The neuroendocrine hormone PRL acts as a progression factor during interleukin-2 (IL2) stimulated... more The neuroendocrine hormone PRL acts as a progression factor during interleukin-2 (IL2) stimulated lymphocyte proliferation. Since the sequential expression of cell cycle regulated genes occurs during this process, we examined the contribution of IL2 and PRL to specific RNA accumulation. Stimulation of the cloned T cell line L2 with IL2 and PRL induced the sequential expression of interferon regulatory factor-1, c-myc, proliferating cell nuclear antigen, thymidine kinase, cyclin B, and histone H3. Stimulation of L2 cells with PRL alone, however, induced only the expression of interferon regulatory factor-1. Depletion of PRL, through the use of an anti-PRL antiserum, inhibited IL2 driven proliferation and the expression of cyclin B and histone H3. These results demonstrate that PRL may regulate T cell proliferation by enhancing the expression of some genes necessary for entry into S-phase.
The response of B lymphocytes to stimulation through surface immunoglobulin (sIg) is developmenta... more The response of B lymphocytes to stimulation through surface immunoglobulin (sIg) is developmentally regulated. While mature B lymphocytes from the adult mouse spleen enter S phase of the cell cycle in response to sIg stimulation, immature B lymphocytes from the neonatal mouse spleen or adult mouse bone marrow undergo cell cycle arrest, and then death by apoptosis when given the same stimulus. It is thought that this negative response of immature B lymphocytes to in vitro sIg stimulation translates to clonal deletion in the event that cognate antigen is encountered in vivo. Previous work in our laboratory has uncovered two characteristics of tolerance-sensitive immature B lymphocytes: the inability to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) in response to sIg stimulation, and the lack of src family protein tyrosine kinase (PTK) p59fyn and p55 fgr expression. To further characterize sIg signaling pathways in mature and immature B lymphocytes, genetic and molecular approaches were employed. B lymphocytes from p59fyn- and p55 fgr-deficient mice were studied to determine whether the absence of either p59fyn or p55 fgr could cause disruptions of sIg signaling pathways. Signaling and cell cycle entry in both p59fyn- and p55 fgr-deficient B lymphocytes was normal. We concluded that there is redundancy among the substrates of the B lymphocyte sIg-activated PTKs. Also, the inability of immature B lymphocytes to proliferate following sIg stimulation is not due to the absence of either p59fyn or p55fgr. To investigate the lack of PIP2 hydrolysis in sIg-stimulated immature B lymphocytes, we first needed to identify the PTK which activates phospholipase C-γ (PLCγ) the enzyme which hydrolyzes PIP2 following mature B lymphocyte sIg stimulation. We used a GST fusion protein containing the PLCγ 1 tandem src-homology 2 (SH2) domains to precipitate interacting PTK from B lymphocyte lysates. We determined that p72 syk from lysates of resting and sIg-stimulated B lymphocytes interacts with the SH2 domains of PLCγ 1. PLCγ 1 and p72 syk were also co-immunoprecipitated from lysates of resting and sIg-stimulated B lymphocytes. From these results we conclude that p72 syk can associate with PLCγ in B lymphocytes. We also attempted to map the parts of the p72syk molecule required for interaction with PLCγ
The nuclear translocation of PRL is demonstrated at the immunofluorescence and electron microscop... more The nuclear translocation of PRL is demonstrated at the immunofluorescence and electron microscopic (EM) levels in interleukin-2 (IL2)-stimulated cloned T-cells and concanavalin A-stimulated splenocytes. This translocation occurs 2-10 h after IL2 stimulation, and is reversed by the addition of anti-PRL antiserum into the extracellular culture medium. The nuclear localization of PRL in IL2 stimulated T-cells was confirmed by postembedding immunogold EM. The nuclear uptake of PRL after IL2 stimulation was further documented by EM studies using PRL-colloidal gold conjugates. These studies suggest that the intranuclear PRL is translocated from the extracellular medium via an endosomal/lysosomal pathway over a period of several hours. Finally, the requirement for PRL no later than 6 h after IL2 stimulation is demonstrated through the reversible inhibition of T-cell growth with anti-PRL antiserum.
