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T Jahn

Publications and source records attributed to T Jahn.

14 recordsLinked to original sources
PubMed1999

Binding of 14-3-3 protein to the plasma membrane H(+)-ATPase AHA2 involves the three C-terminal residues Tyr(946)-Thr-Val and requires phosphorylation of Thr(947).

14-3-3 proteins play a regulatory role in a diverse array of cellular functions such as apoptosis, regulation of the cell cycle, and regulation of gene transcription. The phytotoxin fusicoccin specifically induces association of virtually any 14-3-3 protein to plant plasma membrane H(+)-ATPase. The 14-3-3 binding site in the Arabidopsis plasma membrane H(+)-ATPase AHA2 was localized to the three C-terminal residues of the enzyme (Tyr(946)-Thr-Val). Binding of 14-3-3 protein to this target was induced by phosphorylation of Thr(947) (K(D) = 88 nM) and was in practice irreversible in the presence of fusicoccin (K(D) = 7 nM). Mass spectrometry analysis demonstrated that AHA2 expressed in yeast was phosphorylated at Thr(947). We conclude that the extreme end of AHA2 contains an unusual high-affinity binding site for 14-3-3 protein.

14-3-3 Proteins↗
PubMed1999

Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: mapping of residues that when altered give rise to an activated enzyme.

The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants. The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme. Alanine-scanning mutagenesis through 87 consecutive amino acid residues was used to evaluate the role of the C-terminus in autoinhibition of the plasma membrane H+-ATPase AHA2 from Arabidopsis thaliana. Mutant enzymes were expressed in a strain of Saccharomyces cerevisiae with a defective endogenous H+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP. Residues that when altered lead to increased pump activity group together in two regions of the C-terminus. One region stretches from K863 to L885 and includes two residues (Q879 and R880) that are conserved between plant and fungal H+-ATPases. The other region, incorporating S904 to L919, is situated in an extension of the C-terminus unique to plant H+-ATPases. Alteration of residues in both regions led to increased binding of yeast 14-3-3 protein to the plasma membrane of transformed cells. Taken together, our data suggest that modification of residues in two regions of the C-terminal regulatory domain exposes a latent binding site for activatory 14-3-3 proteins.

14-3-3 Proteins↗
PubMed1999

Motor dysfunction and sensorimotor cortex activation changes in schizophrenia: A study with functional magnetic resonance imaging.

Recent studies demonstrate a diminished activation of the sensorimotor cortex and supplementary motor area (SMA) in schizophrenia which may be involved in the pathogenesis of neurological soft signs (NSS). Yet, the question whether a retarded motor performance may account for these changes remained to be clarified. Twelve DSM-III-R schizophrenics and 12 healthy controls were included. All subjects were right-handed. Nine patients received clozapine, two conventional neuroleptics, and one was drug-free. Functional magnetic resonance imaging (fMRI) was obtained in a resting condition and during pronation/supination at three speed levels (low, medium, and high) with motor performance recorded simultaneously using a pronation/supination device. While measures of motor retardation (i.e., repetition rate and amplitude of the movements) did not differ between patients and controls, the variability of performance was significantly (P < 0.05) increased in the patients' group. In addition, patients with schizophrenia showed a significantly (P < 0.05) decreased activation of the sensorimotor cortices. Similar, although nonsignificant (P = 0.09) activation changes were observed in the SMA. Activation differences were more pronounced at a slow speed and in the drug-free patient. These results confirm a diminished sensorimotor cortex and SMA activation and indicate that variability of performance rather than retarded performance per se may correspond to these changes.

Adult↗
PubMed1998

The SH2-containing adapter protein GRB10 interacts with BCR-ABL.

