Biomedical subjects

T Mustelin

Publications and source records attributed to T Mustelin.

At least 37 records · Page 2Linked to original sources

PubMed1999

Crosstalk between cAMP-dependent kinase and MAP kinase through a protein tyrosine phosphatase.

The haematopoietic protein tyrosine phosphatase (HePTP) is a negative regulator of the MAP kinases Erk1, Erk2 and p38. HePTP binds to these kinases through a kinase-interaction motif (KIM) in its non-catalytic amino terminus and inactivates them by dephosphorylating the critical phosphorylated tyrosine residue in their activation loop. Here we show that cyclic-AMP-dependent protein kinase (PKA) phosphorylates serine residue 23 in the KIM of HePTP in vitro and in intact cells. This modification reduces binding of MAP kinases to the KIM, an effect that is prevented by mutation of serine 23 to alanine. The PKA-mediated release of MAP kinase from HePTP is sufficient to activate the kinase and to induce transcription from the c-fos promoter. Expression of a HePTP serine-23-to-alanine mutant inhibits MAP-kinase dissociation and activation and induction of transcription from the c-fos promoter. We conclude that HePTP not only controls the activity of MAP kinases, but also mediates crosstalk between the cAMP system and the MAP-kinase cascade.

Alanine↗

PubMed1999

A novel isoform of the low molecular weight phosphotyrosine phosphatase, LMPTP-C, arising from alternative mRNA splicing.

The low molecular weight protein tyrosine phosphatase (LMPTP) is an 18-kDa enzyme that it distantly related to other protein tyrosine phosphatases. The single gene for LMPTP is known to undergo an alternative splicing event in which exon 3 or exon 4 is excised, resulting in two isoforms termed A and B; the latter is more mobile in SDS/PAGE. In this paper we report the existence of a third isoform, which we call C, in which both exons 3 and 4 are lacking. We find the resulting mRNA to be ubiquitously expressed at levels that exceed those of the mRNAs for isoforms A and B. This mRNA was reverse-transcribed, cloned and sequenced, confirming the direct splicing of exon 2 to exon 5. In-vitro transcription and translation of the cDNA for the novel isoform resulted in the expected 16 kDa protein. This protein was also detected in Jurkat T cells using an antipeptide antiserum. LMPTP-C immunoprecipitated from transfected cells, as well as bacterially produced recombinant LMPTP-C, lacked phosphatase activity. Unlike LMPTP-B, LMPTP-C was not phosphorylated on tyrosine when coexpressed with Lck despite the presence of the two acceptor tyrosines. Finally, whereas c-fos induction by platelet-derived growth factor was inhibited by LMPTP-B, LMPTP-C augmented it. These results suggest that the lack of the 38-amino acid fragment encoded by exon 3 or 4 results in a protein product with a different three-dimensional folding, that lacks a functional catalytic pocket and that may function as a natural antagonist of isoforms A and B.

Alternative Splicing↗

PubMed1999

Jun kinase phosphorylates and regulates the DNA binding activity of an octamer binding protein, T-cell factor beta1.

POU domain proteins have been implicated as key regulators during development and lymphocyte activation. The POU domain protein T-cell factor beta1 (TCFbeta1), which binds octamer and octamer-related sequences, is a potent transactivator. In this study, we showed that TCFbeta1 is phosphorylated following activation via the T-cell receptor or by stress-induced signals. Phosphorylation of TCFbeta1 occurred predominantly at serine and threonine residues. Signals which upregulate Jun kinase (JNK)/stress-activated protein kinase activity also lead to association of JNK with TCFbeta1. JNK associates with the activation domain of TCFbeta1 and phosphorylates its DNA binding domain. The phosphorylation of recombinant TCFbeta1 by recombinant JNK enhances the ability of TCFbeta1 to bind to a consensus octamer motif. Consistent with this conclusion, TCFbeta1 upregulates reporter gene transcription in an activation- and JNK-dependent manner. In addition, inhibition of JNK activity by catalytically inactive MEKK (in which methionine was substituted for the lysine at position 432) also inhibits the ability of TCFbeta1 to drive inducible transcription from the interleukin-2 promoter. These results suggest that stress-induced signals and T-cell activation induce JNK, which then acts on multiple cis sequences by modulating distinct transactivators like c-Jun and TCFbeta1. This demonstrates a coupling between the JNK activation pathway and POU domain proteins and implicates TCFbeta1 as a physiological target in the JNK signal transduction pathway leading to coordinated biological responses.

