Stefanie Kliche - Academia.edu (original) (raw)

Papers by Stefanie Kliche

Scientific Reports, 2015

Palmitoylation is a reversible post-translational modification used to inducibly compartmentalize... more Palmitoylation is a reversible post-translational modification used to inducibly compartmentalize proteins in cellular membranes, affecting the function of receptors and intracellular signaling proteins. The identification of protein "palmitomes" in several cell lines raises the question to what extent this modification is conserved in primary cells. Here we use primary T cells with acyl-biotin exchange and quantitative mass spectrometry to identify a pool of proteins previously unreported as palmitoylated in vivo.

Journal of virology, 1998

In patients with Kaposi's sarcoma (KS), human herpesvirus 8 (HHV-8) can invariably be detecte... more In patients with Kaposi's sarcoma (KS), human herpesvirus 8 (HHV-8) can invariably be detected in KS tumor tissue and, at a lower frequency, in prostate tissue and peripheral blood B lymphocytes. Whereas the majority of KS spindle cells are latently infected by HHV-8, linear HHV-8 genomes characteristic for lytic infection are found predominantly in the peripheral blood cells of KS patients. In this study, we show that HHV-8 can stably infect B lymphocytes in vitro in the presence of Epstein-Barr virus (EBV). We were able to generate immortalized HHV-8(+)/EBV+ lymphoblastoid cell lines (LCLs) derived from peripheral blood mononuclear cells (PBMC) of EBV- and EBV+ donors. In HHV-8(+)/EBV+ LCLs, which have the phenotype of activated B lymphocytes (CD19(+), surface immunoglobulin M, CD23(+), CD30(+), CD80(+)), HHV-8 was still present after more than 25 passages (more than 9 months of culture). Latent viral transcripts and proteins were present in nonstimulated HHV-8(+)/EBV+ LCLs. A...

Results and Problems in Cell Differentiation, 2006

Signal exchange between cells is a key feature of life from humble monads to human beings. Approp... more Signal exchange between cells is a key feature of life from humble monads to human beings. Appropriate communication is of particular importance between cells of multi-cellular organisms. Various basic mechanisms of cellcell communication have evolved during phylogenesis, which were subject to organ, tissue and cell type-specific adaptation. These mechanisms range from long-distance communication via hormones to more and more local processes, e.g. via cytokines, chemokines or neuromodulators/neurotransmitters, and eventually direct physical interactions of molecules anchored at cell surfaces. Accordingly, highly specialized transient or stable cell-cell contact sites have evolved that mediate signaling between cells. With few exceptions (e.g. lipophilic hormones, gases) intercellular communication depends on specific signal detection devices at the cell surface coupled to a signal transduction apparatus that mediates the signal transfer across the cell membrane and activates intracellular effector systems, which generate intracellularly decipherable signals.

Critical Reviews in Immunology, 2014

Adaptor proteins mediate protein-protein interactions in signal transduction cascades. These sign... more Adaptor proteins mediate protein-protein interactions in signal transduction cascades. These signaling molecules are organized in multimolecular complexes that translate information from cell surface receptors into cellular responses. The cytosolic adhesion- and degranulation-promoting adaptor protein (ADAP) is expressed in T cells, natural killer cells, myeloid cells, and platelets. Here we summarize the data about the function of ADAP in these cells with respect to their contribution to the pathogenesis of experimental autoimmune encephalomyelitis. We discuss possible mechanisms of strongly attenuated experimental autoimmune encephalomyelitis in ADAP-deficient mice.

Molecular and Cellular Biology, 2007

One outcome of T-cell receptor (TCR) signaling is increased affinity and avidity of integrins for... more One outcome of T-cell receptor (TCR) signaling is increased affinity and avidity of integrins for their ligands. This occurs through a process known as inside-out signaling, which has been shown to require several molecular components including the adapter proteins ADAP (adhesion and degranulation-promoting adapter protein) and SKAP-55 (55-kDa src kinase-associated phosphoprotein) and the small GTPase Rap1. Herein, we provide evidence

