 responses Timothy Hoey Timothy Hoey Tularik, Inc., South San Francisco, CA, 94080 USA Search for more papers by this author Shangming Zhang Shangming Zhang Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202 and Walther Cancer Institute, Indianapolis, IN, 46208 USA Search for more papers by this author Nathan Schmidt Nathan Schmidt Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202 and Walther Cancer Institute, Indianapolis, IN, 46208 USA Search for more papers by this author Qing Yu Qing Yu Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202 and Walther Cancer Institute, Indianapolis, IN, 46208 USA Search for more papers by this author Shyam Ramchandani Shyam Ramchandani Tularik, Inc., South San Francisco, CA, 94080 USA Search for more papers by this author Xiang Xu Xiang Xu Tularik, Inc., South San Francisco, CA, 94080 USA Search for more papers by this author Lisa K. Naeger Lisa K. Naeger Tularik, Inc., South San Francisco, CA, 94080 USA Search for more papers by this author Ya-Lin Sun Ya-Lin Sun Tularik, Inc., South San Francisco, CA, 94080 USA Search for more papers by this author Mark H. Kaplan Corresponding Author Mark H. Kaplan Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202 and Walther Cancer Institute, Indianapolis, IN, 46208 USA Search for more papers by this author Timothy Hoey Timothy Hoey Tularik, Inc., South San Francisco, CA, 94080 USA Search for more papers by this author Shangming Zhang Shangming Zhang Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202 and Walther Cancer Institute, Indianapolis, IN, 46208 USA Search for more papers by this author Nathan Schmidt Nathan Schmidt Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202 and Walther Cancer Institute, Indianapolis, IN, 46208 USA Search for more papers by this author Qing Yu Qing Yu Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202 and Walther Cancer Institute, Indianapolis, IN, 46208 USA Search for more papers by this author Shyam Ramchandani Shyam Ramchandani Tularik, Inc., South San Francisco, CA, 94080 USA Search for more papers by this author Xiang Xu Xiang Xu Tularik, Inc., South San Francisco, CA, 94080 USA Search for more papers by this author Lisa K. Naeger Lisa K. Naeger Tularik, Inc., South San Francisco, CA, 94080 USA Search for more papers by this author Ya-Lin Sun Ya-Lin Sun Tularik, Inc., South San Francisco, CA, 94080 USA Search for more papers by this author Mark H. Kaplan Corresponding Author Mark H. Kaplan Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202 and Walther Cancer Institute, Indianapolis, IN, 46208 USA Search for more papers by this author Author Information Timothy Hoey1, Shangming Zhang2, Nathan Schmidt2, Qing Yu2, Shyam Ramchandani1, Xiang Xu1, Lisa K. Naeger1, Ya-Lin Sun1 and Mark H. Kaplan 2 1Tularik, Inc., South San Francisco, CA, 94080 USA 2Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202 and Walther Cancer Institute, Indianapolis, IN, 46208 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:4237-4248https://doi.org/10.1093/emboj/cdg393 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Signal transducer and activator of transcription (Stat)4 is a signaling molecule required for normal responses to interleukin-12 (IL-12) and is critically involved in inflammatory responses. We have isolated an alternatively spliced isoform of Stat4, termed Stat4β, which lacks 44 amino acids at the C-terminus, encompassing the putative transcriptional activation domain. To assess the in vivo roles of these Stat4 isoforms, we generated transgenic Stat4-deficient mice expressing Stat4α or Stat4β. Our results indicate that T-cell-specific expression of Stat4α or Stat4β can mediate many aspects of IL-12 signaling including the differentiation of Th1 cells. However, Stat4α is required for normal levels of IL-12-induced interferon-γ production from Th1 cells. Microarray analysis identified 98 genes induced by both Stat4 isoforms, 32 genes induced only by Stat4α and 29 genes induced only by Stat4β. Some induced genes correlate with specific functions including the ability of