Biomedical subjects

John H Kehrl

Publications and source records attributed to John H Kehrl.

At least 19 recordsLinked to original sources

PubMed2006

RGS1 and RGS13 mRNA silencing in a human B lymphoma line enhances responsiveness to chemoattractants and impairs desensitization.

Chemokines bind receptors that are members of the G-protein-coupled receptor family. Chemokine receptors transduce intracellular signals by activating heterotrimeric G-proteins. Acting to limit and modulate heterotrimeric G-protein signaling is a family of proteins, termed regulator of G-protein signaling (RGS). Two of these proteins, RGS1 and RGS13, are well-expressed in germinal center B cells and many Burkitt's lymphoma cell lines. Reducing RGS13 and to a lesser extent RGS1 expression in a Burkitt's lymphoma cell line enhances responsiveness to two chemokines, CXC chemokine ligand 12 (CXCL12) and CXCL13, and reducing both mRNAs augments the responses more dramatically. The double knock-down (KD) cells respond better to restimulation with CXCL12 or CXCL13 after a primary stimulation with CXCL12 than do the control cells. The double-KD cells also exhibit a greater propensity to polarize and to develop multiple small lamellipodia. These results indicate that RGS1 and RGS13 act together to regulate chemokine receptor signaling in human germinal center B lymphocytes and provide evidence that they contribute significantly to the rapid desensitization of the signaling pathway.

B-Lymphocytes↗

PubMed2006

Roles for phosphoinositide 3-kinases, Bruton's tyrosine kinase, and Jun kinases in B lymphocyte chemotaxis and homing.

B lymphocyte chemokine receptors signal to downstream effectors by activating heterotrimeric G proteins. However, many of these effectors remain unknown and the known ones often have ill-defined roles in B cell trafficking. Here we report that pharmacological inhibitors of phosphoinositide 3-kinases (wortmannin, WMN), Bruton's tyrosine kinase (LFM-A13), and Jun kinases (SP600125) all significantly impair CXCL12-induced mouse B cell chemotaxis and that of a human B lymphoma cell line. Examination of two CXCR4-induced signaling pathways revealed that LFM-A13 and WMN blocked Akt activation, while SP600125 and WMN blocked JNK activation. Each of the inhibitors impaired the homing of transferred B cells to peripheral lymph nodes. Intravital imaging of control and inhibitor-treated mouse B cells in the inguinal lymph node high endothelial venules (HEV) demonstrated a 17%, 35%, and 60% reduction in the number of firmly adherent B cells with LFM-A13, SP600125, and WMN, respectively. These results implicate chemokine receptor mediated activation of phosphoinositide 3-kinases in the firm adhesion of mouse B cells within peripheral lymph node HEV, while Bruton's tyrosine kinase and JNK activation are less important and more likely needed during B cell transmigration through the endothelium and/or trafficking into the lymph node parenchyma.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed2006

The mitogen-activated protein kinase kinase kinase kinase GCKR positively regulates canonical and noncanonical Wnt signaling in B lymphocytes.

Wnt ligands bind receptors of the Frizzled (Fz) family to control cell fate, proliferation, and polarity. Canonical Wnt/Fz signaling stabilizes beta-catenin by inactivating GSK3beta, leading to the translocation of beta-catenin to the nucleus and the activation of Wnt target genes. Noncanonical Wnt/Fz signaling activates RhoA and Rac, and the latter triggers the activation of c-Jun N-terminal kinase (JNK). Here, we show that exposure of B-lymphocytes to Wnt3a-conditioned media activates JNK and raises cytosolic beta-catenin levels. Both the Rac guanine nucleotide exchange factor Asef and the mitogen-activated protein kinase kinase kinase kinase germinal center kinase-related enzyme (GCKR) are required for Wnt-mediated JNK activation in B cells. In addition, we show that GCKR positively affects the beta-catenin pathway in B cells. Reduction of GCKR expression inhibits Wnt3a-induced phosphorylation of GSK3beta at serine 9 and decreases the accumulation of cytosolic beta-catenin. Furthermore, Wnt signaling induces an interaction between GCKR and GSK3beta. Our findings demonstrate that GCKR facilitates both canonical and noncanonical Wnt signaling in B lymphocytes.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Chemoattractant receptor signaling and the control of lymphocyte migration.

