Shc Phosphorylation Research Articles

ROLE OF MITOGEN-ACTIVATED PROTEIN KINASE IN CHOLINERGIC STIMULATION OF CONJUNCTIVAL GOBLET CELL SECRETION.

Conjunctival goblet cells are a primary source for the mucous layer of the tear film. We showed previously that conjunctival goblet cell mucin secretion is under neural control as activation of afferent sensory nerves in the cornea stimulates mucin secretion.1 This neural reflex is mediated either by the parasympathetic and sympathetic nerves that surround the goblet cells, or by antidromic stimulation of the sensory nerves in the conjunctiva and collateral sensory nerves from the cornea. Exogenous addition of the parasympathetic agonist carbachol or the parasympathetic neuropeptide VIP stimulated conjunctival goblet cell secretion.2,3 Cholinergic agonists released from parasympathetic nerves bind to and activate G protein linked- M2 and M3 muscarinic receptors. In most tissues, activation of these receptors stimulates phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 binds to specific receptors on the endoplasmic reticulum to increase intracellular Ca2+. The increased intracellular Ca2+ either directly, or in combination with Ca2+/calmodulin-dependent protein kinases, activates mucin secretion. DAG in turn stimulates protein kinase C (PKC). In many tissues, cholinergic agonists also activate the mitogen-activated protein kinase (MAPK) signaling pathway used by growth factor receptors, which usually regulate cell growth and proliferation. Activation of the MAPK pathway by cholinergic agonists can occur through several mechanisms. One such mechanism is through transactivation of the EGF receptor in an EGF-independent manner.4 The activated recepter then serves as a scaffold for recruitment of Grb2-SoS complex via tyrosine phosphorylation of Shc.

Distinct Roles of the Adaptor Protein Shc and Focal Adhesion Kinase in Integrin Signaling to ERK

It has been proposed that integrins activate ERK through the adaptor protein Shc independently of focal adhesion kinase (FAK) or through FAK acting on multiple target effectors, including Shc. We show that disruption of the actin cytoskeleton by cytochalasin D causes a complete inhibition of FAK but does not inhibit Shc signaling and activation of ERK. We have then generated primary fibroblasts carrying a targeted deletion of the segment of beta(1) subunit cytoplasmic domain required for activation of FAK. Analysis of these cells indicates that FAK is not necessary for efficient tyrosine phosphorylation of Shc, association of Shc with Grb2, and activation of ERK in response to matrix adhesion. In addition, integrin-mediated activation of FAK does not appear to be required for signaling to ERK following growth factor stimulation. To examine if FAK could contribute to the activation of ERK in a cell type-specific manner through the Rap1/B-Raf pathway, we have used Swiss-3T3 cells, which in contrast to primary fibroblasts express B-Raf. Dominant negative studies indicate that Shc mediates the early phase and peak, whereas FAK, p130(CAS), Crk, and Rap1 contribute to the late phase of integrin-dependent activation of ERK in these cells. In addition, introduction of B-Raf enhances and sustains integrin-mediated activation of ERK in wild-type primary fibroblasts but not in those carrying the targeted deletion of the beta(1) cytoplasmic domain. Thus, the Shc and FAK pathways are activated independently and function in a parallel fashion. Although not necessary for signaling to ERK in primary fibroblasts, FAK may enhance and prolong integrin-mediated activation of ERK through p130(CAS), Crk, and Rap1 in cells expressing B-Raf.

Phosphatidylinositol-3 kinase regulates p21ras activation during IgE-mediated stimulation of human basophils

