Interleukin-2 induces β2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)

β2 integrin molecules are involved in a multitude of cellular events, including adhesion, migration, and cellular activation. Here, we studied the influence of β2 integrins on interleukin-2 (IL-2)-mediated signal transduction in human CD4+ T cell lines obtained from healthy donors and a leukocyte adhesion deficiency (LAD) patient. We show that IL-2 induces tyrosine phosphorylation of a 125-kDa protein and homotypic adhesion in β2 integrin (CD18)-positive but not in β2-integrin-negative T cells. EDTA, an inhibitor of integrin adhesion, blocks IL-2-induced tyrosine phosphorylation of the 125-kDa protein but not other proteins in β2-integrin-positive T cells. Likewise, a β2 integrin (CD18) antibody selectively inhibits induction of the 125-kDa phosphotyrosine protein, whereas cytokine-mediated tyrosine phosphorylation of other proteins is largely unaffected. Immunoprecipitation experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in β2-integrin-positive but not in β2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fakB-tyrosine phosphorylation in β2-integrin-positive T cells. In parallel experiments, IL-2 does not induce or augment tyrosine phosphorylation of p125FAK. In conclusion, our data indicate that IL-2 induces β2-integrin-dependent signal transduction events involving the tyrosine kinase substrate fakB.

Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action

The phosphorylation of insulin receptor substrate 1 (IRS-1) on tyrosine residues by the insulin receptor (IR) tyrosine kinase is involved in most of the biological responses of insulin. IRS-1 mediates insulin signaling by recruiting SH2 proteins through its multiple tyrosine phosphorylation sites. The phosphorylation of IRS-1 on serine/threonine residues also occurs in cells; however, the particular protein kinase(s) promoting this type of phosphorylation are unknown. Here we report that glycogen synthase kinase 3 (GSK-3) is capable of phosphorylating IRS-1 and that this modification converts IRS-1 into an inhibitor of IR tyrosine kinase activity in vitro. Expression of wild-type GSK-3 or an “unregulated” mutant of the kinase (S9A) in CHO cells overexpressing IRS-1 and IR, resulted in increased serine phosphorylation levels of IRS-1, suggesting that IRS-1 is a cellular target of GSK-3. Furthermore, insulin-induced tyrosine phosphorylation of IRS-1 and IR was markedly suppressed in cells expressing wild-type or the S9A mutant, indicating that expression of GSK-3 impairs IR tyrosine kinase activity. Taken together, our studies suggest a new role for GSK-3 in attenuating insulin signaling via its phosphorylation of IRS-1 and may provide new insight into mechanisms important in insulin resistance.

Reduction in levels of the cyclin-dependent kinase inhibitor p27kip-1 coupled with transforming growth factor β neutralization induces cell-cycle entry and increases retroviral transduction of primitive human hematopoietic cells

Successful gene therapy depends on stable transduction of hematopoietic stem cells. Target cells must cycle to allow integration of Moloney-based retroviral vectors, yet hematopoietic stem cells are quiescent. Cells can be held in quiescence by intracellular cyclin-dependent kinase inhibitors. The cyclin-dependent kinase inhibitor p15INK4B blocks association of cyclin-dependent kinase (CDK)4/cyclin D and p27kip-1 blocks activity of CDK2/cyclin A and CDK2/cyclin E, complexes that are mandatory for cell-cycle progression. Antibody neutralization of β transforming growth factor (TGFβ) in serum-free medium decreased levels of p15INK4B and increased colony formation and retroviral-mediated transduction of primary human CD34+ cells. Although TGFβ neutralization increased colony formation from more primitive, noncycling hematopoietic progenitors, no increase in M-phase-dependent, retroviral-mediated transduction was observed. Transduction of the primitive cells was augmented by culture in the presence of antisense oligonucleotides to p27kip-1 coupled with TGFβ-neutralizing antibodies. The transduced cells engrafted immune-deficient mice with no alteration in human hematopoietic lineage development. We conclude that neutralization of TGFβ, plus reduction in levels of the cyclin-dependent kinase inhibitor p27, allows transduction of primitive and quiescent hematopoietic progenitor populations.

