Role of the intracellular domain of the human type I interferon receptor 2 chain (IFNAR2c) in interferon signaling. Expression of IFNAR2c truncation mutants in U5A cells

A human cell line (U5A) lacking the type I interferon (IFN) receptor chain 2 (IFNAR2c) was used to determine the role of the IFNAR2c cytoplasmic domain in regulating IFN-dependent STAT activation, interferon-stimulated gene factor 3 (ISGF3) and c-sis-inducible factor (SIF) complex formation, gene expression, and antiproliferative effects. A panel of U5A cells expressing truncation mutants of IFNAR2c on their cell surface were generated for study. Janus kinase (JAK) activation was detected in all mutant cell lines; however, STAT1 and STAT2 activation was observed only in U5A cells expressing full-length IFNAR2c and IFNAR2c truncated at residue 462 (R2.462). IFNAR2c mutants truncated at residues 417 (R2. 417) and 346 (R2.346) or IFNAR2c mutant lacking tyrosine residues in its cytoplasmic domain (R2.Y-F) render the receptor inactive. A similar pattern was observed for IFN-inducible STAT activation, STAT complex formation, and STAT-DNA binding. Consistent with these data, IFN-inducible gene expression was ablated in U5A, R2.Y-F, R2.417, and R2.346 cell lines. The implications are that tyrosine phosphorylation and the 462-417 region of IFNAR2c are independently obligatory for receptor activation. In addition, the distal 53 amino acids of the intracellular domain of IFNAR2c are not required for IFN-receptor mediated STAT activation, ISFG3 or SIF complex formation, induction of gene expression, and inhibition of thymidine incorporation. These data demonstrate for the first time that both tyrosine phosphorylation and a specific domain of IFNAR2c are required in human cells for IFN-dependent coupling of JAK activation to STAT phosphorylation, gene induction, and antiproliferative effects. In addition, human and murine cells appear to require different regions of the cytoplasmic domain of IFNAR2c for regulation of IFN responses.     

Autoinhibition of Jak2 tyrosine kinase is dependent on specific regions in its pseudokinase domain

Jak tyrosine kinases have a unique domain structure containing a kinase domain (JH1) adjacent to a catalytically inactive pseudokinase domain (JH2). JH2 is crucial for inhibition of basal Jak activity, but the mechanism of this regulation has remained elusive. We show that JH2 negatively regulated Jak2 in bacterial cells, indicating that regulation is an intrinsic property of Jak2. JH2 suppressed basal Jak2 activity by lowering the V(max) of Jak2, whereas JH2 did not affect the K(m) of Jak2 for a peptide substrate. Three inhibitory regions (IR1-3) within JH2 were identified. IR3 (residues 758-807), at the C terminus of JH2, directly inhibited JH1, suggesting an inhibitory interaction between IR3 and JH1. Molecular modeling of JH2 showed that IR3 could form a stable alpha-helical fold, supporting that IR3 could independently inhibit JH1. IR2 (725-757) in the C-terminal lobe of JH2, and IR1 (619-670), extending from the N-terminal to the C-terminal lobe, enhanced IR3-mediated inhibition of JH1. Disruption of IR3 either by mutations or a small deletion increased basal Jak2 activity, but abolished interferon-gamma-inducible signaling. Together, the results provide evidence for autoinhibition of a Jak family kinase and identify JH2 regions important for autoregulation of Jak2.     

Identification of tyrosine residues within the intracellular domain of the erythropoietin receptor crucial for STAT5 activation.

FDCP-1 cells are hematopoietic progenitor cells which require interleukin-3 for survival and proliferation. FDCP-1 cells stably transfected with the murine erythropoietin receptor cDNA survive and proliferate in the presence of erythropoietin. Erythropoietin induces the activation of the short forms (80 kDa) of STAT5 in the cells. Erythropoietin-induced activation of STAT5 was strongly reduced in cells expressing mutated variants of the erythropoietin receptors in which tyrosine residues in their intracellular domain have been eliminated. We determined that the erythropoietin receptor tyrosine residues 343 and 401 are independently necessary for STAT5 activation. The amino acid sequences surrounding these two tyrosine residues are very similar. Peptides comprising either phosphorylated Tyr343 or phosphorylated Tyr401, but not their unphosphorylated counterparts, inhibited the STAT5 activation. We propose that these two tyrosine residues of the erythropoietin receptor constitute docking sites for the STAT5 SH2 domain. The growth stimulus mediated by erythropoietin was decreased in cells expressing erythropoietin receptors lacking both Tyr343 and Tyr401. This suggests that STAT5 activation could be involved in the growth control of FDCP-1 cells.     