Abstract: The surface immunoglobulin M (sIgM)-associated src family protein tyrosine kinases (PTK... more Abstract: The surface immunoglobulin M (sIgM)-associated src family protein tyrosine kinases (PTKs) 55b1k, p59’5”, and p53/56 ” become activated in B cells within seconds following sIgM cross-linking. Studies us-ing protein tyrosine kinase (PTK) inhibitors have demonstrated that PTK activity is crucial for down-stream events such as calcium flux, inositol phospholipid hydrolysis, and cell cycle entry. The roles that the in-dividual src family PTKs play in sIgM signaling are largely unknown, however. In order to determine whether p59’) ” plays a distinct role in sIgM signal trans-duction, the signaling capabilities of B cells isolated from fyn “knockout ” mice were evaluated. We observed that in the absence of 59fn, there was no demonstrable com-promise of the sIgM-coupled signaling events measured (tyrosine phosphorylation, inositol phospholipid hydrol-ysis, and Ca2 flux). We propose that either 59f s not involved in coupling sIgM to these specific signaling pathways or that other P...
The surface immunoglobulin M (sIgM)-associated src family protein tyrosine kinases (PTKs) p55blk,... more The surface immunoglobulin M (sIgM)-associated src family protein tyrosine kinases (PTKs) p55blk, p59fyn, and p53/56lyn become activated in B cells within seconds following sIgM cross-linking. Studies using protein tyrosine kinase (PTK) inhibitors have demonstrated that PTK activity is crucial for downstream events such as calcium flux, inositol phospholipid hydrolysis, and cell cycle entry. The roles that the individual src family PTKs play in sIgM signaling are largely unknown, however. In order to determine whether p59fyn plays a distinct role in sIgM signal transduction, the signaling capabilities of B cells isolated from fyn “knockout” mice were evaluated. We observed that in the absence of p59fyn, there was no demonstrable compromise of the slgM-coupled signaling events measured (tyrosine phosphorylation, inositol phospholipid hydrolysis, and Ca2+ flux). We propose that either p59fyn is not involved in coupling sIgM to these specific signaling pathways or that other PTKs are a...
The response of B lymphocytes to stimulation through surface immunoglobulin (sIg) is developmenta... more The response of B lymphocytes to stimulation through surface immunoglobulin (sIg) is developmentally regulated. While mature B lymphocytes from the adult mouse spleen enter S phase of the cell cycle in response to sIg stimulation, immature B lymphocytes from the neonatal mouse spleen or adult mouse bone marrow undergo cell cycle arrest, and then death by apoptosis when given the same stimulus. It is thought that this negative response of immature B lymphocytes to in vitro sIg stimulation translates to clonal deletion in the event that cognate antigen is encountered in vivo. Previous work in our laboratory has uncovered two characteristics of tolerance-sensitive immature B lymphocytes: the inability to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) in response to sIg stimulation, and the lack of src family protein tyrosine kinase (PTK) p59fyn and p55 fgr expression. To further characterize sIg signaling pathways in mature and immature B lymphocytes, genetic and molecular approaches were employed. B lymphocytes from p59fyn- and p55 fgr-deficient mice were studied to determine whether the absence of either p59fyn or p55 fgr could cause disruptions of sIg signaling pathways. Signaling and cell cycle entry in both p59fyn- and p55 fgr-deficient B lymphocytes was normal. We concluded that there is redundancy among the substrates of the B lymphocyte sIg-activated PTKs. Also, the inability of immature B lymphocytes to proliferate following sIg stimulation is not due to the absence of either p59fyn or p55fgr. To investigate the lack of PIP2 hydrolysis in sIg-stimulated immature B lymphocytes, we first needed to identify the PTK which activates phospholipase C-γ (PLCγ) the enzyme which hydrolyzes PIP2 following mature B lymphocyte sIg stimulation. We used a GST fusion protein containing the PLCγ 1 tandem src-homology 2 (SH2) domains to precipitate interacting PTK from B lymphocyte lysates. We determined that p72 syk from lysates of resting and sIg-stimulated B lymphocytes interacts with the SH2 domains of PLCγ 1. PLCγ 1 and p72 syk were also co-immunoprecipitated from lysates of resting and sIg-stimulated B lymphocytes. From these results we conclude that p72 syk can associate with PLCγ in B lymphocytes. We also attempted to map the parts of the p72syk molecule required for interaction with PLCγ
We report the complete sequence of Drosophila alpha-spectrin and show that it is similar to verte... more We report the complete sequence of Drosophila alpha-spectrin and show that it is similar to vertebrate nonerythroid spectrins. As in vertebrates, the alpha subunit consists of two large domains of repetitive sequence (segments 1-9 and 11-19) separated by a short nonrepetitive sequence (segment 10). The 106-residue repetitive segments are defined by a consensus sequence of 54 residues. Chicken alpha-spectrin (Wasenius, V.-M., M. Saraste, P. Salven, M. Eramaa, L. Holm, V.-P. Lehto. 1989. J. Cell Biol. 108:79-93) shares 50 of these consensus positions. Through comparison of spectrin and alpha-actinin sequences, we describe a second lineage of spectrin segments (20 and 21) that differs from the 106-residue segments by an 8-residue insertion and by lack of many of the consensus residues. We present a model of spectrin evolution in which the repetitive lineage of spectrin segments and the nonrepetitive lineage of segments found in spectrin and alpha-actinin arose by separate multiplicatio...
