Tyrosine Phosphorylation Of P130 Research Articles

Illuminating DEPDC1B in Multi-pronged Regulation of Tumor Progression.

DEPDC1B (aliases BRCC3, XTP8, XTP1) is a DEP (Dishevelled, Egl-1, Pleckstrin) and Rho-GAP-like domains containing predominately membrane-associated protein. Earlier, we and others have reported that DEPDC1B is a downstream effector of Raf-1 and long noncoding RNA lncNB1, and an upstream positive effector of pERK. Consistently, DEPDC1B knockdown is associated with downregulation of ligand-stimulated pERK expression. We demonstrate here that DEPDC1B N-terminus binds to the p85 subunit of PI3K, and DEPDC1B overexpression results in decreased ligand-stimulated tyrosine phosphorylation of p85 and downregulation of pAKT1. Collectively, we propose that DEPDC1B is a novel cross-regulator of AKT1 and ERK, two of the prominent pathways of tumor progression. Our data showing high levels of DEPDC1B mRNA and protein during the G2/M phase have significant implications in cell entry into mitosis. Indeed, DEPDC1B accumulation during the G2/M phase has been associated with disassembly of focal adhesions and cell de-adhesion, referred to as a DEPDC1B-mediated de-adhesion mitotic checkpoint. DEPDC1B is a direct target of transcription factor SOX10, and SOX10-DEPDC1B-SCUBE3 axis has been associated with angiogenesis and metastasis. The Scansite analysis of the DEPDC1B amino acid sequence shows binding motifs for three well-established cancer therapeutic targets CDK1, DNA-PK, and aurora kinase A/B. These interactions and functionalities, if validated, may further implicate DEPDC1B in regulation of DNA damage-repair and cell cycle progression processes. Finally, a survey of the publicly available datasets indicates that high DEPDC1B expression is a viable biomarker in breast, lung, pancreatic and renal cell carcinomas, and melanoma. Currently, the systems and integrative biology of DEPDC1B is far from comprehensive. Future investigations are necessary in order to understand how DEPDC1B might impact AKT, ERK, and other networks, albeit in a context-dependent manner, and influence the actionable molecular, spatial, and temporal vulnerabilities within these networks in cancer cells.

Conjugation of SUMO to p85 leads to a novel mechanism of PI3K regulation.

Class IA phosphatidylinositol 3-kinases (PI3Ks) are composed of p110 catalytic and p85 regulatory subunits. How regulatory subunits modulate PI3K activity remains only partially understood. Here we identified SUMO (small ubiquitin-related modifier) as a new player modulating this regulation. We demonstrate that both p85β and p85α are conjugated to SUMO1 and SUMO2. We identified two lysine residues located at the inter-SH2 domain on p85β, a critical region required for inhibition of p110, as being required for SUMO conjugation. A SUMOylation-defective mutant p85β shows higher activation of the PI3K pathway, and increased cell migration and transformation. Moreover, the cancer-related KS459del mutant in p85α was less efficiently SUMOylated compared with the wild-type protein. Finally, our results show that SUMO modulates p85 tyrosine phosphorylation, a modification correlating with PI3K pathway activation. Thus, SUMO reduces the levels of tyrosine-phosphorylated-p85 while loss of SUMOylation results in increased tyrosine phosphorylation of p85. In summary, we identify SUMO as a new important player in the regulation of the PI3K pathway through modulation of p85.

Pro-Tumorigenic Phosphorylation of p120 Catenin in Renal and Breast Cancer.

Altered protein expression and phosphorylation are common events during malignant transformation. These perturbations have been widely explored in the context of E-cadherin cell-cell adhesion complexes, which are central in the maintenance of the normal epithelial phenotype. A major component of these complexes is p120 catenin (p120), which binds and stabilizes E-cadherin to promote its adhesive and tumor suppressing function. However, p120 is also an essential mediator of pro-tumorigenic signals driven by oncogenes, such as Src, and can be phosphorylated at multiple sites. Although alterations in p120 expression have been extensively studied by immunohistochemistry (IHC) in the context of tumor progression, little is known about the status and role of p120 phosphorylation in cancer. Here we show that tyrosine and threonine phosphorylation of p120 in two sites, Y228 and T916, is elevated in renal and breast tumor tissue samples. We also show that tyrosine phosphorylation of p120 at its N-terminus, including at the Y228 site is required for its pro-tumorigenic potential. In contrast, phosphorylation of p120 at T916 does not affect this p120 function. However, phosphorylation of p120 at T916 interferes with epitope recognition of the most commonly used p120 antibody, namely pp120. As a result, this antibody selectively underrepresents p120 levels in tumor tissues, where p120 is phosphorylated. Overall, our data support a role of p120 phosphorylation as a marker and mediator of tumor transformation. Importantly, they also argue that the level and localization of p120 in human cancer tissues immunostained with pp120 needs to be re-evaluated.

