Biomedical subjects

Y Eugene Chin

Publications and source records attributed to Y Eugene Chin.

9 recordsLinked to original sources

PubMed2006

Stat3 cleavage by caspases: impact on full-length Stat3 expression, fragment formation, and transcriptional activity.

Stat3 and its isoforms belong to a family of cytoplasmic transcription factors that affect the synthesis of various proteins. Caspases are cysteinyl-aspartate proteases that function under apoptotic and non-apoptotic conditions. We now report that, in addition to transcriptional splicing, Stat3 fragmentation can be mediated by caspases. Caspase activation in DU145 cells was achieved by staurosporine (STS) exposure, and Western analysis revealed a reduction in full-length Stat3 (fl-Stat3) expression that was caspase-mediated. This proteolytic relationship was further studied by exposing purified Stat3 protein to a mixture of active caspases under cell-free conditions. This demonstrated that caspases directly cleaved Stat3 and Stat3 cleavage was accompanied by the apparent formation of cleavage fragment(s). Stat3 cleavage fragments, reflecting multiple caspase cleavage sites, also were observed in vitro following STS exposure in DU145 cells and in HEK293T cells transfected to express Stat3 truncation mutants. The impact of cleavage on Stat3 transcriptional activity next was assessed and revealed that cleavage of fl-Stat3 was accompanied by reductions in Stat3-DNA binding, Stat3-driven reporter protein (luciferase) activity, and the expression of selected Stat3-dependent genes. Further, reduced Stat3 expression correlated with increased sensitivity to apoptotic stimuli. In concomitant experiments, reporter activity was assessed in Stat3 truncation mutant-expressing HEK293T cells and revealed that, under non-apoptotic conditions, expression of different Stat3 fragments induced differential effects on Stat3-driven luciferase activity. These findings demonstrate that fl-Stat3 undergoes proteolytic processing by caspases that reduces its expression and leads to the formation of cleavage fragments that may modulate Stat3 transcriptional activity.

Amino Acid Chloromethyl Ketones↗

PubMed2005

Stat3 dimerization regulated by reversible acetylation of a single lysine residue.

Upon cytokine treatment, members of the signal transducers and activators of transcription (STAT) family of proteins are phosphorylated on tyrosine and serine sites within the carboxyl-terminal region in cells. We show that in response to cytokine treatment, Stat3 is also acetylated on a single lysine residue, Lys685. Histone acetyltransferase p300-mediated Stat3 acetylation on Lys685 was reversible by type I histone deacetylase (HDAC). Use of a prostate cancer cell line (PC3) that lacks Stat3 and PC3 cells expressing wild-type Stat3 or a Stat3 mutant containing a Lys685-to-Arg substitution revealed that Lys685 acetylation was critical for Stat3 to form stable dimers required for cytokine-stimulated DNA binding and transcriptional regulation, to enhance transcription of cell growth-related genes, and to promote cell cycle progression in response to treatment with oncostatin M.

Acetylation↗

PubMed2005

Ankyrin repeat and SOCS box 3 (ASB3) mediates ubiquitination and degradation of tumor necrosis factor receptor II.

Ankyrin repeat and SOCS box (ASB) family members have a C-terminal SOCS box and an N-terminal ankyrin-related sequence of variable repeats belonging to the SOCS superfamily. While SH2-domain-bearing SOCS proteins are mainly involved in the negative feedback regulation of the protein tyrosine kinase-STAT pathway in response to a variety of cytokines, the roles of ASB family members remain largely unknown. To investigate ASB functions, we screened for ASB3-interacting factors by using antibody array technology and identified tumor necrosis factor receptor II (TNF-R2) as an ASB3 binding target. ASB3 expression and activities are required for (i) TNF-R2 ubiquitination both in vivo and in vitro, (ii) TNF-R2 proteolysis via the proteasome pathway, and (iii) the inhibition of TNF-R2-mediated Jun N-terminal protein kinase (JNK) activation. While the ankyrin repeats of ASB3 interact with the C-terminal 37 amino acids of TNF-R2, the SOCS box of ASB3 is responsible for recruiting the E3 ubiquitin ligase adaptors Elongins-B/C, leading to TNF-R2 ubiquitination on multiple lysine residues within its C-terminal region. Downregulation of ASB3 expression by a small interfering RNA inhibited TNF-R2 degradation and potentiated TNF-R2-mediated cytotoxicity. The data presented here implicate ASB3 as a negative regulator of TNF-R2-mediated cellular responses to TNF-alpha by direct targeting of TNF-R2 for ubiquitination and proteasome-mediated degradation.