Amino Acid Sequence↗

PubMed1998

Lymphocyte activation: the coming of the protein tyrosine phosphatases.

The molecular mechanisms of signal transduction have been at the focus of intense scientific research world-wide. As a result, our understanding of protein tyrosine kinase-mediated signaling has advanced at an unprecedented pace during the past decade. In contrast, the study of protein tyrosine phosphatases is in its infancy, but is currently gathering momentum and is predicted to become a "hot topic" in the field within the next few years. This review summarizes the current state-of-the art in our understanding of the structure, regulation and role of protein tyrosine phosphatases in lymphocyte activation.

Animals↗

PubMed1998

Negative regulation of T cell antigen receptor signal transduction by hematopoietic tyrosine phosphatase (HePTP).

The hematopoietic tyrosine phosphatase (HePTP) is predominantly expressed in thymocytes and T lymphocytes and at lower levels in other hematopoietic cells. Expression of the gene is enhanced by the T cell growth factor interleukin-2, suggesting a role for HePTP in T cell proliferation or differentiation. We report that HePTP blocks T cell antigen receptor (TCR)-induced transcriptional activation of a reporter gene driven by a nuclear factor of activated T cells(NFAT)/AP-1 element taken from the interleukin-2 gene promoter. This effect was specific to HePTP and was abolished by a mutation (C270S) that impaired its phosphatase activity. Co-expression of HePTP also reduced TCR-induced activation of the mitogen-activated protein kinase Erk2 and the TCR-induced appearance of phosphorylated Erk. In contrast, HePTP did not affect the activation of the N-terminal c-Jun kinase, Jnk. Together these findings suggest that HePTP plays an active negative role in TCR signaling by dephosphorylating one or several signaling molecules between the receptor and the mitogen-activated protein kinase pathway.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1998

Modification of phosphatidylinositol 3-kinase SH2 domain binding properties by Abl- or Lck-mediated tyrosine phosphorylation at Tyr-688.

In cells expressing the oncogenic Bcr-Abl tyrosine kinase, the regulatory p85 subunit of phosphatidylinositol 3-kinase is phosphorylated on tyrosine residues. We report that this phosphorylation event is readily catalyzed by the Abl and Lck protein-tyrosine kinases in vitro, by Bcr-Abl or a catalytically activated Lck-Y505F in co-transfected COS cells, and by endogenous kinases in transfected Jurkat T cells upon triggering of their T cell antigen receptor. Using these systems, we have mapped a major phosphorylation site to Tyr-688 in the C-terminal SH2 domain of p85. Tyrosine phosphorylation of p85 in vitro or in vivo was not associated with detectable change in the enzymatic activity of the phosphatidylinositol 3-kinase heterodimer, but correlated with a strong reduction in the binding of some, but not all, phosphoproteins to the SH2 domains of p85. This provides an additional candidate to the list of SH2 domains regulated by tyrosine phosphorylation and may explain why association of phosphatidylinositol 3-kinase with some cellular ligands is transient or of lower stoichiometry than anticipated.

Fusion Proteins, bcr-abl↗

PubMed1998

Phosphorylation of the Grb2- and phosphatidylinositol 3-kinase p85-binding p36/38 by Syk in Lck-negative T cells.

