Cytoplasmic Tyrosine Residues Research Articles

Intracellular trafficking of variant chicken kidney AE1 anion exchangers: role Of alternative NH(2) termini in polarized sorting and Golgi recycling.

The variant chicken kidney AE1 anion exchangers differ only at the NH(2) terminus of their cytoplasmic domains. Transfection studies have indicated that the variant chicken AE1-4 anion exchanger accumulates in the basolateral membrane of polarized MDCK kidney epithelial cells, while the AE1-3 variant, which lacks the NH(2)-terminal 63 amino acids of AE1-4, primarily accumulates in the apical membrane. Mutagenesis studies have shown that the basolateral accumulation of AE1-4 is dependent upon two tyrosine residues at amino acids 44 and 47 of the polypeptide. Interestingly, either of these tyrosines is sufficient to direct efficient basolateral sorting of AE1-4. However, in the absence of both tyrosine residues, AE1-4 accumulates in the apical membrane of MDCK cells. Pulse-chase studies have shown that after delivery to the cell surface, newly synthesized AE1-4 is recycled to the Golgi where it acquires additional N-linked sugar modifications. This Golgi recycling activity is dependent upon the same cytoplasmic tyrosine residues that are required for the basolateral sorting of this variant transporter. Furthermore, mutants of AE1-4 that are defective in Golgi recycling are unable to associate with the detergent insoluble actin cytoskeleton and are rapidly turned over. These studies, which represent the first description of tyrosine-dependent cytoplasmic sorting signal for a type III membrane protein, have suggested a critical role for the actin cytoskeleton in regulating AE1 anion exchanger localization and stability in this epithelial cell type.

Eph receptors and ephrins restrict cell intermingling and communication.

Eph proteins are receptors with tyrosine-kinase activity which, with their ephrin ligands, mediate contact-dependent cell interactions that are implicated in the repulsion mechanisms that guide migrating cells and neuronal growth cones to specific destinations. Ephrin-B proteins have conserved cytoplasmic tyrosine residues that are phosphorylated upon interaction with an EphB receptor, and may transduce signals that regulate a cellular response. Because Eph receptors and ephrins have complementary expression in many tissues during embryogenesis, bidirectional activation of Eph receptors and ephrin-B proteins could occur at interfaces of their expression domains, for example at segment boundaries in the vertebrate hindbrain. Previous work has implicated Eph receptors and ephrin-B proteins in the restriction of cell intermingling between hindbrain segments. We therefore analysed whether complementary expression of Eph receptors and ephrins restricts cell intermingling, and whether this requires bidirectional or unidirectional signalling. Here we report that bidirectional but not unidirectional signalling restricts the intermingling of adjacent cell populations, whereas unidirectional activation is sufficient to restrict cell communication through gap junctions. These results reveal that Eph receptors and ephrins regulate two aspects of cell behaviour that can stabilize a distinct identity of adjacent cell populations.

Differential influence of tyrosine residues of the common receptor β subunit on multiple signals induced by human GM-CSF

Background: GM-CSF induces the proliferation and differentiation of a broad range of hematopoietic cells. Stimulation with this cytokine results in the induction of rapid tyrosine phosphorylation of cellular proteins, including the common β subunit of its receptor. However, the significance of β subunit phosphorylation with regard to regulation of signaling molecules and the functions of GM-CSF is less well understood. Objective: To investigate the role of cytoplasmic tyrosine residues, we performed mutational analyses of the β subunit. Methods: A series of β subunit mutants with 1, some, or all of 8 cytoplasmic tyrosines converted to phenylalanines were constructed. These β subunit mutants were expressed in murine BA/F3 cells together with the wild-type human α subunit, and their potentials to transmit signals were examined. Results: A mutant β subunit lacking all cytoplasmic tyrosines (Fall) activated JAK2, but not Shc, SHP-2, the Raf/ERK cascade, signal transducer and activator of transcription 5 (STAT5), or the c- fos promoter, nor did it effectively induce proliferation. Adding back each tyrosine to Fall revealed that (1) Tyr577 was necessary and sufficient for Shc phosphorylation; (2) Tyr577, Tyr612, and Tyr695 were involved in activation of SHP-2, the Raf/ERK cascade, and c- fos transcription; and (3) all tyrosines, but particularly Tyr612, Tyr695, Tyr750, and Tyr806, facilitated STAT5 activation. Impaired cell growth was also partly restored by any of the tyrosines. Interestingly, the Fall mutant was still capable of suppressing apoptosis and maintaining cell viability. Conclusion: These results provide evidence that β subunit tyrosines possess distinct, yet overlapping, roles in the activation of multiple signaling pathways induced by GM-CSF. In addition, GM-CSF may activate both β subunit tyrosine-dependent and -independent pathways, with the latter being sufficient to transmit survival signals. (J Allergy Clin Immunol 1999;103:S462-70.)