Mechanical stretch induces vascular permeability factor in human mesangial cells: Mechanisms of signal transduction

Hemodynamic abnormalities have been implicated in the pathogenesis of the increased glomerular permeability to protein of diabetic and other glomerulopathies. Vascular permeability factor (VPF) is one of the most powerful promoters of vascular permeability. We studied the effect of stretch on VPF production by human mesangial cells and the intracellular signaling pathways involved. The application of mechanical stretch (elongation 10%) for 6 h induced a 2.4-fold increase over control in the VPF mRNA level (P < 0.05). There was a corresponding 3-fold increase in VPF protein level by 12 h (P < 0.001), returning to the baseline by 24 h. Stretch-induced VPF secretion was partially prevented both by the protein kinase C (PKC) inhibitor H7 (50 μM: 72% inhibition, P < 0.05) and by pretreatment with phorbol ester (phorbol-12-myristate-13 acetate 10−7 M: 77% inhibition, P < 0.05). A variety of protein tyrosine kinase (PTK) inhibitors, genistein (20 μg/ml), herbimycin A (3.4 μM), and a specific pp60src peptide inhibitor (21 μM) also significantly reduced, but did not entirely prevent, stretch-induced VPF protein secretion (respectively 63%, 80%, and 75% inhibition; P < 0.05 for all). The combination of both PKC and PTK inhibition completely abolished the VPF response to mechanical stretch (100% inhibition, P < 0.05). Stretch induces VPF gene expression and protein secretion in human mesangial cells via PKC- and PTK-dependent mechanisms.

Ceramide-induced inhibition of T lymphocyte voltage-gated potassium channel is mediated by tyrosine kinases

The n-type K+ channel (n-K+, Kv1.3) in lymphocytes has been recently implicated in the regulation of Fas-induced programmed cell death. Here, we demonstrate that ceramide, a lipid metabolite synthesized upon Fas receptor ligation, inhibits n-K+ channel activity and induces a tyrosine phosphorylation of the Kv1.3 protein in Jurkat T lymphocytes. Tyrosine phosphorylation of the n-K+ channel correlated with an activation of the Src-like tyrosine kinase p56lck upon cellular treatment with the ceramide analog C6-ceramide. Because genetic deficiency of p56lck or inhibition of Src-like tyrosine kinases by herbimycin A prevented ceramide-mediated n-K+ channel inhibition and tyrosine phosphorylation, we propose a ceramide-initiated activation of p56lck resulting in tyrosine phosphorylation and inhibition of the n-K+ channel protein.

Oncogenic stimulation recruits cyclin-dependent kinase in the cell cycle start in rat fibroblast

The rat fibroblast NRK cells are transformed reversibly by a combination of growth factors. When stimulated with serum, NRK cells rely on cyclin-dependent kinase 4 (Cdk4) for their S phase entry. However, when stimulated with serum containing oncogenic growth factors, they come to rely on either Cdk4 or Cdk6, and their S phase entry cannot be blocked unless both Cdk4 and Cdk6 are immunodepleted. Such change of dependence does not occur in the NRK cell mutants defective in an oncogenic signal pathway and, therefore, deficient in anchorage-independent cell cycle start ability, correlating Cdk6 dependence with this remarkable, cancer-associated phenotype. However, both Cdk4 and Cdk6 are activated upon serum stimulation, and neither the amounts of Cdk6, Cdk4, cyclin D1, and cyclin-dependent kinase inhibitors nor the activities or subcellular localization of Cdk6 and Cdk4 are significantly influenced by oncogenic stimulation. Thus, oncogenic stimulation invokes Cdk6 to participate in a critical step of the cell cycle start in a rat fibroblast, but by a mechanism seemingly unrelated to the regulation of the kinase. Given that many hematopoietic cells employ predominantly Cdk6 for the cell cycle start and perform anchorage-independent growth by nature, our results raise the possibility that the oncogenic stimulation-induced anchorage-independent cell cycle start of NRK is elicited by a mechanism similar to the one used for hematopoietic cell proliferation.

c-Abl-induced apoptosis, but not cell cycle arrest, requires mitogen-activated protein kinase kinase 6 activation

c-Abl is a ubiquitously expressed protein tyrosine kinase activated by DNA damage and implicated in two responses: cell cycle arrest and apoptosis. The downstream pathways by which c-Abl induces these responses remain unclear. We examined the effect of overexpression of c-Abl on the activation of mitogen-activated protein kinase pathways and found that overexpression of c-Abl selectively stimulated p38, while having no effect on c-Jun N-terminal kinase or on extracellular signal-regulated kinase. c-Abl-induced p38 activation was primarily mediated by mitogen-activated protein kinase kinase (MKK)6. A C-terminal truncation mutant of c-Abl showed no activity for stimulating p38 and MKK6, while a kinase-deficient c-Abl mutant still retained a residual activity. We tested different forms of c-Abl for their ability to induce apoptosis and found that apoptosis induction correlated with the activation of the MKK6-p38 kinase pathway. Importantly, dominant-negative MKK6, but not dominant-negative MKK3 or p38, blocked c-Abl-induced apoptosis. Because overexpression of p38 blocks cell cycle G1/S transition, we also tested whether the MKK6-p38 pathway is required for c-Abl-induced cell cycle arrest, and we found that neither MKK6 nor p38 dominant-negative mutants could relieve c-Abl-induced cell cycle arrest. Finally, DNA damage-induced MKK6 and p38 activation was diminished in c-Abl null fibroblasts. Our study suggests that c-Abl is required for DNA damage-induced MKK6 and p38 activation, and that activation of MKK6 by c-Abl is required for c-Abl-induced apoptosis but not c-Abl-induced cell cycle arrest.