Encounter with antigen by newly developing antigen receptor-positive B cells leads to negative se... more Encounter with antigen by newly developing antigen receptor-positive B cells leads to negative selection. This process positions the B cell antigen receptor (BCR) in a central role for initiating the process of negative selection and suggests developmental regulation of its signaling. The observation that immature B cells are more susceptible to negative selection than are mature B cells has been demonstrated in a number of in vitro and in vivo model systems and support the idea of developmental regulation of BCR-initiated responses. Since identical antigen receptors are expressed on immature and mature B cells, the critical fate-determining distinction between these developmental stages must lie downstream of the receptor-ligand interaction itself, in the form of different BCR-linked signaling processes or with different secondary events occurring subsequent to BCR cross-linking. To address the first possibility, our laboratory and others have sought to define the differences in BCR-mediated signal transduction in immature and mature B lymphocytes. In this review article we will discuss current in vitro systems to study this question in primary, nontransformed murine B lymphocytes. In addition, we will discuss our previously published work in order to illustrate how these model systems have been useful in beginning to unravel the molecular basis for immune B cell negative selection and tolerance.
The nuclear translocation of PRL is demonstrated at the immunofluorescence and electron microscop... more The nuclear translocation of PRL is demonstrated at the immunofluorescence and electron microscopic (EM) levels in interleukin-2 (IL2)-stimulated cloned T-cells and concanavalin A-stimulated splenocytes. This translocation occurs 2-10 h after IL2 stimulation, and is reversed by the addition of anti-PRL antiserum into the extracellular culture medium. The nuclear localization of PRL in IL2 stimulated T-cells was confirmed by postembedding immunogold EM. The nuclear uptake of PRL after IL2 stimulation was further documented by EM studies using PRL-colloidal gold conjugates. These studies suggest that the intranuclear PRL is translocated from the extracellular medium via an endosomal/lysosomal pathway over a period of several hours. Finally, the requirement for PRL no later than 6 h after IL2 stimulation is demonstrated through the reversible inhibition of T-cell growth with anti-PRL antiserum.
The neuroendocrine hormone PRL acts as a progression factor during interleukin-2 (IL2) stimulated... more The neuroendocrine hormone PRL acts as a progression factor during interleukin-2 (IL2) stimulated lymphocyte proliferation. Since the sequential expression of cell cycle regulated genes occurs during this process, we examined the contribution of IL2 and PRL to specific RNA accumulation. Stimulation of the cloned T cell line L2 with IL2 and PRL induced the sequential expression of interferon regulatory factor-1, c-myc, proliferating cell nuclear antigen, thymidine kinase, cyclin B, and histone H3. Stimulation of L2 cells with PRL alone, however, induced only the expression of interferon regulatory factor-1. Depletion of PRL, through the use of an anti-PRL antiserum, inhibited IL2 driven proliferation and the expression of cyclin B and histone H3. These results demonstrate that PRL may regulate T cell proliferation by enhancing the expression of some genes necessary for entry into S-phase.
