Thomas Smithgall - Academia.edu (original) (raw)

Papers by Thomas Smithgall

Molecular and cellular biology, 2004

The c-Fes protein-tyrosine kinase (Fes) has been implicated in the differentiation of vascular en... more The c-Fes protein-tyrosine kinase (Fes) has been implicated in the differentiation of vascular endothelial, myeloid hematopoietic, and neuronal cells, promoting substantial morphological changes in these cell types. The mechanism by which Fes promotes morphological aspects of cellular differentiation is unknown. Using COS-7 cells as a model system, we observed that Fes strongly colocalizes with microtubules in vivo when activated via coiled-coil mutation or by coexpression with an active Src family kinase. In contrast, wild-type Fes showed a diffuse cytoplasmic localization in this system, which correlated with undetectable kinase activity. Coimmunoprecipitation and immunofluorescence microscopy showed that the N-terminal Fes/CIP4 homology (FCH) domain is involved in Fes interaction with soluble unpolymerized tubulin. However, the FCH domain was not required for colocalization with polymerized microtubules in vivo. In contrast, a functional SH2 domain was essential for microtubule l...

Molecular and cellular biology, 1999

The cytoplasmic protein-tyrosine kinase Fes has been implicated in cytokine signal transduction, ... more The cytoplasmic protein-tyrosine kinase Fes has been implicated in cytokine signal transduction, hematopoiesis, and embryonic development. Previous work from our laboratory has shown that active Fes exists as a large oligomeric complex in vitro. However, when Fes is expressed in mammalian cells, its kinase activity is tightly repressed. The Fes unique N-terminal sequence has two regions with strong homology to coiled-coil-forming domains often found in oligomeric proteins. Here we show that disruption or deletion of the first coiled-coil domain upregulates Fes tyrosine kinase and transforming activities in Rat-2 fibroblasts and enhances Fes differentiation-inducing activity in myeloid leukemia cells. Conversely, expression of a Fes truncation mutant consisting only of the unique N-terminal domain interfered with Rat-2 fibroblast transformation by an activated Fes mutant, suggesting that oligomerization is essential for Fes activation in vivo. Coexpression with the Fes N-terminal reg...

Proceedings of The National Academy of Sciences, 2006

G protein-coupled receptors induce EGF receptor (EGFR) signaling, leading to the proliferation an... more G protein-coupled receptors induce EGF receptor (EGFR) signaling, leading to the proliferation and invasion of cancer cells. Elucidation of the mechanism of EGFR activation by G protein-coupled receptors may identify new signaling paradigms. A gastrin-releasing peptide (GRP)/GRP receptor-mediated autocrine pathway was previously described in squamous cell carcinoma of head and neck. In the present study, we demonstrate that TNF- converting

Signal transducers and activators of transcription (STATs) were originally identified as key comp... more Signal transducers and activators of transcription (STATs) were originally identified as key components of signaling pathways involved in mediating responses to IFNs. Previous studies showed that the Src oncoprotein constitutively activates one STAT family member, Stat3. In this study, we investigated STAT activation in a panel of rodent fibroblast cell lines stably transformed by diverse viral oncoproteins. Using a temperature-sensitive

The development of more effective prevention and treatment strategies for solid tumors is limited... more The development of more effective prevention and treatment strategies for solid tumors is limited by an incomplete understanding of the critical growth pathways that are activated in carcinogenesis. Signal transducers and activators of transcription (STAT) proteins have been linked to transformation and tumor progression. Studies to date have not eluci- dated clear and distinct roles for Stat5 genes (Stat5a and

American Journal of Obstetrics and Gynecology, 2015

Primary human trophoblasts were previously shown to be resistant to viral infection, and able to ... more Primary human trophoblasts were previously shown to be resistant to viral infection, and able to confer this resistance to nontrophoblast cells. Can trophoblasts protect nontrophoblastic cells from infection by viruses or other intracellular pathogens that are implicated in perinatal infection? Isolated primary term human trophoblasts were cultured for 48-72 hours. Diverse nonplacental human cell lines (U2OS, human foreskin fibroblast, TZM-bl, MeWo, and Caco-2) were preexposed to either trophoblast conditioned medium, nonconditioned medium, or miR-517-3p for 24 hours. Cells were infected with several viral and nonviral pathogens known to be associated with perinatal infections. Cellular infection was defined and quantified by plaque assays, luciferase assays, microscopy, and/or colonization assays. Differences in infection were assessed by Student t test or analysis of variance with Bonferroni correction. Infection by rubella and other togaviruses, human immunodeficiency virus-1, and varicella zoster was attenuated in cells preexposed to trophoblast-conditioned medium (P < .05), and a partial effect by the chromosome 19 microRNA miR-517-3p on specific pathogens. The conditioned medium had no effect on infection by Toxoplasma gondii or Listeria monocytogenes. Our findings indicate that medium conditioned by primary human trophoblasts attenuates viral infection in nontrophoblastic cells. Our data point to a trophoblast-specific antiviral effect that may be exploited therapeutically.

