Protein tyrosine phosphatase activation during nerve growth factor-induced neuronal differentiation of PC12 cells.

We have studied the role of protein tyrosine phosphatases (PTPases) during neuronal differentiation of PC12 cells. Nerve growth factor (NGF), a well-characterized differentiating agent for these cells, led to a decrease in DNA synthesis within 24 h. This was accompanied by a 2- to 3-fold increase in the activity of PTPases, measured as the dephosphorylation of polyacidic or polybasic substrates phosphorylated on tyrosine. PTPase activation was independent of cell density and proportional to NGF concentration, with a half-maximal effect occurring at 0.35 nM. High-performance liquid chromatography size exclusion chromatography revealed that PTPases with molecular masses of 550, 300, and 60 kilodaltons were activated in response to NGF. Additional studies showed that the presence of NGF made PC12 cells refractory to the mitogenic effect of epidermal growth factor. Our data indicate that NGF-induced neuronal differentiation and growth arrest in PC12 cells are associated with activation of several PTPases. We speculate that PTPase activation in response to NGF may inhibit the mitogenic actions of other growth factors.     

Intermittent hypoxia-induced protein phosphatase 2A activation reduces PC12 cell proliferation and differentiation

Background

Intermittent hypoxia (IH) plays a critical role in sleep breathing disorder-associated hippocampus impairments, including neurocognitive deficits, irreversible memory and learning impairments. IH-induced neuronal injury in the hippocampus may result from reduced precursor cell proliferation and the relative numbers of postmitotic differentiated neurons. However, the mechanisms underlying IH-induced reactive oxygen species (ROS) generation effects on cell proliferation and neuronal differentiation remain largely unknown.

Results

ROS generation significantly increased after 1–4 days of IH without increased pheochromocytoma-12 (PC12) cell death, which resulted in increased protein phosphatase 2A (PP2A) mRNA and protein levels. After 3–4 days of IH, extracellular signal-regulated kinases 1/2 (ERK1/2) protein phosphorylation decreased, which could be reversed by superoxide dismutase (SOD), 1,10-phenanthroline (Phe), the PP2A phosphorylation inhibitors, okadaic acid (OKA) and cantharidin, and the ERK phosphorylation activator nicotine (p < 0.05). In particular, the significantly reduced cell proliferation and increased proportions of cells in the G₀/G₁ phase after 1–4 days of IH (p < 0.05), which resulted in decreased numbers of PC12 cells, could be reversed by treatment with SOD, Phe, PP2A inhibitors and an ERK activator. In addition, the numbers of nerve growth factor (NGF)-induced PC12 cells with neurite outgrowths after 3–4 days of IH were less than those after 4 days of RA, which was also reversed by SOD, Phe, PP2A inhibitors and an ERK activator.

Conclusions

Our results suggest that IH-induced ROS generation increases PP2A activation and subsequently downregulates ERK1/2 activation, which results in inhibition of PC12 cell proliferation through G₀/G₁ phase arrest and NGF-induced neuronal differentiation.     

Role of SHP-2 tyrosine phosphatase in the DNA damage-induced cell death response

SHP-2, a ubiquitously expressed Src hmology 2 (SH2) domain-containing tyrosine phosphatase, plays a critical role in the regulation of growth factor and cytokine signal transduction. Here we report a novel function of this phosphatase in DNA damage-induced cellular responses. Mutant embryonic fibroblast cells lacking functional SHP-2 showed significantly decreased apoptosis in response to DNA damage. Following cisplatin treatment, induction of p73 and its downstream effector p21(Cip1) was essentially blocked in SHP-2 mutant cells. Further investigation revealed that activation of the nuclear tyrosine kinase c-Abl, an essential mediator in DNA damage induction of p73, was impaired in the mutant cells, suggesting a functional requirement of SHP-2 in c-Abl activation. Consistent with this observation, the effect of overexpression of c-Abl kinase in SHP-2 mutant cells on sensitizing the cells to DNA damage-induced death was abolished. Additionally, we found that in embryonic fibroblast cells 30-40% of SHP-2 was localized in the nuclei, and that a fraction of nuclear SHP-2 was constitutively associated with c-Abl via its SH3 domain. Phosphatase activity of nuclear but not cytoplasmic SHP-2 was significantly enhanced in response to DNA damage. These results together suggest a novel nuclear function for SHP-2 phosphatase in the regulation of DNA damage-induced apoptotic responses.     