This review focuses on mechanisms by which chemoattractant receptors activate downstream signaling pathways in lymphocytes. An emphasis is placed on heterotrimeric G protein signaling with a discussion of the specific heterotrimeric G-proteins involved in lymphocyte chemotaxis and motility and the role of regulator of G protein signaling (RGS) proteins in controlling the activation of downstream effectors. Also considered are those direct downstream effectors known to function in lymphocyte chemotaxis and/or motility. The consequences of targeting genes suspected, known, or serendipitously found to be involved in chemokine receptor signaling pathways form much of a basis for the review. When needed for clarification, reference to studies of chemoattractant signaling in model organisms and in neutrophils will be compared and contrasted to studies in lymphocytes. Finally, the emergence of tools to image lymphocyte in vitro and in vivo will be mentioned as they are increasing helpful for the analysis of lymphocyte trafficking and amendable to the study of chemokine receptor signaling.

Animals↗

PubMed2005

B cells productively engage soluble antigen-pulsed dendritic cells: visualization of live-cell dynamics of B cell-dendritic cell interactions.

Interactions between B lymphocytes and Ag-bearing dendritic cells (DC) likely occur at inflammatory sites and within lymphoid organs. To better understand these interactions we imaged B cells (TgB) from hen egg lysozyme (HEL) transgenic mice and DC pulsed with HEL (DC-HEL) in collagen matrices. Analysis of live-cell dynamics revealed autonomous movements and repeated encounters between TgB cells and DC-HEL that are best described by a "kiss-run and engage" model, whereas control B cells had only short-lived interactions. Ag localized at contact sites between TgB cells and DC-HEL, and both cell types rearranged their actin cytoskeletons toward the contact zone. The interaction of a TgB cell with a HEL-bearing DC triggered strong Ca2+ transients in the B cells. Thus, B cells can productively interact with DC displaying their cognate Ag.

Animals↗

PubMed2005

Rgs1 and Gnai2 regulate the entrance of B lymphocytes into lymph nodes and B cell motility within lymph node follicles.

Signaling by G protein-coupled receptors coupled to Galpha(i) assists in triggering lymphocyte movement into and out of lymph nodes. Here, we show that modulating the signaling output from these receptors dramatically alters B cell trafficking. Intravital microscopy of adoptively transferred B cells from wild-type and Rgs1-/- mice revealed that Rgs1-/- B cells stick better to lymph node high endothelial venules, home better to lymph nodes, and move more rapidly within lymph node follicles than do wild-type B cells. In contrast, B cells from Gnai2-/- mice enter lymph nodes poorly and move more slowly than do wild-type B cells. The Gnai2-/- mice often lack multiple peripheral lymph nodes, and their B cells respond poorly to chemokines, indicating that Galpha(i1) and Galpha(i3) poorly compensate for the loss of Galpha(i2). These results demonstrate opposing roles for Rgs1 and Gnai2 in B cell trafficking into and within lymph nodes.

Animals↗

PubMed2004

RGS14 is a centrosomal and nuclear cytoplasmic shuttling protein that traffics to promyelocytic leukemia nuclear bodies following heat shock.