PLoS ONE, 2014

Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of sign... more Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo. Citation: Moroco JA, Craigo JK, Iacob RE, Wales TE, Engen JR, et al. (2014) Differential Sensitivity of Src-Family Kinases to Activation by SH3 Domain Displacement. PLoS ONE 9(8): e105629.

B63. PULMONARY CIRCULATION: VASCULAR SMOOTH MUSCLE, 2011

Oncogene, 2001

We have previously reported that the Jak2 tyrosine kinase but not Jak1 is tyrosine phosphorylated... more We have previously reported that the Jak2 tyrosine kinase but not Jak1 is tyrosine phosphorylated in the absence of IL-3 in Bcr–Abl positive M3.16 cells, which are rendered IL-3 independent by BCR–ABL gene expression. We have explored the involvement of Jak2 tyrosine phosphorylation in Bcr-Abl oncogenic effects. Our results indicate that Jak2 became tyrosine-phosphorylated in a number of cell lines

Proceedings of the National Academy of Sciences, 2009

Protein dynamics are inextricably linked to protein function but there are few techniques that al... more Protein dynamics are inextricably linked to protein function but there are few techniques that allow protein dynamics to be conveniently interrogated. For example, mutations and translocations give rise to aberrant proteins such as Bcr-Abl where changes in protein conformation and dynamics are believed to result in deregulated kinase activity that provides the oncogenic signal in chronic myelogeous leukemia. Although crystal structures of the down-regulated c-Abl kinase core have been reported, the conformational impact of mutations that render Abl resistant to smallmolecule kinase inhibitors are largely unknown as is the allosteric interplay of the various regulatory elements of the protein. Hydrogen exchange mass spectrometry (HX MS) was used to compare the conformations of wild-type Abl with a nonmyristoylated form and with 3 clinically relevant imatinib resistance mutants (T315I, Y253H and E255V). A HX-resistant core localized to the interface between the SH2 and kinase domains, a region known to be important for maintaining the down-regulated state. Conformational differences upon demyristoylation were consistent with the SH2 domain moving to the top of the small lobe of the kinase domain as a function of activation. There were conformational changes in the T315I mutant but, surprisingly, no major changes in conformation were detected in either the Y253H or the E255V mutants. Taken together, these results provide evidence that allosteric interactions and conformational changes play a major role in Abl kinase regulation in solution. Similar analyses could be performed on any protein to provide mechanistic details about conformational changes and protein function.

Oncogene, 2002

Chronic myelogenous leukemia (CML) is defined by the presence of the Philadelphia (Ph) chromosome... more Chronic myelogenous leukemia (CML) is defined by the presence of the Philadelphia (Ph) chromosome, which results in the expression of the 210 kDa Bcr -Abl tyrosine kinase. Bcr-Abl constitutively activates several signaling proteins important for the proliferation and survival of myeloid progenitors, including the Src family kinases Hck and Lyn, the Stat5 transcription factor and upstream components of the Ras/Erk pathway. Recently, we found that kinase-defective Hck blocks Bcr -Abl-induced transformation of DAGM myeloid leukemia cells to cytokine independence, suggesting that activation of the Src kinase family may be essential to oncogenic signaling by Bcr -Abl. To investigate the contribution of Src kinases to Bcr -Abl signaling in vivo, we used the pyrrolo-pyrimidine Src kinase inhibitors PP2 and A-419259. Treatment of the Ph + CML cell lines K-562 and Meg-01 with either compound resulted in growth arrest and induction of apoptosis, while the Ph 7 leukemia cell lines TF-1 and HEL were unaffected over the same concentration ranges. Suppression of Ph + cell growth by PP2 and A-419259 correlated with a decrease in Src kinase autophosphorylation. Both inhibitors blocked Stat5 and Erk activation, consistent with the suppressive effects of the compounds on survival and proliferation. In contrast, the phosphotyrosine content of Bcr -Abl and its endogenous substrate CrkL was unchanged at inhibitor concentrations that induced apoptosis, blocked oncogenic signaling and inhibited Src kinases. These data implicate the Src kinase family in Stat5 and Erk activation downstream of Bcr -Abl, and identify myeloid-specific Src kinases as potential drug targets in CML.

Oncogene, 2000

Hematopoiesis involves a complex array of growth factors that regulate the survival and prolifera... more Hematopoiesis involves a complex array of growth factors that regulate the survival and proliferation of immature progenitors, in¯uence dierentiation commitment, and modulate end-stage cell functions. This minireview is focused on the role of Stat activation in the development of myeloid cells in response to hematopoietic cytokines. Much of the evidence implicating Stats in these cellular processes comes from studies of mutant cytokine receptors selectively uncoupled from Stat activation, dominant-inhibitory Stat mutants, and mice with targeted disruptions of Stat genes. Together these approaches provide strong evidence that Stat activation, particularly of Stat3 and Stat5, plays an important role in myeloid dierentiation and survival.