Amino Acid Sequence↗

PubMed2004

Antibodies immobilized as arrays to profile protein post-translational modifications in mammalian cells.

Previously, we demonstrated that antibodies printed on a solid support were able to detect protein-protein interaction in mammalian cells. Here we further developed the antibody array system for detecting proteins with various post-translational modifications in mammalian cells. In this novel approach, immunoprecipitated proteins were labeled with fluorescent dye followed by incubation over antibody arrays. Targeted proteins, captured by the antibodies immobilized on PVDF membrane or glass slide, were detected by means of near infrared fluorescent scanner or fluorescent microscopy. To demonstrate the application of the antibody arrays in protein post-translational modifications, we profiled protein tyrosine phosphorylation, ubiquitination, and acetylation in mammalian cells under different conditions. Our results indicate that antibody array technology can provide a powerful means of profiling a large number of proteins with different post-translational modifications in cells.

3T3 Cells↗

PubMed2004

Identification of the linker-SH2 domain of STAT as the origin of the SH2 domain using two-dimensional structural alignment.

The availability of large volumes of genomic sequences presents an unprecedented proteomic challenge to characterize the structure and function of various protein motifs. Primary structural alignment is often unable to accurately identify a given motif due to sequence divergence; however, with the aid of secondary structural prediction for analysis, it becomes feasible to explore protein motifs on a proteome-wide scale. Here we report the use of secondary structural alignment to characterize the Src homology 2 (SH2) domains of both conventional and divergent sequences and divide them into two groups, Src-type and STAT-type. In addition to the basic "alphabetabetabetaalpha" structure (betaBeta), the Src-type SH2 domain contains an extra beta-strand (betaE or betaE-betaF motif). Alternatively, the linker domain-conjugated SH2 domain in STAT contains the alphaB' motif. Combining BLAST data from betaBeta core motif sequences with predicted secondary structural alignment, we have screened for SH2 domains in various eukaryotic model systems including Arabidopsis, Dictyostelium, and Saccharomyces. Two novel genes carrying the linker-SH2 domain of STAT were discovered and subsequently cloned from Arabidopsis. These genes, designated as STAT-type linker-SH2 domain factors (STATL), are found in a wide array of vascular and nonvascular plants, suggesting that the linker-SH2 domain evolved prior to the divergence of plants and animals. Using this approach, we expanded the number of putative SH2 domain-bearing genes in Dictyostelium and comparatively studied the secondary structural profiles of both typical and atypical SH2 domains. Our results indicate that the linker-SH2 domain of the transcription factor STAT is one of the most ancient and fully developed functional domains, serving as a template for the continuing evolution of the SH2 domain essential for phosphotyrosine signal transduction.

Amino Acid Sequence↗

PubMed2004

Central role of the threonine residue within the p+1 loop of receptor tyrosine kinase in STAT3 constitutive phosphorylation in metastatic cancer cells.

The receptor tyrosine kinases (RTKs) RET, MET, and RON all carry the Met(p+1loop)-->Thr point mutation (i.e., 2B mutation), leading to the formation of tumors with high metastatic potential. Utilizing a novel antibody array, we identified constitutive phosphorylation of STAT3 in cells expressing the 2B mutation but not wild-type RET. MET or RON with the 2B mutation also constitutively phosphorylated STAT3. Members of the EPH, the only group of wild-type RTK that carry Thr(p+1loop) residue, are often expressed unexpectedly in different types of cancers. Ectopic expression of wild-type but not Thr(p+1loop)-->Met substituted EPH family members constitutively phosphorylated STAT3. In both RTK(Metp+1loop) with 2B mutation and wild-type EPH members the Thr(p+1loop) residue is required for constitutive kinase autophosphorylation and STAT3 recruitment. In multiple endocrine neoplasia 2B (MEN-2B) patients expressing RET(M918T), nuclear enrichment of STAT3 and elevated expression of CXCR4 was detected in metastatic thyroid C-cell carcinoma in the liver. In breast adenocarcinoma cell lines expressing multiple EPH members, STAT3 constitutively bound to the promoters of MUC1, MUC4, and MUC5B genes. Inhibiting STAT3 expression resulted in reduced expression of these metastasis-related genes and inhibited mobility. These findings provide insight into Thr(p+1loop) residue in RTK autophosphorylation and constitutive activation of STAT3 in metastatic cancer cells.

Amino Acid Sequence↗

PubMed2003

Stat1 functions as a cytoplasmic attenuator of Runx2 in the transcriptional program of osteoblast differentiation.