Stat4β, but not Stat4α, to mediate IL-12-stimulated proliferation. Thus, Stat4α and Stat4β have distinct roles in mediating responses to IL-12. Introduction Signal transducer and activator of transcription (Stat)4 plays a critical role in inflammatory immune responses. Stat4 is activated in response to interleukin-12 (IL-12) (Bacon et al., 1995; Jacobson et al., 1995), which promotes T-helper type 1 (Th1) cell development, interferon (IFN)-γ production and cell-mediated immune responses (Trinchieri, 1995). Gene targeting experiments have demonstrated the essential role of Stat4 in IL-12-mediated immune function (Kaplan et al., 1996; Thierfelder et al., 1996). Studies using Stat4-deficient mice have demonstrated the critical role of Stat4 in generating immune responses in inflammatory and infectious disease (Simpson et al., 1998; Holz et al., 1999; Stamm et al., 1999; Cai et al., 2000; Tarleton et al., 2000; Afanasyeva et al., 2001; Chitnis et al., 2001; Matsukawa et al., 2001; Tekkanat et al., 2001). Thus, Stat4 is a critical mediator of inflammatory responses to several cytokines. STATs are a class of transcription factors that play a critical role in cellular growth, differentiation and immune responses. The seven STAT proteins are activated by various extracellular signals (reviewed in Darnell, 1997; O'Shea, 1997; Leonard and O'Shea, 1998). Analysis of IFN-α and IFN-γ signaling initially revealed the mechanism of STAT activation (Darnell, 1997). In unstimulated cells, STAT proteins are monomers present in the cytoplasm. After cytokine signaling and subsequent tyrosine phosphorylation of the receptor, STATs bind to the intracellular domain of the receptor and become tyrosine phosphorylated. This modification of the STATs occurs within minutes of cytokine stimulation, and begins to decline 1–2 h later. The phosphorylated STATs dimerize and then migrate to the nucleus where they regulate gene expression by binding to specific promoter elements. Characterization of the STATs reveals several important functional domains (Darnell, 1997). The conserved N-terminal region of STATs is important for tetramer formation and cooperative DNA binding (Vinkemeier et al., 1996, 1998; Xu et al., 1996). This region has also been implicated in tyrosine phosphorylation of STATs in response to cytokine signaling (Murphy et al., 2000) and the inactivation of the tyrosine-phosphorylated Stat1 (Shuai et al., 1996). The STATs contain a novel DNA-binding domain located in the middle of the protein. The SH2 domain mediates interaction with the cytoplasmic region of cytokine receptor after ligand binding and is also required for STAT dimerization. The transcriptional activation domain of the STATs lies at the C-terminal end of the molecules (Wen et al., 1995; Mikita et al., 1996; Qureshi et al., 1996; Wang et al., 1996). Stat1, Stat3 and Stat5 are each expressed in two alternatively spliced isoforms that vary at the C-terminal domain (Schindler et al., 1992; Schaefer et al., 1995; Wang et al., 1996, 2000). The full-length proteins are referred to as α, while the truncated forms are designated β. The β isoforms of Stat1, 3 and 5 have been shown to function as dominant-negative factors (Shuai et al., 1993; Caldenhoven et al., 1996; Wang et al., 2000). However, at some promoters, Stat3β activates transcription from composite elements in combination with c-jun and has roles distinct from those of Stat3α in vivo (Schaefer et al., 1995, 1997; Zhang et al., 1999; Yoo et al., 2002). In this report, we show that the Stat4 gene is also expressed as an alternatively spliced isoform, Stat4β, which lacks 44 amino acids at the C-terminus. Alternatively spliced transcripts and protein for both Stat4 isoforms were detected in cell lines and primary cells. Using transgenic mice, we show that both Stat4α and Stat4β are able to activate transcription in primary cells though the isoforms have both overlapping and distinct roles in mediating the biological effects of IL-12. Results Isolation of Stat4β cDNA clones To determine whether Stat4 is expressed in different