Association of tyrosine phosphatase SHP-2 with F-actin at low cell densities

SHP-2 is an intracellular SH2 domain-containing protein-tyrosine phosphatase with an essential role in cell signaling. Here we demonstrate that localization of SHP-2 is regulated by cell density in a cell adhesion-dependent manner. When cells were plated at low densities, SHP-2 was distributed in Triton X-100-insoluble fractions, whereas it was totally soluble when cells were plated at high densities or when low density cells approached confluency. In all cases, the total protein level of SHP-2 was not changed. Fluorescent cell staining revealed that SHP-2 was co-localized with actin stress fibers to the cell peripheral at low cell densities but was diffused in the entire cytoplasm at high cell densities. Transient transfection of cells with truncated forms of SHP-2 demonstrated that the catalytic domain of the enzyme was responsible for the density-regulated distribution of SHP-2, but the catalytic activity was not required. An in vitro co-sedimentation study demonstrated direct binding of full-length and SH2 domain-truncated forms of SHP-2 to F-actin. The data indicate that SHP-2 is regulated by cell density and that it may have a role in assembling and disassembling of the actin network.     

Involvement of the SHP-1 tyrosine phosphatase in regulation of T cell selection.

The selection events shaping T cell development in the thymus represent the outcome of TCR-driven intracellular signaling cascades evoked by Ag receptor interaction with cognate ligand. In view of data indicating TCR-evoked thymocyte proliferation to be negatively modulated by the SHP-1 tyrosine phosphatase, a potential role for SHP-1 in regulating selection processes was investigated by analysis of T cell development in H-Y TCR transgenic mice rendered SHP-1 deficient by introduction of the viable motheaten mutation or a dominant negative SHP-1-encoding transgene. Characterization of thymocyte and peripheral T cell populations in H-Y TCR-viable motheaten mice revealed TCR-evoked proliferation as well as the positive and negative selection of H-Y-specific thymocytes to be enhanced in these mice, thus implicating SHP-1 in the negative regulation of each of these processes. T cell selection processes were also augmented in H-Y TCR mice carrying a transgene driving lymphoid-restricted expression of a catalytically inert, dominant-negative form of SHP-1. SHP-1-negative effects on thymocyte TCR signaling were not influenced by co-cross-linking of the CD28 costimulatory and/or CTLA-4 inhibitory receptors and appear, accordingly, to be realized independently of these comodulators. These observations indicate that SHP-1 raises the signaling threshold required for both positive and negative selection and reveal the inhibitory effects of SHP-1 on TCR signaling to be cell autonomous. The demonstrated capacity for SHP-1 to inhibit TCR-evoked proliferation and selection indicate SHP-1 modulatory effects on the magnitude of TCR-generated signal to be a key factor in determining the cellular consequences of TCR-ligand interaction.     

Regulation of the SHP-2 tyrosine phosphatase by a novel cholesterol- and cell confluence-dependent mechanism

Endothelial cells approaching confluence exhibit marked decreases in tyrosine phosphorylation of receptor tyrosine kinases and adherens junctions proteins, required for cell cycle arrest and adherens junctions stability. Recently, we demonstrated a close correlation in endothelial cells between membrane cholesterol and tyrosine phosphorylation of adherens junctions proteins. Here, we probe the mechanistic basis for this correlation. We find that as endothelial cells reach confluence, the tyrosine phosphatase SHP-2 is recruited to a low-density membrane fraction in a cholesterol-dependent manner. Binding of SHP-2 to this fraction was not abolished by phenyl phosphate, strongly suggesting that this binding was mediated by other regions of SHP-2 beside its SH2 domains. Annexin II, previously implicated in cholesterol trafficking, was associated in a complex with SHP-2, and both proteins localized to adhesion bands in confluent endothelial monolayers. These studies reveal a novel, cholesterol-dependent mechanism for the recruitment of signaling proteins to specific plasma membrane domains via their interactions with annexin II.     

Role of the SHP-2 tyrosine phosphatase in cytokine-induced signaling and cellular response

Cytokines and growth factors are important extracellular regulatory proteins. They exert their biological functions through binding to their cognate receptors on the cell surface and triggering intracellular signaling cascades. However, the intracellular signaling mechanisms of cytokines and growth factors are not well understood. Accumulating evidence has shown that protein phosphorylation and dephosphorylation carried out by protein kinases and protein phosphatases are fundamental biochemical events in intracellular signal transduction. SHP-2, a Src homology (SH) 2 domain-containing protein tyrosine phosphatase (PTP), is widely involved in a variety of signaling pathways triggered by cytokines and growth factors, including the MAP kinase, Jak-Stat, and PI3 kinase pathways. Recent studies have clearly demonstrated that this phosphatase plays an important role in transducing signals relayed from the cell surface to the nucleus, and is a critical intracellular regulator in cytokine and growth factor-induced cell survival, proliferation, and differentiation.     