Cross-linking of IgE or a bacterial product (f-Met-Leu-Phe; FMLP) induces the release of leukotriene C4 (LTC4) and histamine in human basophils. However, the signaling mechanisms in human basophils are only partially understood. It has been demonstrated that extracellular signal-regulated kinases (ERK1/2) specifically regulate the pathway for LTC4 generation, but not for histamine release and interleukin-4 production. More recent studies have suggested that tyrosine kinase (syk)-mediated phosphorylation of shc is responsible for the ras-ERK cascade via the formation of shc-Grb2-Sos2 following stimulation with anti-IgE antibody, but not FMLP, in human basophils. However, while characterizing the role of phosphatidylinositol (PI)-3 kinase in signaling pathways leading to basophil mediator release, it was noted that this pathway might also regulate p21ras activation. Anti-IgE antibody, but not FMLP, resulted in phosphorylation of p85 (regulatory subunit of PI3 kinase), suggesting activation of PI3 kinase. Inhibition of PI3 kinase by selective inhibitor (LY294002) abolished anti-IgE antibody- but not FMLP-induced phosphorylation of MEK1 (MAPK kinase/ERK kinase) and ERKs while inhibiting LTC4 generation as well as histamine release. IgE-mediated activation of ras (upstream of MEK-ERK) was also inhibited. But, further upstream, phosphorylation of syk and of shc and inducible association between shc and Grb2 were not affected. Furthermore, the IgE-mediated cytosolic calcium response ([Ca(++)](i)) was also diminished. These results suggest that functional responses may be dependent on the activity of PI3 kinase, which regulates at least 2 important signaling pathways: by regulating activation of ras for the MEK-ERK pathway and the increase in [Ca(++)](i).

Phosphatidylinositol-3 kinase regulates p21ras activation during IgE-mediated stimulation of human basophils

AbstractCross-linking of IgE or a bacterial product (f-Met-Leu-Phe; FMLP) induces the release of leukotriene C4 (LTC4) and histamine in human basophils. However, the signaling mechanisms in human basophils are only partially understood. It has been demonstrated that extracellular signal-regulated kinases (ERK1/2) specifically regulate the pathway for LTC4 generation, but not for histamine release and interleukin-4 production. More recent studies have suggested that tyrosine kinase (syk)-mediated phosphorylation of shc is responsible for the ras-ERK cascade via the formation of shc-Grb2-Sos2 following stimulation with anti-IgE antibody, but not FMLP, in human basophils. However, while characterizing the role of phosphatidylinositol (PI)-3 kinase in signaling pathways leading to basophil mediator release, it was noted that this pathway might also regulate p21ras activation. Anti-IgE antibody, but not FMLP, resulted in phosphorylation of p85 (regulatory subunit of PI3 kinase), suggesting activation of PI3 kinase. Inhibition of PI3 kinase by selective inhibitor (LY294002) abolished anti-IgE antibody- but not FMLP-induced phosphorylation of MEK1 (MAPK kinase/ERK kinase) and ERKs while inhibiting LTC4 generation as well as histamine release. IgE-mediated activation of ras (upstream of MEK-ERK) was also inhibited. But, further upstream, phosphorylation of syk and of shc and inducible association between shc and Grb2 were not affected. Furthermore, the IgE-mediated cytosolic calcium response ([Ca++]i) was also diminished. These results suggest that functional responses may be dependent on the activity of PI3 kinase, which regulates at least 2 important signaling pathways: by regulating activation of ras for the MEK-ERK pathway and the increase in [Ca++]i.

Ret oncogene signal transduction via a IRS-2/PI 3-kinase/PKB and a SHC/Grb-2 dependent pathway: possible implication for transforming activity in NIH3T3 cells

Multiple endocrine neoplasia 2A (MEN 2A) is an inherited disease caused by mutations of the Ret proto-oncogene. Although many different Ret mutations have been described, little is known about the signaling pathways triggered by the Ret oncogene. In this study, we have determined the signaling properties of a Ret-9bp duplication encoding amino acids 634-636, which was recently identified in a patient with all clinical features of the MEN 2A syndrome. The Ret-9bp duplication leads to constitutive activation of the Ret tyrosine kinase. Furthermore, Ret-9bp increased mitogenic and transforming activity demonstrated by thymidine incorporation as well as colony formation in soft agar. Studying intracellular signaling pathways, which may be involved in malignant transformation of Ret-9bp expressing NIH3T3 cells, we could demonstrate Ret-9bp dependent phosphorylation of insulin receptor substrate-2 (IRS-2) with consecutive activation of phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (PKB/AKT). Moreover, Ret-9bp induces phosphorylation of SHC resulting in growth factor receptor binding protein-2 (Grb-2) binding and activation of the mitogen activating protein (MAP) kinase pathway. In addition to these postreceptor cytoplasmic signaling events, we have studied nuclear signal by Ret-9bp and found activation of c-jun and jun-D, two members of the jun/AP-1 family of transcription factors. In summary, an oncogenic 9bp duplication of Ret causes Ret dimer formation and ligand independent activation of the tyrosine kinase. Besides the signaling steps leading to MAPK activation, we could demonstrate that Ret-9bp induced constitutive activation of a signaling pathway involving IRS-2, PI 3-kinase and PKB/AKT which could transduce the oncogenic Ret signal to increased gene transcription via activation of the jun/AP-1 transcription factor family.

