scholarly journals The SH2 domain of P210BCR/ABL is not required for the transformation of hematopoietic factor-dependent cells

Blood ◽  
1995 ◽  
Vol 86 (10) ◽  
pp. 3897-3904 ◽  
Author(s):  
RL Jr Ilaria ◽  
RA Van Etten
Keyword(s):  
Cell Lines ◽  
Protein Interactions ◽  
Specific Protein ◽  
Sh2 Domain ◽  
Myelogenous Leukemia ◽  
Mutant Proteins ◽  
Abl Tyrosine Kinase ◽  
Negative Effect ◽  
Dependent Cell

Src-homology region 2 (SH2) domains, by binding to tyrosine- phosphorylated sequences, mediate specific protein-protein interactions important in diverse signal transduction pathways. Previous studies have shown that activated forms of the Abl tyrosine kinase, including P210BCR/ABL of human chronic myelogenous leukemia, require the SH2 domain for the transformation of fibroblasts. To determine whether SH2 is also required for Bcr/Abl to transform hematopoietic cells, we have studied two SH2 domain mutations in P210BCR/ABL: a point mutation in the conserved FLVRES motif (P210/R1033K), which interferes with phosphotyrosine-binding by SH2, and a complete deletion of SH2 (P210/delta SH2). Despite a negative effect on intrinsic Abl kinase activity, both P210 SH2 mutants were still able to transform the hematopoietic factor-dependent cell lines Ba/F3 and FDC-P1 to growth factor independence. Unexpectedly, both mutants showed greater transforming activity than wild-type P210 in a quantitative transformation assay, probably as a consequence of increased stability of the SH2 mutant proteins in vivo. Cells transformed by both P210 SH2 mutants were leukemogenic in synaptic mice and P210/r1053K mice exhibited a distinct disease phenotype, reminiscent of that induced by v-Abl. These results demonstrate that while the Abl SH2 domain is essential for BCR/ABL transformation of fibroblasts, it is dispensable for the transformation of hematopoietic factor-dependent cell lines.

scholarly journals Novel Roles of SH2 and SH3 Domains in Lipid Binding

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1191
Author(s):  
Szabolcs Sipeki ◽  
Kitti Koprivanacz ◽  
Tamás Takács ◽  
Anita Kurilla ◽  
Loretta László ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Specific Protein ◽  
Lipid Binding ◽  
Sh2 Domain ◽  
Essential Property ◽  
Cellular Regulation ◽  
Sh3 Domains ◽  
Protein Partners ◽  
Interaction Domains ◽  
Central Paradigm

Signal transduction, the ability of cells to perceive information from the surroundings and alter behavior in response, is an essential property of life. Studies on tyrosine kinase action fundamentally changed our concept of cellular regulation. The induced assembly of subcellular hubs via the recognition of local protein or lipid modifications by modular protein interactions is now a central paradigm in signaling. Such molecular interactions are mediated by specific protein interaction domains. The first such domain identified was the SH2 domain, which was postulated to be a reader capable of finding and binding protein partners displaying phosphorylated tyrosine side chains. The SH3 domain was found to be involved in the formation of stable protein sub-complexes by constitutively attaching to proline-rich surfaces on its binding partners. The SH2 and SH3 domains have thus served as the prototypes for a diverse collection of interaction domains that recognize not only proteins but also lipids, nucleic acids, and small molecules. It has also been found that particular SH2 and SH3 domains themselves might also bind to and rely on lipids to modulate complex assembly. Some lipid-binding properties of SH2 and SH3 domains are reviewed here.