A Switch of G Protein-Coupled Receptor Binding Preference from Phosphoinositide 3-Kinase (PI3K)–p85 to Filamin A Negatively Controls the PI3K Pathway

Frequent oncogenic alterations occur in the phosphoinositide 3-kinase (PI3K) pathway, urging identification of novel negative controls. We previously reported an original mechanism for restraining PI3K activity, controlled by the somatostatin G protein-coupled receptor (GPCR) sst2 and involving a ligand-regulated interaction between sst2 with the PI3K regulatory p85 subunit. We here identify the scaffolding protein filamin A (FLNA) as a critical player regulating the dynamic of this complex. A preexisting sst2-p85 complex, which was shown to account for a significant basal PI3K activity in the absence of ligand, is disrupted upon sst2 activation. FLNA was here identified as a competitor of p85 for direct binding to two juxtaposed sites on sst2. Switching of GPCR binding preference from p85 toward FLNA is determined by changes in the tyrosine phosphorylation of p85- and FLNA-binding sites on sst2 upon activation. It results in the disruption of the sst2-p85 complex and the subsequent inhibition of PI3K. Knocking down FLNA expression, or abrogating FLNA recruitment to sst2, reversed the inhibition of PI3K and of tumor growth induced by sst2. Importantly, we report that this FLNA inhibitory control on PI3K can be generalized to another GPCR, the mu opioid receptor, thereby providing an unprecedented mechanism underlying GPCR-negative control on PI3K.

Evidence that phosphatidylinositol 3-kinase is involved in sperm-induced tyrosine kinase signaling in Xenopus egg fertilization

BackgroundStudies have examined the function of PI 3-kinase in the early developmental processes that operate in oocytes or early embryos of various species. However, the roles of egg-associated PI 3-kinase and Akt, especially in signal transduction at fertilization, are not well understood.ResultsHere we show that in Xenopus eggs, a potent inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), LY294002 inhibits sperm-induced activation of the tyrosine kinase Src and a transient increase in the intracellular concentration of Ca2+ at fertilization. LY294002 also inhibits sperm-induced dephosphorylation of mitogen-activated protein kinase, breakdown of cyclin B2 and Mos, and first embryonic cleavage, all of which are events of Ca2+-dependent egg activation. In fertilized eggs, an 85-kDa subunit of PI 3-kinase (p85) undergoes a transient translocation to the low-density, detergent-insoluble membranes (membrane microdomains) where Src tyrosine kinase signaling is operating. However, the tyrosine phosphorylation of p85 in fertilized eggs is not as evident as that in H2O2-activated eggs, arguing against the possibility that PI 3-kinase is activated by Src phosphorylation. Nevertheless, sperm-induced activation of PI 3-kinase has been demonstrated by the finding that Akt, a serine/threonine-specific protein kinase, is phosphorylated at threonine-308. The threonine-phosphorylated Akt also localizes to the membrane microdomains of fertilized eggs. Application of bp(V), an inhibitor of PTEN that dephosphorylates PIP3, the enzymatic product of PI 3-kinase, promotes parthenogenetic activation of Xenopus eggs. In vitro kinase assays demonstrate that PIP3 activates Src in a dose-dependent manner.ConclusionsThese results suggest that PI 3-kinase is involved in sperm-induced egg activation via production of PIP3 that would act as a positive regulator of the Src signaling pathway in Xenopus fertilization.