Proceedings of the National Academy of Sciences, 2006
The mammalian Ste20-like Nck-interacting kinase (NIK) and its orthologs Misshapen in Drosophila a... more The mammalian Ste20-like Nck-interacting kinase (NIK) and its orthologs Misshapen in Drosophila and Mig-15 in Caenorhabditis elegans have a conserved function in regulating cell morphology, although through poorly understood mechanisms. We report two previously unrecognized actions of NIK: regulation of lamellipodium formation by growth factors and phosphorylation of the ERM proteins ezrin, radixin, and moesin. ERM proteins regulate cell morphology and plasma membrane dynamics by reversibly anchoring actin filaments to integral plasma membrane proteins. In vitro assays show that NIK interacts directly with ERM proteins, binding their N termini and phosphorylating a conserved C-terminal threonine. In cells, NIK and phosphorylated ERM proteins localize at the distal margins of lamellipodia, and NIK activity is necessary for phosphorylation of ERM proteins induced by EGF and PDGF, but not by thrombin. Lamellipodium extension in response to growth factors is inhibited in cells expressin...
Encounter with antigen by newly developing antigen receptor-positive B cells leads to negative se... more Encounter with antigen by newly developing antigen receptor-positive B cells leads to negative selection. This process positions the B cell antigen receptor (BCR) in a central role for initiating the process of negative selection and suggests developmental regulation of its signaling. The observation that immature B cells are more susceptible to negative selection than are mature B cells has been demonstrated in a number of in vitro and in vivo model systems and support the idea of developmental regulation of BCR-initiated responses. Since identical antigen receptors are expressed on immature and mature B cells, the critical fate-determining distinction between these developmental stages must lie downstream of the receptor-ligand interaction itself, in the form of different BCR-linked signaling processes or with different secondary events occurring subsequent to BCR cross-linking. To address the first possibility, our laboratory and others have sought to define the differences in BCR-mediated signal transduction in immature and mature B lymphocytes. In this review article we will discuss current in vitro systems to study this question in primary, nontransformed murine B lymphocytes. In addition, we will discuss our previously published work in order to illustrate how these model systems have been useful in beginning to unravel the molecular basis for immune B cell negative selection and tolerance.
The neuroendocrine hormone PRL acts as a progression factor during interleukin-2 (IL2) stimulated... more The neuroendocrine hormone PRL acts as a progression factor during interleukin-2 (IL2) stimulated lymphocyte proliferation. Since the sequential expression of cell cycle regulated genes occurs during this process, we examined the contribution of IL2 and PRL to specific RNA accumulation. Stimulation of the cloned T cell line L2 with IL2 and PRL induced the sequential expression of interferon regulatory factor-1, c-myc, proliferating cell nuclear antigen, thymidine kinase, cyclin B, and histone H3. Stimulation of L2 cells with PRL alone, however, induced only the expression of interferon regulatory factor-1. Depletion of PRL, through the use of an anti-PRL antiserum, inhibited IL2 driven proliferation and the expression of cyclin B and histone H3. These results demonstrate that PRL may regulate T cell proliferation by enhancing the expression of some genes necessary for entry into S-phase.