isoforms, a cDNA library prepared from human peripheral blood lymphocytes (PBLs) was screened with a 900 bp probe derived from the 3′ end of the Stat4α cDNA. Several independent clones were isolated that contained a 369 bp insertion within the codon for amino acid 704 (Figure 1A and B). Inclusion of this sequence in the Stat4 message changes one residue and introduces a stop codon immediately downstream of the insertion point. The sequences at the borders of the Stat4β-specific region conform to the GT–AG rule for introns (Mount, 1982), indicating that this sequence corresponds to an intron that can be spliced out to generate the Stat4α isoform encoding 748 amino acids (Figure 1A and B). Inclusion of the β-specific sequence, on the other hand, generates the Stat4β isoform that encodes a protein of 704 amino acids, deleting the 44 C-terminal residues (Figure 1A and B). This cDNA clone is characteristic of a STAT β isoform because the insertion point corresponds exactly to the homologous residue in the Stat1 gene that is the site of alternative splicing for the Stat1β isoform (Yan et al., 1995). Figure 1.Cloning and expression of Stat4 isoforms. (A) Schematic representations of the Stat4α and Stat4β cDNAs are shown. The cross-hatched regions at the 5′ and 3′ ends of the cDNAs indicate the untranslated regions. The transcriptional activation domain (TAD) is denoted by a gray, shaded box, and the other functional domains are denoted by black regions. The numbers above the diagrams indicate the approximate positions in the amino acid sequence of the borders of these domains. The Stat4β form contains an additional exon indicated by the bracket. (B) DNA and protein sequences of the 3′ regions of the Stat4α and Stat4β cDNAs. The amino acid numbers are shown on the right. The β-specific sequence is indicated by lower case letters. Introduction of the β-specific exon introduces a stop codon, designated with an asterisk, just downstream of the splice junction at amino acid 704. (C) RNase protection was performed using a 400 nucleotide probe derived from the Stat4α cDNA (shown in A). The largest protected fragment of 400 nucleotides corresponds to the Stat4α mRNA. Hybridization to the Stat4β mRNA produces two smaller fragments of ∼260 and 140 nucleotides, corresponding to the regions upstream and downstream of the β-specific exon. (D) Nuclear extracts prepared from A139 cells were analyzed by western blot with antibodies specific for either the N- or the C-terminal regions of Stat4 (lanes 1 and 2, respectively). The specificity of the antibodies was confirmed by western blot analysis of extracts from COS cells that had been transiently transfected with Stat4α and Stat4β expression plasmids (2 μg) and immunoblotted with antibodies to the N-terminus (lanes 3 and 4) or the C-terminus (lanes 5 and 6) of Stat4. Experiments in (C) and (D) were performed at least three times. Download figure Download PowerPoint Expression of Stat4β RNase protection analysis demonstrated the expression of both Stat4 isoforms in A139 cells, a T-cell line expressing functional IL-12 receptors (Klein et al., 1996). The antisense RNA probe used in this assay was derived from Stat4α cDNA and spans the alternatively processed region (Figure 1A). Hybridization of the probe with RNA purified from A139 cells revealed three protected species (Figure 1C). The larger species of 400 nucleotides resulted from hybridization to Stat4α mRNA. Hybridization to the Stat4β transcript generated the two smaller fragments of ∼140 and 260 nucleotides, which correspond to the regions upstream and downstream of the β-specific exon (Figure 1A and C). This result indicates that both Stat4α and Stat4β transcripts are expressed in the A139 cell line. Similar results were obtained with human PBLs (unpublished data). Protein expression of the Stat4 isoforms was examined by western blot analysis using nuclear extracts prepared from A139 cells. In this assay, anti-Stat4 antibodies specific for either the N-terminal region or the C-terminal domain were used. Two species of Stat4 