Expression of thrombopoietin receptor and its functional role in human B-precursor leukemia cells with 11q23 translocation or Philadelphia chromosome

Thrombopoietin (TPO) is a hematopoietic growth factor which plays a central role in normal megakaryocytopoiesis and thrombopoiesis. Although the interaction between TPO and its receptor c-Mpl encoded by the c-mpl gene is now known to be implicated in the proliferation and/or differentiation of abnormal myeloid cells and normal hematopoietic stem cells, little is known about a role of the TPO/c-Mpl system in lymphoid leukemia cells. In the present study, we first examined the expression of c-mpl/c-Mpl in 23 human lymphoid leukemic cell lines (T-lineage 4, B-lineage 19) using three distinct methods. The c-mpl mRNA was detectable in as many as 20 cell lines (T-lineage 3, B-lineage 17) by reverse transcriptase-polymerase chain reaction, but its translated product, c-Mpl, was demonstrable by Western blot only in B-lineage cell lines. Flow cytometric analysis revealed the surface c-Mpl expression in 13 of 17 B-lineage cell lines, but its higher expression (>40%) was restricted in nine B-precursor cell lines, eight of which had 11q23 translocation or Philadelphia chromosome (Ph1). We also demonstrated that two of eight cell lines with 11q23 translocation or Ph1 exhibited a significant proliferative response to TPO in the 3H-thymidine uptake and colony-forming assays. Triggering of these cell lines by TPO transiently up-regulated tyrosine phosphorylation of JAK-2 and Shc, indicating that their receptor is functional. Primary leukemia cells separated from patients with B-precursor acute lymphoblastic leukemia with Ph1 or 11q23 translocation also showed the surface c-Mpl expression and a significant responsiveness to TPO. These results suggest that the TPO/c-Mpl interaction may play a physiological role in the growth regulation of B-precursor leukemia cells particularly with specific chromosomal abnormalities.

Insulin Activation of Mitogen-Activated Protein (MAP) Kinase and Akt Is Phosphatidylinositol 3-Kinase-Dependent in Rat Adipocytes

To explore the mechanism of MAP kinase activation in adipocytes, we examined the possible involvement of several candidate signaling proteins. MAP kinase activity was markedly increased 2–4 min after treatment with insulin and declined to basal levels after 20 min. The insulin-dependent tyrosine phosphorylation of IRS-1 in the internal membrane and its association with phosphatidylinositol 3 (PI3) kinase preceded MAP kinase activation. There was little or no tyrosine phosphorylation of Shc or association of Grb2 with Shc or IRS-1. Specific PI3 kinase inhibitors blocked the insulin-mediated activation of MAP kinase. They also decreased the activation of MAP kinase by PMA and EGF but to a much lesser extent. Insulin induced phosphorylation of AKT on serine/threonine residues, and its effect could be blocked by PI3 kinase inhibitors. These results suggest that the insulin-dependent activation of MAP kinase in adipocytes is mediated by the IRS-1/PI3 kinase pathway but not by the Shc/Grb2/SOS pathway.

Angiotensin II-induced growth of vascular smooth muscle cells requires an Src-dependent activation of the epidermal growth factor receptor