The study of protein–protein interactions in bacteria

2012 ◽  
Vol 58 (11) ◽  
pp. 1241-1257 ◽  
Author(s):  
Roberto Velasco-García ◽  
Rocío Vargas-Martínez
Keyword(s):  
Hybrid System ◽  
High Throughput ◽  
Protein Interactions ◽  
Specific Protein ◽  
False Positives ◽  
Interaction Networks ◽  
Protein Protein Interactions ◽  
Extensive Data ◽  
False Positives And Negatives

Many of the functions fulfilled by proteins in the cell require specific protein–protein interactions (PPI). During the last decade, the use of high-throughput experimental technologies, primarily based on the yeast 2-hybrid system, generated extensive data currently located in public databases. This information has been used to build interaction networks for different species. Unfortunately, due to the nature of the yeast 2-hybrid system, these databases contain many false positives and negatives, thus they require purging. A method for confirming these PPI is to test them using a technique that operates in vivo and detects binary PPI. This article comprises an overview of the study of PPI and describes the main techniques that have been used to identify bacterial PPI, prioritizing those that can be used for their verification, and it also mentions a number of PPI that have been identified or confirmed using these methods.

scholarly journals Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase.

1994 ◽  
Vol 14 (5) ◽  
pp. 2883-2894 ◽  
Author(s):  
B J Mayer ◽  
D Baltimore
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Sh3 Domains ◽  
Sh2 Domains ◽  
Abl Tyrosine Kinase ◽  
Transforming Activity

We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.

CNS-9, a Novel Specific Inhibitor of ABL Tyrosine Kinase Overcomes Resistance Mechanism of Imatinib.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 761-761 ◽  
Author(s):  
Shinya Kimura ◽  
Hidekazu Segawa ◽  
Junya Kuroda ◽  
Takeshi Yuasa ◽  
Taira Maekawa
Keyword(s):  
Tyrosine Kinase ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Nude Mice ◽  
Philadelphia Chromosome ◽  
Specific Inhibitor ◽  
Vitro Assay ◽  
Abl Tyrosine Kinase

Abstract Imatinib mesylate (also known as STI-571 and Gleevec) has drastically changed the treatment of Philadelphia chromosome positive (Ph+) leukemias. However, the resistance to imatinib has frequently been reported, particularly in patients with advanced-stage disease. A novel orally bioavailable inhibitor of the ABL tyrosine kinase (TK) named CNS-9 was developed from the 2-(phenylamino)pyrimidine class to overcome resistance mechanisms of imatinib. Inhibition of TK phosphorylation (IC50) on wild type (wt) BCR/ABL in 293T cell line by CNS-9 was 22nM, which was 2-log more potent than imatinib. Importantly, CNS-9 inhibited TK phosphorylation of E255K mutant BCR/ABL with IC50 of 98nM, while imatinib could not inhibit it with clinically relevant concentration. The T315I mutant BCR/ABL protein was resistant to CNS-9 and imatinib. CNS-9 also inhibited TK phosphorylation of platelet-derived growth factor receptor (PDGFR) or c-Kit pathways at the very similar observed IC50s when compared with imatinib, in spite of significant higher potency against ABL. The ability of CNS-9 in vitro to inhibit 101 TK molecules was assayed by KinaseProfilerTM (Upstate), showing also more specific inhibitory activity against ABL than imatinib. The growth of BCR/ABL-positive cell lines K562, KU812, BaF3 harboring wt BCR/ABL (BaF3/wt) and E255K (BaF3/E255K) was inhibited by CNS-9 with IC50 of 5, 3, 17, and 110nM, respectively (Table 1). Generally, CNS-9 was 20 to 30-fold more potent on the growth inhibition than imatinib in these same cell lines. We next investigated the in vivo effect on the leukemic growth inhibition of CNS-9. Nude mice were injected subcutaneously with 3x107 KU812 (wt BCR/ABL) on Day 0. CNS-9 or imatinib were orally administrated twice a day from Day 7 to Day 18. The dosages of CNS-9 and imatinib, which inhibited completely tumor growth were 20mg/kg/day and 200mg/kg/day, respectively, indicating that CNS-9 is 10-fold potent than imatinib in vivo. To examine the in vivo effect of CNS-9 against mutant BCR/ABL, BaF3/wt, BaF3/E255K or BaF3/T315I were engrafted to nude mice and treated with CNS-9 or imatinib. CNS-9 was also 10-fold potent than imatinib against BaF3/wt. Intriguingly, mice harboring BaF3/wt or BaF3/E255K showed significantly prolonged survival when treated with CNS-9. Consistent with in vitro assay, CNS-9 had no effect on T315I, and imatinib was not effective against both E255K and T315I. In conclusion, CNS-9 is substantially more inhibitory and more specifically than imatinib toward BCR/ABL-dependent cell growth both in vitro and in vivo Moreover, CNS-9 may be effective for leukemia patients whose leukemic cells harbor E255K mutant. The efficacy and safety of CNS-9 for Ph+ leukemias should be verified in early phase clinical trials. The IC50s values of leukemic cell lines for CNS-9 and imatinib CNS-9 (nM) imatinib (nM) K562 p210 wt BCR/ABL 5 130 KU812 p210 wt BCR/ABL 3 67 U937 BCR/ABL (−) >1000 >1000 BaF3 p190 wt BCR/ABL 17 360 BaF3 p190 E255K BCR/ABL 110 >1000 BaF3 p190 T315I BCR/ABL >1000 >1000

Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis and Inhibition of Proliferation of Ba/F3 FLT-3 ITD Mutant Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1589-1589
Author(s):  
Jenny E. Hernandez ◽  
Junling Li ◽  
Ru-Qi Wei ◽  
Paul Tapang ◽  
Steven K. Davidsen ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Mutant Cell ◽  
Scid Mice ◽  
Myelogenous Leukemia ◽  
Parp Cleavage ◽  
Mutant Cells

Abstract FLT3 is an receptor tyrosine kinase of the subclass III family that plays a vital role in the regulation of the differentiation, proliferation and survival of normal hematopoietic cells. FLT3 mutations are often found in patients with Acute myelogenous leukemia (AML) and confer poor prognosis. Of these mutations, 15–35% are FLT3 ITD (internal tandem duplication) mutations and 5–7% are point mutations on the FLT3 kinase activation loop (e.g. D835V). Our laboratory is studying the signaling pathways associated with a newly identified multi-targeted tyrosine kinase receptor small molecule inhibitor (RTKI), ABT-869. Recently published work in our laboratory showed that using ABT-869 to treat MV4-11, a human AML FLT-3 ITD mutant cell line, resulted in the inhibition of phosphorylation of FLT-3 with a downstream inhibitory effect on the activation of STAT5, ERK, and Pim-1. Cell viability assays determined that MV-411 cells responded to ABT-869 in a concentration dependent manner (IC50 = 10nM). Apoptosis studies also showed an induction of apoptosis in ABT-869 treated cells. In vivo studies involving xenograft injections of MV-411 cells into SCID mice and subsequent treatment with ABT-869 demonstrated regression of tumor formation. In this study, a Ba/F3 mouse pro-B lymphocytic cell line harboring the FLT-3 ITD or FLT-3 D835V mutation is used as an isolated Flt-3 mutant model system. In vitro, ABT-869 is effective in inhibiting the proliferation of Ba/F3 Flt-3 ITD mutant cells when compared to Ba/F3 Flt-3 D835V mutant and Ba/F3 Flt-3 WT cells. Trypan Blue Exclusion and Alamar Blue assays were used to demonstrate that there is 50% inhibition of growth and proliferation (IC50) of Ba/F3 FLT3 ITD mutant cells at a concentration of 1nM after 48 hours of treatment. Ba/F3 FLT3 D835V mutant cells show an IC50 between 1μM and 10μM after 48 hours of treatment. In contrast, Ba/F3 FLT3 WT cells demonstrate an IC50 of 10μM only after 72 hours of treatment. Annexin V and propidium iodide staining of cells revealed that an increase in apoptosis (41.2%) occurred in Ba/F3 Flt-3 ITD mutant cells treated with 10nM ABT-869 after 24 hours when compared to untreated (6.5%) or vehicle control (6.1%) cells. Staining of Ba/F3 Flt-3 WT treated cell lines revealed no difference in apoptosis when compared to untreated Ba/F3 Flt-3 WT cell only and DMSO controls. PARP cleavage was observed in Ba/F3 FLT-3 ITD mutant cells following treatment with ABT-869 whereas no cleavage was observed with Ba/F3 WT cells treated with ABT-869. In vivo, the activity of ABT-869 treatment of SCID mice injected with Baf3 Flt-3 ITD, Baf3 Flt-3 D835V, or Baf3 Flt-3 WT cells is also being evaluated. Using bioluminescence imaging, it was determined that Ba/F3 FLT-3 ITD mutant and Ba/F3 Flt-3 D835Vmutant cell lines result in metastases and subsequent death in SCID mice after 2 weeks for ITD and 5 weeks for D835V, whereas mice injected with Ba/F3 WT survive longer than 5 weeks. Preliminary data demonstrated that ABT-869 prolonged survival in mice injected with the Ba/F3 FLT3-ITD cells compared to controls. Our preclinical data demonstrate that ABT-869 is effective specifically with FLT-3 ITD mutant cell lines in an isolated system. These studies provide rationale for the treatment of AML patients and the prevention of relapse.