Lipocortin V May Function as a Signaling Protein for Vascular Endothelial Growth Factor Receptor-2/Flk-1

Binding of vascular endothelial growth factor (VEGF) to its receptor, VEGFR-2 (Flk-1/KDR), induces dimerization and activation of the tyrosine kinase domain of the receptor, resulting in autophosphorylation of cytoplasmic tyrosine residues used as docking sites for signaling proteins that relay the signals for cell proliferation, migration, and permeability enhancement. We explored the VEGF/receptor signaling pathway by performing a two-hybrid screen of a rat lung cDNA library in yeast using the intracellular domain of rat VEGFR-2 as bait. Two clones encoding lipocortin V were isolated. Subsequent studies with the yeast two-hybrid assay showed that the complete intracellular domain of VEGFR-2 was required for the interaction. Co-immunoprecipitation of translated proteins confirmed the interaction between the VEGF receptor and lipocortin V. VEGF induced a rapid tyrosine phosphorylation of lipocortin V in human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with antisense oligodeoxyribonucleotide (ODN) for lipocortin V significantly inhibited VEGF-induced cell proliferation, which was accompanied by a decrease in protein synthesis and tyrosine phosphorylation of lipocortin V. Our results indicate that lipocortin V may function as a signaling protein for VEGFR-2 by directly interacting with the intracellular domain of the receptor and appears to be involved in regulation of vascular endothelial cell proliferation mediated by VEGFR-2.

Role of Cytokine Signaling Molecules in Erythroid Differentiation of Mouse Fetal Liver Hematopoietic Cells: Functional Analysis of Signaling Molecules by Retrovirus-Mediated Expression

Erythropoietin (EPO) and its cell surface receptor (EPOR) play a central role in proliferation, differentiation, and survival of erythroid progenitors. Signals induced by EPO have been studied extensively by using erythroid as well as nonerythroid cell lines, and various controversial results have been reported as to the role of signaling molecules in erythroid differentiation. Here we describe a novel approach to analyze the EPO signaling by using primary mouse fetal liver hematopoietic cells to avoid possible artifacts due to established cell lines. Our strategy is based on high-titer retrovirus vectors with a bicistronic expression system consisting of an internal ribosome entry site (IRES) and green fluorescent protein (GFP). By placing the cDNA for a signaling molecule in front of IRES-GFP, virus-infected cells can be viably sorted by fluorescence-activated cell sorter, and the effect of expression of the signaling molecule can be assessed. By using this system, expression of cell-survival genes such as Bcl-2 and Bcl-XL was found to enhance erythroid colony formation from colony-forming unit–erythroid (CFU-E) in response to EPO. However, their expression was not sufficient for erythroid colony formation from CFU-E alone, indicating that EPO induces signals for erythroid differentiation. To examine the role of EPOR tyrosine residues in erythroid differentiation, we introduced a chimeric EGFR-EPOR receptor, which has the extracellular domain of the EGF receptor and the intracellular domain of the EPOR, as well as a mutant EGFR-EPOR in which all the cytoplasmic tyrosine residues are replaced with phenylalanine, and found that tyrosine residues of EPOR are essential for erythroid colony formation from CFU-E. We further analyzed the function of the downstream signaling molecules by expressing modified signaling molecules and found that both JAK2/STAT5 and Ras, two major signaling pathways activated by EPOR, are involved in full erythroid differentiation.