Angiotensin II (Ang II) is a potent stimulus of vascular smooth muscle cell (VSMC) growth. Activation of extracellular signal-regulated kinase (ERK), the archetypal mitogen-activated protein (MAP) kinase, and phosphatidylinositol 3 (PI3) kinase are critical steps in Ang II-induced mitogenic signaling. However, the mechanism involved in the activation of these kinases upon binding of Ang II to its receptor is poorly understood. In the present study, we examined the role of the epidermal growth factor receptor (EGFR) in Ang II signaling in VSMCs employing immunoprecipitation, Western blot analysis, kinase immunocomplex assay, and [3H]-thymidine incorporation. A time-dependent tyrosine phosphorylation of the EGFR in response to Ang II was observed that was mediated by the Ang II type 1 receptor. This transactivation of the EGFR was blocked in the presence of PP1, an inhibitor of the intracellular Src-like tyrosine kinases. The tyrphostin AG 1478, a selective EGFR antagonist, inhibited both Ang II- and EGF-induced tyrosine phosphorylation of the EGFR. Furthermore, Ang II induced the binding of the adaptor protein Shc to the EGFR, leading to phosphorylation of Shc. In addition, the same nanomolar concentrations of AG 1478 that were effective in EGF signaling blocked the Ang II-induced activation of ERK and PI3 kinase in a dose-dependent manner. Proliferation of VSMCs, detected by measurements of DNA synthesis, following stimulation with Ang II was potently inhibited in the presence of AG 1478 or PP1. Our data suggest that EGFR serves as a role in mitogenic signaling following stimulation with Ang I through a ligand-independent and Src-dependent transactivation of the EGFR. Furthermore, we demonstrate this transactivation as a pivotal step in Ang II-induced activation of MAP kinase and PI3 kinase, as well as growth of VSMCs.

IGF-1 Liberates HB-EGF

Receptor cross talk is becoming widely accepted as a normal physiological method of recruiting specific signaling pathways in cells. Growth factor receptors (GFRs) that do not directly associate with or activate specific proteins can still activate the proteins indirectly by enlisting other unrelated GFRs. Roudabush et al . examine insulin-like growth factor-1 (IGF-1) receptor signaling and observe that matrix metalloproteases are involved in releasing heparan-bound EGF to activate the EGF receptor (EGFR). IGF-1-mediated Shc activation in Cos-7 cells occurred through the EGFR pathway, leading to the activation of the MAPKs Erk1 and Erk2, and, subsequently, cell proliferation. However, IGF-1 directly activated insulin receptor substrate-1 (IRS-1) and the PI3K/Akt anti-apoptotic pathway. Thus, the anti-apoptotic pathway is activated independent of EGFR stimulation. Thus it may be possible to design clinically useful drugs to selectively abrogate adverse signals in the anti-apoptotic or proliferative networks activated directly or indirectly by IGF-1. An STKE perspective by Graham Carpenter discusses the role of EGF cross talk in cellular processes. Roudabush, F.L., Pierce, K.L., Maudsley, S., Khan, K.D., and Luttrell, L.M. (2000) Transactivation of the EGF receptor mediates IGF-1-stimulated Shc phosphorylation and ERK1/2 activation in COS-7 cells. J. Biol. Chem. 275 : 22583-22589. [Abstract] [Full Text]

Transactivation of the EGF Receptor Mediates IGF-1-stimulated Shc Phosphorylation and ERK1/2 Activation in COS-7 Cells

The receptor for insulin-like growth factor 1 (IGF-1) mediates multiple cellular responses, including stimulation of both proliferative and anti-apoptotic pathways. We have examined the role of cross talk between the IGF-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR) in mediating responses to IGF-1. In COS-7 cells, IGF-1 stimulation causes tyrosine phosphorylation of the IGF-1R beta subunit, the EGFR, insulin receptor substrate-1 (IRS-1), and the Shc adapter protein. Shc immunoprecipitates performed after IGF-1 stimulation contain coprecipitated EGFR, suggesting that IGF-1R activation induces the assembly of EGFR.Shc complexes. Tyrphostin AG1478, an inhibitor of the EGFR kinase, markedly attenuates IGF-1-stimulated phosphorylation of EGFR, Shc, and ERK1/2 but has no effect on phosphorylation of IGF-1R, IRS-1, and protein kinase B (Akt). Cross talk between IGF-1 and EGF receptors is mediated through an autocrine mechanism involving matrix metalloprotease-dependent release of heparin-binding EGF (HB-EGF), because IGF-1-mediated ERK activation is inhibited both by [Glu(52)]Diphtheria toxin, a specific inhibitor of HB-EGF, and the metalloprotease inhibitor 1,10-phenanthroline. These data demonstrate that IGF-1 stimulation of the IRS-1/PI3K/Akt pathway and the EGFR/Shc/ERK1/2 pathway occurs by distinct mechanisms and suggest that IGF-1-mediated "transactivation" of EGFR accounts for the majority of IGF-1-stimulated Shc phosphorylation and subsequent activation of the ERK cascade.