Oncogene, 2004

The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway is an esse... more The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway is an essential cascade for mediating normal functions of different cytokines in the development of the hematopoietic and immune systems. Chronic exposure to arsenic has been found to cause immunotoxicity and has been associated with the suppression of hematopoiesis (anemia and leukopenia). Here, we report the novel finding of arsenic-mediated inactivation of the JAK-STAT signaling pathway by its direct interaction with JAK tyrosine kinase. Pretreatment with sodium arsenite strongly inhibited IL-6-inducible STAT3 tyrosine phosphorylation in HepG2 cells and did not affect its serine phosphorylation. As a result, sodium arsenite completely abolished STAT activity-dependent expression of suppressors of cytokine signaling (SOCS). Both cellular and subcelluar experiments showed that the inhibition of JAK-STAT signaling resulted from JAK tyrosine kinase's direct interaction with arsenite, and that arsenic's suppression of JAK tyrosine kinase activity also occurred in the interferon c (IFNc) pathway. The ligandindependent inhibition by arsenic indicates that JAK was the direct target of arsenic action. Other inflammatory stimulants, stress agents, and metal cadmium failed to induce similar effects on the tyrosine phosphorylation of STAT3 as arsenic does. Our experiments also revealed that arsenic inactivation of the JAK-STAT pathway occurred independent of arsenic activation of MAP kinases. Taken together, our findings indicate that arsenic directly inhibits JAK tyrosine kinase activity and suggest that this direct interference in the JAK-STAT pathway may play a role in arsenic-associated pathogenesis.

Molecular Biology of the Cell, 2010

؉ T-cells. During the first two days after infection, Nef assembles the 2c-sensitive multi-kinase... more ؉ T-cells. During the first two days after infection, Nef assembles the 2c-sensitive multi-kinase complex to trigger down-regulation of cell-surface MHC-I. By three days postinfection Nef switches to a stoichiometric mode that prevents surface delivery of newly synthesized MHC-I. Pharmacologic inhibition of the multi-kinase cascade prevents the Nef-dependent block in MHC-I transport, suggesting the signaling and stoichiometric modes are causally linked. Together, these studies resolve the seemingly controversial models that describe Nef-induced MHC-I down-regulation and provide new insights into the mechanism of Nef action.

Molecular and Cellular Biology, 2001

The c-fes locus encodes a 93-kDa non-receptor protein tyrosine kinase (Fes) that regulates the gr... more The c-fes locus encodes a 93-kDa non-receptor protein tyrosine kinase (Fes) that regulates the growth and differentiation of hematopoietic and vascular endothelial cells. Unique to Fes is a long N-terminal sequence with two regions of strong homology to coiled-coil oligomerization domains. We introduced leucine-to-proline substitutions into the coiled coils that were predicted to disrupt the coiled-coil structure. The resulting mutant proteins, together with wild-type Fes, were fused to green fluorescent protein and expressed in Rat-2 fibroblasts. We observed that a point mutation in the first coiled-coil domain (L145P) dramatically increased Fes tyrosine kinase and transforming activities in this cell type. In contrast, a similar point mutation in the second coiled-coil motif (L334P) was without effect. However, combining the L334P and L145P mutations reduced transforming and kinase activities by approximately 50% relative to the levels of activity produced with the L145P mutation alone. To study the effects of the coiled-coil mutations in a biologically relevant context, we expressed the mutant proteins in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid leukemia cell line TF-1. In this cellular context, the L145P mutation induced GM-CSF independence, cell attachment, and spreading. These effects correlated with a marked increase in L145P protein autophosphorylation relative to that of wild-type Fes. In contrast, the double coiled-coil mutant protein showed greatly reduced kinase and biological activities in TF-1 cells. These data are consistent with a role for the first coiled coil in the negative regulation of kinase activity and a requirement for the second coiled coil in either oligomerization or recruitment of signaling partners. Gel filtration experiments showed that the unique N-terminal region interconverts between monomeric and oligomeric forms. Single point mutations favored oligomerization, while the double point mutant protein eluted essentially as the monomer. These data provide new evidence for coiled-coil-mediated regulation of c-Fes tyrosine kinase activity and signaling, a mechanism unique among tyrosine kinases.

Journal of Molecular Neuroscience, 1998

NeuroD, a vertebrate homolog of Drosophila atonal gene, plays an important role in the differenti... more NeuroD, a vertebrate homolog of Drosophila atonal gene, plays an important role in the differentiation of neuronal precursors . We have investigated whether NeuroD subserves a similar function in mammalian retinal neurogenesis. Expression of NeuroD is detected in successive stages of retinal neurogenesis and is associated with a differentiating population of retinal cells. The association of NeuroD predominantly with postmitotic precursors in early as well as late neurogenesis suggests that NeuroD expression plays an important role in the terminal differentiation of retinal neurons. This notion is supported by observations that overexpression of NeuroD during late neurogenesis promotes premature differentiation of late-born neurons, rod photoreceptors, and bipolar cells, and that NeuroD can interact specifically with the E-box element in the proximal promoter of the phenotype-specific gene, opsin.