Regulation of signaling in B cells through the phosphorylation of Syk on linker region tyrosines. A mechanism for negative signaling by the Lyn tyrosine kinase

The B cell antigen receptor (BCR) is coupled to the mobilization of Ca(2+) by the protein-tyrosine kinase, Syk. Syk, recruited to the clustered BCR, becomes phosphorylated on three tyrosines (Tyr-317, Tyr-342, and Tyr-346) located within the linker region that separates the C-terminal catalytic domain from the N-terminal tandem Src homology 2 domains. Phosphorylation within the linker region can be either activating or inhibitory to Ca(2+) mobilization depending on the sites that are modified. Syk that is not phosphorylated on linker region tyrosines couples the BCR to Ca(2+) mobilization through a phosphoinositide 3-kinase-dependent pathway. The phosphorylation of Tyr-342 and -346 enhances the phosphorylation and activation of phospholipase C-gamma and the early phase of Ca(2+) mobilization via a phosphoinositide 3-kinase-independent pathway. The phosphorylation of Tyr-317 strongly dampens the Ca(2+) signal. In cells that lack the Src family kinase, Lyn, the phosphorylation of the inhibitory Tyr-317 is suppressed leading to elevated production of inositol 1,4,5-trisphosphate and an amplified Ca(2+) signal. This provides a novel mechanism by which Lyn functions as an inhibitor of BCR-stimulated signaling. Thus, Syk and Lyn combine to determine the pathway through which the BCR is coupled to Ca(2+) mobilization as well as the magnitude and duration of the Ca(2+) flux.     

A minor tyrosine phosphorylation site located within the CAIN domain plays a critical role in regulating tissue-specific transformation by erbB kinase.

Avian c-erbB encodes a protein that is homologous to the human epidermal growth factor receptor. Truncation of the amino-terminal, ligand-binding domain of this receptor results in an oncogene product which is a potent inducing agent for erythroleukemias but not fibrosarcomas in chickens. Here we show that mutation of a single tyrosine residue, p5, in the carboxyl terminus of the erbB oncogene product allows it to become sarcomagenic in vivo and to transform fibroblasts in vitro. Mutations of other autophosphorylation sites do not generate comparable effects. The increased transforming activity of the p5 mutant is accompanied by an elevated level of mitogen-activated protein kinase phosphorylation. By analogy to the human epidermal growth factor receptor, p5 is a minor autophosphorylation site and is located in a domain known to be involved in regulating calcium influx and receptor internalization (CAIN domain). This area of the erbB product has been found to be repeatedly deleted in various sarcomagenic avian erythroblastosis virus isolates. We precisely deleted the CAIN domain and also made point mutations of the acidic residues within the CAIN domain. In both cases, fibroblast-transforming potential is activated. We interpret these data to mean that p5 and its surrounding region negatively regulate fibroblast-transforming and sarcomagenic potential. To our knowledge, this represents the first point mutation of an autophosphorylation site that activates erbB oncogenicity.     

The processing of ligands by the class A scavenger receptor is dependent on signal information located in the cytoplasmic domain

The mechanisms that regulate the transport of the macrophage class A scavenger receptor during ligand uptake were investigated. Kinetic analysis of the changes in receptor phosphorylation demonstrated that serine phosphorylation increased during the internalization of acetyl-low density lipoproteins (LDL) by macrophages. The increase was maximal at about 2.5 min after the initiation of ligand uptake. Oxidized LDL also stimulated serine phosphorylation, but the relative increase was smaller and the time to maximum was shorter. Receptor mutants expressed in Chinese hamster ovary and COS cells showed that elimination of the potential phosphorylation site at Ser(21) increased acetyl-LDL metabolism, whereas inactivation of the site at Ser(49) reduced acetyl-LDL uptake. The increase in uptake by the Ser(21) mutant was due to an increase in surface receptor expression. In contrast, elimination of the site at Ser(49) did not affect receptor expression but slowed receptor internalization. To identify potential internalization signal sequences, beta-turn structure in the cytosolic domain was targeted for mutagenesis. Disruption of one region near Asp(25) inhibited receptor activity. The studies support a model whereby receptor internalization requires the presence of an internalization signal motif but that the rate of receptor internalization is governed by the pattern of receptor phosphorylation induced by the ligand.     

A single tyrosine residue in the amyloid precursor protein intracellular domain is essential for developmental function

The Aβ-precursor protein (APP) intracellular domain is highly conserved and contains many potentially important residues, in particular the (682)YENPTY(687) motif. To dissect the functions of this sequence in vivo, we created an APP knock-in allele mutating Tyr(682) to Gly (Y682G). Crossing this allele to APP-like protein 2 (APLP2) knock-out background showed that mutation of Tyr(682) results in postnatal lethality and neuromuscular synapse defects similar to doubly deficient APP/APLP2 mice. Our results demonstrate that a single residue in the APP intracellular region, Tyr(682), is indispensable for the essential function of APP in developmental regulation.     