The Shc adaptor protein forms interdependent phosphotyrosine-mediated protein complexes in mast cells stimulated with interleukin 3

The Shc adaptor protein possesses 2 distinct phosphotyrosine (pTyr) recognition modules—the pTyr binding (PTB) domain and the Src homology 2 (SH2) domain—and multiple potential sites for tyrosine (Tyr) phosphorylation (Tyr residues 239, 240, and 317). On stimulation of hematopoietic cells with interleukin 3 (IL-3), Shc becomes phosphorylated and may therefore contribute to IL-3 signaling. We investigated the interactions mediated by the Shc modular domains and pTyr sites in IL-3–dependent IC2 premast cells. The Shc PTB domain, rather than the SH2 domain, associated both in vitro and in vivo with the Tyr-phosphorylated β subunit of the IL-3 receptor and with the SH2-containing 5′ inositol phosphatase (SHIP), and it recognized specific NXXpY phosphopeptides from these binding partners. In IL-3–stimulated mast cells, Shc phosphorylation occurred primarily on Tyr239 and 317 and was dependent on a functional PTB domain. Phosphorylated Tyr317, and to a lesser extent, Tyr239, bound the Grb2 adaptor and SHIP. Furthermore, a pTyr317 Shc phosphopeptide selectively recognized Grb2, Sos1, SHIP, and the p85 subunit of phosphatidylinositol 3′ kinase from mast cells, as characterized by mass spectrometry. These results indicate that Shc undergoes an interdependent series of pTyr-mediated interactions in IL-3–stimulated mast cells, resulting in the recruitment of proteins that regulate the Ras pathway and phospholipid metabolism.

The Shc adaptor protein forms interdependent phosphotyrosine-mediated protein complexes in mast cells stimulated with interleukin 3

AbstractThe Shc adaptor protein possesses 2 distinct phosphotyrosine (pTyr) recognition modules—the pTyr binding (PTB) domain and the Src homology 2 (SH2) domain—and multiple potential sites for tyrosine (Tyr) phosphorylation (Tyr residues 239, 240, and 317). On stimulation of hematopoietic cells with interleukin 3 (IL-3), Shc becomes phosphorylated and may therefore contribute to IL-3 signaling. We investigated the interactions mediated by the Shc modular domains and pTyr sites in IL-3–dependent IC2 premast cells. The Shc PTB domain, rather than the SH2 domain, associated both in vitro and in vivo with the Tyr-phosphorylated β subunit of the IL-3 receptor and with the SH2-containing 5′ inositol phosphatase (SHIP), and it recognized specific NXXpY phosphopeptides from these binding partners. In IL-3–stimulated mast cells, Shc phosphorylation occurred primarily on Tyr239 and 317 and was dependent on a functional PTB domain. Phosphorylated Tyr317, and to a lesser extent, Tyr239, bound the Grb2 adaptor and SHIP. Furthermore, a pTyr317 Shc phosphopeptide selectively recognized Grb2, Sos1, SHIP, and the p85 subunit of phosphatidylinositol 3′ kinase from mast cells, as characterized by mass spectrometry. These results indicate that Shc undergoes an interdependent series of pTyr-mediated interactions in IL-3–stimulated mast cells, resulting in the recruitment of proteins that regulate the Ras pathway and phospholipid metabolism.

The docking molecule gab2 is induced by lymphocyte activation and is involved in signaling by interleukin-2 and interleukin-15 but not other common gamma chain-using cytokines.

Interleukin (IL)-2, a critical cytokine with indispensable functions in regulating lymphoid homeostasis, induces the activation of several biochemical pathways. Precisely how these pathways are linked and how they relate to the biological action of IL-2 is incompletely understood. We previously identified SHP-2 (Src homology 2 domain containing phosphatase 2) as an important intermediate in IL-2-dependent MAPK activation and showed its association with a 98-kDa phosphoprotein in response to IL-2. Here, we demonstrate that Gab2, a recently identified adapter molecule, is the major SHP-2 and phosphatidylinositol 3'-kinase-associated 98-kDa protein in normal, IL-2-activated lymphocytes. We further demonstrate that phosphorylation of both Gab2 and SHP-2 is largely dependent upon tyrosine 338 of the IL-2 receptor beta chain. Gab2 can be a substrate of all the three major classes of non-receptor tyrosine kinases associated with the IL-2R, but in terms of IL-2 signaling, JAK3 but not Lck or Syk is essential for Gab2 phosphorylation. We also demonstrate that only IL-2 and IL-15, but not other gammac cytokines induce Gab2 phosphorylation; the ability to phosphorylate Gab2 correlates with Shc phosphorylation and ERK1/ERK2 activation. Finally, we also show that Gab2 levels are regulated by T cell activation, and resting T cells express little Gab2. Therefore, up-regulation and activation of Gab2 may be important in linking the IL-2 receptor to activation of MAPK and may be an important means of achieving specificity in cytokine signaling.