were detected with the anti-N-terminal specific antibodies (Figure 1D, lane 1), while the anti-C-terminal specific antibodies recognized only the slower migrating species (Figure 1D, lane 2). Since the truncated Stat4β isoform would not be recognized by anti-C-terminal Stat4 antibodies, these results suggest that the slower migrating species corresponds to Stat4α while the faster migrating species corresponds to Stat4β. Expression of recombinant Stat4α and Stat4β by transient transfection in COS cells was included as a control for the specificity of the antibodies (Figure 1D, lanes 3–6). These data indicate that both Stat4 isoforms are expressed in human T cells. Generation of Stat4α and Stat4β transgenic mice To determine the ability of Stat4β to activate gene expression in vivo, we generated transgenic mice expressing Stat4α or Stat4β cDNAs regulated by the CD2 locus control region (LCR). This expression vector directs T cell-specific expression (Zhumabekov et al., 1995). Several founders were analyzed and one Stat4α and two Stat4β transgenic lines were selected for extensive analysis. Transgene-positive mice were backcrossed with C57BL/6 Stat4-deficient mice to yield mice expressing transgenic Stat4α or Stat4β without any endogenous Stat4. Expression of transgenic Stat4α or Stat4β did not alter T-cell development as indicated by similar levels of CD4+ and CD8+ T cells in the thymus, spleen and lymph nodes of wild-type, Stat4−/− and Stat4α or Stat4β transgenic mice (unpublished data). Western analysis of total protein extracts from wild-type and transgenic mice examined the expression of Stat4α and Stat4β in primary T cells (Figure 2B). Importantly, the levels of Stat4α and Stat4β in the transgenic mice indicate that the proteins are not overexpressed relative to the endogenous level of Stat4. Analysis of the expression of Stat4 in transgenic mice demonstrated Stat4 in lymphoid tissues, primarily in T cells, with a much lower level of expression in B cells (Figure 2C). Figure 2.Generation of Stat4 transgenic mice. (A) Schematic of the Stat4 transgenic vector. (B) Expression of transgenic Stat4 was assessed by immunoblot using a monoclonal anti-Stat4. Note the slightly faster migration of Stat4β. (C) Total protein extracts from the indicated tissues from a CD2:Stat4β mouse were used for immunoblot analysis. Tissue extracts (50 μg/lane) were probed with a monoclonal anti-Stat4. Immunoblots are representative of 2–4 experiments. Download figure Download PowerPoint Activation of Stat4 isoforms We then examined the tyrosine phosphorylation of the two isoforms following IL-12 treatment in A139 cells. In unstimulated cells, minimal tyrosine phosphorylation of Stat4 protein was observed (Figure 3A). Addition of IL-12 induced tyrosine phosphorylation of both Stat4α and Stat4β, peaking at 2 h and declining thereafter. Both Stat4 isoforms in transgenic mice were activated with kinetics similar to those seen in human cells; however, the Stat4β isoform maintained a high level of activation for a longer period of time (Figure 3B). To determine if degradation of the activated Stat4 proteins was different between the Stat4 isoforms, we treated cells with the proteasome inhibitor MG132 and examined the levels of phosphorylated Stat4. Similar to other studies (Wang et al., 2000), we found that phosphorylated Stat4 was stabilized by MG132 (Figure 3A). Interestingly, Stat4α appeared to be stabilized preferentially by proteasome inhibition while Stat4β was less affected (Figure 3A). We tested whether Stat4α or Stat4β could be ubiquitylated directly by co-transfecting Stat4 isoform-expressing plasmids with a hemagglutinin (HA)-tagged ubiquitin expression plasmid (Musti et al., 1997). Stat4α was found to be more readily ubiquitylated than Stat4β (Figure 3C). Thus, ubiquitin-mediated degradation may be one mechanism for differentially regulating Stat4 isoforms. Figure 3.Distinct activation kinetics of Stat4 isoforms. (A) A139 cells were incubated in the absence or presence of IL-12 (10 ng/ml) for the times indicated and in the presence or