Biomedical subjects

M L Tremblay

Publications and source records attributed to M L Tremblay.

At least 19 recordsLinked to original sources

PubMed2005

Substrate-trapping techniques in the identification of cellular PTP targets.

Tyrosine phosphorylation is negatively regulated by the protein-tyrosine phosphatases (PTPs). In order to find the physiological substrates of these enzymes, diverse PTP mutants that do not possess any catalytic activities but appear to bind tightly to their tyrosine phosphorylated substrates have been designed. Hence, they can be used as tools to pull out their respective substrates from heterogeneous extracts. Named PTP "substrate-trapping" mutants by the Tonks laboratory, they represent a diverse variety of defective PTPs that are epitomized by the Cys to Ser mutant (C/S) where the active cysteine residue of the signature motif is mutated to a serine residue. In addition, new mutants have been developed which are expected to help characterize novel and less abundant substrates. In this article, we review and describe all the different substrate-trapping mutants that have successfully been used or that hold interesting promises. We present their methodology to identify substrates in vivo (co-immunoprecipitation) and in vitro (GST pulldown), and provide a current list of substrates that have been identified using these technologies.

Alanine↗

PubMed2003

Receptor protein tyrosine phosphatase sigma inhibits axonal regeneration and the rate of axon extension.

Transgenic mice lacking receptor protein tyrosine phophatase-sigma (RPTPsigma), a type IIa receptor protein tyrosine phosphatase, exhibit severe neural developmental deficits. Continued expression of RPTPsigma in the adult suggests that it plays a functional role in the mature nervous system. To determine if RPTPsigma might influence axonal regeneration, the time course of regeneration following facial nerve crush in wild-type and RPTPsigma (-/-) mice was compared. Mice lacking RPTPsigma exhibited an accelerated rate of functional recovery. Immunocytochemical examination of wild-type neurons in cell culture showed RPTPsigma protein in the growth cone. To determine if RPTPsigma affects the ability of a neuron to extend an axon, the rate of axon growth in neuronal cultures derived from wild-type and RPTPsigma (-/-) embryonic mice was compared. RPTPsigma did not affect the rate of axon initiation, but the rate of axon extension is enhanced in neurons obtained from RPTPsigma (-/-) mice. These findings indicate that RPTPsigma slows axon growth via a mechanism intrinsic to the neuron and identify a role for RPTPsigma regulating axonal regeneration by motoneurons.

Animals↗

PubMed2002

Mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is essential for embryonic development.

Folate-dependent enzymes are compartmentalized between the cytoplasm and mitochondria of eukaryotes. The role of mitochondrial folate-dependent metabolism and the extent of its contribution to cytoplasmic processes are areas of active investigation. NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) catalyzes the interconversion of 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate in mitochondria of mammalian cells, but its metabolic role is not yet clear. Its expression in embryonic tissues but not in most adult tissues as well as its stringent transcriptional regulation led us to postulate that it may play a role in embryonic development. To investigate the metabolic role of NMDMC, we used a knockout approach to delete the nmdmc gene in mice. Heterozygous mice appear healthy, but homozygous NMDMC knockout mice die in utero. At embryonic day 12.5 (E12.5), homozygous null embryos exhibit no obvious developmental defects but are smaller and pale and die soon thereafter. Mutant fetal livers contain fewer nucleated cells and lack the characteristic redness of wild-type or heterozygous livers. The frequencies of CFU-erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) from fetal livers of E12.5 null mutants were not reduced compared with those of wild-type or heterozygous embryos. It has been assumed that initiation of protein synthesis in mitochondria requires a formylated methionyl-tRNA(fmet). One role postulated for NMDMC is to provide 10-formyltetrahydrofolate as a formyl group donor for the synthesis of this formylmethionyl-tRNA(fmet). To determine if the loss of NMDMC impairs protein synthesis and thus could be a cause of embryonic lethality, mitochondrial translation products were examined in cells in culture. Mitochondrial protein synthesis was unaffected in NMDMC-null mutant cell lines compared with the wild type. These results show that NMDMC is not required to support initiation of protein synthesis in mitochondria in isolated cells but instead demonstrate an essential role for mitochondrial folate metabolism during embryonic development.