RGS14, a member of the regulator of G-protein signaling (RGS) protein family, possesses an N-terminal RGS domain, two Raf-like Ras-binding domains, and a GoLoco motif, which has GDP dissociation inhibitor activity. In this study we show that unique among the known mammalian RGS proteins, RGS14 localizes in centrosomes. Its first Ras-binding domain is sufficient to target RGS14 to centrosomes. RGS14 also shuttles between the cytoplasm and nucleus, and its nuclear export depends on the CRM-1 nuclear export receptor. Mutation of a nuclear export signal or treatment with leptomycin B causes nuclear accumulation of RGS14 and its association with promyelocytic leukemia protein nuclear bodies. Furthermore, a point mutant defective in nuclear export fails to target to centrosomes, suggesting that nuclear cytoplasmic shuttling is necessary for its proper localization. Mild heat stress, but not proteotoxic or transcription-linked stresses, re-localizes the RGS14 from the cytoplasm to promyelocytic leukemia nuclear bodies. Expression of RGS14, but not point mutants that disrupt the functional activity of its RGS domain or GoLoco motif, enhances the reporter gene activity. The multifunctional domains and the dynamic subcellular localization of RGS14 implicate it in a diverse set of cellular processes including centrosome and nuclear functions and stress-induced signaling pathways.

Amino Acid Sequence↗

PubMed2004

Toll-like receptor signaling alters the expression of regulator of G protein signaling proteins in dendritic cells: implications for G protein-coupled receptor signaling.

Conserved structural motifs on pathogens trigger pattern recognition receptors present on APCs such as dendritic cells (DCs). An important class of such receptors is the Toll-like receptors (TLRs). TLR signaling triggers a cascade of events in DCs that includes modified chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein-coupled receptors (GPCRs). One mechanism by which TLR signaling could modify GPCR signaling is by altering the expression of regulator of G protein signaling (RGS) proteins. In this study, we show that human monocyte-derived DCs constitutively express significant amounts of RGS2, RGS10, RGS14, RGS18, and RGS19, and much lower levels of RGS3 and RGS13. Engagement of TLR3 or TLR4 on monocyte-derived DCs induces RGS16 and RGS20, markedly increases RGS1 expression, and potently down-regulates RGS18 and RGS14 without modifying other RGS proteins. A similar pattern of Rgs protein expression occurred in immature bone marrow-derived mouse DCs stimulated to mature via TLR4 signaling. The changes in RGS18 and RGS1 expression are likely important for DC function, because both proteins inhibit G alpha(i)- and G alpha(q)-mediated signaling and can reduce CXC chemokine ligand (CXCL)12-, CC chemokine ligand (CCL)19-, or CCL21-induced cell migration. Providing additional evidence, bone marrow-derived DCs from Rgs1(-/-) mice have a heightened migratory response to both CXCL12 and CCL19 when compared with similar DCs prepared from wild-type mice. These results indicate that the level and functional status of RGS proteins in DCs significantly impact their response to GPCR ligands such as chemokines.

Animals↗

PubMed2004

Pyk2 amplifies epidermal growth factor and c-Src-induced Stat3 activation.

Signal transducers and activators of transcription factors (STATs) mediate many of the cellular responses that occur following cytokine, growth factor, and hormone signaling. STATs are activated by tyrosine and serine phosphorylation, which normally occurs as a tightly regulated process. Dysregulated STAT activity may facilitate oncogenesis, as constitutively activated STATs have been found in many human tumors as well as in v-abl- and v-src-transformed cell lines. Pyk2 is a member of the focal adhesion kinase family and can be activated by c-Src, epidermal growth factor receptor (EGFR), Janus kinase 1, tyrosine kinases, and G-protein-coupled receptor signaling. Although Pyk2 has been implicated in Janus kinase-dependent activation of MAPK and Stat1, no role for Pyk2 in the activation of other STAT proteins has been ascribed. Here, we provide evidence that Pyk2, along with c-Src, facilitates EGFR-mediated Stat3 activation. Pyk2 expression in HeLa cells induces Stat3 reporter gene activation and Stat3 phosphorylation on amino acid residues Tyr-705 and Ser-727. Together Pyk2 and c-Src potently activate Stat3, and Pyk2 enhances Stat3-induced cell proliferation. Moreover, the expression of a dominant negative version of Pyk2 impairs c-Src-induced Stat3 activation and cell proliferation. The treatment of A431 cells with EGF results in the recruitment of c-Src, Pyk2, and Stat3 to the EGFR and the phosphorylation of c-Src, Pyk2, and Stat3. Expression of constructs for dominant negative forms of either Pyk2 or c-Src impair EGF-induced Stat3 phosphorylation. These results indicate that Pyk2 facilitates EGFR- and c-Src-mediated Stat3 activation, thereby implicating Pyk2 activation as a potential co-mediator in triggering Stat3-induced oncogenesis.

