Protein tyrosine phosphatase hPTPN20a is targeted to sites of actin polymerization.

The human genome encodes 38 classical tyrosine-specific PTPs (protein tyrosine phosphatases). Many PTPs have been shown to regulate fundamental cellular processes and several are mutated in human diseases. We report that the product of the PTPN20 gene at the chromosome locus 10q11.2 is alternatively spliced to generate 16 possible variants of the classical human non-transmembrane PTP 20 (hPTPN20). One of these variants, hPTPN20a, was expressed in a wide range of both normal and transformed cell lines. The catalytic domain of hPTPN20 exhibited catalytic activity towards tyrosyl phosphorylated substrates, confirming that it is a bona fide PTP. In serum-starved COS1 cells, hPTPN20a was targeted to the nucleus and the microtubule network, colocalizing with the microtubule-organizing centre and intracellular membrane compartments, including the endoplasmic reticulum and the Golgi apparatus. Stimulation of cells with epidermal growth factor, osmotic shock, pervanadate, or integrin ligation targeted hPTPN20a to actin-rich structures that included membrane ruffles. The present study identifies hPTPN20a as a novel and widely expressed phosphatase with a dynamic subcellular distribution that is targeted to sites of actin polymerization.

Selective regulation of tumor necrosis factor-induced Erk signaling by Src family kinases and the T cell protein tyrosine phosphatase.

The proinflammatory cytokine tumor necrosis factor (TNF) modulates cellular responses through the mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) signaling pathways, but the molecular mechanisms underlying MAPK activation are unknown. T cell protein tyrosine phosphatase (TCPTP) is essential for hematopoietic development and negatively regulates inflammatory responses. Using TCPTP-deficient fibroblasts, we show here that TCPTP regulates TNF-induced MAPK but not NF-kappaB signaling. TCPTP interacted with the adaptor protein TRAF2, and dephosphorylated and inactivated Src tyrosine kinases to suppress downstream signaling through extracellular signal-regulated kinases and production of interleukin 6. These results link TCPTP and Src tyrosine kinases to the selective regulation of TNF-induced MAPK signaling and identify a previously unknown mechanism for modulating inflammatory responses mediated by TNF.

The mammalian testis-specific thioredoxin system.

Redox control of cell physiology is one of the most important regulatory mechanisms in all living organisms. The thioredoxin system, composed of thioredoxin and thioredoxin reductase, has emerged as a key player in cellular redox-mediated reactions. For many years, only one thioredoxin system had been described in higher organisms, ubiquitously expressed in the cytoplasm of eukaryotic cells. However, during the last decade, we and others have identified and characterized novel thioredoxin systems with unique properties, such as organelle-specific localization in mitochondria or endoplasmic reticulum, tissue-specific distribution mostly in the testis, and features novel for thioredoxins, such as microtubule-binding properties. In this review, we will focus on the mammalian testis-specific thioredoxin system that comprises three thioredoxins exclusively expressed in spermatids (named Sptrx-1, Sptrx-2, and Sptrx-3) and an additional thioredoxin highly expressed in testis, but also present in lung and other ciliated tissues (Txl-2). The implications of these findings in the context of male fertility and testicular cancer, as well as evolutionary aspects, will be discussed.

The focal adhesion protein vinexin alpha regulates the phosphorylation and activity of estrogen receptor alpha.

Steroid receptors are transcription factors that regulate hormone-responsive genes and whose activity is controlled by their interaction with numerous other proteins. Observations reported here reveal that estrogen receptors alpha and beta (ERalpha and ERbeta), androgen receptor, and glucocorticoid receptor bind in vitro to vinexin alpha, a multiple SH3 motif-containing protein associated with the cytoskeleton. The SH3 domains are not involved in this interaction. Furthermore, we demonstrate that vinexin alpha stimulates the ligand-induced transactivation function of these receptors, although it is devoid of intrinsic transcriptional activity when tethered to DNA. In addition, the ectopic coexpression of vinexin alpha and ERalpha results in a loss of ERalpha phosphorylation on serines and the partial redistribution of vinexin alpha into the nucleus, where it colocalizes with ERalpha. These results establish a new model of transcriptional regulation where components of the cell-cell and cell-substrate adhesion complexes can regulate the phosphorylation and activity of steroid receptors.