Bone remodeling is central to maintaining the integrity of the skeletal system, wherein the developed bone is constantly renewed by the balanced action of osteoblastic bone formation and osteoclastic bone resorption. In the present study, we demonstrate a novel function of the Stat1 transcription factor in the regulation of bone remodeling. In the bone of the Stat1-deficient mice, excessive osteoclastogenesis is observed, presumably caused by a loss of negative regulation of osteoclast differentiation by interferon (IFN)-beta. However, the bone mass is unexpectedly increased in these mice. This increase is caused by excessive osteoblast differentiation, wherein Stat1 function is independent of IFN signaling. Actually, Stat1 interacts with Runx2 in its latent form in the cytoplasm, thereby inhibiting the nuclear localization of Runx2, an essential transcription factor for osteoblast differentiation. The new function of Stat1 does not require the Tyr 701 that is phosphorylated when Stat1 becomes a transcriptional activator. Our study provides a unique example in which a latent transcription factor attenuates the activity of another transcription factor in the cytoplasm, and reveals a new regulatory mechanism in bone remodeling.

Animals↗

PubMed2002

SHP-2 is a dual-specificity phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues in nuclei.

Signal transducer and activator of transcription (STAT) proteins are both tyrosine- and serine-phosphorylated, mediating signal transduction and gene regulation. Following gene regulation, STAT activity in the nucleus is then terminated by a nuclear protein phosphatase(s), which remains unidentified. Using novel antibody arrays to screen the Stat1-specific protein phosphatase(s), we identified a SHP-2-Stat1 interaction in the A431 cell nucleus. SHP-2 and Stat1 nuclear localization and their association in response to either epidermal growth factor or interferon-gamma (IFNgamma) were confirmed by immunofluorescent staining and affinity precipitation assays. The SHP-2 C-terminal region containing protein-tyrosine phosphatase activity interacted with the C-terminal SH2 transcriptional activation domain of Stat1. In SHP-2-/- mouse fibroblast cells, Stat1 phosphorylation at both the tyrosine residue Tyr(701) and the serine residue Ser(727) by IFNgamma was enhanced and prolonged. Consistently, purified GST-SHP-2 dephosphorylated Stat1 at both tyrosine and serine residues when immunoprecipitated phospho-Stat1 or a peptide corresponding to the sequence surrounding Tyr(P)(701) or Ser(P)(727) of Stat1 was used as the substrate. Overexpression of SHP-2 in 293T cells inhibited IFNgamma-dependent Stat1 phosphorylation and suppressed Stat1-dependent induction of luciferase activity. Our findings demonstrate that SHP-2 is a dual-specificity protein phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues and plays an important role in modulating STAT function in gene regulation.

Active Transport, Cell Nucleus↗

PubMed2002

Identification of both positive and negative domains within the epidermal growth factor receptor COOH-terminal region for signal transducer and activator of transcription (STAT) activation.

The cytoplasmic region of human epidermal growth factor receptor (EGFR) contains an intrinsic tyrosine kinase (697-955) followed by a 231-residue-long COOH-terminal tail (C-tail), which contains multiple tyrosine residues. To examine the role of the EGFR C-tail in signal transducer and activator of transcription (STAT) activation, a series of EGFR C-tail truncations were constructed. Transient transfection of 293 cells with EGFR lacking the C-tail, i.e. Y974DeltaEGFR or Y992DeltaEGFR, led to EGF-independent or constitutive STAT activation, whereas EGF-dependent STAT activation was restored with truncations made COOH-terminal to the next tyrosine residue, i.e. EGFR-Y1045Delta. Transfection with the-truncated form EGFR-Y954Delta resulted in the loss of STAT activation, suggesting that the sequence between Tyr(974) and Tyr(954) is essential for STAT activation. Phosphopeptide competition analysis revealed multiple tyrosine residues within the C-tail that can act as the docking sites for both Stat1 and Stat3. A region that negatively regulated STAT activation was also identified, extending from Tyr(1114) to Glu(1172), consistent with the ability of this region to recruit a suppressor of cytokine signaling factors SOCS1 and SOCS3. When cotransfected with the full-length EGFR, but not Y992DeltaEGFR, SOCS1 or SOCS3 inhibited STAT activation by EGF in 293 cells. This suggests that both SOCS1 and SOCS3 can negatively regulate EGFR activation, presumably by inducing ubiquitination-dependent EGFR degradation upon ligand binding. These findings may therefore offer clues to how the EGF receptor C-tail regulates STAT activity.

Binding Sites↗