Galectin-8 functions as a matricellular modulator of cell adhesion

The interaction of cells with the extracellular matrix regulates cell adhesion and motility. Here we demonstrate that different cell types adhere and spread when cultured in serum-free medium on immobilized galectin-8, a mammalian beta-galactoside-binding protein. At maximal doses, galectin-8 is equipotent to fibronectin in promoting cell adhesion and spreading. Cell adhesion to immobilized galectin-8 is mediated by sugar-protein interactions with integrins, and galectin-8 triggers integrin-mediated signaling cascades including Tyr phosphorylation of focal adhesion kinase and paxillin. Cell adhesion is potentiated in the presence of Mn(2+), whereas it is interrupted in the presence of soluble galectin-8, integrin beta(1) inhibitory antibodies, EDTA, or thiodigalactoside but not by RGD peptides. Furthermore, cells readily adhere onto immobilized monoclonal galectin-8 antibodies, which are equipotent to integrin antibodies in promoting cell adhesion. Cell adhesion to immobilized galectin-8 is partially inhibited by serum proteins, suggesting that complex formation between immobilized galectin-8 and serum components generates a matrix that is less supportive of cell adhesion. Accordingly, cell motility on immobilized galectin-8 readily takes place in the presence of serum. Truncation of the C-terminal half of galectin-8, including one of its two carbohydrate recognition domains, largely abolishes its ability to modulate cell adhesion, indicating that both carbohydrate recognition domains are required to maintain a functional form of galectin-8. Collectively, our findings implicate galectin-8 as a physiological modulator of cell adhesion. When immobilized, it functions as a matrix protein equipotent to fibronectin in promoting cell adhesion by ligation and clustering of cell surface integrin receptors. In contrast, when present in excess as a soluble ligand, galectin-8 (like fibronectin) forms a complex with integrins that negatively regulates cell adhesion. Because of its dual effects on the adhesive properties of the cells and its association with fibronectin, galectin-8 might be considered a novel type of matricellular protein.     

Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.     

Cyclin-dependent kinase inhibitors and JNK act as molecular switches, regulating the choice between growth arrest and apoptosis induced by galectin-8

Galectin-8, a mammalian beta-galactoside binding lectin, functions as an extracellular matrix protein that forms high affinity interactions with integrins. Here we demonstrated that soluble galectin-8 inhibits cell cycle progression and induces growth arrest. These effects cannot be attributed to interference with cell adhesion but can be attributed to a 4-5-fold increase in the cellular content of the cyclin-dependent kinase inhibitor p21, which was already evident following a 4-h incubation of H1299 cells with galectin-8. The increase in p21 levels was preceded by a 3-5-fold increase in JNK and protein kinase B (PKB) activities. Accordingly, SP600125, the inhibitor of JNK, and wortmannin, the inhibitor of phosphatidylinositol 3-kinase, which is the upstream activator of PKB, inhibited the increase in the cellular content of p21. Furthermore, overexpression of a dominant inhibitory form of SEK1, the upstream kinase regulator of JNK, inhibited both JNK activation and p21 accumulation. When p21 expression was inhibited by cycloheximide, galectin-8 directed the cells toward apoptosis, which involves induction of poly(ADP-ribose) polymerase cleavage. Indeed, galectin-8-induced apoptosis was 2-fold higher in HTC (p21-null) cells when compared with parental HTC cells. Because overexpression of galectin-8 attenuates the rate of DNA synthesis, stable colonies that overexpress and secrete galectin-8 can be generated only in cells overexpressing a growth factor receptor, such as the insulin receptor. These results implicate galectin-8 as a modulator of cellular growth through up-regulation of p21. This process involves activation of JNK, which enhances the synthesis of p21, combined with the activation of PKB, which inhibits p21 degradation. These effects of the lectin depended upon protein-sugar interactions and were induced when galectin-8 was present as a soluble ligand or when it was overexpressed in cells.     

Alpha 4 integrin signaling activates phosphatidylinositol 3-kinase and stimulates T cell adhesion to intercellular adhesion molecule-1 to a similar extent as CD3, but induces a distinct rearrangement of the actin cytoskeleton.