Tumor Necrosis Factor α Inhibits Insulin-induced Mitogenic Signaling in Vascular Smooth Muscle Cells

Tumor necrosis factor alpha (TNFalpha) interferes with insulin signaling in adipose tissue and may promote insulin resistance. Insulin binding to the insulin receptor (IR) triggers its autophosphorylation, resulting in phosphorylation of Shc and the downstream activation of p42/p44 extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2), which mediates insulin-induced proliferation in vascular smooth muscle cells (VSMC). Since insulin resistance is a risk factor for vascular disease, we examined the effects of TNFalpha on mitogenic signaling by insulin. In rat aortic VSMC, insulin induced rapid phosphorylation of the IR and Shc and caused a 5.3-fold increase in activated, phosphorylated ERK1/2 at 10 min. Insulin induced a biphasic ERK1/2 activation with a transient peak at 10 min and a sustained late phase after 2 h. Preincubation (30-120 min) with TNFalpha had no effect on insulin-induced IR phosphorylation. In contrast, TNFalpha transiently suppressed insulin-induced ERK1/2 activation. Insulin-induced phosphorylation of Shc was inhibited by TNFalpha in a similar pattern. Since mitogenic signaling by insulin in VSMC requires ERK1/2 activation, we examined the effect of TNFalpha on insulin-induced proliferation. Insulin alone induced a 3.4-fold increase in DNA synthesis, which TNFalpha inhibited by 48%. TNFalpha alone was not mitogenic. Inhibition of ERK1/2 activation with PD98059 also inhibited insulin-stimulated DNA synthesis by 57%. TNFalpha did not inhibit platelet-derived growth factor-induced ERK1/2 activation or DNA synthesis in VSMC. Thus, TNFalpha selectively interferes with insulin-induced mitogenic signaling by inhibiting the phosphorylation of Shc and the downstream activation of ERK1/2.

Signalling pathways of insulin-like growth factor-I that are augmented by cAMP in FRTL-5 cells

We have reported that pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiates the mitogenic response to insulin-like growth factor-I (IGF-I). The present study was undertaken to determine whether the augmentation by cAMP of IGF-I-dependent tyrosine phosphorylation of known IGF-I receptor substrates plays an important role in the cAMP-dependent potentiation of DNA synthesis induced by IGF-I. Pretreatment with TSH or dibutyryl cAMP did not affect the IGF-I-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). In contrast, cAMP pretreatment potentiated the tyrosine phosphorylation of IRS-2 induced by IGF-I, but did not affect the amount of IRS-2. We found that the IGF-I-dependent tyrosine phosphorylation of 66 kDa Shc (Src homology collagen) was markedly increased by cAMP pretreatment, and that this change was mainly due to an increase in the levels of 66 kDa Shc protein. Under these conditions, cAMP pretreatment significantly increased binding of Grb2 (growth-factor-receptor-bound protein 2) to Shc in response to IGF-I, and activation of MAP kinase (mitogen-activated protein kinase) induced by IGF-I was also enhanced by cAMP. The presence of PD98059, an inhibitor of MEK (MAP-kinase/Erk kinase), during treatment with IGF-I partially inhibited the cAMP-dependent augmentation of DNA synthesis in response to IGF-I. On the other hand, cAMP pretreatment increased binding of the phosphoinositide 3-kinase (PI 3-kinase) p85 subunit to IRS-2, which was reflected in PI 3-kinase activity. LY294002, a PI 3-kinase inhibitor, strongly depressed IGF-I-dependent DNA synthesis after pretreatment with and without TSH or dibutyryl cAMP. Our results suggest that the interaction between cAMP-dependent and IGF-I-dependent pathways leads to an augmentation of cell proliferation, which is mediated, at least in part, through the MAP kinase and PI 3-kinase signalling pathways. These effects are mediated by changes in tyrosine phosphorylation of IGF-I receptor substrates, including IRS-2 and Shc.