absence of 40 μM MG132 as indicated. Whole-cell extracts were prepared at the indicated time points. Extracts were resolved by SDS–PAGE, and then blotted with anti-phospho-Stat4 (upper panel). The middle panel represents a longer exposure of the upper panel to highlight Stat4β activation. The filter was then stripped and re-probed with monoclonal anti-Stat4 to demonstrate equal loading (lower panel). Results are representative of three experiments. (B) Nuclear extracts were prepared from splenocytes of Stat4 transgenic mice at the indicated times (h) after IL-12 treatment and analyzed for Stat4 DNA binding activity by DNA pull-down and western analysis. Results are representative of two experiments. (C) Ubiquitylation of Stat4α. COS7 cells were transfected with either Stat4α or Stat4β and an HA-tagged ubiquitin cloned in the pMT123 plasmid (2 μg of each plasmid). Cellular extracts harvested 48 h after transfection were immunoprecipitated with anti-Stat4 and the presence of ubiquitylated Stat4 was tested by western with anti-HA (upper panel). The same blot was stripped and re-probed with anti-Stat4 antibodies (lower panel). Data are representative of three experiments. Download figure Download PowerPoint Stat4 isoform-dependent Th1 differentiation We next wanted to examine further the ability of Stat4α and Stat4β to direct Th1 differentiation and activate IL-12-stimulated transcription in Th1 cells. To analyze gene expression, we first needed to verify target genes in the Stat4-dependent Th1 genetic program since some genes, known to be differentially expressed in Th1 versus Th2 cells, have not been carefully examined for their dependence on Stat4 in Th1 cells. Stat4 heterozygous or Stat4−/− CD4+ cells were differentiated under Th1 or Th2 conditions as indicated in Figure 4A, and either left unstimulated, or stimulated with IL-12 + IL-18 or anti-CD3 for 24 h followed by analysis of gene expression by northern blot. Expression of surface receptors including IL-18R, IL-12Rβ2 and CCR5 was greatly reduced in Stat4-deficient Th1 cultures (Lawless et al., 2000; Iwasaki et al., 2001) (Figure 4A), though expression was higher than in Th2 cells. Expression of the transcription factors ERM and T-bet was also absent or decreased in Stat4-deficient T cells, as previously observed (Ouyang et al., 1999; White et al., 2001). IFN-regulating factor-1 (IRF-1) expression was induced by IL-12 stimulation in Stat4-expressing cells, while induction was not observed in cells lacking Stat4, similar to previous reports in human cells (Coccia et al., 1999; Galon et al., 1999) (Figure 4A). IFN-γ is the hallmark cytokine secreted by Th1 cells, and its expression in response to IL-12 is known to be Stat4 dependent (Kaplan et al., 1996; Thierfelder et al., 1996; Lawless et al., 2000). In these experiments, expression of IFN-γ in response to IL-12 and IL-18 was strongly reduced in the Stat4−/− cells, while induction in Th1 polarized cells in response to anti-CD3 was also reduced, although to a lesser extent. This same pattern was observed for LTα (Figure 4A). However, not all Th1-specific genes are Stat4 dependent as we, and others, have previously demonstrated Lymphotactin (Ltn) and other genes to be expressed normally in Stat4-deficient Th1 cultures (Venkataraman et al., 2000; Zhang et al., 2000b) (Figure 4A). As controls, we observed Th2-specific expression of IL-4, and equal expression of T-cell receptor (TCR) was observed in all genotypes and culture conditions. Thus, there are both Stat4-dependent and -independent aspects of the Th1 genetic program. Figure 4.Stat4β activates the Th1 genetic program. (A) Stat4+/− CD4+ cells were differentiated into Th1 or Th2 cells by activation with anti-CD3 in the presence of 2 ng/ml IL-12 + 10 μg/ml anti-IL-4 (Th1) or 10 ng/ml IL-4 + 10 μg/ml anti-IFN-γ (Th2). Stat4−/− CD4+ cells were differentiated into Th1 cells. After 6 days in culture, cells were left unstimulated or stimulated with 2 ng/ml IL-12 + 50 ng/ml IL-18 or 2 μg/ml plate-bound anti-CD3 for 24 h. RNA was then recovered and expression of the