Role of Cytokine Signaling Molecules in Erythroid Differentiation of Mouse Fetal Liver Hematopoietic Cells: Functional Analysis of Signaling Molecules by Retrovirus-Mediated Expression

AbstractErythropoietin (EPO) and its cell surface receptor (EPOR) play a central role in proliferation, differentiation, and survival of erythroid progenitors. Signals induced by EPO have been studied extensively by using erythroid as well as nonerythroid cell lines, and various controversial results have been reported as to the role of signaling molecules in erythroid differentiation. Here we describe a novel approach to analyze the EPO signaling by using primary mouse fetal liver hematopoietic cells to avoid possible artifacts due to established cell lines. Our strategy is based on high-titer retrovirus vectors with a bicistronic expression system consisting of an internal ribosome entry site (IRES) and green fluorescent protein (GFP). By placing the cDNA for a signaling molecule in front of IRES-GFP, virus-infected cells can be viably sorted by fluorescence-activated cell sorter, and the effect of expression of the signaling molecule can be assessed. By using this system, expression of cell-survival genes such as Bcl-2 and Bcl-XL was found to enhance erythroid colony formation from colony-forming unit–erythroid (CFU-E) in response to EPO. However, their expression was not sufficient for erythroid colony formation from CFU-E alone, indicating that EPO induces signals for erythroid differentiation. To examine the role of EPOR tyrosine residues in erythroid differentiation, we introduced a chimeric EGFR-EPOR receptor, which has the extracellular domain of the EGF receptor and the intracellular domain of the EPOR, as well as a mutant EGFR-EPOR in which all the cytoplasmic tyrosine residues are replaced with phenylalanine, and found that tyrosine residues of EPOR are essential for erythroid colony formation from CFU-E. We further analyzed the function of the downstream signaling molecules by expressing modified signaling molecules and found that both JAK2/STAT5 and Ras, two major signaling pathways activated by EPOR, are involved in full erythroid differentiation.

Recruitment of a Nicotinic Acetylcholine Receptor Mutant Lacking Cytoplasmic Tyrosine Residues in Its β Subunit into Agrin-Induced Aggregates

During synaptogenesis at the vertebrate skeletal neuromuscular junction, acetylcholine receptors (AChRs) form high-density aggregates opposite the presynaptic terminal in response to nerve-derived agrin. Agrin has been shown to stimulate tyrosine phosphorylation of a muscle-specific receptor tyrosine kinase MuSK and of the AChR β subunit, and tyrosine kinase inhibitors and a tyrosine kinase-deficient mutant of MuSK prevent AChR aggregation. To evaluate the role of tyrosine phosphorylation of the AChR β subunit in receptor aggregation, we replaced all three putative cytoplasmic tyrosine residues of the AChR β subunit with phenylalanine residues and expressed the mutant receptors in cultured myotubes. Upon agrin treatment, transfected myotubes formed AChR aggregates that contained receptors with mutant β subunits. Thus, AChRs can be recruited into agrin-induced specializations by protein–protein interactions that do not depend on tyrosine phosphorylation of the AChR β subunit.

Thymocyte activation induces the association of the proto-oncoprotein c-cbl and ras GTPase-activating protein with CD5.

Studies of knockout mice indicate that the glycoprotein CD5, which is expressed on Tcells, most thymocytes and a subset of B cells, down-regulates TCR- and B cell receptor (BCR)-mediated signaling. CD5 is associated with the TCR and BCR, and is phosphorylated on cytoplasmic tyrosine residues following antigen receptor ligation. Cross-linking of CD5 or pervanadate stimulation of thymocytes induces the association of a 120-kDa tyrosine-phosphorylated protein with CD5. The proto-oncoprotein c-cbl associates with CD5 in pervanadate-stimulated thymocytes, and reprecipitation analysis demonstrates that the major proportion of CD5-associated pp120 is c-cbl. The GTPase-activating protein for ras (ras GAP), which is not tyrosine phosphorylated following CD5 cross-linking, associates with CD5 in pervanadate-stimulated thymocytes. Using tyrosine-phosphorylated peptides we show that ras GAP interacts in an SH2-mediated manner with the phosphorylated Y429SQP sequence of CD5. Both c-cbl and ras GAP have been proposed to suppress receptor-mediated signaling, and may contribute to CD5-mediated suppression of TCR or BCR signaling.

