Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulates interleukin 2 production

T cell receptor ζ (TcRζ)/CD3 ligation initiates a signaling cascade that involves src kinases p56lck and ζ-associated protein 70, leading to the phosphorylation of substrates such as TcRζ, Vav, SH2-domain-containing leukocyte protein 76 (SLP-76), cbl, and p120/130. FYN binding protein (FYB or p120/130) associates with p59fyn, the TcRζ/CD3 complex, and becomes tyrosine-phosphorylated in response to receptor ligation. In this study, we report the cDNA cloning of human and murine FYB and show that it is restricted in expression to T cells and myeloid cells and possesses an overall unique hydrophilic sequence with several tyrosine-based motifs, proline-based type I and type II SH3 domain binding motifs, several putative lysine/glutamic acid-rich nuclear localization motifs, and a SH3-like domain. In addition to binding the src kinase p59fyn, FYB binds specifically to the hematopoietic signaling protein SLP-76, an interaction mediated by the SLP-76 SH2 domain. In keeping with this, expression of FYB augmented interleukin 2 secretion from a T cell hybridoma, DC27.10, in response to TcRζ/CD3 ligation. FYB is therefore a novel hematopoietic protein that acts as a component of the FYN and SLP-76 signaling cascades in T cells.

A novel bacterial tyrosine kinase essential for cell division and differentiation

Protein kinases play central roles in the regulation of eukaryotic and prokaryotic cell growth, division, and differentiation. The Caulobacter crescentus divL gene encodes a novel bacterial tyrosine kinase essential for cell viability and division. Although the DivL protein is homologous to the ubiquitous bacterial histidine protein kinases (HPKs), it differs from previously studied members of this protein kinase family in that it contains a tyrosine residue (Tyr-550) in the conserved H-box instead of a histidine residue, which is the expected site of autophosphorylation. DivL is autophosphorylated on Tyr-550 in vitro, and this tyrosine residue is essential for cell viability and regulation of the cell division cycle. Purified DivL also catalyzes phosphorylation of CtrA and activates transcription in vitro of the cell cycle-regulated fliF promoter. Suppressor mutations in ctrA bypass the conditional cell division phenotype of cold-sensitive divL mutants, providing genetic evidence that DivL function in cell cycle and developmental regulation is mediated, at least in part, by the global response regulator CtrA. DivL is the only reported HPK homologue whose function has been shown to require autophosphorylation on a tyrosine, and, thus, it represents a new class of kinases within this superfamily of protein kinases.

CD19 and CD22 expression reciprocally regulates tyrosine phosphorylation of Vav protein during B lymphocyte signaling

B cell development and humoral immune responses are controlled by signaling thresholds established through the B lymphocyte antigen receptor (BCR) complex. BCR signaling thresholds are differentially regulated by the CD22 and CD19 cell surface receptors in vivo. B cells from CD22-deficient mice exhibit characteristics of chronic stimulation and are hyper-responsive to BCR crosslinking with augmented intracellular Ca2+ responses. By contrast, B cells from CD19-deficient mice are hypo-responsive to transmembrane signals. To identify signaling molecules involved in the positive and negative regulation of signaling thresholds, the signal transduction pathways activated after BCR crosslinking were examined in CD22- and CD19-deficient B cells. These comparisons revealed that tyrosine phosphorylation of Vav protein was uniquely augmented after BCR or CD19 crosslinking in CD22-deficient B cells, yet was modest and transient after BCR crosslinking in CD19-deficient B cells. Ligation of CD19 and CD22 in vivo is likely to positively and negatively regulate BCR signaling, respectively, because CD19 crosslinking was more efficient than BCR crosslinking at inducing Vav phosphorylation. However, simultaneous crosslinking of CD19 with the BCR resulted in a substantial decrease in Vav phosphorylation when CD22 was expressed. Thus, the differential regulation of Vav tyrosine phosphorylation by CD19 and CD22 may provide a molecular mechanism for adjusting BCR signaling thresholds.

β2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein

G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced β2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating β2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.

Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1

SOCS-1, a member of the suppressor of cytokine signaling (SOCS) family, was identified in a genetic screen for inhibitors of interleukin 6 signal transduction. SOCS-1 transcription is induced by cytokines, and the protein binds and inhibits Janus kinases and reduces cytokine-stimulated tyrosine phosphorylation of signal transducers and activators of transcription 3 and the gp130 component of the interleukin 6 receptor. Thus, SOCS-1 forms part of a feedback loop that modulates signal transduction from cytokine receptors. To examine the role of SOCS-1 in vivo, we have used gene targeting to generate mice lacking this protein. SOCS-1−/− mice exhibited stunted growth and died before weaning with fatty degeneration of the liver and monocytic infiltration of several organs. In addition, the thymus of SOCS-1−/− mice was reduced markedly in size, and there was a progressive loss of maturing B lymphocytes in the bone marrow, spleen, and peripheral blood. Thus, SOCS-1 is required for in vivo regulation of multiple cell types and is indispensable for normal postnatal growth and survival.

Thiazolidinediones and insulin resistance: Peroxisome proliferatoractivated receptor γ activation stimulates expression of the CAP gene

c-Cbl-associated protein (CAP) is a signaling protein that interacts with both c-Cbl and the insulin receptor that may be involved in the specific insulin-stimulated tyrosine phosphorylation of c-Cbl. The restricted expression of CAP in cells metabolically sensitive to insulin suggests an important potential role in insulin action. The expression of CAP mRNA and proteins are increased in 3T3-L1 adipocytes by the insulin sensitizing thiazolidinedione drugs, which are activators of the peroxisome proliferator-activated receptor γ (PPARγ). The stimulation of CAP expression by PPARγ activators results from increased transcription. This increased expression of CAP was accompanied by a potentiation of insulin-stimulated c-Cbl tyrosine phosphorylation. Administration of the thiazolidinedione troglitazone to Zucker (fa/fa) rats markedly increased the expression of the major CAP isoform in adipose tissue. This effect was sustained for up to 12 weeks of treatment and accompanied the ability of troglitazone to prevent the onset of diabetes and its complications. Thus, CAP is the first PPARγ-sensitive gene identified that participates in insulin signaling and may play a role in thiazolidinedione-induced insulin sensitization.

Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity

The C-C chemokine receptor 5 (CCR5) plays a crucial role in facilitating the entry of macrophage-tropic strains of the HIV-1 into cells, but the mechanism of this phenomenon is completely unknown. To explore the role of CCR5-derived signal transduction in viral entry, we introduced mutations into two cytoplasmic domains of CCR5 involved in receptor-mediated function. Truncation of the terminal carboxyl-tail to eight amino acids or mutation of the highly conserved aspartate-arginine-tyrosine, or DRY, sequence in the second cytoplasmic loop of CCR5 effectively blocked chemokine-dependent activation of classic second messengers, intracellular calcium fluxes, and the cellular response of chemotaxis. In contrast, none of the mutations altered the ability of CCR5 to act as an HIV-1 coreceptor. We conclude that the initiation of signal transduction, the prototypic function of G protein coupled receptors, is not required for CCR5 to act as a coreceptor for HIV-1 entry into cells.

Novel form of crosstalk between G protein and tyrosine kinase pathways

Neuronal Ca2+ channels are inhibited by a variety of transmitter receptors coupled to Go-type GTP-binding proteins. Go has been postulated to work via a direct interaction between an activated G protein subunit and the Ca2+ channel complex. Here we show that the inhibition of sensory neuron N-type Ca2+ channels produced by γ-aminobutyric acid involves a novel, rapidly activating tyrosine kinase signaling pathway that is mediated by Gαo and a src-like kinase. In contrast to other recently described G protein-coupled tyrosine kinase pathways, the Gαo-mediated modulation requires neither protein kinase C nor intracellular Ca2+. The results suggest that this pathway mediates rapid receptor-G protein signaling in the nervous system and support the existence of a previously unrecognized form of crosstalk between G protein and tyrosine kinase pathways.