genes indicated was analyzed by northern blot. Results are representative of two experiments. (B) Wild-type CD2:Stat4α and CD2:Stat4β were differentiated and stimulated as in (A). Results are representative of 4–6 experiments. Supernatants were then recovered from cultures and IFN-γ levels were determined by ELISA. (C) Expression of the indicated genes was assayed by northern blot in Th1 cells derived from Stat4+/−, Stat4−/−, CD2:Stat4α and CD2:Stat4β mice. Cells were treated and RNA isolated as described in (A). Results are representative of three experiments. Download figure Download PowerPoint To test the ability of Stat4α and Stat4β to induce Th1 differentiation, we purified CD4+ cells from wild-type, CD2:Stat4α and CD2:Stat4β mice and differentiated the cells in vitro to the Th1 phenotype by stimulating with anti-CD3 in the presence of IL-12 and anti-IL-4. After 6 days in culture, cells were washed and restimulated to examine IFN-γ production. Figure 4B demonstrates that stimulation of IFN-γ secretion in response to anti-CD3 is equivalent between wild-type and CD2:Stat4α, as previously observed (Broxmeyer et al., 2002). Importantly, IFN-γ production was comparable in CD2:Stat4β cultures following anti-CD3 stimulation. In contrast, stimulation of Th1 cultures with IL-12 demonstrated distinct functions of Stat4 isoforms. Stat4α transgenic cells secreted levels of IFN-γ comparable with control cells, while Stat4β cultures showed decreased IFN-γ secretion (Figure 4B). Thus, while both Stat4 isoforms can activate the Th1 differentiation program, they are not equivalent in direct activation of the IFN-γ gene. To determine if other Th1-specific and Stat4-dependent genes are also expressed normally in CD2:Stat4β cells, we performed northern analysis of Th1 cultures from Stat4+/−, Stat4-deficient, CD2:Stat4α and two founder lines of CD2:Stat4β CD4+ cells left unstimulated or stimulated with IL-12 + IL-18, IL-12 alone or anti-CD3 for 24 h. Th1-specific expression of IL-12Rβ2, T-bet, ERM and LTα was observed to be similar between Stat4α- and Stat4β-expressing transgenic cells compared with Th1 cells (Figure 4C). However, IFN-γ mRNA levels were lower in cells from both Stat4β founder lines compared with either Stat4+/− or Stat4α transgenic Th1 cells (Figure 4C), which agrees with the data on IFN-γ secretion in Figure 4B. Thus, Stat4β can activate the Th1 genetic program and rescue the phenotype of Stat4-deficient Th1 cells. However, Stat4β is not as efficient as Stat4α in directly inducing IFN-γ gene expression. Stat4 isoform-specific gene regulation While a handful of genes have been identified as IL-12 inducible and Stat4 dependent, we wanted to determine if additional genes showed specific regulation by either Stat4α or Stat4β. Microarray analysis was performed using RNA isolated from CD2:Stat4α or CD2:Stat4β Th1 cells that were left unstimulated or stimulated with IL-12 for 18 h. Data are presented as fold induction of expression at 18 h over the expression in the unstimulated condition. Only genes that were induced >2-fold are listed. Ninety-eight genes were activated by both Stat4 isoforms (Table I). However, additional genes were induced specifically by either Stat4α (32 genes) or Stat4β (29 genes) (Tables II and Table III). Surprisingly, no gene expression was decreased by IL-12 treatment. Table 1. Genes induced by both Stat4α and Stat4β Accession No. Gene name Fold induction Stat4α Stat4β Secreted factors and signal transduction M32745 TGF-β3 5.7 5.1 AK013606 Megakaryocyte-associated tyrosine kinase 4.5 5.2 AK008250 Mucin 2 4.4 4.6 AB018002 Death-associated kinase 2 3.7 3.7 AK017818 RagD G protein 3.6 3.7 W74976 Complement component 3 3.6 3.2 AF132851 Ras suppressor factor (RASSF) 3.2 3.1 BB264520 JNK-interacting protein-3a (Jip3) homolog 3.0 2.8 AK005361 Regulator of G protein signaling 16 (RGS16) 2.9 2.8 AK007774 Latent TGF-β3-binding protein 2.6 3.3 AK018113 Angiopoietin-related protein 2 precursor homolog 2.6 2.7 AK017187 Serine/arginine-rich protein-specific kinase 1 (SRPK1) 2.6 2.5 AK016359 ADP-ribosylation