Lymphocyte membrane rafts contain molecules critical for immunoreceptor signaling. Here, we repor... more Lymphocyte membrane rafts contain molecules critical for immunoreceptor signaling. Here, we report identification of a new raft-associated adaptor protein LIME (Lck-interacting molecule) expressed predominantly in T lymphocytes. LIME becomes tyrosine phosphorylated after cross- linking of the CD4 or CD8 coreceptors. Phospho-LIME associates with the Src family kinase Lck and its negative regulator, Csk. Ectopic expression of LIME in Jurkat T

Seminars in Immunology, 2004

Transmembrane adapter proteins (TRAPs) represent a relatively new and unique group of signalling ... more Transmembrane adapter proteins (TRAPs) represent a relatively new and unique group of signalling molecules in hematopoetic cells. They differ from other signalling proteins as they lack any enzymatic or transcriptional activity, instead they possesses multiple tyrosine-based signalling motifs (TBSMs). Triggering of immunoreceptors induces tyrosine phosphorylation of these motifs by members of the Src-, Syk-or Tec-family of protein tyrosine kinases thus enabling the TRAPs to recruit cytosolic adapter and/or effector molecules via their SH2-domains into close proximity to the immunoreceptors, a position from which they can coordinate and modulate signal transduction pathways important for lymphocyte function.

Journal of Molecular Biology - J MOL BIOL, 2006

SH3 domains represent versatile scaffolds within eukaryotic cells by targeting proline-rich seque... more SH3 domains represent versatile scaffolds within eukaryotic cells by targeting proline-rich sequences within intracellular proteins. More recently, binding of SH3 domains to unusual peptide motifs, folded proteins or lipids has been reported. Here we show that the newly defined hSH3 domains of immune cell adapter proteins bind lipid membranes with distinct affinities. The interaction of the hSH3 domains of adhesion and degranulation promoting adapter protein (ADAP) and PRAM-1 (Promyelocytic-Retinoic acid receptor alpha target gene encoding an Adaptor Molecule-1), with phosphatidylcholine-containing liposomes is observed upon incorporation of phosphatidylserine (PS) or phosphoinositides (PIs) into the membrane bilayer. Mechanistically we show that stable association of the N-terminal, amphipathic helix with the β-sheet scaffold favours lipid binding and that the interaction with PI(4,5)P2-containing liposomes is consistent with a single-site, non-cooperative binding mechanism. Functi...

PLoS ONE, 2010

ABSTRACT [This corrects the article on p. e11708 in vol. 5.].

PLoS ONE, 2010

TCR stimulation leads to an increase in cellular adhesion among other outcomes. The adhesion and ... more TCR stimulation leads to an increase in cellular adhesion among other outcomes. The adhesion and degranulation promoting adapter protein (ADAP) is known to be rapidly phosphorylated after T cell stimulation and relays the TCR signal to adhesion molecules of the integrin family. While three tyrosine phosphorylation sites have been characterized biochemically, the binding capabilities and associated functions of several other potential phosphotyrosine motifs remain unclear. Here, we utilize in vitro phosphorylation and mass spectrometry to map novel phosphotyrosine sites in the Cterminal part of human ADAP (486-783). Individual tyrosines were then mutated to phenylalanine and their relevance for cellular adhesion and migration was tested experimentally. Functionally important tyrosine residues include two sites within the folded hSH3 domains of ADAP and two at the C-terminus. Furthermore, using a peptide pulldown approach in combination with stable isotope labeling in cell culture (SILAC) we identified SLP-76, PLCc, PIK3R1, Nck, CRK, Gads, and RasGAP as phospho-dependent binding partners of a central YDDV motif of ADAP. The phosphorylation-dependent interaction between ADAP and Nck was confirmed by yeast two-hybrid analysis, immunoprecipitation and binary pulldown experiments, indicating that ADAP directly links integrins to modulators of the cytoskeleton independent of SLP-76.