Activation of the mitogen-activated protein kinase (MAPK) pathway by the T-cell antigen receptor (TCR) in T cells involves a positive role for phosphatidylinositol 3-kinase (PI3K) activity. We recently reported that over-expression of the Syk protein tyrosine kinase in the Lck-negative JCaM1 cells enabled the TCR to induce a normal activation of the Erk2 MAPK and enhanced transcription of a reporter gene driven by the nuclear factor of activated T cells and AP-1. Because this system allows us to analyse the targets for Syk in receptor-mediated signalling, we examined the role of PI3K in signalling events between the TCR-regulated Syk and the downstream activation of Erk2. We report that inhibition of PI3K by wortmannin or an inhibitory p85 construct, p85deltaiSH2, reduced the TCR-induced Syk-dependent activation of Erk2, as well as the appearance of phospho-Erk and phospho-Mek. At the same time, expression of Syk resulted in the activation-dependent phosphorylation of three proteins that bound to the src homology 2 (SH2) domains of PI3K p85. The strongest of these bands had an apparent molecular mass of 36-38 kDa on SDS gels, and it was quantitatively removed from the lysates by adsorption to a fusion protein containing the SH2 domain of Grb2. The appearance of this band was Syk dependent, and it was seen only upon triggering of the TCR complex. Thus, p36/38 was phosphorylated by Syk or a Syk-regulated kinase, and this protein may provide a link to the recruitment and activation of PI3K, as well as to the Ras-MAPK pathway, in TCR-triggered T cells.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Constitutive activation of a slowly migrating isoform of Stat3 in mycosis fungoides: tyrphostin AG490 inhibits Stat3 activation and growth of mycosis fungoides tumor cell lines.

Mycosis fungoides (MF) is a low-grade cutaneous T cell lymphoma of unknown etiology. In this report, the Jak/Stat (Janus kinase/signal transducer and activator of transcription) signaling pathway was investigated in tumor cell lines established from skin biopsy specimens from a patient with MF. Jaks link cytokine receptors to Stats, and abnormal Jak/Stat signaling has been observed in some hemopoietic cancers. In MF tumor cells, a slowly migrating isoform of Stat3, Stat3(sm), was found to be constitutively activated, i.e., (i) Stat3(sm) was constitutively phosphorylated on tyrosine residues, and tyrosine phosphorylation was not enhanced by growth factor stimulation; (ii) band shift assays and immunoprecipitations of DNA/Stat complexes showed constitutive DNA-binding properties of Stat3(sm); and (iii) Stat3(sm) was constitutively associated with Jak3. The abnormal activation of Stat3(sm) was highly specific. Thus, neither the fast migrating isoform of Stat3 (Stat3(fm)) nor other Stats (Stat1, Stat2, and Stat4 through Stat6) were constitutively activated. The Jak kinase inhibitor, tyrphostin AG490, blocked the constitutive activation of Stat3(sm) and inhibited spontaneous as well as interleukin 2-induced growth of MF tumor cells. In conclusion, we have provided evidence for an abnormal Jak/Stat signaling and growth regulation in tumor cells obtained from affected skin of an MF patient.

Cell Division↗

PubMed1997

Temporal phases in apoptosis defined by the actions of Src homology 2 domains, ceramide, Bcl-2, interleukin-1beta converting enzyme family proteases, and a dense membrane fraction.

We have begun to explore the mechanisms of apoptosis using a cell-free system based on extracts from Xenopus eggs. Nuclei assembled or placed in these extracts undergo the morphological changes typical of apoptosis and eventually disintegrate. We used this system to investigate the potential involvement in apoptosis of proteins containing Src homology 2 (SH2) domains, which are known to interact with specific tyrosine-phosphorylated ligands. SH2 domains from a number of signaling proteins, including Lck, Src, and Abl, inhibited apoptosis when present at concentrations of 10-100 nM. The inhibition was dependent on specific interaction with endogenous tyrosine-phosphorylated ligands. A synthetic peptide ligand for Src family SH2 domains also inhibited apoptosis in a phosphotyrosine-dependent manner. Kinetic analysis defined three phases in the apoptotic process occurring in this cell-free system. SH2 domains and ceramide act throughout the first 60-90 min of the process (the "initiation" phase). Next, Bcl-2, interleukin-1beta converting enzyme family(CPP32-like) proteases, and the heavy membrane fraction act in a period occurring approximately 90-120 min after the start of incubation (the "sentencing" phase). In the final phase ("execution"), the process of active nuclear destruction ensues.

Animals↗

PubMed1997

Role of Tyr518 and Tyr519 in the regulation of catalytic activity and substrate phosphorylation by Syk protein-tyrosine kinase.