factor-like protein 5 (Arl5) homolog 2.6 2.4 AK010318 Stanniocalcin 2 2.4 2.5 AF061744 FYN-binding protein 2.4 2.5 AK014991 Death-associated protein 1 (DAP-1) homolog 2.4 2.9 AK002400 ADP-ribosylation factor GTPase-activating protein 1 homolog 2.4 2.4 AF117340 Mitogen-activated protein kinase kinase kinase 1 2.4 2.4 AK005456 Tumor protein D52 2.3 2.4 M33960 Plasminogen activator inhibitor, type I 2.1 2.0 Cell cycle AK003389 Cyclin I 2.6 2.7 AK010928 Cyclin-dependent kinase 2 2.6 2.8 AK008585 Cyclin ANIA-6B homolog 2.2 2.2 DNA/RNA metabolism and transcription factors AK008707 AF-9 homolog 3.0 2.7 AK011088 G1-related zinc finger protein 2.8 2.4 AK007880 Groucho-related protein 2.8 2.7 AK010506 Pre-B-cell leukemia transcription factor 4 2.8 3.0 AK004238 Trif gene 2.8 2.5 AK017655 Luc7 homolog 2.7 2.6 AI595019 Suppressor of ty3 homolog (SUPT3H) homolog 2.5 2.7 AK012829 Hypothetical nuclear factor SBBI22 (Zn finger) homolog 2.5 2.9 AK008242 CBF1-interacting corepressor CIR homolog 2.5 2.4 AK017984 Polycomb complex protein BMI-1 homolog 2.4 2.3 AF091234 Btg-associated nuclear protein (BANP) 2.2 2.2 AK010477 DNA polymerase δ smallest subunit P12 homolog 2.1 2.1 Cell surface receptors AK018582 G1-related zinc finger protein G1RP homolog sim to (GRAIL) 4.6 5.4 AK004650 Plakophilin 2A homolog 4.6 5.9 L48015 Activin A receptor, type II-like 1 4.3 4.3 AK017275 Melanoma antigen, family D, 1 3.7 4.2 V01527 Histocompatibility 2, class II antigen A, β1 3.4 4.0 AK015705 Transmembrane 4 superfamily member 9 3.4 3.4 AF038572 Jagged 2 3.1 3.0 AK004539 Receptor (calcitonin) activity-modifying protein 1 (RAMP1) 2.7 3.0 U03736 Copper efflux ATPase homolog 2.6 2.6 AK010968 Erythropoietin receptor 2.6 2.6 U06670 Very low density lipoprotein receptor 2.5 2.7 L23423 Integrin α7 2.5 2.4 NM_010609 Potassium channel, subfamily K, member 8 2.4 2.7 AK010094 Nitrophenylphosphatase homolog 2.2 2.7 L12120 Interleukin 10 receptor α 2.2 2.3 X67469 Low-density lipoprotein receptor-related protein 2.2 2.2 Vesicle formation and trafficking L33726 Fascin homolog 1 4.5 5.0 BB222822 Calpain 5 3.2 3.7 AK011678 Vesicle-associated membrane protein 4 2.4 2.8 AK003515 ER lumen protein-retaining receptor 2 (KDEL receptor 2) homolog 2.4 2.1 AJ272203 Profilin 2 2.2 2.5 AK004761 Lysosomal apyrase-like protein (LALP70) homolog 2.2 2.4 AK011355 Peroxisomal biogenesis factor 13 2.2 2.2 Cellular metabolism AK013167 1,4-α-glucan branching enzyme homolog 4.2 4.8 AK009667 ERO1 4.6 4.7 M74495 Adenylosuccinate synthetase 1 4.0 3.5 AK002783 Acid phosphatase 6 3.3 4.0 U16163 Proline 4-hydroxylase, αII polypeptide 3.7 3.5 AF288783 Glycogen phosphorylase 3.7 3.3 AK019539 Lipin 1 3.3 3.1 AI787918 Pyruvate dehydrogenase kinase homolog 3.0 2.9 NM_011961 Procollagen lysine, 2-oxoglutarate 5-dioxygenase 2 2.7 3.1 AK012811 F-box and leucine-rich repeat protein 12 2.8 2.6 AK012060 Ubiquitin E3 ligase SMURF2 homolog 2.8 2.6 AK005680 DnaJ (Hsp40) homolog 2.7 2.5 AK005077 Aldolase 3, C isoform 2.6 2.6 L29123 Ferredoxin 1 2.5 2.5 AK012017 Tripeptidyl peptidase II 2.4 2.3 AK004042 Acetyl-coenzyme A synthetase 2 2.3 2.4 AK002531 Spermidine/spermine N1-acetyl transferase 2.3 2.1 BE573662 Inducible 6-phosphofructo-2-kinase 2.2 2.2 Y00964 Hexosaminidase B 2.1 2.4 Unknown AK004548 N-myc downstream regulated 1 3.7 4.0 AK003295 DJ434O11.1 (novel protein) homolog 3.3 3.9 AK012716 P33ING2 homolog 3.0 3.3 AK009377 Hypoxia-induced gene 2 3.0 2.8 AF022992 Period homolog 3.0 3.1 AK004851 Gene 33 polypeptide homolog 3.1 2.9 BE573435 Y029_human hypothetical protein KIAA0029 homolog 3.0 2.9 AK003156 NPD017 homolog 3.0 3.1 AK009866 PDZ domain-containing protein 3.0 2.9 AK003466 Immediate early response 3 2.9 2.9 AK016342 Putative ovary-specific acidic protein homolog 2.9 3.1 AK011325 Neighbor of A-kinase anchoring protein 95 2.4 2.1 BE623294 Wolf–Hirschhorn syndrome candidate 1-like 1 protein homolog 2.4 2.2 AK017846 PR-domain-containing protein 16 homolog 2.3 2.6 AK013649 HDCMC04P homolog 2.3 2.4 AK003918 Reticulocabin precursor homolog 2.3 2.5 AK018058 BM024 homolog 2.3 2.2 AK014511 RIS homolog 2.2 2.9