Bcr-Abl is an oncogenic tyrosine kinase expressed in tumor cells of CML and a subset of ALL which in its unregulated and activated state is thought to cause cell transformation and leukemia. Bcr-Abl contains several autophosphorylation sites which serve as potential docking sites for SH2-containing signaling molecules. Mutational analysis has indicated that these autophosphorylation sites play a critical role in the transforming capability of Bcr-Abl. It has been shown that the SH2-containing adapter protein Grb2 binds to the autophosphorylation site Tyr(p)177 whereby it couples Bcr-Abl to the Ras pathway. The biological consequences of this interaction, however, are presently unclear. A Tyr177-mutated Bcr-Abl which lacks the ability to interact with the Grb2-SH2 domain still transforms myeloid cells and generates tumors in nude mice. We performed a yeast two-hybrid screen to identify signaling proteins which bind to distinct Bcr-Abl autophosphorylation sites. Autophosphorylation of Bcr-Abl in yeast was accomplished by using the DNA binding protein LexA which permits dimerization and crossphosphorylation of the fused bait. Using a LexA-Bcr-Abl full length fusion protein as bait, we identified several SH2-containing proteins. Among them we confirmed molecules already shown by others to interact with Bcr-Abl, in vivo, including Grb2, PI-3-kinase and Crk indicating that dimerization in yeast leads to autophosphorylation of tyrosine residues crucial for Bcr-Abl signaling in vivo. More importantly, we identified the SH2-containing protein Grb10 as a new binding partner for Bcr-Abl. This binding occurs in a phosphotyrosine-dependent manner at Bcr sites of Bcr-Abl. Both Abl and Bcr alone, as well as a kinase-defective Bcr-Abl, failed to interact with Grb10 in yeast. Mutational analysis uncovered a new SH2 binding site in Bcr-Abl located between Bcr aa242-446, which is different from the Grb2 binding site. Binding could be demonstrated in vitro and also in vivo as shown by co-immunoprecipitation analysis in CML cells. Using a temperature sensitive Bcr-Abl stably overexpressed in hematopoetic cells, we demonstrated that complex formation of Grb10 with Bcr-Abl was kinase activation-dependent in vivo. Notably, a Bcr-Abl mutant protein (Bcr/1-242-Abl) which lacks the ability to interact with Grb10 partially alleviated IL-3 dependence of Ba/F3 cells, indicating that the Grb10/Bcr-Abl interaction is important for Bcr-Abl-induced IL-3 independence of Ba/F3 cells. In addition, the Bcr/1-242-Abl mutant has a reduced capacity to induce focus formation in fibroblasts.

Absorption↗
PubMed1998

The 14-3-3 proteins associate with the plant plasma membrane H(+)-ATPase to generate a fusicoccin binding complex and a fusicoccin responsive system.

The plasma membrane H(+)-ATPase in higher plants has been implicated in nutrient uptake, phloem loading, elongation growth and establishment of turgor. Although a C-terminal regulatory domain has been identified, little is known about the physiological factors involved in controlling the activity of the enzyme. To identify components which play a role in the regulation of the plant H(+)-ATPase, a fusicoccin responsive yeast expressing Arabidopsis plasma membrane H(+)-ATPase AHA2 was employed. By testing the fusicoccin binding activity of yeast membranes, the C-terminal regulatory domain of AHA2 was found to be part of a functional fusicoccin receptor, a component of which was the 14-3-3 protein. ATP hydrolytic activity of AHA2 expressed in yeast internal membranes was activated by all tested isoforms of the 14-3-3 protein of yeast and Arabidopsis, but only in the presence of fusicoccin, and activation was prevented by a phosphoserine peptide representing a known 14-3-3 protein binding motif in Raf-1. The results demonstrate that the 14-3-3 protein is an activator molecule of the H(+)-ATPase and provides the first evidence of a protein involved in activation of plant plasma membrane H(+)-ATPase.

14-3-3 Proteins↗
PubMed1997

Interleukin-4 signaling in B lymphocytes from patients with X-linked severe combined immunodeficiency.