Tyrosine Phosphorylation of the β3 Cytoplasmic Domain Mediates Integrin-Cytoskeletal Interactions

Tyrosine phosphorylation of the beta3 subunit of the major platelet integrin alphaIIb beta3 has been shown to occur during thrombin-induced platelet aggregation (1). We now show that a wide variety of platelet stimuli induced beta3 tyrosine phosphorylation, but that this phosphorylation occurred only following platelet aggregation. Several lines of evidence suggest that the beta3 cytoplasmic domain tyrosine residues and/or their phosphorylation function to mediate interactions between beta3 integrins and cytoskeletal proteins. First, phospho-beta3 was retained preferentially in a Triton X-100 insoluble cytoskeletal fraction of thrombin-aggregated platelets. Second, in vitro experiments show that the cytoskeletal protein, myosin, associated in a phosphotyrosine-dependent manner with a diphosphorylated peptide corresponding to residues 740-762 of beta3. Third, mutation of both tyrosines in the beta3 cytoplasmic domain to phenylalanines markedly reduced beta3-dependent fibrin clot retraction. Thus, our data indicate that platelet aggregation is both necessary and sufficient for beta3 tyrosine phosphorylation, and this phosphorylation results in the physical linkage of alphaIIb beta3 to the cytoskeleton. We hypothesize that this linkage may involve direct binding of the phosphorylated integrin to the contractile protein myosin in order to mediate transmission of force to the fibrin clot during the process of clot retraction.

Distinct Involvement of β3 Integrin Cytoplasmic Domain Tyrosine Residues 747 and 759 in Integrin-mediated Cytoskeletal Assembly and Phosphotyrosine Signaling

We have investigated the structural requirements of the beta3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during alphav beta3-mediated cell spreading. Using CHO cells transfected with various beta3 mutants, we demonstrate a close correlation between alphav beta3-mediated cell spreading and tyrosine phosphorylation of FAK and paxillin, and highlight a distinct involvement of the NPLY747 and NITY759 motifs in these signaling processes. Deletion of the NITY759 motif alone was sufficient to completely prevent alphav beta3-dependent focal contact formation, cell spreading, and FAK/paxillin phosphorylation. The single Y759A substitution induced a strong inhibitory phenotype, while the more conservative, but still phosphorylation-defective, Y759F mutation restored wild type receptor function. Alanine substitution of the highly conserved Tyr747 completely abolished alphav beta3-dependent formation of focal adhesion plaques, cell spreading, and FAK/paxillin phosphorylation, whereas a Y747F substitution only partially restored these events. As none of these mutations affected receptor-ligand interaction, our results suggest that the structural integrity of the NITY759 motif, rather than the phosphorylation status of Tyr759 is important for beta3-mediated cytoskeleton reorganization and tyrosine phosphorylation of FAK and paxillin, while the presence of Tyr at residue 747 within the NPLY747 motif is required for optimal beta3 post-ligand binding events.

Arrest of Vascular Development Correlates with the Failure of Platelet Endothelial Factor-1 (PECAM-1) Dephosphorylation in Hyperglycemia-Induced Embryopathy • 366