Aminohydrolases↗

PubMed2001

TYK2 and JAK2 are substrates of protein-tyrosine phosphatase 1B.

The reversible tyrosine phosphorylation of proteins, modulated by the coordinated actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs), regulates the cellular response to a wide variety of stimuli. It is established that protein kinases possess discrete sets of substrates and that substrate recognition is often dictated by the presence of consensus phosphorylation sites. Here, we have extended this concept to the PTPs and demonstrated that (E/D)-pY-pY-(R/K) is a consensus substrate recognition motif for PTP1B. We have shown that JAK2 and TYK2 are substrates of PTP1B and that the substrate recognition site within theses kinases is similar to the site of dephosphorylation previously identified within the insulin receptor. A substrate-trapping mutant of PTP1B formed a stable interaction with JAK2 and TYK2 in response to interferon stimulation. Expression of wild type or substrate-trapping mutant PTP1B inhibited interferon-dependent transcriptional activation. Finally, mouse embryo fibroblasts deficient in PTP1B displayed subtle changes in tyrosine phosphorylation, including hyperphosphorylation of JAK2. The closely related JAK family member, JAK1, which does not match the consensus dephosphorylation site, was not recognized as a substrate. These data illustrate that PTP1B may be an important physiological regulator of cytokine signaling and that it may be possible to derive consensus substrate recognition motifs for other members of the PTP family, which may then be used to predict novel physiological substrates.

Amino Acid Sequence↗

PubMed2001

Ubiquinone is necessary for mouse embryonic development but is not essential for mitochondrial respiration.

Ubiquinone (UQ) is a lipid found in most biological membranes and is a co-factor in many redox processes including the mitochondrial respiratory chain. UQ has been implicated in protection from oxidative stress and in the aging process. Consequently, it is used as a dietary supplement and to treat mitochondrial diseases. Mutants of the clk-1 gene of the nematode Caenorhabditis elegans are fertile and have an increased life span, although they do not produce UQ but instead accumulate a biosynthetic intermediate, demethoxyubiquinone (DMQ). DMQ appears capable to partially replace UQ for respiration in vivo and in vitro. We have produced a vertebrate model of cells and tissues devoid of UQ by generating a knockout mutation of the murine orthologue of clk-1 (mclk1). We find that mclk1-/- embryonic stem cells and embryos accumulate DMQ instead of UQ. As in the nematode mutant, the activity of the mitochondrial respiratory chain of -/- embryonic stem cells is only mildly affected (65% of wild-type oxygen consumption). However, mclk1-/- embryos arrest development at midgestation, although earlier developmental stages appear normal. These findings indicate that UQ is necessary for vertebrate embryonic development but suggest that mitochondrial respiration is not the function for which UQ is essential when DMQ is present.

Animals↗

PubMed2001

Murine embryonic fibroblasts lacking TC-PTP display delayed G1 phase through defective NF-kappaB activation.

Previous results suggested a potential role for T-cell protein tyrosine phosphatase (TC-PTP) in cell proliferation. However, no conclusive data has supported such a function in the modulation of this process. In order to clarify this issue, we isolated TC-PTP-/- murine embryonic fibroblasts (MEFs) as well as cell lines to characterize the role of TC-PTP in the control of cell proliferation and cell cycle. Both TC-PTP-/- primary MEFs and cell lines proliferate slower than TC-PTP+/+ cells. We also demonstrated that TC-PTP-/- cells have a slow progression through the G1 phase of the cell cycle. Further characterization of the G1 defect indicates that the kinetics of cyclin D1 induction was delayed and that p27(KIP1) remains at higher levels for an extended period of time. Moreover, cells lacking TC-PTP showed a delayed activation of CDK2. This slow progression through the early G1-phase resulted in decreased phosphorylation of the RB protein and subsequent delay into the S phase transition. In contrast, no further defects were detected in other phases of the cell cycle. Survey of the potential signaling pathways leading to this delayed cyclin D1 expression indicated that NF-kappaB activation was compromised and that IKKbeta activity was also reduced following PDGF stimulation. Reintroduction of wild-type TC-PTP into the TC-PTP-/- cells rescued the defective proliferation, cyclin D1 expression, NF-kappaB activation as well as IkappaB phosphorylation. Together, these results confirm that TC-PTP plays a positive role in the progression of early G1 phase of the cell cycle through the NF-kappaB pathway.