Journal of Molecular Biology, 1999

Protein dynamics play an important role in protein function and regulation of enzymatic activity.... more Protein dynamics play an important role in protein function and regulation of enzymatic activity. To determine how additional interactions with surrounding structure affects local protein dynamics, we have used hydrogen exchange and mass spectrometry to investigate the SH2 and SH3 domains of the protein tyrosine kinase Hck. Exchange rates of isolated Hck SH3 and SH2 domains were compared with rates for the same domains when part of a larger SH(3 2) construct. Increased deuterium incorporation was observed for the SH3 domain in the joint construct, particularly near the SH2 interface and the short sequence that connects SH3 to SH2, implying greater¯exibility of SH3 when it is part of SH(3 2). Slow cooperative unfolding of the SH3 domain occurred at the same rate in isolated SH3 as in the SH(3 2) construct, suggesting a functional signi®cance for this unfolding. The SH2 domain displayed relatively smaller changes in¯exibility when part of the SH(3 2) construct. These results suggest that the domains in¯uence each other. Further, our results imply a link between functional regulation and structural dynamics of SH3 and SH2 domains. Abbreviations used: SH3, Src homology 3; SH2, Src homology 2; PTK, protein tyrosine kinase; Abl, Abelson tyrosine kinase; PI3K, phosphatidylinositol 3-kinase; Hck, hematopoietic cell kinase; NHs, backbone amide hydrogens; HX, hydrogen exchange; SH(32), construct of tandem Hck SH3 and SH2 domains; SH3-A, the Hck SH3 domain expressed alone; SH2-A, the Hck SH2 domain expressed alone; SH3-J, the Hck SH3 domain when part of the SH(32) construct; SH2-J, the Hck SH2 domain when part of the SH(32) construct; GAP, GTPase activating protein.

Journal of Molecular Biology, 2007

Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoidspec... more Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoidspecific Src-family kinase Lck. The Tip:Lck interaction is essential for transformation and oncogenesis in HVS-infected cells. As there are no structural data for Tip, hydrogen exchange mass spectrometry was used to investigate the conformation of a nearly full-length form (residues 1-187) of Tip from HVS strain C484. Disorder predictions suggested that Tip would be mostly unstructured so great care was taken to ascertain whether recombinant Tip was functional. Circular dichroism and gel filtration analysis indicated an extended, unstructured protein. In vitro and in vivo binding and kinase assays confirmed that purified, recombinant Tip interacted with Lck, was capable of strongly activating Lck kinase activity and was multiply phosphorylated by Lck. Hydrogen exchange mass spectrometry of Tip then showed that the majority of backbone amide hydrogens became deuterated after only 10 seconds of labeling. Such a result suggested that Tip was almost totally unstructured in solution. Digestion of deuterium labeled Tip revealed some regions with minor protection from exchange. Overall, it was found that although recombinant Tip is still functional and capable of binding and activating its target Lck, it is largely unstructured.

Journal of Molecular Biology, 2007

Activation of Src family kinases by HIV-1 Nef may play an important role in the pathogenesis of H... more Activation of Src family kinases by HIV-1 Nef may play an important role in the pathogenesis of HIV/AIDS. Here we investigated whether diverse Nef sequences universally activate Hck, a Src family member expressed in macrophages and other HIV-1 target cells. In general, we observed that Hck activation is a highly conserved Nef function. However, we identified an unusual Nef variant from an HIV-positive individual that did not develop AIDS which failed to activate Hck despite the presence of conserved residues linked to Hck SH3 domain binding and kinase activation. Amino acid sequence alignment with active Nef proteins revealed differences in regions not previously implicated in Hck activation, including a large internal flexible loop absent from available Nef structures. Substitution of these residues in active Nef compromised Hck activation without affecting SH3 domain binding. These findings show that residues at a distance from the SH3 domain binding site allosterically influence Nef interactions with a key effector protein linked to AIDS progression.

Journal of Molecular Biology, 2011

HIV-1 has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family ... more HIV-1 has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family of host-cell enzymes. The HIV-1 virion infectivity factor, one of several HIV accessory proteins, targets APOBEC3 proteins for proteasomal degradation and down-regulates their expression at the mRNA level. Despite the importance of Vif for HIV-1 infection, there is little conformational data on Vif alone or in complex with other cellular factors due to incompatibilities with many structural techniques and difficulties in producing suitable quantities of protein for biophysical analysis. As an alternative, we have turned to hydrogen exchange mass spectrometry (HX MS), a conformational analysis method well suited for proteins that are difficult to study using X-ray crystallography and/or NMR. HX MS was used to probe the solution conformation of recombinant full-length HIV-1 Vif. Vif specifically interacted with the previously identified binding partner Hck and was able to cause kinase activation suggesting that the Vif studied by HX MS retained a biochemically competent conformation relevant to Hck interaction. HX MS analysis of Vif alone revealed low deuteration levels in the N-terminal portion indicating that this region contained structured or otherwise protected elements. In contrast, high deuteration levels in the C-terminal portion of Vif indicated that this region was likely unstructured in the absence of cellular interacting proteins. Several regions within Vif displayed conformational heterogeneity in solution including the APOBEC3G/F binding site and HCCH zinc finger. Taken together, these HX MS results provide new insights into the solution conformation of Vif.