PLoS ONE, 2008

Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation ... more Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation of the TCR/CD3complex. In rodents these reagents induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. Unexpectedly, the humanized CD28 superagonist TGN1412 caused severe and life threatening adverse effects during a recently conducted phase I clinical trail. The underlying molecular mechanisms are as yet unclear. We show that TGN1412 as well as the commercially available CD28 superagonist ANC28.1 induce a delayed but extremely sustained calcium response in human naïve and memory CD4 + T cells but not in cynomolgus T lymphocytes. The sustained Ca ++-signal was associated with the activation of multiple intracellular signaling pathways and together these events culminated in the rapid de novo synthesis of high amounts of pro-inflammatory cytokines, most notably IFN-c and TNF-a. Importantly, sustained transmembranous calcium flux, activation of Src-kinases as well as activation of PI3K were found to be absolutely required for CD28SA-mediated production of IFN-c and IL-2. Collectively, our data suggest a molecular basis for the severe side effects caused by TGN1412 and impinge upon the relevance of non-human primates as preclinical models for reagents that are supposed to modify the function of human T cells.

Oncogene, 2005

Constitutive expression of cyclin D1 is a frequent abnormality in human cancer and sustains the t... more Constitutive expression of cyclin D1 is a frequent abnormality in human cancer and sustains the transformed phenotype. We have previously demonstrated that cyclin D1 is constitutively expressed in human BON neuroendocrine tumour cells due to an autocrine insulinlike growth factor-I (IGF-I) loop. Here we examine the regulation of cyclin D1 expression by endogenously released IGF-I in BON cells. Cyclin D1 expression in these cells was found to be dependent on phosphatidylinositol 3-kinase (PI3-K), but independent of the extracellular signal-regulated kinase cascade. Ras-and Rac-GTPases were found to be upstream activators of cyclin D1 expression, whereas protein kinase B/AKT and nuclear factor kappa B (NFjB) could be established as downstream mediators of cyclin D1 transcription in response to endogenously released IGF-I in these cells. In addition, the Ras/PI3-K/AKT/Rac/NFjB/cyclin D1 signaling cascade triggered by endogenously released IGF-I is sufficient to sustain Rb phosphorylation and cdk4 kinase activity in BON cells. In conclusion, our data provide the first comprehensive map of the signaling events elicited by endogenously released IGF-I leading to constitutive cyclin D1 expression in human neuroendocrine tumour cells.

Molecular Immunology, 2014

Molecular Immunology, 2004

Molecular Cell, 2001

The induction of a transformed cellular phenotype by viruses requires the modulation of signaling... more The induction of a transformed cellular phenotype by viruses requires the modulation of signaling pathways through viral proteins. We show here that the phenotypic changes induced by the kaposin A protein of human herpesvirus 8 are mediated through its direct interaction with cytohesin-1, a guanine nucleotide exchange factor for ARF GTPases and regulator of integrin-mediated cell adhesion. Focus formation, stress fiber dissolution, and activation of the ERK-1/2 MAP kinase signal cascade were reverted by the cytohesin-1 E157K mutant, which is deficient in catalyzing guanine nucleotide exchange. Furthermore, liposome-embedded kaposin A specifically stimulates cytohesin-1 dependent GTP binding of myristoylated ARF1 in vitro. These results suggest a previously unknown involvement of ARF GTPases in the control of cellular functions by herpesviruses.

Molecular and Cellular Biology, 2006

have collectively been termed inside-out signaling. The molecular basis for inside-out signaling ... more have collectively been termed inside-out signaling. The molecular basis for inside-out signaling is not yet completely understood. Here, we show that a signaling module comprising the cytosolic adapter proteins ADAP and SKAP55 is involved in TCR-mediated inside-out signaling and, moreover, that the interaction between ADAP and SKAP55 is mandatory for integrin activation. Disruption of the ADAP/SKAP55 module leads to displacement of the small GTPase Rap1 from the plasma membrane without influencing its GTPase activity. These findings suggest that the ADAP/SKAP55 complex serves to recruit activated Rap1 to the plasma membrane. In line with this hypothesis is the finding that membrane targeting of the ADAP/SKAP55 module induces T-cell adhesion in the absence of TCR-mediated stimuli. However, it appears as if the ADAP/SKAP55 module can exert its signaling function outside of the classical raft fraction of the cell membrane.