Interleukin-4 (IL-4) is an important cytokine for B and T lymphocyte function and mediates its effects via a receptor that contains gammac. B cells derived from patients with X-linked severe combined immunodeficiency (X-SCID) are deficient in gammac and provide a useful model in which to dissect the role of this subunit in IL-4-mediated signaling. We found that although IL-4 stimulation of X-SCID B cells did not result in Janus tyrosine kinase-3 (JAK3) phosphorylation, other IL-4 substrates including JAK1 and IRS-1 were phosphorylated. Additionally, we detected signal transducers and activators of transcription 6 (STAT6) tyrosine phosphorylation and DNA binding activity in X-SCID B cells with a wide range of gammac mutations. However, reconstitution of these X-SCID B cells with gammac enhanced IL-4-mediated responses including STAT6 phosphorylation and DNA binding activity and resulted in increased CD23 expression. Thus, gammac is not necessary to trigger IL-4-mediated responses in B cells, but its presence is important for optimal IL-4-signaling. These results suggest that two distinct IL-4 signaling pathways exist.

B-Lymphocytes↗
PubMed1997

Differential activation of the tyrosine kinases ZAP-70 and Syk after Fc gamma RI stimulation.

Engagement of the high-affinity IgG Fc receptor (Fc gamma RI) activates a signal transduction pathway involving tyrosine phosphorylation of associated kinases. We compared the activation of the related protein tyrosine kinases (PTKs), Syk and ZAP-70, in Fc gamma RI-mediated signaling. Cross-linking of the Fc gamma RI multimeric receptor in monocytic cells results in tyrosine phosphorylation of the Fc epsilon RI gamma subunit and association of Syk with this complex. We stably introduced ZAP-70 via a retroviral vector into two monocytic cell lines, U937 and THP-1, which normally do not express ZAP-70. Neither Syk nor MAP kinase activation was affected by the presence of ZAP-70. Although transduced ZAP-70 had in vitro kinase activity and associated with Fc epsilon RI gamma after receptor aggregation, it was not tyrosine phosphorylated. In contrast, both ZAP-70 and Syk were phosphorylated in a T-cell line in which their respective levels of expression were similar to those detected in U937/ZAP-70 cells. Therefore, these results suggest that requirements for Syk and ZAP-70 phosphorylation are distinct in a monocytic cell context.

Cell Line↗
PubMed1997

The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase.

Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H(+)-ATPase isolated from fusicoccin-treated maize shoots was copurified with the 14-3-3 protein (as determined by protein gel blotting), and the H(+)-ATPase was recovered in an activated state. In the absence of fusicoccin treatment, H(+)-ATPase and the 14-3-3 protein were well separated, and the H(+)-ATPase was recovered in a nonactivated form. Trypsin treatment removed the 10-kD C-terminal region from the H(+)-ATPase as well as the 14-3-3 protein. Using the yeast two-hybrid system, we could show a direct interaction between Arabidopsis 14-3-3 GF14-phi and the last 98 C-terminal amino acids of the Arabidopsis AHA2 plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase.

14-3-3 Proteins↗
PubMed1996

Reconstitution of T cell receptor signaling in ZAP-70-deficient cells by retroviral transduction of the ZAP-70 gene.

A variant of severe combined immunodeficiency syndrome (SCID) with a selective inability to produce CD8 single positive T cells and a signal transduction defect in peripheral CD4+ cells has recently been shown to be the result of mutations in the ZAP-70 gene. T cell receptor (TCR) signaling requires the association of the ZAP-70 protein tyrosine kinase with the TCR complex. Human T cell leukemia virus type I-transformed CD4+ T cell lines were established from ZAP-70-deficient patients and normal controls. ZAP-70 was expressed and appropriately phosphorylated in normal T cell lines after TCR engagement, but was not detected in T cell lines from ZAP-70-deficient patients. To determine whether signaling could be reconstituted, wild-type ZAP-70 was introduced into deficient cells with a ZAP-70 retroviral vector. High titer producer clones expressing ZAP-70 were generated in the Gibbon ape leukemia virus packaging line PG13. After transduction, ZAP-70 was detected at levels equivalent to those observed in normal cells, and was appropriately phosphorylated on tyrosine after receptor engagement. The kinase activity of ZAP-70 in the reconstituted cells was also appropriately upregulated by receptor aggregation. Moreover, normal and transduced cells, but not ZAP-70-deficient cells, were able to mobilize calcium after receptor ligation, indicating that proximal TCR signaling was reconstituted. These results indicate that this form of SCID may be corrected by gene therapy.