Expression of multiple insulin and insulin-like growth factor receptor genes in salmon gill cartilage

In mammals, one of the major actions of insulin-like growth factor I (IGF-I) is to increase skeletal growth by stimulating new cartilage formation. IGF-I stimulates chondrocytes in vitro to synthesize new cartilage matrix, measured by enhanced uptake of 35S-sulfate, but the addition of insulin does not produce a similar effect except when added at high concentrations. However, recent studies have shown that, in teleosts, both insulin and IGF-I are potent activators of 35S-sulfate uptake in gill cartilage. To further characterize the growth-promoting activities of these hormones in fish, we have used reverse transcriptase-linked PCR to analyze the expression of insulin receptor family genes in salmon gill cartilage. Partial cDNA sequences encoding the tyrosine kinase domains from six distinct members of the IR gene family were obtained, and sequence comparisons revealed that four of the cDNAs encoded amino acid sequences that were highly homologous to human IR whereas the encoded sequences from two of the cDNAs were more similar to the human type I IGF receptor (IGF-R). Furthermore, a comparative reverse transcriptase-linked PCR assay revealed that the four putative IR mRNAs expressed in toto in gill cartilage were 56% of that found in liver whereas the expressed amount of the two IGF-R mRNAs was 9-fold higher compared with liver. These results suggest that the chondrogenic actions of insulin and IGF-I in fish are mediated by the ligands binding to their cognate receptors. However, further studies will be required to characterize the binding properties and relative contribution of the individual IR and IGF-R genes.

Eps15 Is Recruited to the Plasma Membrane upon Epidermal Growth Factor Receptor Activation and Localizes to Components of the Endocytic Pathway during Receptor Internalization

Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment, suggesting sustained phosphorylation in endocytic compartments. Our results are consistent with a model in which eps15 undergoes cycles of association:dissociation with membranes and suggest multiple roles for this protein in the endocytic pathway.

The Multisubstrate Docking Site of the MET Receptor Is Dispensable for MET-mediated RAS Signaling and Cell Scattering

The scatter factor/hepatocyte growth factor regulates scattering and morphogenesis of epithelial cells through activation of the MET tyrosine kinase receptor. In particular, the noncatalytic C-terminal tail of MET contains two autophosphorylation tyrosine residues, which form a multisubstrate-binding site for several cytoplasmic effectors and are thought to be essential for signal transduction. We show here that a MET receptor mutated on the four C-terminal tyrosine residues, Y1311F, Y1347F, Y1354F, and Y1363F, can induce efficiently a transcriptional response and cell scattering, whereas it cannot induce cell morphogenesis. Although the mutated receptor had lost its ability to recruit and/or activate known signaling molecules, such as GRB2, SHC, GAB1, and PI3K, by using a sensitive association–kinase assay we found that the mutated receptor can still associate and phosphorylate a ∼250-kDa protein. By further examining signal transduction mediated by the mutated MET receptor, we established that it can transmit efficient RAS signaling and that cell scattering by the mutated MET receptor could be inhibited by a pharmacological inhibitor of the MEK-ERK (MAP kinase kinase–extracellular signal-regulated kinase) pathway. We propose that signal transduction by autophosphorylation of the C-terminal tyrosine residues is not the sole mechanism by which the activated MET receptor can transmit RAS signaling and cell scattering.

Transduction of Basolateral-to-Apical Signals across Epithelial Cells: Ligand-stimulated Transcytosis of the Polymeric Immunoglobulin Receptor Requires Two Signals

Transcytosis of the polymeric immunoglobulin receptor (pIgR) is stimulated by binding of its ligand, dimeric IgA (dIgA). During this process, dIgA binding at the basolateral surface of the epithelial cell transmits a signal to the apical region of the cell, which in turn stimulates the transport of dIgA–pIgR complex from a postmicrotubule compartment to the apical surface. We have previously reported that the signal of stimulation was controlled by a protein-tyrosine kinase (PTK) activated upon dIgA binding. We now show that this signal of stimulation moves across the cell independently of pIgR movement or microtubules and acts through the tyrosine kinase activity by releasing Ca++ from inositol trisphosphate–sensitive intracellular stores. Surprisingly we have found that a second independent signal is required to achieve dIgA-stimulated transcytosis of pIgR. This second signal depends on dIgA binding to the pIgR solely at the basolateral surface and the ability of pIgR to dimerize. This enables pIgR molecules that have bound dIgA at the basolateral surface to respond to the signal of stimulation once they reach the postmicrotubule compartment. We propose that the use of two signals may be a general mechanism by which signaling receptors maintain specificity along their signaling and trafficking pathways.