Molecular and cellular biology, 2004

The c-Fes protein-tyrosine kinase (Fes) has been implicated in the differentiation of vascular en... more The c-Fes protein-tyrosine kinase (Fes) has been implicated in the differentiation of vascular endothelial, myeloid hematopoietic, and neuronal cells, promoting substantial morphological changes in these cell types. The mechanism by which Fes promotes morphological aspects of cellular differentiation is unknown. Using COS-7 cells as a model system, we observed that Fes strongly colocalizes with microtubules in vivo when activated via coiled-coil mutation or by coexpression with an active Src family kinase. In contrast, wild-type Fes showed a diffuse cytoplasmic localization in this system, which correlated with undetectable kinase activity. Coimmunoprecipitation and immunofluorescence microscopy showed that the N-terminal Fes/CIP4 homology (FCH) domain is involved in Fes interaction with soluble unpolymerized tubulin. However, the FCH domain was not required for colocalization with polymerized microtubules in vivo. In contrast, a functional SH2 domain was essential for microtubule l...

Molecular and cellular biology, 1999

The cytoplasmic protein-tyrosine kinase Fes has been implicated in cytokine signal transduction, ... more The cytoplasmic protein-tyrosine kinase Fes has been implicated in cytokine signal transduction, hematopoiesis, and embryonic development. Previous work from our laboratory has shown that active Fes exists as a large oligomeric complex in vitro. However, when Fes is expressed in mammalian cells, its kinase activity is tightly repressed. The Fes unique N-terminal sequence has two regions with strong homology to coiled-coil-forming domains often found in oligomeric proteins. Here we show that disruption or deletion of the first coiled-coil domain upregulates Fes tyrosine kinase and transforming activities in Rat-2 fibroblasts and enhances Fes differentiation-inducing activity in myeloid leukemia cells. Conversely, expression of a Fes truncation mutant consisting only of the unique N-terminal domain interfered with Rat-2 fibroblast transformation by an activated Fes mutant, suggesting that oligomerization is essential for Fes activation in vivo. Coexpression with the Fes N-terminal reg...

Proceedings of The National Academy of Sciences, 2006

G protein-coupled receptors induce EGF receptor (EGFR) signaling, leading to the proliferation an... more G protein-coupled receptors induce EGF receptor (EGFR) signaling, leading to the proliferation and invasion of cancer cells. Elucidation of the mechanism of EGFR activation by G protein-coupled receptors may identify new signaling paradigms. A gastrin-releasing peptide (GRP)/GRP receptor-mediated autocrine pathway was previously described in squamous cell carcinoma of head and neck. In the present study, we demonstrate that TNF- converting

Signal transducers and activators of transcription (STATs) were originally identified as key comp... more Signal transducers and activators of transcription (STATs) were originally identified as key components of signaling pathways involved in mediating responses to IFNs. Previous studies showed that the Src oncoprotein constitutively activates one STAT family member, Stat3. In this study, we investigated STAT activation in a panel of rodent fibroblast cell lines stably transformed by diverse viral oncoproteins. Using a temperature-sensitive

The development of more effective prevention and treatment strategies for solid tumors is limited... more The development of more effective prevention and treatment strategies for solid tumors is limited by an incomplete understanding of the critical growth pathways that are activated in carcinogenesis. Signal transducers and activators of transcription (STAT) proteins have been linked to transformation and tumor progression. Studies to date have not eluci- dated clear and distinct roles for Stat5 genes (Stat5a and

American Journal of Obstetrics and Gynecology, 2015

Primary human trophoblasts were previously shown to be resistant to viral infection, and able to ... more Primary human trophoblasts were previously shown to be resistant to viral infection, and able to confer this resistance to nontrophoblast cells. Can trophoblasts protect nontrophoblastic cells from infection by viruses or other intracellular pathogens that are implicated in perinatal infection? Isolated primary term human trophoblasts were cultured for 48-72 hours. Diverse nonplacental human cell lines (U2OS, human foreskin fibroblast, TZM-bl, MeWo, and Caco-2) were preexposed to either trophoblast conditioned medium, nonconditioned medium, or miR-517-3p for 24 hours. Cells were infected with several viral and nonviral pathogens known to be associated with perinatal infections. Cellular infection was defined and quantified by plaque assays, luciferase assays, microscopy, and/or colonization assays. Differences in infection were assessed by Student t test or analysis of variance with Bonferroni correction. Infection by rubella and other togaviruses, human immunodeficiency virus-1, and varicella zoster was attenuated in cells preexposed to trophoblast-conditioned medium (P < .05), and a partial effect by the chromosome 19 microRNA miR-517-3p on specific pathogens. The conditioned medium had no effect on infection by Toxoplasma gondii or Listeria monocytogenes. Our findings indicate that medium conditioned by primary human trophoblasts attenuates viral infection in nontrophoblastic cells. Our data point to a trophoblast-specific antiviral effect that may be exploited therapeutically.