The Natural Product Borrelidin Induces UPR and Apoptosis in AML Cell Lines

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4260-4260
Author(s):  
Leah Jackson ◽  
Shelby Bechler ◽  
Justin Miller ◽  
Amy Brownell ◽  
Danielle Garshott ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Natural Product ◽  
Cell Lines ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Xenograft Model ◽  
Myelogenous Leukemia ◽  
Ic50 Values

Abstract Abstract 4260 Acute Myelogenous Leukemia (AML) is the most common form of leukemia. Current therapies are intense and even those fortunate enough to achieve remission often relapse extending extremely poor prognoses to these patient. The most commonly used therapeutics, namely cytarabine aribinoside, the anthracyclines and etoposide, are decades old and target ubiquitous cellular processes. We have previously reported that small molecules and natural products that activate and exacerbate the unfolded protein response (UPR) can effectively and selectively induce cell death in a wide variety of solid tumor cells. We hypothesized that the UPR might be a viable new therapeutic target in AML and sought to determine whether or not the novel UPR-inducing natural product borrelidin might be used as such an agent. A luminescent proliferation assay performed with panel of four AML cell lines treated with the ER stress-inducing antibiotic tunicamycin (Tm) revealed that three of the cell lines displayed IC50 values between 0.47–2.5μ M, doses of Tm which are known to induce a low to moderate level of ER stress. We then repeated the experiment with the more general UPR-inducing natural product borrelidin, which has been shown to have potent anti-inflammatory properties in several murine assays in vivo. All four cell lines were sensitive to borrelidin, displaying IC50 values between 0.032–0.29 μ M. Time course assays performed with borrelidin revealed 4–20 fold increases in active caspase 3 and 7 indicating borrelidin-induced AML decreases in cell proliferation might be the result of apoptosis. Quantitative reverse-transcription real time PCR performed with mRNA isolated from two AML cell lines revealed an increase in the UPR-related transcripts CHOP, ATF4, and GADD34 and the cell death genes Noxa, Puma, DR5 and Bim confirming that borrelidin could induce the UPR and apoptosis in AML cells. Studies currently underway in our laboratory will determine the ability of borrelidin and other UPR-inducing agents to reduce leukemic burden in an in vivo xenograft model. Disclosures: No relevant conflicts of interest to declare.

NK Cell Line Killing of Leukemia Cells Is Enhanced By Reverse Antibody Dependent Cell Mediated Cytotoxicity (R-ADCC) Via NKp30 and NKp44 and Target Fcγ Receptor II (CD32)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2444-2444 ◽  
Author(s):  
Brent A. Williams ◽  
Xinghua Wang ◽  
Bertrand Routy ◽  
Richard Cheng ◽  
Sonam Maghera ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Cell Line ◽  
Cell Lines ◽  
Nk Cell ◽  
P Value ◽  
Fcγ Receptor ◽  
Isotype Control ◽  
Nsg Mice ◽  
Dependent Cell