CSK Tyrosine-Protein Kinase↗

PubMed2004

Regulation of chemokine-induced lymphocyte migration by RGS proteins.

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by inducing the G-protein alpha (Galpha) subunit to exchange guanosine diphosphate for guanosine triphosphate. Regulators of G-protein signaling (RGS) proteins enhance the deactivation of Galpha subunits, thereby reducing the activation of downstream effectors. Several members of the RGS family are expressed in lymphocytes. Among RGS proteins with the highest levels of expression are RGS1, RGS2, RGS10, RGS13, RGS14, RGS16, and RGS19. Perhaps the most important G-protein-coupled receptors in lymphocytes potentially subject to regulation by RGS proteins are the chemokine receptors. By signaling through these receptors, chemokines help orchestrate immune cell trafficking both during the development of the immune system and during responses to exogenous or infectious agents. Thus, the level and regulation of RGS proteins in lymphocytes likely significantly impact lymphocyte migration and function. This article provides some tools for the analysis of RGS protein expression in lymphocytes and outlines a number of methods for the analysis of the effects of RGS proteins on lymphocyte migration and chemokine receptor signaling.

Animals↗

PubMed2004

The G12 family of heterotrimeric G proteins and Rho GTPase mediate Sonic hedgehog signalling.

Sonic hedgehog (Shh) is a secreted morphogen crucial for cell fate decision, cellular proliferation, and patterning during vertebrate development. The intracellular Shh signalling is transduced by Smoothened (Smo), a seven-transmembrane spanning protein that belongs to the G-protein coupled receptor family. Among four families of Galpha subunits, Galphai has been thought to be responsible for transducing Shh signalling, while several lines of evidence indicated that other signalling pathways may be involved. We found that the G12 family of heterotrimeric G proteins and the small GTPase RhoA are involved in Shh/Smo-mediated cellular responses, including stimulation of target gene promoter and inhibition of neurite outgrowth of neuroblastoma cells. We also found that the G12/RhoA pathway is responsible for Smo-induced nuclear import of GLI3 which is thought to transduce Shh signals to nucleus. Furthermore, misexpression of a G12-specific GTPase-activating protein in rat neural tubes leads to pertubation of motor neurone and interneurone development, mimicking the effects of decreased Shh signalling. These results show that Shh signalling is mediated in part by activating G12 family coupled signalling pathways. The participation of RhoA, a pivotal molecular switch in many signal transduction pathways, may help explain how Shh can trigger a variety of cellular responses.

Animals↗

PubMed2004

Abnormal B-cell responses to chemokines, disturbed plasma cell localization, and distorted immune tissue architecture in Rgs1-/- mice.

Normal lymphoid tissue development and function depend upon chemokine-directed cell migration. Since chemokines signal through heterotrimeric G-protein-coupled receptors, RGS proteins, which act as GTPase-activating proteins for Galpha subunits, likely fine tune the cellular responses to chemokines. Here we show that Rgs1(-/-) mice possess B cells that respond excessively and desensitize improperly to the chemokines CXCL12 and CXCL13. Many of the B-cell follicles in the spleens of Rgs1(-/-) mice have germinal centers even in the absence of immune stimulation. Furthermore, immunization of these mice leads to exaggerated germinal center formation; partial disruption of the normal architecture of the spleen and Peyer's patches; and abnormal trafficking of immunoglobulin-secreting cells. These results reveal the importance of a regulatory mechanism that limits and desensitizes chemokine receptor signaling.

Animals↗

PubMed2004

In vitro and in vivo assays of B-lymphocyte migration.