PLoS ONE, 2014

Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of sign... more Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo. Citation: Moroco JA, Craigo JK, Iacob RE, Wales TE, Engen JR, et al. (2014) Differential Sensitivity of Src-Family Kinases to Activation by SH3 Domain Displacement. PLoS ONE 9(8): e105629.

B63. PULMONARY CIRCULATION: VASCULAR SMOOTH MUSCLE, 2011

Oncogene, 2001

We have previously reported that the Jak2 tyrosine kinase but not Jak1 is tyrosine phosphorylated... more We have previously reported that the Jak2 tyrosine kinase but not Jak1 is tyrosine phosphorylated in the absence of IL-3 in Bcr–Abl positive M3.16 cells, which are rendered IL-3 independent by BCR–ABL gene expression. We have explored the involvement of Jak2 tyrosine phosphorylation in Bcr-Abl oncogenic effects. Our results indicate that Jak2 became tyrosine-phosphorylated in a number of cell lines

Proceedings of the National Academy of Sciences, 2009

Protein dynamics are inextricably linked to protein function but there are few techniques that al... more Protein dynamics are inextricably linked to protein function but there are few techniques that allow protein dynamics to be conveniently interrogated. For example, mutations and translocations give rise to aberrant proteins such as Bcr-Abl where changes in protein conformation and dynamics are believed to result in deregulated kinase activity that provides the oncogenic signal in chronic myelogeous leukemia. Although crystal structures of the down-regulated c-Abl kinase core have been reported, the conformational impact of mutations that render Abl resistant to smallmolecule kinase inhibitors are largely unknown as is the allosteric interplay of the various regulatory elements of the protein. Hydrogen exchange mass spectrometry (HX MS) was used to compare the conformations of wild-type Abl with a nonmyristoylated form and with 3 clinically relevant imatinib resistance mutants (T315I, Y253H and E255V). A HX-resistant core localized to the interface between the SH2 and kinase domains, a region known to be important for maintaining the down-regulated state. Conformational differences upon demyristoylation were consistent with the SH2 domain moving to the top of the small lobe of the kinase domain as a function of activation. There were conformational changes in the T315I mutant but, surprisingly, no major changes in conformation were detected in either the Y253H or the E255V mutants. Taken together, these results provide evidence that allosteric interactions and conformational changes play a major role in Abl kinase regulation in solution. Similar analyses could be performed on any protein to provide mechanistic details about conformational changes and protein function.

Oncogene, 2002

Chronic myelogenous leukemia (CML) is defined by the presence of the Philadelphia (Ph) chromosome... more Chronic myelogenous leukemia (CML) is defined by the presence of the Philadelphia (Ph) chromosome, which results in the expression of the 210 kDa Bcr -Abl tyrosine kinase. Bcr-Abl constitutively activates several signaling proteins important for the proliferation and survival of myeloid progenitors, including the Src family kinases Hck and Lyn, the Stat5 transcription factor and upstream components of the Ras/Erk pathway. Recently, we found that kinase-defective Hck blocks Bcr -Abl-induced transformation of DAGM myeloid leukemia cells to cytokine independence, suggesting that activation of the Src kinase family may be essential to oncogenic signaling by Bcr -Abl. To investigate the contribution of Src kinases to Bcr -Abl signaling in vivo, we used the pyrrolo-pyrimidine Src kinase inhibitors PP2 and A-419259. Treatment of the Ph + CML cell lines K-562 and Meg-01 with either compound resulted in growth arrest and induction of apoptosis, while the Ph 7 leukemia cell lines TF-1 and HEL were unaffected over the same concentration ranges. Suppression of Ph + cell growth by PP2 and A-419259 correlated with a decrease in Src kinase autophosphorylation. Both inhibitors blocked Stat5 and Erk activation, consistent with the suppressive effects of the compounds on survival and proliferation. In contrast, the phosphotyrosine content of Bcr -Abl and its endogenous substrate CrkL was unchanged at inhibitor concentrations that induced apoptosis, blocked oncogenic signaling and inhibited Src kinases. These data implicate the Src kinase family in Stat5 and Erk activation downstream of Bcr -Abl, and identify myeloid-specific Src kinases as potential drug targets in CML.

Oncogene, 2000

Hematopoiesis involves a complex array of growth factors that regulate the survival and prolifera... more Hematopoiesis involves a complex array of growth factors that regulate the survival and proliferation of immature progenitors, in¯uence dierentiation commitment, and modulate end-stage cell functions. This minireview is focused on the role of Stat activation in the development of myeloid cells in response to hematopoietic cytokines. Much of the evidence implicating Stats in these cellular processes comes from studies of mutant cytokine receptors selectively uncoupled from Stat activation, dominant-inhibitory Stat mutants, and mice with targeted disruptions of Stat genes. Together these approaches provide strong evidence that Stat activation, particularly of Stat3 and Stat5, plays an important role in myeloid dierentiation and survival.