Dynamic regulation of beta(2) integrin-dependent adhesion is critical for a wide array of T cell functions. We previously showed that binding of high-affinity alpha(4)beta(1) integrins to VCAM-1 strengthens alpha(L)beta(2) integrin-mediated adhesion to ICAM-1. In this study, we compared beta(2) integrin-mediated adhesion of T cells to ICAM-1 under two different functional contexts: alpha(4) integrin signaling during emigration from blood into tissues and CD3 signaling during adhesion to APCs and target cells. Cross-linking either alpha(4) integrin or CD3 on Jurkat T cells induced adhesion to ICAM-1 of comparable strength. Adhesion was dependent on phosphatidylinositol (PI) 3-kinase but not p44/42 mitogen-activated protein kinase (extracellular regulated kinase 1/2), because it was inhibited by wortmannin and LY294002 but not U0126. These data suggest that PI 3-kinase is a ubiquitous regulator of beta(2) integrin-mediated adhesion. A distinct morphological change consisting of Jurkat cell spreading and extension of filopodia was induced by alpha(4) integrin signaling. In contrast, CD3 induced radial rings of cortical actin polymerization. Inhibitors of PI 3-kinase and extracellular regulated kinase 1/2 did not affect alpha(4) integrin-induced rearrangement of the actin cytoskeleton, but treatment with ionomycin, a Ca(2+) ionophore, modulated cell morphology by reducing filopodia and promoting lamellipodia formation. Qualitatively similar morphological and adhesive changes to those observed with Jurkat cells were observed following alpha(4) integrin or CD3 stimulation of human peripheral blood T cells.     

EGFR and beta1 integrins utilize different signaling pathways to activate Akt

Akt, also called PKB, is a serine/threonine kinase that plays a major role in cell survival. It can be activated by several cellular receptors, including integrins and growth factor receptors, in PI3K-dependent manners. In this study, we analyzed the two current models for Akt activation upon beta1 integrin-mediated adhesion: via focal adhesion kinase and via transactivation of the EGF receptor. Distinct differences in the pathways leading to phosphorylation and activation of Akt from stimulated beta1 integrins and EGF receptor were observed, including opposing sensitivity to the tyrosine kinase inhibitors PP2 and Gefitinib. Using knockout cells and integrin mutant cells, we show that beta1 integrins can induce phosphorylation of Akt at Ser473 and Thr308 and Akt kinase activity independently of the EGF receptor activity, focal adhesion kinase, and the Src family members. In contrast to stimulation with EGF, beta1 integrin-mediated adhesion did not induce Akt tyrosine phosphorylation. Moreover, tyrosine phosphorylation of Akt was found not to be required for its catalytic activity. The results identify a previously unrecognized mechanism by which beta1 integrins activate the PI3K/Akt pathway.     

The EphA8 Receptor Regulates Integrin Activity through p110γ Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via alpha(5)beta(1)- or beta(3) integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110gamma isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110gamma. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110gamma mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110gamma PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion.     

Mechanism of zinc-induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3beta in SH-SY5Y neuroblastoma cells

We have previously reported an aberrant accumulation of activated protein kinase B (PKB), glycogen synthase kinase (GSK)-3beta, extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38 and p70 S6 kinase (p70S6K) in neurons bearing neurofibrillary tangles (NFTs) in Alzheimer's disease (AD). However, the mechanism by which these tau candidate kinases are involved in the regulation of p70S6K and GSK-3beta phosphorylation is unknown. In the current study, 100 microM zinc sulfate was used, and influences of various components of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways on p70S6K and GSK-3beta phosphorylation have been investigated in serum-deprived SH-SY5Y neuroblastoma cells. We found that zinc could induce an increase of phosphorylated (p) p70S6K, p-PKB, p-GSK-3beta, p-ERK1/2, p-JNK and p-p38, especially in long-term treatment (4-8 h). Treatment with different inhibitors including rapamycin, wortmannin, LY294002, and U0126, and their combinations, indicated that phosphorylation of p70S6K and GSK-3beta is regulated by rapamycin-dependent, PI3K and MAPK pathways. Furthermore, phosphorylation of p70S6K and GSK-3beta affected levels of tau unphosphorylated at the Tau-1 site and phosphorylated at the PHF-1 site, and p70S6K phosphorylation affected the total tau level. Thus, 100 microM zinc might activate PKB, GSK-3beta, ERK1/2, JNK, p38 and p70S6K, that are consequently involved in tau changes in SH-SY5Y cells.     