Concerted activity of tyrosine phosphatase SHP-2 and focal adhesion kinase in regulation of cell motility

The coordinated interplay of substrate adhesion and deadhesion is necessary for cell motility. Using MCF-7 cells, we found that insulin-like growth factor I (IGF-I) induces the adhesion of MCF-7 to vitronectin and collagen in a dose- and time-dependent manner, suggesting that IGF-I triggers the activation of different integrins. On the other hand, IGF-I promotes the association of insulin receptor substrate 1 with the focal adhesion kinase (FAK), paxillin, and the tyrosine phosphatase SHP-2, resulting in FAK and paxillin dephosphorylation. Abrogation of SHP-2 catalytic activity with a dominant-negative mutant (SHP2-C>S) abolishes IGF-I-induced FAK dephosphorylation, and cells expressing SHP2-C>S show reduced IGF-I-stimulated chemotaxis compared with either mock- or SHP-2 wild-type-transfected cells. This impairment of cell migration is recovered by reintroduction of a catalytically active SHP-2. Interestingly, SHP-2-C>S cells show a larger number of focal adhesion contacts than wild-type cells, suggesting that SHP-2 activity participates in the integrin deactivation process. Although SHP-2 regulates mitogen-activated protein kinase activity, the mitogen-activated protein kinase kinase inhibitor PD-98059 has only a marginal effect on MCF-7 cell migration. The role of SHP-2 as a general regulator of cell chemotaxis induced by other chemotactic agents and integrins is discussed.     

The SHP—2 tyrosine phosphatase：Signaling mechanisms and biological functions

Cellular biological avtivities are tightly controlled by intracellular signaling processes initiated by extracellular signals.Protein tyrosine phosphatases,which remove phosphate groups from phosphorylated signaling molecules,play equally important tyrosine roles as protein tyrosine kinases in signal transduction.SHP-2 a cytoplasmic SH2 domain containing protein tyrosine phosphatase,is involved in the signaling pathways of a variety of growth factors and cytokines.Recent studies have clearly demonstrated that this phosphatase plays an important role in transducing signal relay from the cell surface to the nucleus,and is a critical intracellular regulator in mediating cell proliferation and differentiation.     

Site-specific incorporation of a phosphotyrosine mimetic reveals a role for tyrosine phosphorylation of SHP-2 in cell signaling.

The regulation of protein tyrosine phosphatase (PTPase) SHP-2 is proposed to involve tyrosine phosphorylation on two tail tyrosine residues. Using "expressed protein ligation", nonhydrolyzable phosphotyrosine analogs were introduced at known phosphorylation sites in SHP-2. Biochemical analysis suggests that a phosphonate at Tyr542 interacts intramolecularly with the N-terminal SH2 domain to relieve basal inhibition of the PTPase, whereas a phosphonate at Tyr-580 stimulates the PTPase activity by interaction with the C-terminal SH2 domain. Microinjection experiments indicate that a single phosphorylation of Tyr-542 of SHP-2 is sufficient to activate the MAP kinase pathway in living cells. These studies support a novel mechanism explaining how tyrosine phosphorylation of a PTPase is important in signal transduction.     

A model of activation of the protein tyrosine phosphatase SHP-2 by the human leptin receptor

Signalling through the leptin receptor has been shown to activate the SH2 domain-containing tyrosine phosphatase SHP-2 through tyrosine phosphorylation. The human leptin receptor contains five tyrosine residues in the cytoplasmic domain that may become phosphorylated. We show here using BIAcore studies, wherein binding of peptides to SHP-2 was detected, that peptides corresponding to sequences containing phosphotyrosines 974 and 986 (LR974P and LR986P, respectively) from the leptin receptor cytoplasmic domain were the only two peptides that bound to the enzyme. Binding of LR974P to SHP-2 was inhibited in a dose-dependent fashion by orthovanadate, whereas binding of LY986P was not, indicating that the enzyme binds to these peptides through different sites. Only the leptin receptor-derived peptide corresponding to tyrosine 974 was dephosphorylated by recombinant purified SHP-2. Time courses of the reaction were complex, and fitted a two exponent rate equation. Preincubation of SHP-2 with LR986P markedly activated the enzyme at early time points and time courses of the activated enzyme fitted a single exponential first order rate equation. We propose that LR974P binds to the active site of SHP-2, whereas LR986P may bind to the N- and C-terminal SH2 domains of SHP-2, thus activating the phosphatase activity. These data support a model in which SHP-2 binds to phosphotyrosine 986 in the activated leptin receptor and is activated to dephosphorylate phosphotyrosine 974, downregulating signalling events emanating from SH2 domain-containing proteins that bind here.     