Animals↗

PubMed2001

Targeted ablation of the 25-hydroxyvitamin D 1alpha -hydroxylase enzyme: evidence for skeletal, reproductive, and immune dysfunction.

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)2D], is synthesized from its precursor 25 hydroxyvitamin D [25(OH)D] via the catalytic action of the 25(OH)D-1alpha-hydroxylase [1alpha(OH)ase] enzyme. Many roles in cell growth and differentiation have been attributed to 1,25(OH)2D, including a central role in calcium homeostasis and skeletal metabolism. To investigate the in vivo functions of 1,25(OH)2D and the molecular basis of its actions, we developed a mouse model deficient in 1alpha(OH)ase by targeted ablation of the hormone-binding and heme-binding domains of the 1alpha(OH)ase gene. After weaning, mice developed hypocalcemia, secondary hyperparathyroidism, retarded growth, and the skeletal abnormalities characteristic of rickets. These abnormalities are similar to those described in humans with the genetic disorder vitamin D dependent rickets type I [VDDR-I; also known as pseudovitamin D-deficiency rickets (PDDR)]. Altered non-collagenous matrix protein expression and reduced numbers of osteoclasts were also observed in bone. Female mutant mice were infertile and exhibited uterine hypoplasia and absent corpora lutea. Furthermore, histologically enlarged lymph nodes in the vicinity of the thyroid gland and a reduction in CD4- and CD8-positive peripheral T lymphocytes were observed. Alopecia, reported in vitamin D receptor (VDR)-deficient mice and in humans with VDDR-II, was not seen. The findings establish a critical role for the 1alpha(OH)ase enzyme in mineral and skeletal homeostasis as well as in female reproduction and also point to an important role in regulating immune function.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed2001

Attenuation of adhesion-dependent signaling and cell spreading in transformed fibroblasts lacking protein tyrosine phosphatase-1B.

Previous biochemical evidence has yielded conflicting models for the role of protein tyrosine phosphatase-1B (PTP-1B) in the regulation of integrin signaling. Thus, to establish the physiological relevance for such a role, we employed a genetic approach by generating embryonic fibroblasts from PTP-1B knockout mice. Both primary fibroblasts and their derived cell lines were used in this study. Immortalization of wild-type primary cells with the SV40 Large T antigen resulted in a dramatic increase in the endogenous expression of PTP-1B, suggesting a role during transformation. Moreover, the absence of PTP-1B in the transformed cell lines led to a more pronounced effect on different pathways of fibronectin-mediated signaling compared with the untransformed state. Specifically, p130(Cas) phosphorylation, Erk activation as well as cell spreading were delayed in PTP-1B-deficient cells, compared with their wild-type counterparts. Interestingly, this attenuation in integrin-mediated events closely resembles that of Src-deficient fibroblasts. Indeed, PTP-1B deficient, transformed fibroblasts held in suspension do exhibit a hyperphosphorylation of the inhibitory site (Tyr-527) of Src, compared with their wild-type counterparts. These results establish PTP-1B as a positive physiological regulator of integrin signaling in transformed cells, acting upstream of Src Tyr-527 dephosphorylation that leads to several adhesion-dependent events.

Animals↗

PubMed2001

Adipose tissue reduction in mice lacking the translational inhibitor 4E-BP1.