Expression of a rapid,low-voltage threshold K current in insulin-secreting cells is dependent on intracellular calcium buffering

Summary Depolarization-activated outward currents ranging in amplitude from 100–1000 pA were studied in cultured, insulinsecreting HIT cells and mouse B-cells using the whole-cell patch clamp. Outward current was identified as a K current since it was blocked by K channel blockers and its tail current reversed nearE _K. The K currents of HIT cells dialyzed with internal solutions containing 0.1–10mm EGTA with no added calcium (Ca), or 10mm EGTA with 2mm added Ca, activated rapidly with depolarization. However, the stronger Ca buffer BAPTA (5mm; no added Ca) blocked the rapidly activating current to reveal an underlying more slowly activating K current. With intracellular EGTA, application of the Ca channel blocker cadmium mimicked the effect of intracellular BAPTA. These data suggest that the rapid K current was mediated by low-voltage threshold, Ca-activated K channels while the slower K current was mediated by high threshold delayed rectifier K channels. Mouse B-cells also had both K current components. Dialyzing these cells with either BAPTA (5mm, no added Ca) or high EGTA (10mm with 2mm Ca) blocked the rapid Ca-activated K current observed when cells were filled with 0.1 to 1mm EGTA. It is concluded that the extent of Ca-activated K current activation in either HIT or adult mouse B-cells depends on the degree of intracellular Ca buffering.     

Recognition of a basic AP-2 binding motif within the C2B domain of synaptotagmin is dependent on multimerization

Synaptotagmin is a multifunctional membrane protein that may regulate exo-endocytic cycling of synaptic vesicles at the presynaptic plasmalemma. Its C2B domain has been postulated to interact with a variety of effector molecules including acidic phospholipids, phosphoinositides, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), calcium channels, and the clathrin adaptor complex AP-2. Here we report that a basic motif within the C2B domain is required and sufficient for binding to AP-2 via its mu2 subunit and that this interaction is dependent on multimerization of the AP-2 binding site. Moreover, we show that upon fusion to a plasma membrane reporter protein this sequence is sufficient to target the chimeric molecule for internalization. We hypothesize that basic motifs within multimeric membrane proteins may represent a novel type of clathrin/AP-2-dependent endocytosis signal.     

Contribution of the tyrosines to the structure and function of the human U1A N-terminal RNA binding domain.

RNA binding domains (RBDs) are members of a large family of proteins that share minimal sequence conservation but adopt an alpha beta sandwich global fold. Defining the contributions of specific amino acids to RBD structure and RNA binding is critical to understanding the functions of these proteins. In these experiments with the human U1A N-terminal RNA binding domain (RBD1), the contributions from each of its four tyrosines to protein structure, stability, and RNA binding were measured. Each tyrosine was substituted with phenylalanine and one other selected residue, and the resulting proteins were characterized by chemical denaturation to measure their unfolding free energy, by binding free energies to the wild-type RNA hairpin, and by 19F NMR to probe for structural changes. Features of the protein identified in these experiments include a possible tyrosine/lysine contact in an alpha-helix, which may be an example of an energetically favorable aromatic/amino side chain interaction. One long loop of the protein, which shows unusual 15N backbone and tyrosine side-chain dynamics, is implicated in protein:protein association. The diverse interactions of the four tyrosine residues in the organization of RBD1 illustrate how each member of this family of proteins will have unique molecular details that contribute to function.     

Juxtamembrane tyrosine residues couple the Eph family receptor EphB2/Nuk to specific SH2 domain proteins in neuronal cells.

Eph-related receptor tyrosine kinases have been implicated in the control of axonal navigation and fasciculation. To investigate the biochemical mechanisms underlying such functions, we have expressed the EphB2 receptor (formerly Nuk/Cek5/Sek3) in neuronal NG108-15 cells, and have observed the tyrosine phosphorylation of multiple cellular proteins upon activation of EphB2 by its ligand, ephrin-B1 (formerly Elk-L/Lerk2). The activated EphB2 receptor induced the tyrosine phosphorylation of a 62-64 kDa protein (p62[dok]), which in turn formed a complex with the Ras GTPase-activating protein (RasGAP) and SH2/SH3 domain adaptor protein Nck. RasGAP also bound through its SH2 domains to tyrosine-phosphorylated EphB2 in vitro, and complexed with activated EphB2 in vivo. We have localized an in vitro RasGAP-binding site to conserved tyrosine residues Y604 and Y610 in the juxtamembrane region of EphB2, and demonstrated that substitution of these amino acids abolishes ephrin-B1-induced signalling events in EphB2-expressing NG108-15 cells. These tyrosine residues are followed by proline at the + 3 position, consistent with the binding specificity of RasGAP SH2 domains determined using a degenerate phosphopeptide library. These results identify an EphB2-activated signalling cascade involving proteins that potentially play a role in axonal guidance and control of cytoskeletal architecture.     