The development and functional activities of B lymphocytes critically depend on their migratory capacity. Both in vitro and in vivo assays can be used to assess the migratory ability of B cells. Filter-based transwell assays measure both spontaneous and chemoattractant-induced cell migration in vitro. Flow cytometric analysis of labeled cell subsets present in the input and migratory cells from transwell assays allows the assessment of the migratory capacity of specific cell types in a complex mixture of cells. A similar approach can be used to assess the migratory capacity of B cells transfected with a green fluorescent fusion protein. Despite the success of the transwell assay, attempts to image directionally migrating B cells have been limited. The assessment of the in vivo migratory capacity of genetically modified or pharmacologically treated B cells usually involves their adoptive transfer into recipient mice. The localization of transferred cells in lymphoid organs can be determined by immunohistochemistry and/or flow cytometric analysis of harvested tissues or cells. Because the proper localization of B cells in lymphoid organs is a multistep process, abnormal positioning can occur even in the absence of an intrinsic defect in cell migration.

Animals↗

PubMed2004

G-protein-coupled receptor signaling, RGS proteins, and lymphocyte function.

The positioning of lymphocytes in immune organs and the migration of lymphocytes that occurs during normal immune surveillance and following immune activation depends on appropriate signaling through receptors that couple to heterotrimeric G-proteins. In addition other mediators that affect lymphocyte function, such as histamine, purine nucleosides, C5A, prostaglandins, leukotrienes, serotonin, epinephrine, opioids, and certain phospholipids, also signal through G-protein-coupled receptors (GPCRs). Downstream of heterotrimeric G-proteins are a limited number of downstream effectors, which, in turn, activate a large number of other signaling molecules, many of which are shared with other signaling pathways, such as those activated by antigen receptors, coreceptors, and adhesion receptors. Crucial to signaling through GPCRs are finely developed regulatory systems, which control the activation of heterotrimeric G-proteins and their interactions with their immediate downstream effectors. This review will focus on the overall importance of GPCR signaling in lymphocyte function and an upstream regulatory system present in lymphocytes, which fine tunes heterotrimeric G-protein signaling.

Animals↗

PubMed2003

The aorta and heart differentially express RGS (regulators of G-protein signalling) proteins that selectively regulate sphingosine 1-phosphate, angiotensin II and endothelin-1 signalling.

Normal cardiovascular development and physiology depend in part upon signalling through G-protein-coupled receptors (GPCRs), such as the angiotensin II type 1 (AT(1)) receptor, sphingosine 1-phosphate (S1P) receptors and endothelin-1 (ET-1) receptor. Since regulator of G-protein signalling (RGS) proteins function as GTPase-activating proteins for the G alpha subunit of heterotrimeric G-proteins, these proteins undoubtedly have functional roles in the cardiovascular system. In the present paper, we show that human aorta and heart differentially express RGS1, RGS2, RGS3S (short-form), RGS3L (long-form), PDZ-RGS3 (PDZ domain-containing) and RGS4. The aorta prominently expresses mRNAs for all these RGS proteins except PDZ-RGS3. Various stimuli that are critical for both cardiovascular development and function regulate dynamically the mRNA levels of several of these RGS proteins in primary human aortic smooth muscle cells. Both RGS1 and RGS3 inhibit signalling through the S1P(1) (formerly known as EDG-1), S1P(2) (formerly known as EDG-5) and S1P(3) (formerly known as EDG-3) receptors, whereas RGS2 and RGS4 selectively attenuate S1P(2)-and S1P(3)-receptor signalling respectively. All of the tested RGS proteins inhibit AT(1)-receptor signalling, whereas only RGS3 and, to a lesser extent, RGS4 inhibit ET(A)-receptor signalling. The conspicuous expression of RGS proteins in the cardiovascular system and their selective effects on relevant GPCR-signalling pathways provide additional evidence that they have functional roles in cardiovascular development and physiology.

Angiotensin II↗

PubMed2003

Identification of RGS2 and type V adenylyl cyclase interaction sites.