Oncogene, 2004

The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway is an esse... more The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway is an essential cascade for mediating normal functions of different cytokines in the development of the hematopoietic and immune systems. Chronic exposure to arsenic has been found to cause immunotoxicity and has been associated with the suppression of hematopoiesis (anemia and leukopenia). Here, we report the novel finding of arsenic-mediated inactivation of the JAK-STAT signaling pathway by its direct interaction with JAK tyrosine kinase. Pretreatment with sodium arsenite strongly inhibited IL-6-inducible STAT3 tyrosine phosphorylation in HepG2 cells and did not affect its serine phosphorylation. As a result, sodium arsenite completely abolished STAT activity-dependent expression of suppressors of cytokine signaling (SOCS). Both cellular and subcelluar experiments showed that the inhibition of JAK-STAT signaling resulted from JAK tyrosine kinase's direct interaction with arsenite, and that arsenic's suppression of JAK tyrosine kinase activity also occurred in the interferon c (IFNc) pathway. The ligandindependent inhibition by arsenic indicates that JAK was the direct target of arsenic action. Other inflammatory stimulants, stress agents, and metal cadmium failed to induce similar effects on the tyrosine phosphorylation of STAT3 as arsenic does. Our experiments also revealed that arsenic inactivation of the JAK-STAT pathway occurred independent of arsenic activation of MAP kinases. Taken together, our findings indicate that arsenic directly inhibits JAK tyrosine kinase activity and suggest that this direct interference in the JAK-STAT pathway may play a role in arsenic-associated pathogenesis.

Molecular Biology of the Cell, 2010

؉ T-cells. During the first two days after infection, Nef assembles the 2c-sensitive multi-kinase... more ؉ T-cells. During the first two days after infection, Nef assembles the 2c-sensitive multi-kinase complex to trigger down-regulation of cell-surface MHC-I. By three days postinfection Nef switches to a stoichiometric mode that prevents surface delivery of newly synthesized MHC-I. Pharmacologic inhibition of the multi-kinase cascade prevents the Nef-dependent block in MHC-I transport, suggesting the signaling and stoichiometric modes are causally linked. Together, these studies resolve the seemingly controversial models that describe Nef-induced MHC-I down-regulation and provide new insights into the mechanism of Nef action.

Molecular and Cellular Biology, 2001

The c-fes locus encodes a 93-kDa non-receptor protein tyrosine kinase (Fes) that regulates the gr... more The c-fes locus encodes a 93-kDa non-receptor protein tyrosine kinase (Fes) that regulates the growth and differentiation of hematopoietic and vascular endothelial cells. Unique to Fes is a long N-terminal sequence with two regions of strong homology to coiled-coil oligomerization domains. We introduced leucine-to-proline substitutions into the coiled coils that were predicted to disrupt the coiled-coil structure. The resulting mutant proteins, together with wild-type Fes, were fused to green fluorescent protein and expressed in Rat-2 fibroblasts. We observed that a point mutation in the first coiled-coil domain (L145P) dramatically increased Fes tyrosine kinase and transforming activities in this cell type. In contrast, a similar point mutation in the second coiled-coil motif (L334P) was without effect. However, combining the L334P and L145P mutations reduced transforming and kinase activities by approximately 50% relative to the levels of activity produced with the L145P mutation alone. To study the effects of the coiled-coil mutations in a biologically relevant context, we expressed the mutant proteins in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid leukemia cell line TF-1. In this cellular context, the L145P mutation induced GM-CSF independence, cell attachment, and spreading. These effects correlated with a marked increase in L145P protein autophosphorylation relative to that of wild-type Fes. In contrast, the double coiled-coil mutant protein showed greatly reduced kinase and biological activities in TF-1 cells. These data are consistent with a role for the first coiled coil in the negative regulation of kinase activity and a requirement for the second coiled coil in either oligomerization or recruitment of signaling partners. Gel filtration experiments showed that the unique N-terminal region interconverts between monomeric and oligomeric forms. Single point mutations favored oligomerization, while the double point mutant protein eluted essentially as the monomer. These data provide new evidence for coiled-coil-mediated regulation of c-Fes tyrosine kinase activity and signaling, a mechanism unique among tyrosine kinases.

Journal of Molecular Neuroscience, 1998

NeuroD, a vertebrate homolog of Drosophila atonal gene, plays an important role in the differenti... more NeuroD, a vertebrate homolog of Drosophila atonal gene, plays an important role in the differentiation of neuronal precursors . We have investigated whether NeuroD subserves a similar function in mammalian retinal neurogenesis. Expression of NeuroD is detected in successive stages of retinal neurogenesis and is associated with a differentiating population of retinal cells. The association of NeuroD predominantly with postmitotic precursors in early as well as late neurogenesis suggests that NeuroD expression plays an important role in the terminal differentiation of retinal neurons. This notion is supported by observations that overexpression of NeuroD during late neurogenesis promotes premature differentiation of late-born neurons, rod photoreceptors, and bipolar cells, and that NeuroD can interact specifically with the E-box element in the proximal promoter of the phenotype-specific gene, opsin.