The response of B lymphocytes to stimulation through surface immunoglobulin (sIg) is developmenta... more The response of B lymphocytes to stimulation through surface immunoglobulin (sIg) is developmentally regulated. While mature B lymphocytes from the adult mouse spleen enter S phase of the cell cycle in response to sIg stimulation, immature B lymphocytes from the neonatal mouse spleen or adult mouse bone marrow undergo cell cycle arrest, and then death by apoptosis when given the same stimulus. It is thought that this negative response of immature B lymphocytes to in vitro sIg stimulation translates to clonal deletion in the event that cognate antigen is encountered in vivo. Previous work in our laboratory has uncovered two characteristics of tolerance-sensitive immature B lymphocytes: the inability to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) in response to sIg stimulation, and the lack of src family protein tyrosine kinase (PTK) p59fyn and p55 fgr expression. To further characterize sIg signaling pathways in mature and immature B lymphocytes, genetic and molecular approaches were employed. B lymphocytes from p59fyn- and p55 fgr-deficient mice were studied to determine whether the absence of either p59fyn or p55 fgr could cause disruptions of sIg signaling pathways. Signaling and cell cycle entry in both p59fyn- and p55 fgr-deficient B lymphocytes was normal. We concluded that there is redundancy among the substrates of the B lymphocyte sIg-activated PTKs. Also, the inability of immature B lymphocytes to proliferate following sIg stimulation is not due to the absence of either p59fyn or p55fgr. To investigate the lack of PIP2 hydrolysis in sIg-stimulated immature B lymphocytes, we first needed to identify the PTK which activates phospholipase C-γ (PLCγ) the enzyme which hydrolyzes PIP2 following mature B lymphocyte sIg stimulation. We used a GST fusion protein containing the PLCγ 1 tandem src-homology 2 (SH2) domains to precipitate interacting PTK from B lymphocyte lysates. We determined that p72 syk from lysates of resting and sIg-stimulated B lymphocytes interacts with the SH2 domains of PLCγ 1. PLCγ 1 and p72 syk were also co-immunoprecipitated from lysates of resting and sIg-stimulated B lymphocytes. From these results we conclude that p72 syk can associate with PLCγ in B lymphocytes. We also attempted to map the parts of the p72syk molecule required for interaction with PLCγ
The nuclear translocation of PRL is demonstrated at the immunofluorescence and electron microscop... more The nuclear translocation of PRL is demonstrated at the immunofluorescence and electron microscopic (EM) levels in interleukin-2 (IL2)-stimulated cloned T-cells and concanavalin A-stimulated splenocytes. This translocation occurs 2-10 h after IL2 stimulation, and is reversed by the addition of anti-PRL antiserum into the extracellular culture medium. The nuclear localization of PRL in IL2 stimulated T-cells was confirmed by postembedding immunogold EM. The nuclear uptake of PRL after IL2 stimulation was further documented by EM studies using PRL-colloidal gold conjugates. These studies suggest that the intranuclear PRL is translocated from the extracellular medium via an endosomal/lysosomal pathway over a period of several hours. Finally, the requirement for PRL no later than 6 h after IL2 stimulation is demonstrated through the reversible inhibition of T-cell growth with anti-PRL antiserum.
Abstract: The surface immunoglobulin M (sIgM)-associated src family protein tyrosine kinases (PTK... more Abstract: The surface immunoglobulin M (sIgM)-associated src family protein tyrosine kinases (PTKs) 55b1k, p59’5”, and p53/56 ” become activated in B cells within seconds following sIgM cross-linking. Studies us-ing protein tyrosine kinase (PTK) inhibitors have demonstrated that PTK activity is crucial for down-stream events such as calcium flux, inositol phospholipid hydrolysis, and cell cycle entry. The roles that the in-dividual src family PTKs play in sIgM signaling are largely unknown, however. In order to determine whether p59’) ” plays a distinct role in sIgM signal trans-duction, the signaling capabilities of B cells isolated from fyn “knockout ” mice were evaluated. We observed that in the absence of 59fn, there was no demonstrable com-promise of the sIgM-coupled signaling events measured (tyrosine phosphorylation, inositol phospholipid hydrol-ysis, and Ca2 flux). We propose that either 59f s not involved in coupling sIgM to these specific signaling pathways or that other P...
The surface immunoglobulin M (sIgM)-associated src family protein tyrosine kinases (PTKs) p55blk,... more The surface immunoglobulin M (sIgM)-associated src family protein tyrosine kinases (PTKs) p55blk, p59fyn, and p53/56lyn become activated in B cells within seconds following sIgM cross-linking. Studies using protein tyrosine kinase (PTK) inhibitors have demonstrated that PTK activity is crucial for downstream events such as calcium flux, inositol phospholipid hydrolysis, and cell cycle entry. The roles that the individual src family PTKs play in sIgM signaling are largely unknown, however. In order to determine whether p59fyn plays a distinct role in sIgM signal transduction, the signaling capabilities of B cells isolated from fyn “knockout” mice were evaluated. We observed that in the absence of p59fyn, there was no demonstrable compromise of the slgM-coupled signaling events measured (tyrosine phosphorylation, inositol phospholipid hydrolysis, and Ca2+ flux). We propose that either p59fyn is not involved in coupling sIgM to these specific signaling pathways or that other PTKs are a...