Endocytic Clathrin-coated Pit Formation Is Independent of Receptor Internalization Signal Levels

The mechanisms responsible for coated pit formation in cells remain unknown, but indirect evidence has argued both for and against a critical role of receptor cytoplasmic domains in the process. If the endocytic motifs of receptors are responsible for recruiting AP2 to the plasma membrane, thereby driving coated pit formation, then the level of constitutively internalized receptors at the membrane would be expected to govern the steady-state level of coated pits in cells. Here we directly test this hypothesis for broad classes of receptors containing three distinct constitutive internalization signals. Chimeric proteins consisting of an integral membrane reporter protein (Tac) coupled to cytoplasmic domains bearing tyrosine-, di-leucine-, or acidic cluster/casein kinase II-based internalization signals were overexpressed to levels that saturated the internalization pathway. Quantitative confocal immunofluorescence microscopy indicated that the number of plasma membrane clathrin-coated pits and the concentration of their structural components were invariant when comparing cells expressing saturating levels of the chimeric receptors to nonexpressing cells or to cells expressing only the Tac reporter lacking cytoplasmic internalization signals. Biochemical analysis showed that the distribution of coat proteins between assembled coated pits and soluble pools was also not altered by receptor overexpression. Finally, the cellular localizations of AP2 and AP1 were similarly unaffected. These results provide a clear indication that receptor endocytic signals do not determine coated pit levels by directly recruiting AP2 molecules. Rather, the findings support a model in which coated pit formation proceeds through recruitment and activation of AP2, likely through a limited number of regulated docking sites that act independently of endocytic signals.

Integrin-mediated Signaling Events in Human Endothelial Cells

Vascular endothelial cells are important in a variety of physiological and pathophysiological processes. The growth and functions of vascular endothelial cells are regulated both by soluble mitogenic and differentiation factors and by interactions with the extracellular matrix; however, relatively little is known about the role of the matrix. In the present study, we investigate whether integrin-mediated anchorage to a substratum coated with the extracellular matrix protein fibronectin regulates growth factor signaling events in human endothelial cells. We show that cell adhesion to fibronectin and growth factor stimulation trigger distinct initial tyrosine phosphorylation events in endothelial cells. Thus, integrin-dependent adhesion of endothelial cells leads to tyrosine phosphorylation of both focal adhesion kinase and paxillin, but not of several growth factor receptors. Conversely, EGF stimulation causes receptor autophosphorylation, with no effect on focal adhesion kinase or paxillin tyrosine phosphorylation. Adhesion to fibronectin, in the absence of growth factors, leads to activation of MAPK. In addition, adhesion to fibronectin also potentiates growth factor signaling to MAPK. Thus, polypeptide growth factor activation of MAPK in anchored cells is far more effective than in cells maintained in suspension. Other agonists known to activate MAPK were also examined for their ability to activate MAPK in an anchorage-dependent manner. The neuropeptide bombesin, the bioactive lipid lysophosphatidic acid (LPA), and the cytokine tumor necrosis factor α, which signal through diverse mechanisms, were all able to activate MAPK to a much greater degree in fibronectin-adherent cells than in suspended cells. In addition, tumor necrosis factor α activation of c-Jun kinase (JNK) was also much more robust in anchored cells. Together, these data suggest a cooperation between integrins and soluble mitogens in efficient propagation of signals to downstream kinases. This cooperation may contribute to anchorage dependence of mitogenic cell cycle progression.

Radiation-induced Release of Transforming Growth Factor α Activates the Epidermal Growth Factor Receptor and Mitogen-activated Protein Kinase Pathway in Carcinoma Cells, Leading to Increased Proliferation and Protection from Radiation-induced Cell Death

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0–10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90–240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor α receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor α (TGFα) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFα cleavage 120–180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFα. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFα. Neutralization of TGFα function by an anti-TGFα antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFα–EGFR–MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.