The Syk protein-tyrosine kinase is expressed in many hematopoietic cells and is involved in signaling from various receptors for antigen and Fc portions of IgG and IgE. After cross-linking of these receptors, Syk is rapidly phosphorylated on tyrosine residues. We have previously reported that Syk expressed in COS cells is predominantly phosphorylated at both Tyr518 and Tyr519 at its putative autophosphorylation site. In this study, we have examined the role of each of these two residues for the catalytic activity of Syk in vitro and for the Syk-induced phosphorylation of cellular proteins in intact cells. Mutation of either residue had minor effects on the catalytic activity of Syk, and even the double mutant [F518, F519]Syk was about 60% as active as the wild-type enzyme. In intact cells, however, all three mutants consistently failed to induce the extensive tyrosine phosphorylation of cellular proteins typically observed with wild-type Syk. We have recently shown that the doubly phosphorylated Y518/Y519 site is also the site for association of Syk with the SH2 domain of the Lck kinase, which suggests that although phosphates at Y518/Y519 may enhance the catalytic activity of Syk, its interaction with Src family protein-tyrosine kinases is at least equally important for the induction of downstream substrate phosphorylation.

Amino Acid Sequence↗

PubMed1997

Involvement of phosphatidylinositol 3-kinase in NFAT activation in T cells.

Phosphatidylinositol 3-kinase (PI3-K) has been implicated in the regulation of cell proliferation in many cell types. We have previously shown that in T cells the PI3-K inhibitor, wortmannin, interferes with activation of the mitogen-activated kinase, Erk2, after T cell receptor (TcR) stimulation. To further explore the involvement of PI3-K in T cell activation, we created a set of potentially dominant negative PI3-K constructs comprising individual or tandem domains of the regulatory p85 subunit and tested their effect on downstream signaling events like Erk2 activation and transcription from an NFAT (nuclear factor of activated T cells) element taken from the interleukin-2 promoter. Following TcR stimulation, activation of Erk2 was only inhibited by a previously described truncated form of p85 that cannot bind the catalytic subunit, but not by other constructs of p85. In contrast, several mutant p85 alleles had dramatic effects on NFAT activation. Most interestingly, the N-terminal SH2 domain had an inhibitory effect, whereas a mutant p85 containing only the two SH2 domains enhanced basal NFAT activity in a Ras-dependent manner. Ionomycin induced synergistic activation of NFAT in cells expressing p85 mutants that contained the C-terminal SH2 domain. Analysis of phosphotyrosine-containing proteins bound to truncated p85 constructs revealed cooperative binding of the two SH2 domains but no apparent differences between the N- and C-terminal SH2 domains. Wortmannin did not interfere with NFAT activation, although it inhibited PI3-K and Erk2 activation in the same experiment. These results suggest that PI3-K is involved in NFAT activation through a complex adaptor function of its regulatory subunit and that its lipid kinase activity is dispensable for this effect.

DNA-Binding Proteins↗

PubMed1997

Reconstitution of T cell antigen receptor-induced Erk2 kinase activation in Lck-negative JCaM1 cells by Syk.

The two related protein-tyrosine kinases Syk and Zap are rapidly phosphorylated on tyrosine residues and enzymatically activated upon crosslinking of the T cell antigen receptor. We have previously reported that the activation of Syk is less dependent on the Src family kinase Lck than the activation of Zap. Here we report that overexpression of Syk in the Lck-negative JCaM1 cells enabled the T cell antigen receptor/CD3 complex to induce a normal activation of the mitogen-activated protein kinase (MAPK) pathway and expression of a nuclear factor of activated T cells reporter construct. In contrast, Zap and other protein-tyrosine kinases were unable to reconstitute these signaling pathways when expressed at the same levels. In parallel, Syk was phosphorylated on tyrosine, while Zap was not. The Syk-mediated T cell antigen receptor-induced MAPK activation was detectable within 1 min of receptor stimulation and peaked at 3-5 min. The capacity of Syk to reconstitute the MAPK response required the catalytic activity of Syk, an intact autophosphorylation site (Y518 and Y519), both Src homology 2 domains and it was blocked by the inhibitory N17-mutated dominant-negative Ras construct. A Y341-->F mutant of Syk, which is deficient in its interaction with phospholipase Cy1 and Vav, was less efficient than wild-type Syk. Our results suggest that Syk, in contrast to Zap, can transduce signals from the T cell antigen receptor independently of Lck.