The response of B lymphocytes to stimulation through surface immunoglobulin (sIg) is developmenta... more The response of B lymphocytes to stimulation through surface immunoglobulin (sIg) is developmentally regulated. While mature B lymphocytes from the adult mouse spleen enter S phase of the cell cycle in response to sIg stimulation, immature B lymphocytes from the neonatal mouse spleen or adult mouse bone marrow undergo cell cycle arrest, and then death by apoptosis when given the same stimulus. It is thought that this negative response of immature B lymphocytes to in vitro sIg stimulation translates to clonal deletion in the event that cognate antigen is encountered in vivo. Previous work in our laboratory has uncovered two characteristics of tolerance-sensitive immature B lymphocytes: the inability to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) in response to sIg stimulation, and the lack of src family protein tyrosine kinase (PTK) p59fyn and p55 fgr expression. To further characterize sIg signaling pathways in mature and immature B lymphocytes, genetic and molecular approaches were employed. B lymphocytes from p59fyn- and p55 fgr-deficient mice were studied to determine whether the absence of either p59fyn or p55 fgr could cause disruptions of sIg signaling pathways. Signaling and cell cycle entry in both p59fyn- and p55 fgr-deficient B lymphocytes was normal. We concluded that there is redundancy among the substrates of the B lymphocyte sIg-activated PTKs. Also, the inability of immature B lymphocytes to proliferate following sIg stimulation is not due to the absence of either p59fyn or p55fgr. To investigate the lack of PIP2 hydrolysis in sIg-stimulated immature B lymphocytes, we first needed to identify the PTK which activates phospholipase C-γ (PLCγ) the enzyme which hydrolyzes PIP2 following mature B lymphocyte sIg stimulation. We used a GST fusion protein containing the PLCγ 1 tandem src-homology 2 (SH2) domains to precipitate interacting PTK from B lymphocyte lysates. We determined that p72 syk from lysates of resting and sIg-stimulated B lymphocytes interacts with the SH2 domains of PLCγ 1. PLCγ 1 and p72 syk were also co-immunoprecipitated from lysates of resting and sIg-stimulated B lymphocytes. From these results we conclude that p72 syk can associate with PLCγ in B lymphocytes. We also attempted to map the parts of the p72syk molecule required for interaction with PLCγ
We report the complete sequence of Drosophila alpha-spectrin and show that it is similar to verte... more We report the complete sequence of Drosophila alpha-spectrin and show that it is similar to vertebrate nonerythroid spectrins. As in vertebrates, the alpha subunit consists of two large domains of repetitive sequence (segments 1-9 and 11-19) separated by a short nonrepetitive sequence (segment 10). The 106-residue repetitive segments are defined by a consensus sequence of 54 residues. Chicken alpha-spectrin (Wasenius, V.-M., M. Saraste, P. Salven, M. Eramaa, L. Holm, V.-P. Lehto. 1989. J. Cell Biol. 108:79-93) shares 50 of these consensus positions. Through comparison of spectrin and alpha-actinin sequences, we describe a second lineage of spectrin segments (20 and 21) that differs from the 106-residue segments by an 8-residue insertion and by lack of many of the consensus residues. We present a model of spectrin evolution in which the repetitive lineage of spectrin segments and the nonrepetitive lineage of segments found in spectrin and alpha-actinin arose by separate multiplicatio...
Encounter with antigen by newly developing antigen receptor-positive B cells leads to negative se... more Encounter with antigen by newly developing antigen receptor-positive B cells leads to negative selection. This process positions the B cell antigen receptor (BCR) in a central role for initiating the process of negative selection and suggests developmental regulation of its signaling. The observation that immature B cells are more susceptible to negative selection than are mature B cells has been demonstrated in a number of in vitro and in vivo model systems and support the idea of developmental regulation of BCR-initiated responses. Since identical antigen receptors are expressed on immature and mature B cells, the critical fate-determining distinction between these developmental stages must lie downstream of the receptor-ligand interaction itself, in the form of different BCR-linked signaling processes or with different secondary events occurring subsequent to BCR cross-linking. To address the first possibility, our laboratory and others have sought to define the differences in BCR-mediated signal transduction in immature and mature B lymphocytes. In this review article we will discuss current in vitro systems to study this question in primary, nontransformed murine B lymphocytes. In addition, we will discuss our previously published work in order to illustrate how these model systems have been useful in beginning to unravel the molecular basis for immune B cell negative selection and tolerance.