Protein tyrosine nitration is triggered by nerve growth factor during neuronal differentiation of PC12 cells

Nitric oxide (NO) is a signaling molecule implicated in a spectrum of cellular processes including neuronal differentiation. The signaling pathway triggered by NO in physiological processes involves the activation of soluble guanylate cyclase and S-nitrosylation of proteins, and, as recently proposed, nitration of tyrosine residues in proteins. However, little is known about the mechanisms involved and the target proteins for endogenous NO during the progression of neuronal differentiation. To address this question, we investigated the presence, localization, and subcellular distribution of nitrated proteins during neurotrophin-induced differentiation of PC12 cells. We find that some proteins show basal levels of tyrosine nitration in PC12 cells grown in the absence of nerve growth factor (NGF) and that nitration levels increase significantly after 2 days of incubation with this neurotrophin. Nitrated proteins accumulate over a period of several days in the presence of NGF. We demonstrate that this nitration is coupled to activation of nitric oxide synthase. The subcellular distribution of nitrated proteins changes during PC12 cell differentiation, displaying a shift from the cytosolic to the cytoskeletal fraction and we identified alpha-tubulin as the major target of nitration in PC12 cells by N-terminal sequence and MALDI-TOF analyses. We conclude that tyrosine nitration of proteins could be a novel molecular mechanism involved in the signaling pathway by which NO modulates NGF-induced differentiation in PC12 cells.     

Regulation of early events in integrin signaling by protein tyrosine phosphatase SHP-2

The nontransmembrane protein tyrosine phosphatase SHP-2 plays a critical role in growth factor and cytokine signaling pathways. Previous studies revealed that a fraction of SHP-2 moves to focal contacts upon integrin engagement and that SHP-2 binds to SHP substrate 1 (SHPS-1)/SIRP-1alpha, a transmembrane glycoprotein with adhesion molecule characteristics (Y. Fujioka et al., Mol. Cell. Biol. 16:6887-6899, 1996; M. Tsuda et al., J. Biol. Chem. 273:13223-13229). Therefore, we asked whether SHP2-SHPS-1 complexes participate in integrin signaling. SHPS-1 tyrosyl phosphorylation increased upon plating of murine fibroblasts onto specific extracellular matrices. Both in vitro and in vivo studies indicate that SHPS-1 tyrosyl phosphorylation is catalyzed by Src family protein tyrosine kinases (PTKs). Overexpression of SHPS-1 in 293 cells potentiated integrin-induced mitogen-activated protein kinase (MAPK) activation, and potentiation required functional SHP-2. To further explore the role of SHP-2 in integrin signaling, we analyzed the responses of SHP-2 exon 3(-/-) and wild-type cell lines to being plated on fibronectin. Integrin-induced activation of Src family PTKs, tyrosyl phosphorylation of several focal adhesion proteins, MAPK activation, and the ability to spread on fibronectin were defective in SHP-2 mutant fibroblasts but were restored upon SHP-2 expression. Our data suggest a positive-feedback model in which, upon integrin engagement, basal levels of c-Src activity catalyze the tyrosyl phosphorylation of SHPS-1, thereby recruiting SHP-2 to the plasma membrane, where, perhaps by further activating Src PTKs, SHP-2 transduces positive signals for downstream events such as MAPK activation and cell shape changes.     

A positive role of the PI3-K/Akt signaling pathway in PC12 cell differentiation

Activation of phosphatidylinositol 3-kinase (PI3-K) is considered to be a key event upon stimulation of cells with growth factors. Akt is known to be a downstream target of PI3-K when it is activated by nerve growth factor (NGF). NGF induces cell differentiation of PC12 cells as indicated by neurite outgrowth. In order to investigate the role of PI3-K/Akt in NGF-induced differentiation of PC12 cells, we generated cells ectopically expressing constitutively activated (CA), wild type (WT) and dominant negative (DN) forms of Akt. NGF-induced neurite outgrowth was greatly accelerated in the cells expressing CA-Akt, and dramatically inhibited in those expressing DN-Akt. Pre-treatment with an Akt inhibitor, ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine], inhibited NGF-induced Akt phosphorylation as well as neurite outgrowth but did not markedly affect the activities of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The PI3-K inhibitors wortmannin and LY294002 blocked NGF-induced Akt phosphorylation as well as neurite outgrowth. These results indicate that PI3-K/Akt is a positive regulator of NGF-induced neuronal differentiation in PC12 cells.     