The vascular system is the first organ system to develop in the embryo and its maldevelopment induced by hyperglycemia has serious consequences including embryonic and perinatal lethality. We previously reported that the state of phosphorylation/dephosphorylation of the vascular system specific cell adhesion molecule PECAM-1 correlates with development of vitelline vasculature. PECAM-1 is highly tyrosine phosphorylated prior to vessel formation, and dephosphorylated when the interconnecting circulation between the yolk sac and the embryo is established. We hypothesized that in diabetes-related embryopathy there is alteration of PECAM-1 phosphorylation state and this adversely affects an integrin mediated signal transduction pathway. We utilized an in vitro whole embryo culture system as a model for in situ vasculogenesis. Conceptuses were cultured between 7.5 and 9.5 days p.c. in control (glucose levels: 7.4-9.5 mMol) and in hyperglycemic (25 mMol) rat serum for 48 hr. An arrest of yolk sac vessel development at the primitive capillary plexus stage with aberrant arborization was present in the hyperglycemic condition. This yolk sac vasculopathy always accompanied a malformation of the embryos (malformation in axial rotation, open neural tube, heart defects) when conceptuses were harvested prior to the late headfold stage. A 3 hr period of hyperglycemia was sufficient to elicit yolk sac vasculopathy during this susceptible time of development. Exposure to the same high glucose environment in conceptuses past the late headfold stage was not sufficient to elicit yolk sac vasculopathy. Immunoprecipitation-Western blot analyses revealed that PECAM-1 remained highly tyrosine phosphorylated in the hyperglycemia-exposed malformed conceptuses at the end of organogenesis, in contrast to its dephosphorylated state in normally developing age matched controls. We also found that PECAM-1 in hyperglycemia-exposed conceptuses co-precipitates with the phosphatase SHPTP2. These data indicate that the cell adhesion molecule PECAM-1 is involved in vasculogenesis and its dephosphorylation is delayed/arrested in hyperglycemia-related embryopathy. Thus, modulation of PECAM-1 cytoplasmic tyrosine residues Y663 and Y686 which comprise part of PECAM-1's ITAM domain is postulated to have effect on signaling and/or adaptor molecule binding and subsequent transduction pathways influencing vasculo-and angiogenesis.

IL-10 inhibits macrophage activation and proliferation by distinct signaling mechanisms: evidence for Stat3-dependent and -independent pathways.

Interleukin-10 (IL-10) limits inflammatory responses by inhibiting macrophage activation. In macrophages, IL-10 activates Stat1 and Stat3. We characterized IL-10 responses of the J774 mouse macrophage cell line, and of J774 cells expressing wild-type hIL-10R, mutant hIL-10R lacking two membrane-distal tyrosines involved in recruitment of Stat3 (hIL-10R-TyrFF), a truncated Stat3 (DeltaStat3) which acts as a dominant negative, or an inducibly active Stat3-gyraseB chimera (Stat3-GyrB). A neutralizing anti-mIL-10R monoclonal antibody was generated to block the function of endogenous mIL-10R. IL-10 inhibited proliferation of J774 cells and of normal bone marrow-derived macrophages, but not J774 cells expressing hIL-10RTyrFF. Dimerization of Stat3-GyrB by coumermycin mimicked the effect of IL-10, and expression of DeltaStat3 blocked the anti-proliferative activity of IL-10. For macrophage de-activation responses, hIL10R-TyrFF could not mediate inhibition of lipopolysaccharide-induced TNFalpha, IL-1beta or CD86 expression, while DeltaStat3 did not interfere detectably with these IL-10 responses. Thus signals mediating both anti-proliferative and macrophage de-activation responses to IL-10 require the two membrane-distal tyrosines of IL-10R, but Stat3 appears to function only in the anti-proliferative response.

Definition of the role of tyrosine residues of the common beta subunit regulating multiple signaling pathways of granulocyte-macrophage colony-stimulating factor receptor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces various functions, including the proliferation and differentiation of a broad range of hematopoietic cells. We previously reported that at least two distinct pathways are involved in human GM-CSF receptor signaling; both require the box 1 region of the common beta subunit (beta c). This region is essential for the activation of JAK2, which is necessary for all the biological functions of GM-CSF. The activation of JAK2 by GM-CSF leads to rapid tyrosine phosphorylation of cellular proteins, including the beta c. However, the significance of beta c phosphorylation with regard to the regulation of signaling molecules and the expression of GM-CSF functions is less well understood. Here we investigated the role of the cytoplasmic tyrosine residues of the beta c by using a series of beta c mutants expressed in murine BA/F3 cells. A mutant beta c with all eight cytoplasmic tyrosines converted to phenylalanine (Fall) activated JAK2 but not SHP-2, MAPK cascades, STAT5, or the c-fos promoter in BA/F3 cells, and it did not effectively induce proliferation. Adding back each tyrosine to Fall revealed that Tyr577, Tyr612, and Tyr695 are involved in the activation of SHP-2, MAPK cascades, and c-fos transcription, while every tyrosine, particularly Tyr612, Tyr695, Tyr750, and Tyr806, facilitated STAT5 activation. Impaired growth was also restored, at least partly, by any of the tyrosines. These results provide evidence that beta c tyrosines possess distinct yet overlapping functions in activating multiple signaling pathways induced by GM-CSF.