Molecular and Cellular Biology, 2006

The transmembrane adaptor molecule TRIM is strongly expressed within thymus and in peripheral CD4... more The transmembrane adaptor molecule TRIM is strongly expressed within thymus and in peripheral CD4 ؉ T cells. Previous studies suggested that TRIM is an integral component of the T-cell receptor (TCR)/CD3 complex and might be involved in regulating TCR cycling. To elucidate the in vivo function of TRIM, we generated TRIM-deficient mice by homologous recombination. TRIM ؊/؊ mice develop normally and are healthy and fertile. However, the animals show a mild reduction in body weight that appears to be due to a decrease in the size and/or cellularity of many organs. The morphology and anatomy of nonlymphoid as well as primary and secondary lymphoid organs is normal. The frequency of thymocyte and peripheral T-cell subsets does not differ from control littermates. In addition, a detailed analysis of lymphocyte development revealed that TRIM is not required for either positive or negative selection. Although TRIM ؊/؊ CD4 ؉ T cells showed an augmented phosphorylation of the serine/threonine kinase Akt, the in vitro characterization of peripheral T cells indicated that proliferation, survival, activation-induced cell death, migration, adhesion, TCR internalization and recycling, TCR-mediated calcium fluxes, tyrosine phosphorylation, and mitogenactivated protein family kinase activation are not affected in the absence of TRIM. Similarly, the in vivo immune response to T-dependent and T-independent antigens as well as the clinical course of experimental autoimmune encephalomyelitis, a complex Th1-mediated autoimmune model, is comparable to that of wildtype animals. Collectively, these results demonstrate that TRIM is dispensable for T-cell development and peripheral immune functions. The lack of an evident phenotype could indicate that TRIM shares redundant functions with other transmembrane adaptors involved in regulating the immune response.

Molecular and Cellular Biology, 2005

SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family prot... more SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family protein tyrosine kinase Fyn. Previously, several groups have provided experimental evidence that SKAP-HOM (most likely in cooperation with the cytosolic adaptor protein ADAP) is involved in regulating leukocyte adhesion. To further assess the physiological role of SKAP-HOM, we investigated the immune system of SKAP-HOMdeficient mice. Our data show that T-cell responses towards a variety of stimuli are unaffected in the absence of SKAP-HOM. Similarly, B-cell receptor (BCR)-mediated total tyrosine phosphorylation and phosphorylation of Erk, p38, and JNK, as well as immunoreceptor-mediated Ca 2؉ responses, are normal in SKAP-HOM ؊/؊

Scientific Reports, 2015

Palmitoylation is a reversible post-translational modification used to inducibly compartmentalize... more Palmitoylation is a reversible post-translational modification used to inducibly compartmentalize proteins in cellular membranes, affecting the function of receptors and intracellular signaling proteins. The identification of protein "palmitomes" in several cell lines raises the question to what extent this modification is conserved in primary cells. Here we use primary T cells with acyl-biotin exchange and quantitative mass spectrometry to identify a pool of proteins previously unreported as palmitoylated in vivo.

Journal of virology, 1998

In patients with Kaposi's sarcoma (KS), human herpesvirus 8 (HHV-8) can invariably be detecte... more In patients with Kaposi's sarcoma (KS), human herpesvirus 8 (HHV-8) can invariably be detected in KS tumor tissue and, at a lower frequency, in prostate tissue and peripheral blood B lymphocytes. Whereas the majority of KS spindle cells are latently infected by HHV-8, linear HHV-8 genomes characteristic for lytic infection are found predominantly in the peripheral blood cells of KS patients. In this study, we show that HHV-8 can stably infect B lymphocytes in vitro in the presence of Epstein-Barr virus (EBV). We were able to generate immortalized HHV-8(+)/EBV+ lymphoblastoid cell lines (LCLs) derived from peripheral blood mononuclear cells (PBMC) of EBV- and EBV+ donors. In HHV-8(+)/EBV+ LCLs, which have the phenotype of activated B lymphocytes (CD19(+), surface immunoglobulin M, CD23(+), CD30(+), CD80(+)), HHV-8 was still present after more than 25 passages (more than 9 months of culture). Latent viral transcripts and proteins were present in nonstimulated HHV-8(+)/EBV+ LCLs. A...