Protein tyrosine phosphatase PTP1B suppresses p210 bcr-abl-induced transformation of Rat-1 fibroblasts and promotes differentiation of K562 cells

The bcr-abl chimeric oncoprotein exhibits deregulated protein tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive human leukemias, such as chronic myelogenous leukemia (CML). Recently we have shown that the levels of the protein tyrosine phosphatase PTP1B are enhanced in p210 bcr-abl-expressing cell lines. Furthermore, PTP1B recognizes p210 bcr-abl as a substrate, disrupts the formation of a p210 bcr-abl/Grb2 complex, and inhibits signaling events initiated by this oncoprotein PTK. In this report, we have examined whether PTP1B effects transformation induced by p210 bcr-abl. We demonstrate that expression of either wild-type PTP1B or the substrate-trapping mutant form of the enzyme (PTP1B-D181A) in p210 bcr-abl-transformed Rat-1 fibroblasts diminished the ability of these cells to form colonies in soft agar, to grow in reduced serum, and to form tumors in nude mice. In contrast, TCPTP, the closest relative of PTP1B, did not effect p210 bcr-abl-induced transformation. Furthermore, neither PTP1B nor TCPTP inhibited transformation induced by v-Abl. In addition, overexpression of PTP1B or treatment with CGP57148, a small molecule inhibitor of p210 bcr-abl, induced erythroid differentiation of K562 cells, a CML cell line derived from a patient in blast crisis. These data suggest that PTP1B is a selective, endogenous inhibitor of p210 bcr-abl and is likely to be important in the pathogenesis of CML.

Nerve growth factor acutely reduces chemical transmission by means of postsynaptic TrkA-like receptors in squid giant synapse

Tyrosine phosphorylation has been shown to be an important modulator of synaptic transmission in both vertebrates and invertebrates. Such findings hint toward the existence of extracellular ligands capable of activating this widely represented signaling mechanism at or close to the synapse. Examples of such ligands are the peptide growth factors which, on binding, activate receptor tyrosine kinases. To gain insight into the physiological consequences of receptor tyrosine kinase activation in squid giant synapse, a series of growth factors was tested in this preparation. Electrophysiological, pharmacological, and biochemical analysis demonstrated that nerve growth factor (NGF) triggers an acute and specific reduction of the postsynaptic potential amplitude, without affecting the presynaptic spike generation or presynaptic calcium current. The NGF target is localized at a postsynaptic site and involves a new TrkA-like receptor. The squid receptor crossreacts with antibodies generated against mammalian TrkA, is tyrosine phosphorylated in response to NGF stimulation, and is blocked by specific pharmacological inhibitors. The modulation described emphasizes the important role of growth factors on invertebrate synaptic transmission.

Expression of DFak56, a Drosophila homolog of vertebrate focal adhesion kinase, supports a role in cell migration in vivo

Focal adhesion kinase (FAK) is a highly conserved, cytoplasmic tyrosine kinase that has been implicated in promoting cell migration and transmission of antiapoptotic signals in vertebrate cells. In cultured cells, integrin engagement with the extracellular matrix promotes the recruitment of FAK to focal contacts and increases in its phosphotyrosine content and kinase activity, suggesting FAK is an intracellular mediator of integrin signaling. We have identified a Drosophila FAK homolog, DFak56, that is 33% identical to vertebrate FAK, with the highest degree of homology in domains critical for FAK function, including the kinase and focal adhesion targeting domains, and several protein–protein interaction motifs. Furthermore, when expressed in NIH 3T3 cells, DFak56 both localizes to focal contacts and displays the characteristic elevation of phosphotyrosine content in response to plating the cells on fibronectin. During embryogenesis, DFak56 is broadly expressed, and it becomes elevated in the gut and central nervous system at later stages. Consistent with a role in cell migration, we also observe that DFak56 is abundant in the border cells of developing egg chambers before the onset of, and during, their migration.