Activation of STAT6 is not dependent on phosphotyrosine-mediated docking to the interleukin-4 receptor and can be blocked by dominant-negative mutants of both receptor subunits.

Stimulation of susceptible cells by interleukin-4 leads to activation of signal transducer and activator of transcription (STAT6) through tyrosine phosphorylation and dimerisation, thus directing it to the cell nucleus and rendering it a sequence-specific transcription factor. We functionally reconstituted human interleukin-4 receptor complexes with intracellular truncations of either the alpha or gamma subunits and demonstrate the requirement for elements from both receptor chains for STAT6 activation induced by interleukin-4. By assaying the signalling properties of human interleukin-4-receptor alpha-chain-deletion constructs in both Ba/F3 cells and COS-7 cells, we show that all its cytoplasmic tyrosine residues can be removed without affecting the capability of the receptor complex to trigger STAT6 function with regard to tyrosine phosphorylation, DNA binding, and specific gene transcription. The activation of both STAT6 and janus kinase 1 (JAK1) by the interleukin-4 receptor was completely abolished by disruption of the membrane-proximal 'box1' motif in the interleukin-4 receptor alpha chain. Our results indicate a redundant role of the previously defined phosphotyrosine-containing STAT6 docking site and suggest a mechanism of immediate activation of STAT6 by receptor-associated janus kinase(s). In addition, we demonstrate that dominant negative versions of both interleukin-4 receptor subunits are able to block interleukin-4 induced signalling via STAT6.

Characterization of Phosphotyrosine Binding Motifs in the Cytoplasmic Domain of Platelet/Endothelial Cell Adhesion Molecule-1 (PECAM-1) That Are Required for the Cellular Association and Activation of the Protein-tyrosine Phosphatase, SHP-2

Recent studies have shown that the Src homology-2 (SH2) domain-containing protein-tyrosine phosphatase, SHP-2, associates with the cytoplasmic domain of PECAM-1 as it becomes tyrosine-phosphorylated during platelet aggregation: a process that can be mimicked in part by small synthetic phosphopeptides corresponding to the cytoplasmic domain of PECAM-1 encompassing tyrosine residues Tyr-663 or Tyr-686. To further examine the molecular requirements for PECAM-1/SHP-2 interactions, we generated human embryonic kidney (HEK)-293 cell lines that stably expressed mutant forms of PECAM-1 harboring tyrosine to phenylalanine (Tyr --> Phe) mutations in the cytoplasmic domain. Y663F and Y686F forms of PECAM-1 were tyrosine-phosphorylated to a somewhat lesser extent than wild-type PECAM-1, and a doubly substituted Y663,686F form of PECAM-1 failed to become tyrosine-phosphorylated, suggesting that the PECAM-1 cytoplasmic domain tyrosine residues 596, 636 and 701 do not serve as substrates for cellular kinases. Interestingly, SHP-2 binding was lost when either Tyr-663 or Tyr-686 were changed to phenylalanine, indicating that both residues are required for SHP-2/PECAM-1 association. Although PECAM-1 phosphopeptides NSDVQpY663TEVQV and DTETVpY686SEVRK stimulated the catalytic activity of the phosphatase to a similar extent, surface plasmon resonance studies revealed that the Tyr-663-containing peptide had approximately 10-fold higher affinity for SHP-2 than did the Tyr-686 peptide. Finally, peptido-precipitation analysis showed that the NH2-terminal SH2 domain of SHP-2 reacted preferentially with the Tyr-663 PECAM-1 phosphopeptide, while the Tyr-686 phosphopeptide associated only with the COOH-terminal SH2 domain of the phosphatase. Together, these data provide a molecular model for PECAM-1/SHP-2 interactions that may shed light on the downstream events that follow PECAM-1-mediated interactions of vascular cells.