Results and Problems in Cell Differentiation, 2006

Signal exchange between cells is a key feature of life from humble monads to human beings. Approp... more Signal exchange between cells is a key feature of life from humble monads to human beings. Appropriate communication is of particular importance between cells of multi-cellular organisms. Various basic mechanisms of cellcell communication have evolved during phylogenesis, which were subject to organ, tissue and cell type-specific adaptation. These mechanisms range from long-distance communication via hormones to more and more local processes, e.g. via cytokines, chemokines or neuromodulators/neurotransmitters, and eventually direct physical interactions of molecules anchored at cell surfaces. Accordingly, highly specialized transient or stable cell-cell contact sites have evolved that mediate signaling between cells. With few exceptions (e.g. lipophilic hormones, gases) intercellular communication depends on specific signal detection devices at the cell surface coupled to a signal transduction apparatus that mediates the signal transfer across the cell membrane and activates intracellular effector systems, which generate intracellularly decipherable signals.

Critical Reviews in Immunology, 2014

Adaptor proteins mediate protein-protein interactions in signal transduction cascades. These sign... more Adaptor proteins mediate protein-protein interactions in signal transduction cascades. These signaling molecules are organized in multimolecular complexes that translate information from cell surface receptors into cellular responses. The cytosolic adhesion- and degranulation-promoting adaptor protein (ADAP) is expressed in T cells, natural killer cells, myeloid cells, and platelets. Here we summarize the data about the function of ADAP in these cells with respect to their contribution to the pathogenesis of experimental autoimmune encephalomyelitis. We discuss possible mechanisms of strongly attenuated experimental autoimmune encephalomyelitis in ADAP-deficient mice.

Molecular and Cellular Biology, 2007

One outcome of T-cell receptor (TCR) signaling is increased affinity and avidity of integrins for... more One outcome of T-cell receptor (TCR) signaling is increased affinity and avidity of integrins for their ligands. This occurs through a process known as inside-out signaling, which has been shown to require several molecular components including the adapter proteins ADAP (adhesion and degranulation-promoting adapter protein) and SKAP-55 (55-kDa src kinase-associated phosphoprotein) and the small GTPase Rap1. Herein, we provide evidence

Lymphocyte membrane rafts contain molecules critical for immunoreceptor signaling. Here, we repor... more Lymphocyte membrane rafts contain molecules critical for immunoreceptor signaling. Here, we report identification of a new raft-associated adaptor protein LIME (Lck-interacting molecule) expressed predominantly in T lymphocytes. LIME becomes tyrosine phosphorylated after cross- linking of the CD4 or CD8 coreceptors. Phospho-LIME associates with the Src family kinase Lck and its negative regulator, Csk. Ectopic expression of LIME in Jurkat T

Seminars in Immunology, 2004

Transmembrane adapter proteins (TRAPs) represent a relatively new and unique group of signalling ... more Transmembrane adapter proteins (TRAPs) represent a relatively new and unique group of signalling molecules in hematopoetic cells. They differ from other signalling proteins as they lack any enzymatic or transcriptional activity, instead they possesses multiple tyrosine-based signalling motifs (TBSMs). Triggering of immunoreceptors induces tyrosine phosphorylation of these motifs by members of the Src-, Syk-or Tec-family of protein tyrosine kinases thus enabling the TRAPs to recruit cytosolic adapter and/or effector molecules via their SH2-domains into close proximity to the immunoreceptors, a position from which they can coordinate and modulate signal transduction pathways important for lymphocyte function.

Journal of Molecular Biology - J MOL BIOL, 2006

SH3 domains represent versatile scaffolds within eukaryotic cells by targeting proline-rich seque... more SH3 domains represent versatile scaffolds within eukaryotic cells by targeting proline-rich sequences within intracellular proteins. More recently, binding of SH3 domains to unusual peptide motifs, folded proteins or lipids has been reported. Here we show that the newly defined hSH3 domains of immune cell adapter proteins bind lipid membranes with distinct affinities. The interaction of the hSH3 domains of adhesion and degranulation promoting adapter protein (ADAP) and PRAM-1 (Promyelocytic-Retinoic acid receptor alpha target gene encoding an Adaptor Molecule-1), with phosphatidylcholine-containing liposomes is observed upon incorporation of phosphatidylserine (PS) or phosphoinositides (PIs) into the membrane bilayer. Mechanistically we show that stable association of the N-terminal, amphipathic helix with the β-sheet scaffold favours lipid binding and that the interaction with PI(4,5)P2-containing liposomes is consistent with a single-site, non-cooperative binding mechanism. Functi...