The production of cAMP is controlled on many levels, notably at the level of cAMP synthesis by the enzyme adenylyl cyclase. We have recently identified a new regulator of adenylyl cyclase activity, RGS2, which decreases cAMP accumulation when overexpressed in HEK293 cells and inhibits the in vitro activity of types III, V, and VI adenylyl cyclase. In addition, RGS2 blocking antibodies lead to elevated cAMP levels in olfactory neurons. Here we examine the nature of the interaction between RGS2 and type V adenylyl cyclase. In HEK293 cells expressing type V adenylyl cyclase, RGS2 inhibited Galpha(s)-Q227L- or beta(2)-adrenergic receptor-stimulated cAMP accumulation. Deletion of the N-terminal 19 amino acids of RGS2 abolished its ability to inhibit cAMP accumulation and to bind adenylyl cyclase. Further mutational analysis indicated that neither the C terminus, RGS GAP activity, nor the RGS box domain is required for inhibition of adenylyl cyclase. Alanine scanning of the N-terminal amino acids of RGS2 identified three residues responsible for the inhibitory function of RGS2. Furthermore, we show that RGS2 interacts directly with the C(1) but not the C(2) domain of type V adenylyl cyclase and that the inhibition by RGS2 is independent of inhibition by Galpha(i). These results provide clear evidence for functional effects of RGS2 on adenylyl cyclase activity that adds a new dimension to an intricate signaling network.

Adenylyl Cyclase Inhibitors↗

PubMed2003

Tumor necrosis factor (TNF)-induced germinal center kinase-related (GCKR) and stress-activated protein kinase (SAPK) activation depends upon the E2/E3 complex Ubc13-Uev1A/TNF receptor-associated factor 2 (TRAF2).

Tumor necrosis factor (TNF)-induced activation of apoptosis signal-regulating kinase 1 (ASK1) and germinal center kinases (GCKs) and the subsequent activation of stress-activated protein kinases (SAPKs and c-Jun NH(2)-terminal kinases) requires TNF receptor-associated factor 2 (TRAF2). Although the TRAF2 TRAF domain binds ASK1, GCK, and the highly related kinase GCKR, the RING finger domain is needed for their activation. Here, we report that TNF activates GCKR and the SAPK pathway in a manner that depends upon TRAF2 and Ubc13, a member along with Uev1A of a dimeric ubiquitin-conjugating enzyme complex. Interference with Ubc13 function or expression inhibits both TNF- and TRAF2-mediated GCKR and SAPK activation, but has a minimal effect on ASK1 activation. TNF signaling leads to TRAF2 polyubiquitination and oligomerization and to the oligomerization, ubiquitination, and activation of GCKR, all of which are sensitive to the disruption of Ubc13 function. These results indicate that the assembly of a TRAF2 lysine 63-linked polyubiquitin chain by Ubc13/Uev1A is required for TNF-mediated GCKR and SAPK activation, but may not be required for ASK1 activation.

Enzyme Activation↗

PubMed2003

Pericyte-specific expression of Rgs5: implications for PDGF and EDG receptor signaling during vascular maturation.

RGS proteins finely tune heterotrimeric G-protein signaling. Implying the need for such fine-tuning in the developing vascular system, in situ hybridization revealed a striking and extensive expression pattern of Rgs5 in the arterial walls of E12.5-E17.5 mouse embryos. The distribution and location of the Rgs5-positive cells typified that of pericytes and strikingly overlapped the known expression pattern of platelet-derived growth factor receptor (PDGFR)-beta. Both E14.5 PDGFR-beta- and platelet-derived growth factor (PDGF)-B-deficient mice exhibited markedly reduced levels of Rgs5 in their vascular plexa and small arteries. This likely reflects the loss of pericytes in the mutant mice. RGS5 acts as a potent GTPase activating protein for Gi(alpha) and Gq(alpha) and it attenuated angiotensin II-, endothelin-1-, sphingosine-1-phosphate-, and PDGF-induced ERK-2 phosphorylation. Together these results indicate that RGS5 exerts control over PDGFR-beta and GPCR-mediated signaling pathways active during fetal vascular maturation.

3T3 Cells↗