All nuclear-encoded mRNAs contain a 5' cap structure (m7GpppN, where N is any nucleotide), which is recognized by the eukaryotic translation initiation factor 4E (eIF4E) subunit of the eIF4F complex. The eIF4E-binding proteins constitute a family of three polypeptides that reversibly repress cap-dependent translation by binding to eIF4E, thus preventing the formation of the eIF4F complex. We investigated the biological function of 4E-BP1 by disrupting its gene (Eif4ebp1) in the mouse. Eif4ebp1-/- mice manifest markedly smaller white fat pads than wild-type animals, and knockout males display an increase in metabolic rate. The males' white adipose tissue contains cells that exhibit the distinctive multilocular appearance of brown adipocytes, and expresses the uncoupling protein 1 (UCP1), a specific marker of brown fat. Consistent with these observations, translation of the peroxisome proliferator-activated receptor-gamma co-activator 1 (PGC1), a transcriptional co-activator implicated in mitochondrial biogenesis and adaptive thermogenesis, is increased in white adipose tissue of Eif4ebp1-/- mice. These findings demonstrate that 4E-BP1 is a novel regulator of adipogenesis and metabolism in mammals.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Inactivation of the acid labile subunit gene in mice results in mild retardation of postnatal growth despite profound disruptions in the circulating insulin-like growth factor system.

Insulin-like growth factors (IGFs) I and II are important regulators of cell proliferation and differentiation. After birth, plasma IGFs, representing mostly liver-derived IGFs, circulate in ternary complexes of 150 kDa consisting of one molecule each of IGF, IGF-binding protein (IGFBP) 3, and an acid labile subunit (ALS). Onset of ALS synthesis after birth is the primary factor driving the formation of ternary complexes. Capture of IGFs by ALS is thought to allow the development of a plasma reservoir without negative effects such as hypoglycemia and cell proliferation. To evaluate the importance of ALS and ternary complexes, we have created mice in which the ALS gene has been inactivated. The mutation was inherited in a Mendelian manner, without any effects on survival rates and birth weights. A growth deficit was observed in null mice after 3 weeks of life and reached 13% by 10 weeks. This modest phenotype was observed despite reductions of 62 and 88% in the concentrations of plasma IGF-I and IGFBP-3, respectively. Increased turnover accounted for these reductions because indices of synthesis in liver and kidney were not decreased. Surprisingly, absence of ALS did not affect glucose and insulin homeostasis. Therefore, ALS is required for postnatal accumulation of IGF-I and IGFBP-3 but, consistent with findings supporting a predominant role for locally produced IGF-I, is not critical for growth. This model should be useful to determine whether presence of ALS is needed for other actions of liver-derived IGF-I and for maintenance of homeostasis in presence of high circulating levels of IGF-II.

Animals↗

PubMed2000

The T-cell protein tyrosine phosphatase.

In recent years, the T-cell protein tyrosine phosphatase (TC-PTP) has become an important member of the protein-tyrosine phosphatase (PTP) family in two aspects. Firstly, TC-PTP has been reported to act on downstream signalling events initiated by the epidermal growth receptor, suggesting that it may act as an important modulator of receptor tyrosine kinases and mitogenic signalling. Secondly, the finding of immune deficiency and lethality observed in TC-PTP null mice emphasizes the importance of this small PTP in the hematopoietic system. In this review, we provide a summary of the recent literature published on the TC-PTP and its various orthologs. Although much remains to be uncovered, some recent findings on the function of this small PTP suggest that it plays a critical role in regulating mammalian cell signalling.

Amino Acid Sequence↗

PubMed2000

Modulation of insulin signaling by protein tyrosine phosphatases.

Non-insulin-dependent diabetes mellitus (NIDDM) is a worldwide endocrine disorder afflicting persons of all races and age groups. At the molecular level NIDDM is often characterized by impaired insulin action on peripheral tissues. One important mechanism in regulating insulin signaling is mediated by protein tyrosine phosphatases, which may act on the insulin receptor itself and/or its substrates. Understanding the molecular events triggered by insulin has undoubtedly provided important clues in the treatment of NIDDM. In particular, the use of mouse models has helped us to focus on specific gene targets that are involved in the onset and progression of diabetes. Here we present an overview of the biochemical and genetic evidence supporting the role of five protein tyrosine phosphatases in insulin-mediated responses.

Animals↗

PubMed2000

Regulation and role of the acid-labile subunit of the 150-kilodalton insulin-like growth factor complex in the mouse.