Galectin binding to Mgat5-modified N-glycans regulates fibronectin matrix remodeling in tumor cells

Oncogenic signaling stimulates the dynamic remodeling of actin microfilaments and substrate adhesions, essential for cell spreading and motility. Transformation is associated with increased expression of beta1,6GlcNAc-branched N-glycans, products of Golgi beta1,6-acetylglucosaminyltransferase V (Mgat5) and the favored ligand for galectins. Herein we report that fibronectin fibrillogenesis and fibronectin-dependent cell spreading are deficient in Mgat5(-/-) mammary epithelial tumor cells and inhibited in Mgat5(+/+) cells by blocking Golgi N-glycan processing with swainsonine or by competitive inhibition of galectin binding. At an optimum dosage, exogenous galectin-3 added to Mgat5(+/+) cells activates focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K), recruits conformationally active alpha5beta1-integrin to fibrillar adhesions, and increases F-actin turnover. RGD peptide inhibits PI3K-dependent fibronectin matrix remodeling and fibronectin-dependent cell motility, while galectin-3 stimulates and overrides the inhibitory effects of RGD. Antibodies to the galectin-3 N-terminal oligomerization domain stimulate alpha5beta1 activation and recruitment to fibrillar adhesions in Mgat5(+/+) cells, an effect that is blocked by disrupting galectin-glycan binding. Our results demonstrate that fibronectin polymerization and tumor cell motility are regulated by galectin-3 binding to branched N-glycan ligands that stimulate focal adhesion remodeling, FAK and PI3K activation, local F-actin instability, and alpha5beta1 translocation to fibrillar adhesions.     

The role of p42/44 MAPK and protein kinase B in connective tissue growth factor induced extracellular matrix protein production,cell migration,and actin cytoskeletal rearrangement in human mesangial cells

Connective tissue growth factor (CTGF) is a member of an emerging family of immediate-early gene products that coordinate complex biological processes during differentiation and tissue repair. Here we describe the role of CTGF in integrin-mediated adhesive signaling and the production of extracellular matrix components in human mesangial cells. The addition of CTGF to primary mesangial cells induced fibronectin production, cell migration, and cytoskeletal rearrangement. These functional responses were associated with recruitment of Src and phosphorylation of p42/44 MAPK and protein kinase B. The inhibition of CTGF-induced p42/44 MAPK or phosphatidylinositol 3-kinase (PI3K)/protein kinase B pathway activities abrogated the induction of fibronectin expression. In addition, anti-beta(3) integrin antibodies attenuated the activation of both the p42/44 MAPK and protein kinase B and the increase in fibronectin levels. CTGF also induced mesangial cell migration via a beta(3) integrin-dependent mechanism that was similarly sensitive to the inhibition of the p42/44 MAPK and PI3K pathways, and it promoted the adhesion of the mesangial cells to type I collagen via up-regulation of alpha(1) integrin. Transient actin cytoskeletal disassembly was observed following treatment with the ligand over the course of a 24-h period. CTGF induced the loss of focal adhesions from the mesangial cell as evidenced by the loss of punctate vinculin. However, these processes are p42/44 MAPK and PI3K pathway-independent. Our data support the hypothesis that CTGF mediates a number of its biological effects by the induction of signaling processes via beta(3) integrin. However, others such as actin cytoskeleton disassembly are modulated in a beta(3) integrin/MAPK/PI3K-independent manner, indicating that CTGF is a complex pleiotropic factor with the potential to amplify primary pathophysiological responses.     