Cross-linking of FcɛRI causes Ca 2+ mobilization via a sphingosine kinase pathway in a clathrin-dependent manner

Clathrin-coated pits are now recognized to be involved in cell signaling in addition to receptor down-regulation. Here we tried to identify signaling pathways that might be dependent on clathrin. Our initial data with pharmacological inhibitors of formation of clathrin-coated pits or lipid-rafts indicated that Ca 2+ response evoked by cross-linking of the high affinity receptors for IgE (FcɛRI) was dependent on clathrin. To confirm this finding, we created clathrin-knockdown cells by transfecting the mast cell line RBL-2H3 with a shRNA-clathrin heavy chain construct. In these cells, the FcɛRI-mediated Ca 2+ response was almost completely abolished, which was accompanied by the inhibition of sphingosine 1-phosphate (S1P) production with no changes in inositol 1,4,5-trisphosphate (IP 3) production. This suggests that the Ca 2+ signaling pathway via a sphingosine kinase (SK) is dependent on clathrin. Furthermore, antigen-induced tyrosine phosphorylation of p85 and p110 subunits of PI3K was almost completely inhibited in clathrin-knockdown cells. In contrast, antigen-induced tyrosine phosphorylation of phospholipase Cγ was not affected by clathrin-knockdown and tyrosine phosphorylation of Syk and degranulation were partially inhibited in clathrin-knockdown cells. The present study identifies the SK/Ca 2+ pathway to be dependent on clathrin.

Control of T Lymphocyte Signaling by Ly108, a Signaling Lymphocytic Activation Molecule Family Receptor Implicated in Autoimmunity

The signaling lymphocytic activation molecule family of receptors has been implicated in the pathophysiology of autoimmunity in humans and mice. One member of the family, Ly108, was strongly linked to lupus susceptibility in mice. High expression of a Ly108 isoform, Ly108-1, was observed in lymphocytes of lupus-prone mice. Herein, we examined the molecular basis for the influence of Ly108 on lupus susceptibility by studying Ly108 signal transduction in T cells. We observed that Ly108 was able to mediate a tyrosine phosphorylation signal implicating Ly108, Vav-1, and c-Cbl in a manner strictly dependent on engagement of the extracellular domain of Ly108 and co-expression of the Src homology 2 (SH2) domain-containing adaptor signaling lymphocytic activation molecule (SLAM)-associated protein (SAP). Evaluation of T cells from mice carrying mutations in the SAP-FynT pathway indicated that Ly108-triggered protein tyrosine phosphorylation was due to the capacity of SAP to recruit FynT. Importantly, Ly108-1 was more apt at triggering tyrosine phosphorylation signals in T cells when compared with the predominant Ly108 isoform found in non-lupus-prone mice, Ly108-2. This difference was due in part to the presence in Ly108-1 of a unique intra-cytoplasmic tyrosine-based motif that promoted Ly108 signal transduction. Together these data provided a molecular explanation for the involvement of Ly108 in lupus susceptibility in mice.