Blocking the function of tyrosine phosphatase SHP-2 by targeting its Src homology 2 domains

SHP-2 is an Src homology 2 (SH2) domain-containing tyrosine phosphatase with crucial functions in cell signaling and major pathological implications. It stays inactive in the cytosol and is activated by binding through its SH2 domains to tyrosine-phosphorylated receptors on the cell surface. One such cell surface protein is PZR, which contains two tyrosine-based inhibition motifs responsible for binding of SHP-2. We have generated a glutathione S-transferase fusion protein carrying the tandem tyrosine-based inhibition motifs of PZR, and the protein was tyrosine-phosphorylated by co-expressing c-Src in Escherichia coli cells. The purified phosphoprotein displays a strong binding to SHP-2 and causes its activation in vitro. However, when introduced into NIH 3T3 cells by using a protein delivery reagent, it effectively inhibited the activation of ERK1/2 induced by growth factors and serum but not by phorbol ester, in reminiscence of the effects caused by expression of dominant negative SHP-2 mutants and deletion of functional SHP-2. The data suggest that the exogenously introduced PZR protein specifically binds SHP-2, blocks its translocation, and renders it functionally incompetent. This is further supported by the fact that the phosphorylated PZR protein had no inhibitory effects on fibroblasts derived from mice expressing only a mutant SHP-2 protein lacking most of the N-terminal SH2 domain. This study thus provides a novel and highly specific method to interrupt the function of SHP-2 in cells.     

Regulation of calcium-sensitive tyrosine kinase Pyk2 by angiotensin II in endothelial cells. Roles of Yes tyrosine kinase and tyrosine phosphatase SHP-2

Calcium-sensitive tyrosine kinase Pyk2 has been implicated in the regulation of ion channels, cellular adhesion, and mitogenic and hypertrophic reactions. In this study, we have investigated the regulation of Pyk2 by angiotensin II (Ang II) in pulmonary vein endothelial cells. We found that the Ang II-induced tyrosine phosphorylation of Pyk2, which requires the activity of Src family kinase, was specifically regulated by the Src family kinase member, Yes kinase. Moreover, we identified for the first time the constitutive association of Pyk2 with an Src homology 2 (SH2) domain-containing tyrosine phosphatase SHP-2. SHP-2 interacts with Pyk2 through a region other than its SH2 domains. Pyk2 can be dephosphorylated in vitro in SHP-2 immunoprecipitates and in intact cells expressing an NH(2) terminus-truncated form of SHP-2, which lacks the two SH2 domains but has an enhanced phosphatase activity. Ang II activates the endogenous SHP-2. Finally, the SHP-2-mediated dephosphorylation of Pyk2 correlates with the negative effect of SHP-2 on the Ang II-induced activation of extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase. Thus, the balance of Pyk2 tyrosine phosphorylation in response to Ang II is controlled by Yes kinase and by a tyrosine phosphatase SHP-2 in endothelial cells.     

A novel membrane glycoprotein, SHPS-1, that binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion.

Protein tyrosine phosphatases (PTPases), such as SHP-1 and SHP-2, that contain Src homology 2 (SH2) domains play important roles in growth factor and cytokine signal transduction pathways. A protein of approximately 115 to 120 kDa that interacts with SHP-1 and SHP-2 was purified from v-src-transformed rat fibroblasts (SR-3Y1 cells), and the corresponding cDNA was cloned. The predicted amino acid sequence of the encoded protein, termed SHPS-1 (SHP substrate 1), suggests that it is a glycosylated receptor-like protein with three immunoglobulin-like domains in its extracellular region and four YXX(L/V/I) motifs, potential tyrosine phosphorylation and SH2-domain binding sites, in its cytoplasmic region. Various mitogens, including serum, insulin, and lysophosphatidic acid, or cell adhesion induced tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2 in cultured cells. Thus, SHPS-1 may be a direct substrate for both tyrosine kinases, such as the insulin receptor kinase or Src, and a specific docking protein for SH2-domain-containing PTPases. In addition, we suggest that SHPS-1 may be a potential substrate for SHP-2 and may function in both growth factor- and cell adhesion-induced cell signaling.