Aint/Tacc3 is highly expressed in proliferating mouse tissues during development, spermatogenesis, and oogenesis.

Aint was originally identified on the basis of its interaction in vitro with the aryl hydrocarbon nuclear receptor translocator (Arnt). Arnt is a common heterodimerization partner in the basic helix-loop-helix (bHLH)-PER-ARNT-SIM (PAS) protein family and is involved in diverse biological functions. These include xenobiotic metabolism, hypoxic response, and circadian rhythm. In addition, Arnt has a crucial role during development. Aint is a member of a growing family of transforming acidic coiled-coil (TACC) proteins and is the murine homologue of human TACC3. Here we report the spatiotemporal expression of Tacc3 mRNA and protein in embryonic, postnatally developing, and adult mouse tissues using in situ hybridization and immunocytochemistry. Tacc3 mRNA was highly expressed in proliferating cells of several organs during murine development. However, the only adult tissues expressing high levels were testis and ovary. Immunocytochemistry revealed that Tacc3 is a nuclear protein. Our results suggest that Tacc3 has an important role in murine development, spermatogenesis, and oogenesis.

Characterization of human thioredoxin-like 2. A novel microtubule-binding thioredoxin expressed predominantly in the cilia of lung airway epithelium and spermatid manchette and axoneme.

We describe here the cloning and characterization of a novel member of the thioredoxin family, thioredoxin-like protein 2 (Txl-2). The Txl-2 open reading frame codes for a protein of 330 amino acids consisting of two distinct domains: an N-terminal domain typical of thioredoxins and a C-terminal domain belonging to the nucleoside-diphosphate kinase family, separated by a small interface domain. The Txl-2 gene spans approximately 28 kb, is organized into 11 exons, and maps at locus 3q22.3-q23. A splicing variant lacking exon 5 (Delta 5Txl-2) has also been isolated. By quantitative real time PCR we demonstrate that Txl-2 mRNA is ubiquitously expressed, with testis and lung having the highest levels of expression. Unexpectedly, light and electron microscopy analyses show that the protein is associated with microtubular structures such as lung airway epithelium cilia and the manchette and axoneme of spermatids. Using in vitro translated proteins, we demonstrate that full-length Txl-2 weakly associates with microtubules. In contrast, Delta 5Txl-2 specifically binds with very high affinity brain microtubule preparations containing microtubule-binding proteins. Importantly, Delta 5Txl-2 also binds to pure microtubules, proving that it possesses intrinsic microtubule binding capability. Taken together, Delta 5Txl-2 is the first thioredoxin reported to bind microtubules and might therefore be a novel regulator of microtubule physiology.

Different regulation of the LXRalpha promoter activity by isoforms of CCAAT/enhancer-binding proteins.

LXRs have recently been shown to regulate key enzymes in cholesterol degradation, reverse transport of cholesterol from peripheral cells, cholesterol uptake and lipogenesis. The LXRalpha promoter was thus studied to investigate if LXRalpha gene expression is under the regulation of transcription factors involved in adipogenesis. We report that the C/EBP transcription factor interacts with the promoter of the LXRalpha gene. In in vitro footprinting experiments, protein extracts from several tissues gave footprints covering a putative C/EBP recognition site. Transfection experiments and EMSA showed a direct effect of these transcription factors on the LXRalpha promoter. C/EBPalpha upregulated expression of the reporter gene in an NIH 3T3-L1 preadipocyte cell line, while C/EBPbeta and C/EBPdelta had no effect. In liver hepatoma Fao II and Cos-7 kidney cells, both C/EBPalpha and C/EBPbeta downregulated expression of the reporter gene while C/EBPdelta induced activity, indicating that the functional consequences of C/EBP isoform interactions with the LXRalpha promoter are dependent on the cellular context. Monitoring of the LXR mRNA levels during adipose tissue differentiation showed that LXRbeta is constitutively expressed during the entire differentiation process while LXRalpha is induced upon addition of differentiation mix.