After birth, the acid-labile subunit (ALS) associates in the circulation with insulin-like growth factor (IGF)-I or -II and with IGF binding protein-3 (IGFBP-3) to form a 150-kilodalton complex. This association leads to the retention of IGFs in the vascular system and promotes their endocrine actions. ALS is synthesized almost exclusively in liver, and both hepatic ALS mRNA and circulating levels are increased by growth hormone (GH). Three major areas of study were pursued to better understand the regulation of ALS synthesis and its role in the circulating IGF system. First, the mouse ALS gene was isolated and shown to be organized into two exons and a single intron on chromosome 17. Second, using transient transfection studies in the rat H4-II-E hepatoma cell line and primary rat hepatocytes, the region of the mouse promoter that is responsive to GH was mapped to a nine-base pair cis-element resembling a gamma-interferon-activated sequence. The activation of the mouse ALS gene by GH is mediated by the binding of STAT5 isoforms to this sequence. Finally, an ALS knockout model was created by inactivating the ALS gene in mouse embryonic stem cells. Mice that are homozygous for the mutation grow at a slower rate after birth. This growth depression is associated with large decreases in the plasma concentrations of both IGF-I and IGFBP-3, indicating the critical role of ALS in the regulation of circulating levels of these proteins. Studies of this model will lead to a better understanding of the circulating IGF system.

Animals↗

PubMed2000

Cytoskeletal protein PSTPIP1 directs the PEST-type protein tyrosine phosphatase to the c-Abl kinase to mediate Abl dephosphorylation.

A search for c-Abl interacting proteins resulted in the recovery of PSTPIP1, originally identified as a binding protein of the PEST-type protein tyrosine phosphatases (PTP). PSTPIP1 was phosphorylated by c-Abl, and growth factor-induced PSTPIP1 phosphorylation was diminished in Abl null fibroblasts. PSTPIP1 was able to bridge c-Abl to the PEST-type PTPs. Several experiments suggest that the PEST-type PTPs negatively regulate c-Abl activity: c-Abl was hyperphosphorylated in PTP-PEST-deficient cells; disruption of the c-Abl-PSTPIP1-PEST-type PTP ternary complex by overexpression of PSTPIP1 mutants increased c-Abl phosphotyrosine content; and PDGF-induced c-Abl kinase activation was prolonged in PTP-PEST-deficient cells. Dephosphorylation of c-Abl by PEST-type PTP represents a novel mechanism by which c-Abl activity is regulated.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Promoter analysis of the murine T-cell protein tyrosine phosphatase gene.

The T-cell protein tyrosine phosphatase (TC PTP) is expressed ubiquitously at all stages of mammalian development. However, mRNA levels fluctuate in a cell-cycle-dependent manner, reaching peak levels in late G1, and rapidly decreasing in S phase. Furthermore, TC PTP being present in higher amounts in lymphoid tissues, we have recently shown that it is essential for proper maintenance of both the bone marrow micro-environment and B- and T-cell functions. In order to better understand the elements controlling the expression pattern of this gene, we have isolated and characterized approx. 4kb of the murine TC PTP promoter. DNA sequencing of the proximal 5' region revealed the absence of both TATAA and CAAT boxes. Primer extension analysis and S1 nuclease mapping techniques identified multiple transcription initiation sites. Functional promoter activity was determined using transfection experiments of promoter deletion constructs fused to a CAT reporter construct. Our results indicate that the minimal promoter sequence required for functional expression is contained within the first 147bp of the TC PTP promoter. In addition, consistent with the cell-cycle-dependent expression of TC PTP, we localized a domain between 492 and 1976bp from the transcription initiation site through which repression occurs. In conclusion, although initiator-driven transcription allows for ubiquitous expression of TC PTP, we define general transcription motifs present within the promoter that may mediate specific modulations of the TC PTP gene.

3T3 Cells↗

PubMed1999

Intact LIM 3 and LIM 4 domains of paxillin are required for the association to a novel polyproline region (Pro 2) of protein-tyrosine phosphatase-PEST.