Stimulation of fascin spikes by thrombospondin-1 is mediated by the GTPases Rac and Cdc42

Cell adhesion to extracellular matrix is an important physiological stimulus for organization of the actin-based cytoskeleton. Adhesion to the matrix glycoprotein thrombospondin-1 (TSP-1) triggers the sustained formation of F-actin microspikes that contain the actin-bundling protein fascin. These structures are also implicated in cell migration, which may be an important function of TSP-1 in tissue remodelling and wound repair. To further understand the function of fascin microspikes, we examined whether their assembly is regulated by Rho family GTPases. We report that expression of constitutively active mutants of Rac or Cdc42 triggered localization of fascin to lamellipodia, filopodia, and cell edges in fibroblasts or myoblasts. Biochemical assays demonstrated prolonged activation of Rac and Cdc42 in C2C12 cells adherent to TSP-1 and activation of the downstream kinase p21-activated kinase (PAK). Expression of dominant-negative Rac or Cdc42 in C2C12 myoblasts blocked spreading and formation of fascin spikes on TSP-1. Spreading and spike assembly were also blocked by pharmacological inhibition of F-actin turnover. Shear-loading of monospecific anti-fascin immunoglobulins, which block the binding of fascin to actin into cytoplasm, strongly inhibited spreading, actin cytoskeletal organization and migration on TSP-1 and also affected the motility of cells on fibronectin. We conclude that fascin is a critical component downstream of Rac and Cdc42 that is needed for actin cytoskeletal organization and cell migration responses to thrombospondin-1.     

Galectin-8 binds specific beta1 integrins and induces polarized spreading highlighted by asymmetric lamellipodia in Jurkat T cells

Integrin-mediated encounters of T cells with extracellular cues lead these cells to adhere to a variety of substrates and acquire a spread phenotype needed for their tissue incursions. We studied the effects of galectin-8 (Gal-8), a beta-galactoside binding lectin, on Jurkat T cells. Immobilized Gal-8 bound alpha1beta1, alpha3beta1 and alpha5beta1 but not alpha2beta1 and alpha4beta1 and adhered these cells with similar kinetics to immobilized fibronectin (FN). Function-blocking experiments with monoclonal anti-integrin antibodies suggested that alpha5beta1 is the main mediator of cell adhesion to this lectin. Gal-8, but not FN, induced extensive cell spreading frequently leading to a polarized phenotype characterized by an asymmetric lamellipodial protrusion. These morphological changes involved actin cytoskeletal rearrangements controlled by PI3K, Rac-1 and ERK1/2 activity. Gal-8-induced Rac-1 activation and binding to alpha1 and alpha5 integrins have not been described in any other cellular system. Strikingly, Gal-8 was also a strong stimulus on Jurkat cells in suspension, triggering ERK1/2 activation that in most adherent cells is instead dependent on cell attachment. In addition, we found that patients with systemic lupus erythematosus (SLE), a prototypic autoimmune disorder, produce Gal-8 autoantibodies that impede both its binding to integrins and cell adhesion. These are the first function-blocking autoantibodies reported for a member of the galectin family. These results indicate that Gal-8 constitutes a novel extracellular stimulus for T cells, able to bind specific beta1 integrins and to trigger signaling pathways conducive to cell spreading. Gal-8 could modulate a wide range of T cell-driven immune processes that eventually become altered in autoimmune disorders.     

The putative morphogen, DIF-1, of Dictyostelium discoideum activates Akt/PKB in human leukemia K562 cells.