Animals↗

PubMed1997

Differential T cell signaling induced by antagonist peptide-MHC complexes and the associated phenotypic responses.

Certain changes in TCR contact residues have been shown to have profound effects on the capacity of a peptide Ag to stimulate a T cell response. Although some of these changes apparently lead to a complete loss of the ability to interact with the TCR, others result in partial agonist activity (e.g., cytokine production without proliferation) or antagonist activity (i.e., the capacity to inhibit the engagement to the TCR by Ag). We show MHC class II-restricted antagonist activity was associated with a differential pattern of early tyrosine phosphorylation events that was characterized by a preponderance of phosphorylation of low molecular mass TCRzeta and the failure to phosphorylate Zap-70. These early tyrosine phosphorylation patterns are the same as those previously described for partial agonists. Thus, a partial agonist phenotype such as anergy induction cannot be ascribed in a causal manner to this pattern of tyrosine phosphorylation. We further extend the studies of signal transduction elicited by agonist and antagonist peptides by characterizing differential recruitment of Zap-70 associated with TCRzeta isoforms and differential phosphorylation of p120 proto-oncogene c-Cbl. Another early event following TCR engagement by Ag, down-modulation of the TCR, was studied with antagonist peptides. We show that antagonist peptides do not cause TCR down-modulation. This failure may represent a mechanism by which antagonists inhibit antigen-mediated stimulation of T cells.

Amino Acid Sequence↗

PubMed1997

Regulation of the low molecular weight phosphotyrosine phosphatase by phosphorylation at tyrosines 131 and 132.

Activation of resting T lymphocytes is initiated by rapid but transient tyrosine phosphorylation of a number of cellular proteins. Several protein tyrosine kinases and protein tyrosine phosphatases are known to be important for this response. Here we report that normal T lymphocytes express the B isoform of low molecular weight protein tyrosine phosphatase B (LMPTP-B). The cDNA was cloned from Jurkat T cells, and an antiserum was raised against it. LMPTP immunoprecipitated from resting Jurkat T cells was found to be tyrosine phosphorylated. On stimulation of the cells through their T cell antigen receptor, the phosphotyrosine content of LMPTP-B declined rapidly. In co-transfected COS cells, Lck and Fyn caused phosphorylation of LMPTP, whereas Csk, Zap, and Jak2 did not. Most of the phosphate was located at Tyr-131, and some was also located at Tyr-132. Incubation of wild-type LMPTP with Lck and adenosine 5'-O-(thiotriphosphate) caused a 2-fold increase in the activity of LMPTP. Site-directed mutagenesis showed that Tyr-131 is important for the catalytic activity of LMPTP, and that thiophosphorylation of Tyr-131, and to a lesser degree Tyr-132, is responsible for the activation.

3T3 Cells↗

PubMed1997

Bcr - Abl-mediated resistance to apoptosis is independent of PI 3-kinase activity.

The Bcr - Abl tyrosine kinase is responsible for the oncogenic phenotype observed in Philadelphia chromosome-positive leukemia and induces resistance to apoptotic cell death in a variety of cell types. Recent evidence supports the hypothesis that these two properties of Bcr - Abl are derived from cooperative but distinct signaling pathways. Phosphatidylinositol 3-kinase (PI3K), which has been suggested to associate with and become activated by Bcr - Abl, has been shown to be required for Bcr - Abl-mediated cell growth. Also, PI3K has been implicated in resistance to apoptosis induced by some growth factors. We therefore examined the role of PI 3-kinase in the anti-apoptotic effect of Bcr - Abl. First, we confirmed that expression of p185(bcr - abl) in HL-60 cells, which renders these cells resistant to apoptosis, induces tyrosine phosphorylation of the p85 subunit of PI3K. Consistent with this result, we observed a 20-fold increase in PI3K activity upon immunoprecipitation of tyrosine-phosphorylated proteins from cells expressing Bcr - Abl versus control cells. Nevertheless, treatment of HL-60.p185(bcr - abl) cells with wortmannin, a potent inhibitor of PI3K, eliminated PI3K activity but did not interfere with the resistance of these cells to apoptosis. Similar results were obtained with the CML line K562 and with the BaF3.p185 (bcr - abl) line. We conclude that while PI3K participates in the anti-apoptotic response mediated by some growth factors and also seems to be important for the growth of Bcr-Abl-positive cells, it does not play any role in Bcr - Abl-mediated resistance to apoptosis.