The nuclear translocation of PRL is demonstrated at the immunofluorescence and electron microscop... more The nuclear translocation of PRL is demonstrated at the immunofluorescence and electron microscopic (EM) levels in interleukin-2 (IL2)-stimulated cloned T-cells and concanavalin A-stimulated splenocytes. This translocation occurs 2-10 h after IL2 stimulation, and is reversed by the addition of anti-PRL antiserum into the extracellular culture medium. The nuclear localization of PRL in IL2 stimulated T-cells was confirmed by postembedding immunogold EM. The nuclear uptake of PRL after IL2 stimulation was further documented by EM studies using PRL-colloidal gold conjugates. These studies suggest that the intranuclear PRL is translocated from the extracellular medium via an endosomal/lysosomal pathway over a period of several hours. Finally, the requirement for PRL no later than 6 h after IL2 stimulation is demonstrated through the reversible inhibition of T-cell growth with anti-PRL antiserum.
The neuroendocrine hormone PRL acts as a progression factor during interleukin-2 (IL2) stimulated... more The neuroendocrine hormone PRL acts as a progression factor during interleukin-2 (IL2) stimulated lymphocyte proliferation. Since the sequential expression of cell cycle regulated genes occurs during this process, we examined the contribution of IL2 and PRL to specific RNA accumulation. Stimulation of the cloned T cell line L2 with IL2 and PRL induced the sequential expression of interferon regulatory factor-1, c-myc, proliferating cell nuclear antigen, thymidine kinase, cyclin B, and histone H3. Stimulation of L2 cells with PRL alone, however, induced only the expression of interferon regulatory factor-1. Depletion of PRL, through the use of an anti-PRL antiserum, inhibited IL2 driven proliferation and the expression of cyclin B and histone H3. These results demonstrate that PRL may regulate T cell proliferation by enhancing the expression of some genes necessary for entry into S-phase.
Proceedings of the National Academy of Sciences, 2006
The mammalian Ste20-like Nck-interacting kinase (NIK) and its orthologs Misshapen in Drosophila a... more The mammalian Ste20-like Nck-interacting kinase (NIK) and its orthologs Misshapen in Drosophila and Mig-15 in Caenorhabditis elegans have a conserved function in regulating cell morphology, although through poorly understood mechanisms. We report two previously unrecognized actions of NIK: regulation of lamellipodium formation by growth factors and phosphorylation of the ERM proteins ezrin, radixin, and moesin. ERM proteins regulate cell morphology and plasma membrane dynamics by reversibly anchoring actin filaments to integral plasma membrane proteins. In vitro assays show that NIK interacts directly with ERM proteins, binding their N termini and phosphorylating a conserved C-terminal threonine. In cells, NIK and phosphorylated ERM proteins localize at the distal margins of lamellipodia, and NIK activity is necessary for phosphorylation of ERM proteins induced by EGF and PDGF, but not by thrombin. Lamellipodium extension in response to growth factors is inhibited in cells expressin...
Encounter with antigen by newly developing antigen receptor-positive B cells leads to negative se... more Encounter with antigen by newly developing antigen receptor-positive B cells leads to negative selection. This process positions the B cell antigen receptor (BCR) in a central role for initiating the process of negative selection and suggests developmental regulation of its signaling. The observation that immature B cells are more susceptible to negative selection than are mature B cells has been demonstrated in a number of in vitro and in vivo model systems and support the idea of developmental regulation of BCR-initiated responses. Since identical antigen receptors are expressed on immature and mature B cells, the critical fate-determining distinction between these developmental stages must lie downstream of the receptor-ligand interaction itself, in the form of different BCR-linked signaling processes or with different secondary events occurring subsequent to BCR cross-linking. To address the first possibility, our laboratory and others have sought to define the differences in BCR-mediated signal transduction in immature and mature B lymphocytes. In this review article we will discuss current in vitro systems to study this question in primary, nontransformed murine B lymphocytes. In addition, we will discuss our previously published work in order to illustrate how these model systems have been useful in beginning to unravel the molecular basis for immune B cell negative selection and tolerance.
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Papers by Amy Sillman