Cytoplasmic Domain of E-selectin Contains a Non-tyrosine Endocytosis Signal

E-selectin is an activation-dependent, endothelial cell-restricted adhesion molecule that is internalized and degraded rapidly once expressed on the cell surface. Tyrosine-containing structural motifs play an important role in the internalization of a number of integral proteins, and the membrane-proximal E-selectin cytoplasmic tyrosine residue (Tyr582) conforms to the endocytosis motif proposed previously. To determine the endocytosis motif in E-selectin, we selectively introduced truncation, substitution, and deletion mutations to the cytoplasmic tail of E-selectin. We analyzed the internalization kinetics of surface-expressed wild-type and mutant E-selectin constructs in transiently transfected Chinese hamster ovary cells using 125I-labeled E-selectin monoclonal antibody (125I-P6E2) in an acid elution assay. Interestingly, truncation immediately membrane proximal to Tyr582 (DeltaDGS construct) did not alter internalization kinetics significantly (DeltaDGS versus wild-type, mean surface half-life = 42 versus 45 min, respectively). Thus, it appears that the tyrosine residues are not required for internalization of E-selectin. Additional analyses indicated that Ser581 was necessary but alone was insufficient for surface E-selectin endocytosis. Thus, we conclude that there exists a novel non-tyrosine-containing endocytosis signal in the cytoplasmic tail which involves Ser581 and residues membrane-proximal to it.

Sequential activation of phoshatidylinositol 3-kinase and phospholipase C-γ2 by the M-CSF receptor is necessary for differentiation signaling

Binding of macrophage colony stimulating factor (M-CSF) to its receptor (Fms) induces dimerization and activation of the tyrosine kinase domain of the receptor, resulting in autophosphorylation of cytoplasmic tyrosine residues used as docking sites for SH2-containing signaling proteins that relay growth and development signals. To determine whether a distinct signaling pathway is responsible for the Fms differentiation signal versus the growth signal, we sought new molecules involved in Fms signaling by performing a two-hybrid screen in yeast using the autophosphorylated cytoplasmic domain of the wild-type Fms receptor as bait. Clones containing SH2 domains of phospholipase C-gamma2 (PLC-gamma2) were frequently isolated and shown to interact with phosphorylated Tyr721 of the Fms receptor, which is also the binding site of the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase). At variance with previous reports, M-CSF induced rapid and transient tyrosine phosphorylation of PLC-gamma2 in myeloid FDC-P1 cells and this activation required the activity of the PI3-kinase pathway. The Fms Y721F mutation strongly decreased this activation. Moreover, the Fms Y807F mutation decreased both binding and phosphorylation of PLC-gamma2 but not that of p85. Since the Fms Y807F mutation abrogates the differentiation signal when expressed in FDC-P1 cells and since this phenotype could be reproduced by a specific inhibitor of PLC-gamma, we propose that a balance between the activities of PLC-gamma2 and PI3-kinase in response to M-CSF is required for cell differentiation.

Analysis of CD28 cytoplasmic tail tyrosine residues as regulators and substrates for the protein tyrosine kinases, EMT and LCK.