The focal adhesion protein p130(Cas) was identified as a substrate for the protein-tyrosine phosphatase (PTP)-PEST, and the specificity of this interaction is mediated by a dual mechanism involving a Src homology 3 domain-mediated binding and PTP domain recognition. Recently, paxillin was also demonstrated to interact with PTP-PEST (Shen, Y., Schneider, G., Cloutier, J. F., Veillette, A., and Schaller, M. D. (1998) J. Biol. Chem. 273, 6474-6481). In the present study, we show that amino acids 344-397 of PTP-PEST are sufficient for the binding to paxillin. We demonstrate that a proline-rich segment of PTP-PEST (Pro 2), 355PPEPHPVPPILTPSPPSAFP374, is essential for this interaction in vivo. Furthermore, mutation of proline residues within the Pro 2 motif reveal that proline 362 is critical for the binding of paxillin. Conversely, using deletion and point mutants of paxillin, LIM 3 and 4 domains were both found to be necessary for binding of PTP-PEST. Finally, using a "substrate trapping" approach, we demonstrate that, unlike p130(Cas), paxillin is not a substrate for PTP-PEST. In conclusion, we show that a novel proline-rich motif found in PTP-PEST serves as a ligand for the LIM domains of paxillin. Interestingly, the focal adhesion targeting of paxillin is mediated by LIM 3. Thus, we propose that PTP-PEST, by a competition with the ligand of paxillin in the focal adhesion complex, could contribute to the removal of paxillin from the adhesion sites and consequently promote focal adhesion turnover.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Hic-5, a paxillin homologue, binds to the protein-tyrosine phosphatase PEST (PTP-PEST) through its LIM 3 domain.

The Hic-5 protein is encoded by a transforming growth factor-beta1- and hydrogen peroxide-inducible gene, hic-5, and has striking similarity to paxillin, especially in their C-terminal LIM domains. Like paxillin, Hic-5 is localized in focal adhesion plaques in association with focal adhesion kinase in cultured fibroblasts. We carried out yeast two-hybrid screening to identify cellular factors that form a complex with Hic-5 using its LIM domains as a bait, and we identified a cytoplasmic tyrosine phosphatase (PTP-PEST) as one of the partners of Hic-5. These two proteins are associated in mammalian cells. From in vitro binding experiments using deletion and point mutations, it was demonstrated that the essential domain in Hic-5 for the binding was LIM 3. As for PTP-PEST, one of the five proline-rich sequences found on PTP-PEST, Pro-2, was identified as the binding site for Hic-5 in in vitro binding assays. Paxillin also binds to the Pro-2 domain of PTP-PEST. In conclusion, Hic-5 may participate in the regulation of signaling cascade through its interaction with distinct tyrosine kinases and phosphatases.

Animals↗

PubMed1999

Protein tyrosine phosphatase-PEST regulates focal adhesion disassembly, migration, and cytokinesis in fibroblasts.

In this article, we show that, in transfected COS-1 cells, protein tyrosine phosphatase (PTP)-PEST translocates to the membrane periphery following stimulation by the extracellular matrix protein fibronectin. When plated on fibronectin, PTP-PEST (-/-) fibroblasts display a strong defect in motility. 3 h after plating on fibronectin, the number and size of vinculin containing focal adhesions were greatly increased in the homozygous PTP-PEST mutant cells as compared with heterozygous cells. This phenomenon appears to be due in part to a constitutive increase in tyrosine phosphorylation of p130(CAS), a known PTP-PEST substrate, paxillin, which associates with PTP-PEST in vitro, and focal adhesion kinase (FAK). Another effect of this constitutive hyperphosphorylation, consistent with the focal adhesion regulation defect, is that (-/-) cells spread faster than the control cell line when plated on fibronectin. In the PTP-PEST (-/-) cells, an increase in affinity for the SH2 domains of Src and Crk towards p130(CAS) was also observed. In (-/-) cells, we found a significant increase in the level of tyrosine phosphorylation of PSTPIP, a cleavage furrow-associated protein that interacts physically with all PEST family members. An effect of PSTPIP hyperphosphorylation appears to be that some cells remain attached at the site of the cleavage furrow for an extended period of time. In conclusion, our data suggest PTP-PEST plays a dual role in cell cytoskeleton organization, by promoting the turnover of focal adhesions required for cell migration, and by directly or indirectly regulating the proline, serine, threonine phosphatase interacting protein (PSTPIP) tyrosine phosphorylation level which may be involved in regulating cleavage furrow formation or disassembly during normal cell division.

Animals↗