The differentiation-inducing factor-1 (DIF-1) is a putative morphogen that induces stalk-cell formation in the lower eukaryote Dictyostelium discoideum. This molecule has been shown to inhibit cell growth and induce erythroid differentiation in human leukemia K562 cells. In the present study, to clarify the mechanism of the actions of DIF-1, we examined the effect of DIF-1 on Akt/protein kinase B (PKB) in K562 cells. Akt/PKB is a serine/threonine kinase that plays a pivotal role in the regulation of cell survival and differentiation in a variety of cells. A nonphosphorylated (inactive) form of Akt/PKB was ordinarily expressed in K562 cells. However, Akt/PKB was phosphorylated and potently activated within several hours of incubation with 5-30 microM DIF-1, and this activation was inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase). Calcium-increasing agents thapsigargin and A23187 also activated Akt/PKB slightly, which was inhibited by wortmannin. By contrast, calcium-reducing agents TMB-8 and EGTA together with A23187 inhibited the DIF-1-induced activation of Akt/PKB. PMA (PKC activator) also activated Akt/PKB but this activation was not inhibited by wortmannin. DIF-1 exhibited no marked effect on the activation of PKCalpha, beta, and gamma, which were activated by PMA. These results indicate that DIF-1 activates Akt/PKB possibly via cytosolic calcium and subsequent activation of PI3-kinase and also that PMA activates Akt/PKB in a PI3-kinase-independent manner.     

Ligand density and integrin repertoire regulate cellular response to LPA

Engagement of integrin receptors by the extracellular matrix (ECM) protein fibronectin (FN) activates intracellular signaling, cytoskeletal reorganization and cellular tension. The soluble factor lysophosphatidic acid (LPA) acts through Rho GTPase and its effector Rho kinase (ROCK) to enhance α5β1 integrin-mediated cell spreading on the Arg-Gly-Asp (RGD) cell-binding domain of FN. A second cell-binding site for α4 integrins resides in the CS1 segment of the alternatively spliced V region of FN. We show here that LPA treatment of α4β1-expressing CHOα4 cells on FN induced a significant decrease in spread cell area. LPA also decreased apoptosis induced by serum-deprivation in CHOα4 and human A375 melanoma cells in an α4β1-dependent manner. Improvement in cell viability and changes in cell morphology were dependent on ROCK and on the number of substrate binding sites for α4β1. LPA signaling combined with α4β1-mediated adhesion appears to sustain cell viability in situations where FN matrix is limiting. Such cooperation may impact dynamic cellular events such as wound healing, fibrosis, and metastasis.     

c-kit receptor signaling through its phosphatidylinositide-3'-kinase-binding site and protein kinase C: role in mast cell enhancement of degranulation, adhesion, and membrane ruffling.

In bone marrow-derived mast cells (BMMCs), the Kit receptor tyrosine kinase mediates diverse responses including proliferation, survival, chemotaxis, migration, differentiation, and adhesion to extracellular matrix. In connective tissue mast cells, a role for Kit in the secretion of inflammatory mediators has been demonstrated as well. We recently demonstrated a role for phosphatidylinositide-3' (PI 3)-kinase in Kit-ligand (KL)-induced adhesion of BMMCs to fibronectin. Herein, we investigated the mechanism by which Kit mediates enhancement of Fc epsilon RI-mediated degranulation, cytoskeletal rearrangements, and adhesion in BMMCs. Wsh/Wsh BMMCs lacking endogenous Kit expression, were transduced to express normal and mutant Kit receptors containing Tyr-->Phe substitution at residues 719 and 821. Although the normal Kit receptor fully restored KL-induced responses in Wsh/Wsh BMMCs, Kit gamma 719F, which fails to bind and activate PI 3-kinase, failed to potentiate degranulation and is impaired in mediating membrane ruffling and actin assembly. Inhibition of PI 3-kinase with wortmannin or LY294002 also inhibited secretory enhancement and cytoskeletal rearrangements mediated by Kit. In contrast, secretory enhancement and adhesion stimulated directly through protein kinase C (PKC) do not require PI 3-kinase. Calphostin C, an inhibitor of PKC, blocked Kit-mediated adhesion to fibronectin, secretory enhancement, membrane ruffling, and filamentous actin assembly. Although cytochalasin D inhibited Kit-mediated filamentous actin assembly and membrane ruffling, secretory enhancement and adhesion to fibronectin were not affected by this drug. Therefore, Kit-mediated cytoskeletal rearrangements that are dependent on actin polymerization can be uncoupled from the Kit-mediated secretory and adhesive responses. Our results implicate receptor-proximal PI 3-kinase activation and activation of a PKC isoform in Kit-mediated secretory enhancement, adhesion, and cytoskeletal reorganization.     