Abstract Introduction: We are studying NK cell immunotherapy to treat acute myeloid leukemia (AML) and have focused on NK-92 and KHYG-1, CD16(-) human malignant NK cell lines. Phase I NK-92 trials show minimal toxicity; KHYG-1 has not been tested in humans. Here, we investigated modulation of cytotoxicity of NK cell lines against primary AML blasts and cell lines with monoclonal antibodies (mAb) directed against natural cytotoxicity receptors. Methods: NK cytotoxicity was assessed with a standard 4 hour Cr51 release assay at an effector to target (E:T) ratio of 10:1. NK lines were incubated with and without isotype control and mAbs against NKp30, NKp44 at various doses (0.001-10 µg/ml) for 1 hour and washed with medium prior to cytotoxicity assays. The student’s t-test was used to compare cytotoxicity data. Target cells were incubated with 100 µCi Cr51 and cell supernatants assayed on a gamma counter. NK targets (leukemic and esophageal cancer) were evaluated for Fcγ receptor expression by flow cytometry. To test the cytotoxic effect on in vivo proliferation, OCI/AML5 cells were co-incubated with irradiated KHYG-1 (iKHYG-1) +/-1 µg/ml NKp30 pretreatment for 4 hours at a 10:1 E:T ratio and injected ip into NOD/SCID gamma null (NSG) mice with survival as an endpoint analyzed with the log rank test. Results: NK-92 and KHYG-1 were both highly cytotoxic against K562 with moderate killing of OCI/AML3 and KG1 and KG1a. OCI/AML5 was highly sensitive to killing by NK-92, but resistant to KHYG-1. Pretreatment of NK-92 with mAbs against NKp30, NKp44 (10 µg/ml) yielded small increases in cytotoxicity against leukemic cell lines with NKp30 pretreatment only. Pretreatment of KHYG-1 with 10 µg/ml of anti-NKp30 or anti-NKp44 mediated fold increases in cytotoxicity above isotype control against 4 leukemia cell line targets and 4 primary AML samples (Table1). Anti-NKp30 and anti-NKp44 pretreatment of NK-92 and KHYG-1 did not enhance killing of a panel of esophageal cancer cell lines. Immunophenotyping cancer cell lines showed high expression of Fcγ receptor II (CD32), but very low expression of Fcγ receptor I (CD64) or III (CD16) on leukemia lines (K562, OCI/AML3, OCI/AML5, KG1 and KG1a), and no expression of Fcγ receptors on esophageal lines (OE-33, FLO-1, KYAE-1, SKGT-4). Regression analysis of the relationship between cytotoxic enhancement and CD32 expression of targets revealed a strong correlation for NKp30 (p<0.01; R2=0.71) and NKp44 (p<0.01; R2=0.64) pretreated KHYG-1. NSG mice injected with 2x106 OCI/AML5 cells developed progressive malignant ascites at 9 weeks requiring sacrifice, unaffected by iKHYG-1 (p=0.92). However, NKp30 pretreated iKHYG-1 improved survival versus no therapy (p<0.05) or iKHYG-1 (p<0.05) cohorts. Conclusion: We show a novel means to enhance cytotoxicity of NK cell lines many fold against primary AML cells by pretreatment with mAbs against NKp30 and NKp44. The mechanism of enhanced KHYG-1 cytotoxicity is from bound NKp30 or NKp44 becoming crosslinked when the Fc portion binds the Fcγ receptor II (CD32) on targets. This is the first demonstration of reverse antibody-dependent cell-mediated cytotoxicity (R-ADCC) with a NK cell line leading to enhanced killing of AML primary blasts in vitro and the first demonstration of R-ADCC in an in vivo model. Table 2: Effect of NKp30 or NKp44 pretreatment on KHYG-1 cytotoxicity against primary AML samples Fold lysis and p values Primary AML samples 5890 080078 0909 080179 Fold change lysis NKp30 1.7 15.7 2.7 4.9 Fold change lysis NKp44 0.9 16.3 2.8 6.2 p value NKp30 <0.05 =0.0001 <0.0001 <0.001 p value NKp44 0.9 <0.001 <0.001 <0.001 Disclosures No relevant conflicts of interest to declare.