PLoS ONE, 2010

ABSTRACT [This corrects the article on p. e11708 in vol. 5.].

PLoS ONE, 2010

TCR stimulation leads to an increase in cellular adhesion among other outcomes. The adhesion and ... more TCR stimulation leads to an increase in cellular adhesion among other outcomes. The adhesion and degranulation promoting adapter protein (ADAP) is known to be rapidly phosphorylated after T cell stimulation and relays the TCR signal to adhesion molecules of the integrin family. While three tyrosine phosphorylation sites have been characterized biochemically, the binding capabilities and associated functions of several other potential phosphotyrosine motifs remain unclear. Here, we utilize in vitro phosphorylation and mass spectrometry to map novel phosphotyrosine sites in the Cterminal part of human ADAP (486-783). Individual tyrosines were then mutated to phenylalanine and their relevance for cellular adhesion and migration was tested experimentally. Functionally important tyrosine residues include two sites within the folded hSH3 domains of ADAP and two at the C-terminus. Furthermore, using a peptide pulldown approach in combination with stable isotope labeling in cell culture (SILAC) we identified SLP-76, PLCc, PIK3R1, Nck, CRK, Gads, and RasGAP as phospho-dependent binding partners of a central YDDV motif of ADAP. The phosphorylation-dependent interaction between ADAP and Nck was confirmed by yeast two-hybrid analysis, immunoprecipitation and binary pulldown experiments, indicating that ADAP directly links integrins to modulators of the cytoskeleton independent of SLP-76.

PLoS ONE, 2008

Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation ... more Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation of the TCR/CD3complex. In rodents these reagents induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. Unexpectedly, the humanized CD28 superagonist TGN1412 caused severe and life threatening adverse effects during a recently conducted phase I clinical trail. The underlying molecular mechanisms are as yet unclear. We show that TGN1412 as well as the commercially available CD28 superagonist ANC28.1 induce a delayed but extremely sustained calcium response in human naïve and memory CD4 + T cells but not in cynomolgus T lymphocytes. The sustained Ca ++-signal was associated with the activation of multiple intracellular signaling pathways and together these events culminated in the rapid de novo synthesis of high amounts of pro-inflammatory cytokines, most notably IFN-c and TNF-a. Importantly, sustained transmembranous calcium flux, activation of Src-kinases as well as activation of PI3K were found to be absolutely required for CD28SA-mediated production of IFN-c and IL-2. Collectively, our data suggest a molecular basis for the severe side effects caused by TGN1412 and impinge upon the relevance of non-human primates as preclinical models for reagents that are supposed to modify the function of human T cells.

Oncogene, 2005

Constitutive expression of cyclin D1 is a frequent abnormality in human cancer and sustains the t... more Constitutive expression of cyclin D1 is a frequent abnormality in human cancer and sustains the transformed phenotype. We have previously demonstrated that cyclin D1 is constitutively expressed in human BON neuroendocrine tumour cells due to an autocrine insulinlike growth factor-I (IGF-I) loop. Here we examine the regulation of cyclin D1 expression by endogenously released IGF-I in BON cells. Cyclin D1 expression in these cells was found to be dependent on phosphatidylinositol 3-kinase (PI3-K), but independent of the extracellular signal-regulated kinase cascade. Ras-and Rac-GTPases were found to be upstream activators of cyclin D1 expression, whereas protein kinase B/AKT and nuclear factor kappa B (NFjB) could be established as downstream mediators of cyclin D1 transcription in response to endogenously released IGF-I in these cells. In addition, the Ras/PI3-K/AKT/Rac/NFjB/cyclin D1 signaling cascade triggered by endogenously released IGF-I is sufficient to sustain Rb phosphorylation and cdk4 kinase activity in BON cells. In conclusion, our data provide the first comprehensive map of the signaling events elicited by endogenously released IGF-I leading to constitutive cyclin D1 expression in human neuroendocrine tumour cells.