CD4-Positive T-Lymphocytes↗
PubMed1996

Correction of interleukin-2 receptor function in X-SCID lymphoblastoid cells by retrovirally mediated transfer of the gamma-c gene.

X-SCID, the most common form of human SCID, is due to mutations in the common gamma chain gene (gamma-c) that encodes an essential component of the cytokine receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. Activation of the Janus family tyrosine kinases Jak1 and Jak3 is necessary for appropriate signalling through the IL-2 receptor (IL-2R). Neither Jak1 nor Jak3 was phosphorylated after IL-2 stimulation of an Epstein-Barr virus-transformed cell line (LCL) from an X-SCID patient with a gamma-c null mutation. However, we now show that appropriate IL-2R function can be restored in an X-SCID LCL by transduction of a wild-type gamma-c gene. A retroviral vector, G1gamma-cSvNa, was constructed and produced in the PG13 packaging line. Transduced X-SCID LCL expressed the G1gamma-cSvNa transcript. IL-2 stimulation of the transduced cell line resulted in appropriate tyrosine phosphorylation of both Jak1 and Jak3. Thus, retroviral-mediated transduction of normal gamma-c can reconstitute downstream signalling through the IL-2R in X-SCID cell lines, suggesting that gene therapy may be a treatment for this disease.

B-Lymphocytes↗
PubMed1996

Monitoring of task performance during functional magnetic resonance imaging of sensorimotor cortex at 1.5 T.

Functional magnetic resonance imaging (fMRI) has found widespread clinical interest. Difficulties in clinical use of the fMRI technique arise, considering the lack of knowledge about activation task performance. This accounts especially for sensorimotor activation studies, in which performance of the sensorimotor activation task is-if at all-usually rated visually by subjective or semiquantitative methods (i.e., defining categories of performance such as neurological soft signs scales). Recently, instrumental methods for the measurement and analysis of motor performance have been developed. Pronation/supination (hand rotation) movement was shown to be an easily measurable and promising motor task. We have adapted a mechanic device (pronation/supination device, PSD) to monitor motor performance during the fMRI experiment. In a feasibility study, an investigation of fMRI activation strength dependence of sensorimotor cortices and supplementary motor area upon task frequency (25, 50, and 75 cycles/min) was carried out on 10 right-handed healthy volunteers. Furthermore, the authors report the observation of stimulus-induced activation changes in the cerebellum during pronation/supination movement.

Hand↗
PubMed1993

Relationship between crossover and modality shift effects in sequential reaction time performance of schizophrenic patients.

On the basis of reaction time measurement, two well-known experimental indicators of attentional dysfunctions in schizophrenia were studied. These are the crossover effect (COE), induced by alternating preparatory intervals, and the modality shift effect (MSE), induced by alternating stimulus modalities. For the first time, the relationship between these indicators was examined. Correlations of both indicators with clinical variables such as psychopathology, subjective complaints, duration of illness and medication were also analyzed. Subjects were 50 schizophrenic inpatients and 50 healthy controls, 21 women and 29 men in each group. While earlier results for COE and MSE were replicable, no correlation between the two was found, leading to the conclusion that they involve different aspects of attention not necessarily impaired to the same extent in all patients.

Adolescent↗