The critical role of Shc in insulin-like growth factor-I-mediated mitogenesis and differentiation in 3T3-L1 preadipocytes.

Insulin-like growth factor-I (IGF-I) stimulates mitogenesis in proliferating preadipocytes, but when cells reach confluence and become growth arrested, IGF-I stimulates differentiation into adipocytes. IGF-I induces signaling pathways that involve IGF-I receptor-mediated tyrosine phosphorylation of Shc and insulin receptor substrate 1 (IRS-1). Either of these adaptor proteins can lead to activation of the three-kinase cascade ending in activation of the extracellular signal-regulated kinase 1 and -2 (ERK-1 and -2) mitogen-activated protein kinases (MAPKs). Several lines of evidence suggest that activation of MAPK inhibits 3T3-L1 preadipocyte differentiation. We have shown that IGF-I stimulation of MAPK activity is lost as 3T3-L1 preadipocytes begin to differentiate. This change in MAPK signaling coincides with loss of IGF-I-mediated Shc, but not IRS-1, tyrosine phosphorylation. We hypothesized that down-regulation of MAPK via loss of proximal signaling through Shc is an early component in the IGF-I switch from mitogenesis to differentiation in 3T3-L1 preadipocytes. Treatment of subconfluent cells with the MEK inhibitor PD098059 inhibited both IGF-I-activation of MAPK as well as 3H-thymidine incorporation. PD098059, in the presence of differentiation-inducing media, accelerated differentiation in subconfluent cells as measured by expression of adipocyte protein-2 (aP-2), peroxisome proliferator-activated receptor gamma (PPARgamma) and lipoprotein lipase (LPL). Transient transfection of subconfluent cells with Shc-Y317F, a dominant-negative mutant, attenuated IGF-I-mediated MAPK activation, inhibited DNA synthesis, and accelerated expression of differentiation markers aP-2, PPARgamma, and LPL. We conclude that signaling through Shc to MAPK plays a critical role in mediating IGF-I-stimulated 3T3-L1 mitogenesis. Our results suggest that loss of the ability of IGF-I to activate Shc signaling to MAPK may be an early component of adipogenesis in 3T3-L1 cells.

The m3 muscarinic acetylcholine receptor is coupled to mitogen-activated protein kinase via protein kinase C and epidermal growth factor receptor kinase

The acetylcholine analogue carbachol rapidly activated mitogen-activated protein kinase (MAPK), and caused tyrosine phosphorylation of the adapter protein p52 Shc and the epidermalgrowth factor (EGF) receptor, in human embryonic kidney cells stably expressing m3 muscarinic receptors. The protein kinase C (PKC) inhibitor GF109203X caused a significant partial inhibition of m3 receptor-mediated activation of MAPK. The PKC-independent MAPK activity elicited by carbachol in the presence of GF109203X was reproducibly abolished by AG1478, an inhibitor of EGF-receptor tyrosine kinase activity, and by the Src tyrosine kinase inhibitor PP1. In a subset of these experiments, GF109203X concomitantly increased carbachol-induced tyrosine phosphorylation of p52 Shc and the EGF receptor. In co-stimulation experiments, carbachol and EGF activated MAPK in a non-additive fashion; moreover, EGF-induced association of Shc with the phosphorylated EGF receptor was inhibited by carbachol. This effect of carbachol was blocked by GF109203X. The results indicate that MAPK activation by m3 receptor stimulation is regulated by two pathways; one dependent on PKC, and the other mediated via the EGF receptor and Src. Moreover, the EGF-receptor-dependent pathway may be subject to negative-feedback regulation via m3 receptor-coupled activation of PKC.