粘着斑激酶在TNF-α作用下影响SMMC-7721细胞PKB的表达水平

 探讨在肿瘤坏死因子α(TNF-α)作用下 ,粘着斑激酶 (focal adhesion kinase,FAK)对蛋白激酶 B(PKB)蛋白水平的影响 .利用构建、转染 FAK反义质粒来特异性降低 SMMC- 772 1细胞的FAK含量 ,及用 Western杂交的方法来检测 PKB的蛋白含量 .文献报道 TNF- α能够激活磷脂酰肌醇 3-激酶 (PI3K)而使 PKB发生磷酸化 .但是至于 TNF-α对 PKB蛋白水平的影响目前并无报道 .研究发现 ,当用 wortmannin特异性抑制 PI3K活性后可以显著降低 PKB的蛋白含量 .提示PI3K对维持 PKB的基础蛋白水平是必需的 .但是 TNF- α本身对 PKB的蛋白水平无明显影响 .而当用不同浓度的 TNF- α和 wortmannin处理 SMMC- 772 1细胞时 ,发现 PKB的蛋白含量随着TNF-α浓度的增加而降低 .提示 TNF-α可能除了通过 PI3K外 ,还可能通过另一条途径来下调PKB的表达 .而当用 FAK反义质粒转染 SMMC- 772 1细胞后 (FAK下降了 60 % ) ,发现在当用不同浓度的 TNF- α处理的情况下 ,FAK反义质粒转染株 AS- 772 1细胞的 PKB含量降低为对照的70 % ;而在用 TNF-α和 wortmannin处理的情况下 ,下降为对照的 40 %～ 60 % .TNF-α能够通过PI3K及另一未知途径来影响 PKB的蛋白水平 .而 FAK在 TNF- α作用下能够不通过 PI3K来影响PKB的蛋白水平 .     

Identification of WNK1 as a substrate of Akt/protein kinase B and a negative regulator of insulin-stimulated mitogenesis in 3T3-L1 cells

Insulin signaling through protein kinase Akt/protein kinase B (PKB), a downstream element of the phosphatidylinositol 3-kinase (PI3K) pathway, regulates diverse cellular functions including metabolic pathways, apoptosis, mitogenesis, and membrane trafficking. To identify Akt/PKB substrates that mediate these effects, we used antibodies that recognize phosphopeptide sites containing the Akt/PKB substrate motif (RXRXX(p)S/T) to immunoprecipitate proteins from insulin-stimulated adipocytes. Tryptic peptides from a 250-kDa immunoprecipitated protein were identified as the protein kinase WNK1 (with no lysine) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, consistent with a recent report that WNK1 is phosphorylated on Thr60 in response to insulin-like growth factor I. Insulin treatment of 3T3-L1 adipocytes stimulated WNK1 phosphorylation, as detected by immunoprecipitation with antibody against WNK1 followed by immunoblotting with the anti-phosphoAkt substrate antibody. WNK1 phosphorylation induced by insulin was unaffected by rapamycin, an inhibitor of p70 S6 kinase pathway but abolished by the PI3K inhibitor wortmannin. RNA interference-directed depletion of Akt1/PKB alpha and Akt2/PKB beta attenuated insulin-stimulated WNK1 phosphorylation, but depletion of protein kinase C lambda did not. Whereas small interfering RNA-induced loss of WNK1 protein did not significantly affect insulin-stimulated glucose transport in 3T3-L1 adipocytes, it significantly enhanced insulin-stimulated thymidine incorporation by about 2-fold. Furthermore, depletion of WNK1 promoted serum-stimulated cell proliferation of 3T3-L1 preadipocytes, as evidenced by a 36% increase in cell number after 48 h in culture. These data suggest that WNK1 is a physiologically relevant target of insulin signaling through PI3K and Akt/PKB and functions as a negative regulator of insulin-stimulated mitogenesis.