Constitutive tyrosine phosphorylation of the inhibitory paired Ig-like receptor PIR-B

PIR-A and PIR-B are activating and inhibitory Ig-like receptors on murine B lymphocytes, dendritic cells, and myeloid-lineage cells. The inhibitory function of PIR-B is mediated via its cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, whereas PIR-A pairs with the Fc receptor common γ chain to form an activating receptor complex. In these studies, we observed constitutive tyrosine phosphorylation of PIR-B molecules on macrophages and B lymphocytes, irrespective of the cell activation status. Splenocyte PIR-B molecules were constitutively associated with the SHP-1 protein tyrosine phosphatase and Lyn protein tyrosine kinase. In Lyn-deficient mice, PIR-B tyrosine phosphorylation was greatly reduced. Unexpectedly, tyrosine phosphorylation of PIR-B was not observed in most myeloid and B cell lines but could be induced by ligation of the PIR molecules. Finally, the phosphorylation status of PIR-B was significantly reduced in MHC class I-deficient mice, although not in mice deficient in TAP1 or MHC class II expression. These findings suggest a physiological inhibitory role for PIR-B that is regulated by endogenous MHC class I-like ligands.

Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor

Nerve growth factor (NGF) prevents apoptosis through stimulation of the TrkA receptor protein tyrosine kinase. The downstream activation of phosphatidylinositol 3-kinase (PI 3-kinase) is essential for the inhibition of apoptosis, although this enzyme does not bind to and is not directly activated by TrkA. We have found that the addition of NGF to PC-12 cells resulted in the phosphorylation of the Grb2-associated binder-1 (Gab1) docking protein and induced the association of several SH2 domain-containing proteins, including PI 3-kinase. A substantial fraction of the total cellular PI 3-kinase activity was associated with Gab1. PC-12 cells that overexpressed Gab1 show a decreased requirement for the amount of NGF necessary to inhibit apoptosis. The expression of a Gab1 mutant that lacked the binding sites for PI 3-kinase enhanced apoptosis and diminished the protective effect of NGF. Hence, Gab1 has a major role in connecting TrkA with PI 3-kinase activation and for the promotion of cell survival by NGF.

Different Tyrosine Autophosphorylation Requirements in Fibroblast Growth Factor Receptor-1 Mediate Urokinase-Type Plasminogen Activator Induction and Mitogenesis

Among the seven tyrosine autophosphorylation sites identified in the intracellular domain of tyrosine kinase fibroblast growth factor receptor-1 (FGFR1), five of them are dispensable for FGFR1-mediated mitogenic signaling. The possibility of dissociating the mitogenic activity of basic FGF (FGF2) from its urokinase-type plasminogen activator (uPA)-inducing capacity both at pharmacological and structural levels prompted us to evaluate the role of these autophosphorylation sites in transducing FGF2-mediated uPA upregulation. To this purpose, L6 myoblasts transfected with either wild-type (wt) or various FGFR1 mutants were evaluated for the capacity to upregulate uPA production by FGF2. uPA was induced in cells transfected with wt-FGFR1, FGFR1-Y463F, -Y585F, -Y730F, -Y766F, or -Y583/585F mutants. In contrast, uPA upregulation was prevented in L6 cells transfected with FGFR1-Y463/583/585/730F mutant (FGFR1–4F) or with FGFR1-Y463/583/585/730/766F mutant (FGFR1–5F) that retained instead a full mitogenic response to FGF2; however, preservation of residue Y730 in FGFR1-Y463/583/585F mutant (FGFR1–3F) and FGFR1-Y463/583/585/766F mutant (FGFR1–4Fbis) allows the receptor to transduce uPA upregulation. Wild-type FGFR1, FGFR1–3F, and FGFR1–4F similarly bind to a 90-kDa tyrosine-phosphorylated protein and activate Shc, extracellular signal-regulated kinase (ERK)2, and JunD after stimulation with FGF2. These data, together with the capacity of the ERK kinase inhibitor PD 098059 to prevent ERK2 activation and uPA upregulation in wt-FGFR1 cells, suggest that signaling through the Ras/Raf-1/ERK kinase/ERK/JunD pathway is necessary but not sufficient for uPA induction in L6 transfectants. Accordingly, FGF2 was able to stimulate ERK1/2 phosphorylation and cell proliferation, but not uPA upregulation, in L6 cells transfected with the FGFR1-Y463/730F mutant, whereas the FGFR1-Y583/585/730F mutant was fully active. We conclude that different tyrosine autophosphorylation requirements in FGFR1 mediate cell proliferation and uPA upregulation induced by FGF2 in L6 cells. In particular, phosphorylation of either Y463 or Y730, dispensable for mitogenic signaling, represents an absolute requirement for FGF2-mediated uPA induction.

The Small Mr Ras-like GTPase Rap1 and the Phospholipase C Pathway Act to Regulate Phagocytosis in Dictyostelium discoideum

The function of the small-Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes in Dictyostelium and the oxidative burst in mammalian neutrophils. To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type [Rap1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(+) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C–specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.

Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.