Signalling pathways of insulin-like growth factor-I that are augmented by cAMP in FRTL-5 cells

We have reported that pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiates the mitogenic response to insulin-like growth factor-I (IGF-I). The present study was undertaken to determine whether the augmentation by cAMP of IGF-I-dependent tyrosine phosphorylation of known IGF-I receptor substrates plays an important role in the cAMP-dependent potentiation of DNA synthesis induced by IGF-I. Pretreatment with TSH or dibutyryl cAMP did not affect the IGF-I-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). In contrast, cAMP pretreatment potentiated the tyrosine phosphorylation of IRS-2 induced by IGF-I, but did not affect the amount of IRS-2. We found that the IGF-I-dependent tyrosine phosphorylation of 66 kDa Shc (Src homology collagen) was markedly increased by cAMP pretreatment, and that this change was mainly due to an increase in the levels of 66 kDa Shc protein. Under these conditions, cAMP pretreatment significantly increased binding of Grb2 (growth-factor-receptor-bound protein 2) to Shc in response to IGF-I, and activation of MAP kinase (mitogen-activated protein kinase) induced by IGF-I was also enhanced by cAMP. The presence of PD98059, an inhibitor of MEK (MAP-kinase/Erk kinase), during treatment with IGF-I partially inhibited the cAMP-dependent augmentation of DNA synthesis in response to IGF-I. On the other hand, cAMP pretreatment increased binding of the phosphoinositide 3-kinase (PI 3-kinase) p85 subunit to IRS-2, which was reflected in PI 3-kinase activity. LY294002, a PI 3-kinase inhibitor, strongly depressed IGF-I-dependent DNA synthesis after pretreatment with and without TSH or dibutyryl cAMP. Our results suggest that the interaction between cAMP-dependent and IGF-I-dependent pathways leads to an augmentation of cell proliferation, which is mediated, at least in part, through the MAP kinase and PI 3-kinase signalling pathways. These effects are mediated by changes in tyrosine phosphorylation of IGF-I receptor substrates, including IRS-2 and Shc.

Signal transduction and transforming properties of the TEL-TRKC fusions associated with t(12;15)(p13;q25) in congenital fibrosarcoma and acute myelogenous leukemia.

The TEL-TRKC fusion is expressed as a consequence of t(12;15)(p13;q25), and is associated with two human cancers: congenital fibrosarcoma and acute myelogenous leukemia (AML). We report that the T/T(F) and T/T(L) fusion variants associated with congenital fibrosarcoma and AML, respectively, are constitutively tyrosine phosphorylated, and confer factor-independent growth to the murine hematopoietic cell line Ba/F3. Retroviral transduction of T/T(L) causes a rapidly fatal myeloproliferative disease in a murine bone marrow transplant (BMT) model, whereas T/T(F) causes a long-latency, pre-B-cell lymphoblastic lymphoma. TEL-TRKC variants are potent activators of the MAP kinase pathway, but neither variant activates Stat5 or other Stat family members. T/T(L), but not T/T(F), induces tyrosine phosphorylation of phospholipase Cgamma (PLCgamma), phosphoinositol-3 kinase and SHC. However, mutation analysis demonstrates that PLCgamma tyrosine phos phorylation by T/T(L) is dispensable for induction of the myeloproliferative phenotype by T/T(L). Collectively, these data demonstrate that the TEL-TRKC fusion variants are oncoproteins that activate the MAP kinase pathway, and do not require activation of either PLCgamma or Stat5 for efficient induction of a myeloproliferative phenotype in the murine BMT model.

A new feature of Mpl receptor: ligand-induced transforming activity in FRE rat fibroblasts.

Mpl is the receptor for thrombopoietin, the primary regulator of platelet production by megakaryocytes. Upon stimulation by its ligand, Mpl receptor induces proliferation and differentiation of hematopoietic cell lines of various origins. In this paper, we show that Mpl is also able to transform FRE rat fibroblasts in the presence of MGDF (pegylated Megakaryocyte Growth and Development Factor), a modified form of its ligand. We also demonstrate that upon MGDF stimulation Mpl receptor activates the classical transduction pathways described for hematopoietic cell lines in FRE cells. Introduction of Mpl deletion mutants in FRE cells allowed us to demonstrate that the C-terminal region of the Mpl intracytoplasmic domain, which is involved in hematopoietic differentiation, is necessary for the transformation process. Within that region, site-directed mutagenesis showed that the Y112 residue, which is required for Shc phosphorylation, is essential for rat fibroblast transformation by Mpl/MGDF, suggesting the involvement of Shc in Mpl-mediated transformation. Interestingly, we showed that transformation correlated with strong and sustained MAPK activation. Neither Jak2, Stat3 nor Stat5 phosphorylation was sufficient to induce the transformation process. Taken altogether, our results suggest the oncogenicity of Mpl in fibroblastic cells in the presence of its ligand.