Journal of Molecular Biology, 1999

Protein dynamics play an important role in protein function and regulation of enzymatic activity.... more Protein dynamics play an important role in protein function and regulation of enzymatic activity. To determine how additional interactions with surrounding structure affects local protein dynamics, we have used hydrogen exchange and mass spectrometry to investigate the SH2 and SH3 domains of the protein tyrosine kinase Hck. Exchange rates of isolated Hck SH3 and SH2 domains were compared with rates for the same domains when part of a larger SH(3 2) construct. Increased deuterium incorporation was observed for the SH3 domain in the joint construct, particularly near the SH2 interface and the short sequence that connects SH3 to SH2, implying greater¯exibility of SH3 when it is part of SH(3 2). Slow cooperative unfolding of the SH3 domain occurred at the same rate in isolated SH3 as in the SH(3 2) construct, suggesting a functional signi®cance for this unfolding. The SH2 domain displayed relatively smaller changes in¯exibility when part of the SH(3 2) construct. These results suggest that the domains in¯uence each other. Further, our results imply a link between functional regulation and structural dynamics of SH3 and SH2 domains. Abbreviations used: SH3, Src homology 3; SH2, Src homology 2; PTK, protein tyrosine kinase; Abl, Abelson tyrosine kinase; PI3K, phosphatidylinositol 3-kinase; Hck, hematopoietic cell kinase; NHs, backbone amide hydrogens; HX, hydrogen exchange; SH(32), construct of tandem Hck SH3 and SH2 domains; SH3-A, the Hck SH3 domain expressed alone; SH2-A, the Hck SH2 domain expressed alone; SH3-J, the Hck SH3 domain when part of the SH(32) construct; SH2-J, the Hck SH2 domain when part of the SH(32) construct; GAP, GTPase activating protein.

Journal of Molecular Biology, 2007

Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoidspec... more Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoidspecific Src-family kinase Lck. The Tip:Lck interaction is essential for transformation and oncogenesis in HVS-infected cells. As there are no structural data for Tip, hydrogen exchange mass spectrometry was used to investigate the conformation of a nearly full-length form (residues 1-187) of Tip from HVS strain C484. Disorder predictions suggested that Tip would be mostly unstructured so great care was taken to ascertain whether recombinant Tip was functional. Circular dichroism and gel filtration analysis indicated an extended, unstructured protein. In vitro and in vivo binding and kinase assays confirmed that purified, recombinant Tip interacted with Lck, was capable of strongly activating Lck kinase activity and was multiply phosphorylated by Lck. Hydrogen exchange mass spectrometry of Tip then showed that the majority of backbone amide hydrogens became deuterated after only 10 seconds of labeling. Such a result suggested that Tip was almost totally unstructured in solution. Digestion of deuterium labeled Tip revealed some regions with minor protection from exchange. Overall, it was found that although recombinant Tip is still functional and capable of binding and activating its target Lck, it is largely unstructured.

Journal of Molecular Biology, 2007

Activation of Src family kinases by HIV-1 Nef may play an important role in the pathogenesis of H... more Activation of Src family kinases by HIV-1 Nef may play an important role in the pathogenesis of HIV/AIDS. Here we investigated whether diverse Nef sequences universally activate Hck, a Src family member expressed in macrophages and other HIV-1 target cells. In general, we observed that Hck activation is a highly conserved Nef function. However, we identified an unusual Nef variant from an HIV-positive individual that did not develop AIDS which failed to activate Hck despite the presence of conserved residues linked to Hck SH3 domain binding and kinase activation. Amino acid sequence alignment with active Nef proteins revealed differences in regions not previously implicated in Hck activation, including a large internal flexible loop absent from available Nef structures. Substitution of these residues in active Nef compromised Hck activation without affecting SH3 domain binding. These findings show that residues at a distance from the SH3 domain binding site allosterically influence Nef interactions with a key effector protein linked to AIDS progression.

Journal of Molecular Biology, 2011

HIV-1 has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family ... more HIV-1 has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family of host-cell enzymes. The HIV-1 virion infectivity factor, one of several HIV accessory proteins, targets APOBEC3 proteins for proteasomal degradation and down-regulates their expression at the mRNA level. Despite the importance of Vif for HIV-1 infection, there is little conformational data on Vif alone or in complex with other cellular factors due to incompatibilities with many structural techniques and difficulties in producing suitable quantities of protein for biophysical analysis. As an alternative, we have turned to hydrogen exchange mass spectrometry (HX MS), a conformational analysis method well suited for proteins that are difficult to study using X-ray crystallography and/or NMR. HX MS was used to probe the solution conformation of recombinant full-length HIV-1 Vif. Vif specifically interacted with the previously identified binding partner Hck and was able to cause kinase activation suggesting that the Vif studied by HX MS retained a biochemically competent conformation relevant to Hck interaction. HX MS analysis of Vif alone revealed low deuteration levels in the N-terminal portion indicating that this region contained structured or otherwise protected elements. In contrast, high deuteration levels in the C-terminal portion of Vif indicated that this region was likely unstructured in the absence of cellular interacting proteins. Several regions within Vif displayed conformational heterogeneity in solution including the APOBEC3G/F binding site and HCCH zinc finger. Taken together, these HX MS results provide new insights into the solution conformation of Vif.