An RGS for PI3K

Immunoglobulin E (IgE) triggers the activation of the FcεRI immune receptor, which signals through immunoreceptor-based tyrosine activation motifs, in response to allergens. FcεRI activation on mast cells produces an increase in intracellular calcium concentration, which causes degranulation and the release of inflammatory mediators such as histamine. The signaling pathway from the FcεRI to calcium involves the tyrosine kinases Lyn and Syk, the adaptor proteins Gab2 and Grb2, phosphoinositide 3-kinase (PI3K), and phospholipase C-mediated production of inositol 1,4,5-trisphosphate (IP 3 ). G protein-coupled receptors, such as adenosine or prostaglandin receptors, may amplify IgE-mediated mast cell activation. Bansal et al . found that RGS13, a member of the R4 subfamily of regulator of G protein signaling (RGS) proteins, provided an inhibitory input into IgE-mediated mast cell degranulation through a mechanism involving the p85 subunit of PI3K instead of a G protein. RGS proteins are best known for attenuating G protein signaling through their actions as GTPase-activating proteins (GAPs) for specific Gα subunits and for interfering with the interactions between Gα subunits and downstream effectors. Bansal et al . studied bone marrow-derived mast cells (BMMCs) from Rgs13 knockout ( Rgs13 -KO) mice and found that antigen-elicited degranulation was greater and inhibition of G proteins with pertussis toxin did not alter this enhanced response, suggesting that G proteins were not mediating the effect. Furthermore, reconstitution of the Rgs13 -deficient BMMCs with mutant forms of RGS13 that could not interact with G proteins prevented the enhanced degranulation response. The Rgs13 -KO mice also exhibited elevated cutaneous allergic response compared with wild-type mice, yet did not show altered mast cell numbers or morphology. Using glutathione S -transferase fused to RGS13, the p85 subunit of PI3K was identified from lysates of BMMCs as a tyrosine-phosphorylated protein that interacted with RGS13. The interaction between RGS13 and p85 was direct and occurred with recombinant proteins, although it did require p85 tyrosine phosphorylation (achieved by incubation with Lyn). Rgs13 -deficient BMMCs showed increased production of IP 3 , phosphorylation of Akt, and increased intracellular calcium concentrations in response to antigen. Transduction of wild-type BMMCs with a cell-permeable RGS13 construct inhibited antigen-stimulated calcium flux and PI3K activity. Immunoprecipitation experiments with BMMCS transduced with the cell-permeable RGS13 indicated that RGS13 interfered with the recruitment of p85 to Gab2 of the FcεRI signaling complex. G. Bansal, Z. Xie, S. Rao, K. H. Nocka, K. M. Druey, Suppression of immunoglobulin E-mediated allergic responses by regulator of G protein signaling 13. Nat. Immunol. 9 , 73-80 (2008). [PubMed]

Differential regulation of class IA phosphoinositide 3-kinase catalytic subunits p110α and β by protease-activated receptor 2 and β-arrestins

PAR-2 (protease-activated receptor 2) is a GPCR (G-protein-coupled receptor) that can elicit both G-protein-dependent and -independent signals. We have shown previously that PAR-2 simultaneously promotes Galphaq/Ca2+-dependent activation and beta-arrestin-1-dependent inhibition of class IA PI3K (phosphoinositide 3-kinase), and we sought to characterize further the role of beta-arrestins in the regulation of PI3K activity. Whereas the ability of beta-arrestin-1 to inhibit p110alpha (PI3K catalytic subunit alpha) has been demonstrated, the role of beta-arrestin-2 in PI3K regulation and possible differences in the regulation of the two catalytic subunits (p110alpha and p110beta) associated with p85alpha (PI3K regulatory subunit) have not been examined. In the present study we have demonstrated that: (i) PAR-2 increases p110alpha- and p110beta-associated lipid kinase activities, and both p110alpha and p110beta are inhibited by over-expression of either beta-arrestin-1 or -2; (ii) both beta-arrestin-1 and -2 directly inhibit the p110alpha catalytic subunit in vitro, whereas only beta-arrestin-2 directly inhibited p110beta; (iii) examination of upstream pathways revealed that PAR-2-induced PI3K activity required the small GTPase Cdc (cell-division cycle)42, but not tyrosine phosphorylation of p85; and (iv) beta-arrestins inhibit PAR-2-induced Cdc42 activation. Taken together, these results indicated that beta-arrestins could inhibit PAR-2-stimulated PI3K activity, both directly and through interference with upstream pathways, and that the two beta-arrestins differ in their ability to inhibit the p110alpha and p110beta catalytic subunits. These results are particularly important in light of the growing interest in PAR-2 as a pharmacological target, as commonly used biochemical assays that monitor G-protein coupling would not screen for beta-arrestin-dependent signalling events.

Tyrosine phosphorylation of a 38‐kDa capacitation‐associated buffalo (Bubalus bubalis) sperm protein is induced by L‐arginine and regulated through a cAMP/PKA‐independent pathway