Characterization of Grb4, an adapter protein interacting with Bcr-Abl

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 618-624 ◽  
Author(s):  
Sunita Coutinho ◽  
Thomas Jahn ◽  
Marc Lewitzky ◽  
Stephan Feller ◽  
Peter Hutzler ◽  
...  
Keyword(s):  
Myelogenous Leukemia ◽  
Adapter Protein ◽  
Intact Cells ◽  
Abl Tyrosine Kinase ◽  
Abl Kinases ◽  
Src Homology ◽  
Dependent Interaction

We report here the characterization of an adapter protein identified in a yeast 2-hybrid screen with the use of Bcr-Abl as the bait. Grb4 bound to Bcr-Abl in a variety of systems, both in vitro and in vivo, and is an excellent substrate of the Bcr-Abl tyrosine kinase. The association of Grb4 and Bcr-Abl in intact cells was mediated by an src homology (SH)2–mediated phosphotyrosine-dependent interaction as well as an SH3-mediated phosphotyrosine-independent interaction. Grb4 has 68% homology to the adapter protein Nck and has similar but distinct binding specificities in K562 lysates. Subcellular localization studies indicate that Grb4 localizes to both the nucleus and the cytoplasm. Coexpression of kinase-active Bcr-Abl with Grb4 resulted in the translocation of Grb4 from the cytoplasm and the nucleus to the cytoskeleton to colocalize with Bcr-Abl. In addition, expression of Grb4 with kinase-active Bcr-Abl resulted in a redistribution of actin-associated Bcr-Abl. Finally, coexpression of Grb4 and oncogenic v-Abl strongly inhibited v-Abl–induced AP-1 activation. Together, these data indicate that Grb4 in conjunction with Bcr-Abl may be capable of modulating the cytoskeletal structure and negatively interfering with the signaling of oncogenic Abl kinases. Grb4 may therefore play a role in the molecular pathogenesis of chronic myelogenous leukemia. (Blood. 2000;96:618-624)

scholarly journals The Alpha Chain of the Nascent Polypeptide-Associated Complex Functions as a Transcriptional Coactivator

1998 ◽  
Vol 18 (3) ◽  
pp. 1303-1311 ◽  
Author(s):  
Wagner V. Yotov ◽  
Alain Moreau ◽  
René St-Arnaud
Keyword(s):  
Affinity Chromatography ◽  
Protein Interactions ◽  
Transcriptional Control ◽  
Specific Protein ◽  
Transcriptional Coactivator ◽  
Protein Blotting ◽  
Nascent Polypeptide ◽  
Alpha Chain ◽  
Tata Box Binding Protein

ABSTRACT We report the characterization of clone 1.9.2, a gene expressed in mineralizing osteoblasts. Remarkably, clone 1.9.2 is the murine homolog of the alpha chain of the nascent polypeptide-associated complex (α-NAC). Based on sequence similarities between α-NAC/1.9.2 and transcriptional regulatory proteins and the fact that the heterodimerization partner of α-NAC was identified as the transcription factor BTF3b (B. Wiedmann, H. Sakai, T. A. Davis, and M. Wiedmann, Nature 370:434–440, 1994), we investigated a putative role for α-NAC/1.9.2 in transcriptional control. The α-NAC/1.9.2 protein potentiated by 10-fold the activity of the chimeric activator GAL4/VP-16 in vivo. The potentiation was shown to be mediated at the level of gene transcription, because α-NAC/1.9.2 increased GAL4/VP-16-mediated mRNA synthesis without affecting the half-life of the GAL4/VP-16 fusion protein. Moreover, the interaction of α-NAC/1.9.2 with a transcriptionally defective mutant of GAL4/VP-16 was severely compromised. Specific protein-protein interactions between α-NAC/1.9.2 and GAL4/VP-16 were demonstrated by gel retardation, affinity chromatography, and protein blotting assays, while interactions with TATA box-binding protein (TBP) were detected by immunoprecipitation, affinity chromatography, and protein blotting assays. Based on these interactions that define the coactivator class of proteins, we conclude that the α-NAC/1.9.2 gene product functions as a transcriptional coactivator.

Sign in / Sign up

Export Citation Format

Share Document