The CD28 cell surface receptor provides an important costimulatory signal for T cells necessary for their response to Ag. Early events in CD28 signaling include recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase) and activation of the protein tyrosine kinases (PTKs), LCK and EMT. Recruitment and activation of PI3-kinase is known to be dependent upon phosphorylation of tyrosine 173 of the CD28 cytoplasmic tail contained within a YMNM motif. By contrast, little is known of which residues of the CD28 tail, including tyrosines, are required for the activation of PTKs. To address this we studied the ability of truncation mutants and tyrosine to phenylalanine substitution mutants of the CD28 cytoplasmic tail to activate LCK and EMT in Jurkat T leukemia cells. Our results indicate that 1) activation of EMT is partially dependent upon tyrosine 173 of the CD28 tail, although it does not require PI3-kinase activation; 2) activation of LCK is independent of CD28 cytoplasmic tail tyrosine residues; and 3) elements sufficient for the activation of both kinases are contained within the first half of the tail. In addition we studied the CD28 tail as a substrate for both PTKs in in vitro kinase assays. We demonstrate that EMT can phosphorylate all four tyrosines of the CD28 tail, in contrast to LCK, which phosphorylates only tyrosine 173. Together with evidence that in vivo, tyrosines other than tyrosine 173 become phosphorylated following CD28 stimulation, this finding suggests that, like LCK, one function of EMT during CD28 signaling is phosphorylation of the receptor.

Tyrosine Residues in the Granulocyte Colony-stimulating Factor (G-CSF) Receptor Mediate G-CSF-induced Differentiation of Murine Myeloid Leukemic (M1) Cells

The cytoplasmic tyrosine residues of many growth factor receptors have been shown to be important for receptor signal transduction via the recruitment of proteins containing phosphotyrosine-binding domains. This study demonstrates the importance of specific tyrosine residues in the granulocyte colony-stimulating factor (G-CSF) receptor cytoplasmic domain in G-CSF-induced macrophage cell differentiation. Site-directed mutagenesis was used to generate a series of G-CSF receptor (G-CSF-R) mutants in which the tyrosine residues were replaced with phenylalanine either singly or in combination. The mouse myeloid leukemic cell line (M1) transfected with G-CSF-R cDNA can be induced to differentiate into macrophages in response to G-CSF. The effect of the tyrosine mutations on this differentiation response was assessed by examining cell morphology and differentiation in soft agar colony assays. Although three of the four cytoplasmic tyrosine residues appeared to contribute to the differentiation response, mutation of a single residue (Tyr744) significantly reduced the ability of the M1 cells to differentiate. The STAT family of signaling molecules (Stat1, Stat3, and Stat5) were activated by G-CSF in M1 cells expressing those G-CSF-R tyrosine mutants unable to mediate G-CSF-induced differentiation. Furthermore, activation of STAT proteins was shown to occur in the absence of all four cytoplasmic tyrosine residues, suggesting an alternative mechanism for STAT activation other than direct interaction with receptor phosphotyrosines.

Identification of growth hormone receptor (GHR) tyrosine residues required for GHR phosphorylation and JAK2 and STAT5 activation.

To determine whether GH receptor (GHR) cytoplasmic tyrosine residue(s) and tyrosine phosphorylation are required for signal transduction, we have substituted the eight porcine (p) GHR cytoplasmic tyrosines with phenylalanine individually or in a stepwise manner from the C terminus. Conversely, the eight tyrosines were individually regenerated in a non-tyrosine-containing pGHR analog. Mutated pGHR cDNAs were transfected into mouse L cells (MLCs) and cell lines were established. Each individual tyrosine-substituted pGHR analogs was able to activate STAT5 (signal transducer and activator of transcription 5; previously termed pp95) at levels comparable to those of wild type pGHR. Analyses of these pGHR analogs revealed that a single tyrosine residue at position 487, 534, 566, or 627 is sufficient for STAT5 phosphorylation. This result suggested that a redundancy in tyrosine residue requirement may be employed in GH-mediated signal transduction. Also, we found that the requirement of tyrosine residues for STAT5 phosphorylation directly correlated with their phosphorylation status. Combining both STAT5 and GHR tyrosine phosphorylation results, we have deduced that Y332, Y487, Y534, Y566, and Y627 are pGHR tyrosine phosphorylation sites. Additionally, Janus kinase 2 was activated by GH in all pGHR tyrosine-substituted analogs, including one containing no intracellular tyrosines, which agrees with a previous report that Janus kinase 2 activation is independent of GHR tyrosine phosphorylation.