Identification of a region within the ErbB2/HER2 intracellular domain that is necessary for ligand-independent association

Ligand-independent ErbB2 activation occurs principally by two distinct mechanisms: overexpression and mutation. Overexpression of ErbB2 at the plasma membrane drives receptor self-association in a concentration-dependent manner, which in turn leads to constitutive receptor activation. Subsets of human breast cancers contain a molecular alteration that leads to erbB2 gene amplification and subsequent protein overexpression. Although not recognized to occur in human cancers, mutation can also lead to increased ErbB2 association. A well characterized mutant of the rodent ortholog neu involves substitution of glutamate for valine within the transmembrane domain. In each case, a number of explanations have been proposed to explain the resulting ErbB2 activation. These include stabilization of receptor oligomers, release of negative constraints, and altered receptor conformations. Here we define a short amino acid segment comprising amino acids 966-968 in the intracellular domain that seemingly disrupts receptor-receptor association that is driven either by overexpression or mutation in the transmembrane region. Because of the hydrophobic nature of these amino acids (VVI), we propose that alteration of this segment likely results in a global conformational change in an area that has been proposed previously to be a dimerization motif for ErbB homomeric association.     

Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS

Background  

The enzymes responsible for the synthesis of poly-ADP-ribose are named poly-ADP-ribose polymerases (PARP). PARP-2 is a nuclear protein, which regulates a variety of cellular functions that are mainly controlled by protein-protein interactions. A previously described non-conventional bipartite nuclear localization sequence (NLS) lies in the amino-terminal DNA binding domain of PARP-2 between amino acids 1–69; however, this targeting sequence has not been experimentally examined or validated.     

Expression of peptidylarginine deiminase in the uterine epithelial cells of mouse is dependent on estrogen.

The effects of the steroid hormones estrogen and progesterone on peptidylarginine deiminase protein-L-arginine iminohydrolase, EC 3.5.3.15) levels in adult ovariectomized mouse uterus were studied. The amount of the enzyme in the uterus was considerably diminished by ovariectomy. When the mice were injected with a variety of estrogenic compounds, 17 beta-estradiol-3-benzoate, which was the most potent stimulator of uterine cell proliferation among the estrogens tested, dramatically elevated the enzyme formation of the uterus in a dose- and time-dependent fashion. Results of immunohistochemistry with the antiserum against mouse peptidylarginine deiminase demonstrated that the induction of the enzyme by the estradiol exclusively occurred at the luminal and glandular epithelia, corresponding with the previous findings in the normal estrous cycle. Furthermore, administration of the estradiol significantly increased the content of mRNA coding for peptidylarginine deiminase in uterus, indicating the evidence of regulation in pretranslation. On the other hand, progesterone alone did not restore the enzyme level of the ovariectomized mouse, but moderated the action of estrogen when given in concert with estrogen. Thus, the expression of peptidylarginine deiminase in luminal and glandular epithelia of mouse uterus is controlled by the amount of the steroid hormones estrogen and progesterone.     

Gas6-mediated signaling is dependent on the engagement of its gamma-carboxyglutamic acid domain with phosphatidylserine

Gas6 is a vitamin K-dependent protein containing gamma-carboxyglutamic acid (Gla) at its N-terminus and a receptor binding domain at its C-terminus. Gas6-Axl binding is necessary but not sufficient to support endothelial cell survival as decarboxylated gas6 inhibits the pro-survival function of gas6 by binding and inhibiting Axl, even though decarboxylated gas6 cannot support endothelial cell survival itself. It is hypothesized that interactions between the Gla domain of gas6 and phosphatidylserine (PS), though not required for gas6 binding to Axl, are necessary for gas6-Axl function. In support of this hypothesis are results showing that (1) two specific inhibitors of Gla-PS interactions, namely soluble PS and Annexin V, abrogate gas6-mediated endothelial cell survival and (2) Soluble PS inhibits Akt activation, a downstream intracellular event triggered by gas6-Axl binding. In conclusion, we propose a heretofore unknown function of Gla, where Gla-PS binding on the N-terminus of gas6 is necessary for a gas6 function mediated through its binding to Axl via its C-terminus.