Molecular Immunology, 2014

Molecular Immunology, 2004

Molecular Cell, 2001

The induction of a transformed cellular phenotype by viruses requires the modulation of signaling... more The induction of a transformed cellular phenotype by viruses requires the modulation of signaling pathways through viral proteins. We show here that the phenotypic changes induced by the kaposin A protein of human herpesvirus 8 are mediated through its direct interaction with cytohesin-1, a guanine nucleotide exchange factor for ARF GTPases and regulator of integrin-mediated cell adhesion. Focus formation, stress fiber dissolution, and activation of the ERK-1/2 MAP kinase signal cascade were reverted by the cytohesin-1 E157K mutant, which is deficient in catalyzing guanine nucleotide exchange. Furthermore, liposome-embedded kaposin A specifically stimulates cytohesin-1 dependent GTP binding of myristoylated ARF1 in vitro. These results suggest a previously unknown involvement of ARF GTPases in the control of cellular functions by herpesviruses.

Molecular and Cellular Biology, 2006

have collectively been termed inside-out signaling. The molecular basis for inside-out signaling ... more have collectively been termed inside-out signaling. The molecular basis for inside-out signaling is not yet completely understood. Here, we show that a signaling module comprising the cytosolic adapter proteins ADAP and SKAP55 is involved in TCR-mediated inside-out signaling and, moreover, that the interaction between ADAP and SKAP55 is mandatory for integrin activation. Disruption of the ADAP/SKAP55 module leads to displacement of the small GTPase Rap1 from the plasma membrane without influencing its GTPase activity. These findings suggest that the ADAP/SKAP55 complex serves to recruit activated Rap1 to the plasma membrane. In line with this hypothesis is the finding that membrane targeting of the ADAP/SKAP55 module induces T-cell adhesion in the absence of TCR-mediated stimuli. However, it appears as if the ADAP/SKAP55 module can exert its signaling function outside of the classical raft fraction of the cell membrane.

Molecular and Cellular Biology, 2006

The transmembrane adaptor molecule TRIM is strongly expressed within thymus and in peripheral CD4... more The transmembrane adaptor molecule TRIM is strongly expressed within thymus and in peripheral CD4 ؉ T cells. Previous studies suggested that TRIM is an integral component of the T-cell receptor (TCR)/CD3 complex and might be involved in regulating TCR cycling. To elucidate the in vivo function of TRIM, we generated TRIM-deficient mice by homologous recombination. TRIM ؊/؊ mice develop normally and are healthy and fertile. However, the animals show a mild reduction in body weight that appears to be due to a decrease in the size and/or cellularity of many organs. The morphology and anatomy of nonlymphoid as well as primary and secondary lymphoid organs is normal. The frequency of thymocyte and peripheral T-cell subsets does not differ from control littermates. In addition, a detailed analysis of lymphocyte development revealed that TRIM is not required for either positive or negative selection. Although TRIM ؊/؊ CD4 ؉ T cells showed an augmented phosphorylation of the serine/threonine kinase Akt, the in vitro characterization of peripheral T cells indicated that proliferation, survival, activation-induced cell death, migration, adhesion, TCR internalization and recycling, TCR-mediated calcium fluxes, tyrosine phosphorylation, and mitogenactivated protein family kinase activation are not affected in the absence of TRIM. Similarly, the in vivo immune response to T-dependent and T-independent antigens as well as the clinical course of experimental autoimmune encephalomyelitis, a complex Th1-mediated autoimmune model, is comparable to that of wildtype animals. Collectively, these results demonstrate that TRIM is dispensable for T-cell development and peripheral immune functions. The lack of an evident phenotype could indicate that TRIM shares redundant functions with other transmembrane adaptors involved in regulating the immune response.

Molecular and Cellular Biology, 2005

SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family prot... more SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family protein tyrosine kinase Fyn. Previously, several groups have provided experimental evidence that SKAP-HOM (most likely in cooperation with the cytosolic adaptor protein ADAP) is involved in regulating leukocyte adhesion. To further assess the physiological role of SKAP-HOM, we investigated the immune system of SKAP-HOMdeficient mice. Our data show that T-cell responses towards a variety of stimuli are unaffected in the absence of SKAP-HOM. Similarly, B-cell receptor (BCR)-mediated total tyrosine phosphorylation and phosphorylation of Erk, p38, and JNK, as well as immunoreceptor-mediated Ca 2؉ responses, are normal in SKAP-HOM ؊/؊