Journal Article↗

PubMed1997

Diminished TCR signaling in cutaneous T cell lymphoma is associated with decreased activities of Zap70, Syk and membrane-associated Csk.

Malignant cells of patients with cutaneous T cell lymphoma (CTCL) are of monoclonal origin and of the CD4+/CD45RO+ subset. Since unlike their normal counterparts, triggering of their TCR/CD3 in vitro elicits only a weak mitogenic response, we set out to determine which of the signal transduction molecules initiated by anti-CD3E antibodies are affected in neoplastic cells. The results obtained from analysis of tumor cells from four patients show a general reduction in basal and induced tyrosine phosphorylation of a wide range of signaling proteins. Furthermore, the function of members from distinct families of protein tyrosine kinases was altered in neoplastic cells. The enzymatic activity of the membrane-bound fraction of Csk was suppressed, and its association with other cellular proteins was altered. There was a decline in the amount and activity of Syk, and a slight decrease in the specific activity of Lck kinases. Zap70 tyrosyl phosphorylation was reduced or undetectable and the kinase associated weakly, or not at all, with the TCR zeta chain. We propose that dampened TCR-triggered responses in CTCL are caused by suppression of an array of effector molecules required for coupling cell surface receptors to early and late signaling events.

Adult↗

PubMed1996

Regulation of the Lck SH2 domain by tyrosine phosphorylation.

Src homology 2 (SH2) domains bind to phosphotyrosine (Tyr(P)) residues in specific sequence contexts in other proteins and thereby mediate tyrosine phosphorylationdependent protein-protein interactions. The SH2 domain of the Src family kinase Lck is phosphorylated at tyrosine 192 in T cells upon T cell antigen receptor triggering. We have studied the consequences of this phosphorylation on the properties of the SH2 domain and on the function of Lck in T cell activation. We report that phosphorylation at Tyr192 reduced the capacity of the isolated SH2 domain to bind a high affinity peptide ligand and Tyr(P)-containing cellular proteins. This effect was mimicked by mutation of Tyr192 to an acidic residue. In intact T cells, where Lck participates in T cell antigen receptor signal transduction in an SH2 domain-dependent manner, phosphorylation of Tyr192 correlated with reduced downstream signaling. Our results indicate that tyrosine phosphorylation of the SH2 domain of Lck terminates its high affinity binding to ligands, thereby negatively regulating its participation in T cell antigen receptor signaling. This represents a novel mechanism for the regulation of the function of SH2 domains.

Amino Acid Sequence↗

PubMed1996

Identification of the site in the Syk protein tyrosine kinase that binds the SH2 domain of Lck.

The Syk protein tyrosine kinase (PTK) is expressed in many hematopoietic cells and is involved in signaling from various receptors for antigen and Fc portions of IgG and IgE. Upon cross-linking of these receptors, Syk is rapidly phosphorylated on tyrosine residues and enzymatically activated. We and others have found that the Lck kinase, a member of the Src family of PTKs, binds through its Src homology (SH) 2 domain to tyrosine phosphorylated Syk and to the related Zap kinase. Here we report that this interaction is direct and identify the two tandem tyrosines at the autophosphorylation site of Syk, Tyr518, and Tyr519, as the binding site for the SH2 domain of Lck. Mutation of either or both tyrosines to phenylalanines abrogated binding, while mutation of a second repetition of the motif at Tyr539 and Tyr540, or of the three tyrosines in the C terminus of Syk, did not. The SH2 domain of Lck bound the autophosphorylation site only when both Tyr518 and Tyr519 were phosphorylated. In intact cells the binding of the SH2 domain of Lck correlated with the ability of Syk to induce tyrosine phosphorylation of cellular proteins.

Amino Acid Sequence↗