The aim of the present study was to determine the effect of L-arginine on nitric oxide (NO*) synthesis, capacitation and protein tyrosine phosphorylation in buffalo spermatozoa. Ejaculated buffalo spermatozoa were capacitated in the absence or presence of heparin, or L-arginine or N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS) for 6 h. Capacitating spermatozoa generated NO* both spontaneously and following stimulation with L-arginine and L-NAME quenched such L-arginine-induced NO* production. Immunolocalization of NOS suggested for existence of constitutive NOS in buffalo spermatozoa. L-Arginine (10 mm) was found to be a potent capacitating agent and addition of L-NAME to the incubation media attenuated both L-arginine and heparin-induced capacitation and suggested that NO* is involved in the capacitation of buffalo spermatozoa. Two sperm proteins of M(r) 38 000 (p38) and 20 000 (p20) were tyrosine phosphorylated extensively by both heparin and L-arginine. Of these, the tyrosine phosphorylation of p38 was insensitive to both induction by cAMP agonists as well as inhibition by a protein kinase A (PKA) inhibitor. Further, most of these L-arginine-induced tyrosine phosphorylated proteins were localized to the midpiece and principal piece regions of flagellum of capacitated spermatozoa and suggested that sperm flagellum takes active part during capacitation. These results indicated that L-arginine induces capacitation of buffalo spermatozoa through NO* synthesis and tyrosine phosphorylation of specific sperm proteins involving a pathway independent of cAMP/PKA.

P120-catenin is required for the collective invasion of squamous cell carcinoma cells via a phosphorylation-independent mechanism

Loss of E-cadherin-mediated cell-cell junctions has been correlated with cancer cell invasion and poor patient survival. p120-catenin has emerged as a key player in promoting E-cadherin stability and adherens junction integrity and has been proposed as a potential invasion suppressor by preventing release of cells from the constraints imposed by cadherin-mediated cell-cell adhesion. However, it has been proposed that tyrosine phosphorylation of p120 may contribute to cadherin-dependent junction disassembly during invasion. Here, we use small interfering RNA (siRNA) in A431 cells to show that knockdown of p120 promotes two-dimensional migration of cells. In contrast, p120 knockdown impairs epidermal growth factor-induced A431 invasion into three-dimensional matrix gels or in organotypic culture, whereas re-expression of siRNA-resistant p120, or a p120 isoform that cannot be phosphorylated on tyrosine, restores the collective mode of invasion employed by A431 cells in vitro. Thus, p120 promotes A431 cell invasion in a phosphorylation-independent manner. We show that the collective invasion of A431 cells depends on the presence of cadherin-mediated (P- and E-cadherin) cell-cell contacts, which are lost in cells where p120 expression is knocked down. Furthermore, membranous p120 is maintained in invasive squamous cell carcinomas in tumours suggesting that p120 may be important for the collective invasion of tumours cells in vivo.

Role of protein phosphatase 2A in calcium-dependent chloride secretion by human colonic epithelial cells

EGF inhibits carbachol-induced chloride secretion by regulating a basolateral potassium channel via phosphatidylinositol 3-kinase (PI 3-kinase) and PKCepsilon activation. Although both EGF and carbachol cause tyrosine phosphorylation of p85 of PI 3-kinase, only EGF activates the enzyme. Serine phosphorylation of p85 is thought to suppress the lipid kinase of PI 3-kinase. Our present study examined whether the differential effects of carbachol and EGF on PI 3-kinase activity correspond to varying phosphorylation of p85, and the mechanisms and consequences. T(84) colonic epithelial cells were treated with either EGF or carbachol. Cell lysates were immunoprecipitated with p85 antibody and blotted with either phosphotyrosine or phosphoserine antibodies. Protein phosphatase (PP) 1 and 2A activities were also measured. Both tyrosine and serine residues of p85 were phosphorylated by carbachol, whereas EGF induced only tyrosine phosphorylation. Moreover, EGF abolished carbachol-induced serine phosphorylation of p85 and activated PP2A without affecting PP1. Carbachol did not affect either phosphatase. Calyculin A or okadaic acid pretreatment reversed the inhibitory action of EGF on carbachol-induced chloride secretion and restored serine phosphorylation of p85. Although carbachol recruits p85, it phosphorylates both serine and tyrosine residues so that the lipid kinase of PI 3-kinase is inhibited. EGF results in p85 tyrosine phosphorylation as well as dephosphorylation of serine residues via the activation of PP2A. This explains the differential induction of PI 3-kinase enzyme activity in response to EGF and/or carbachol and has functional implications. Our data provide further insights into negative signals that regulate chloride secretion and into the molecular basis of signaling diversification in the intestinal epithelium.

Acidosis-induced protein tyrosine phosphorylation depends on Ca 2+ influx via voltage-dependent Ca 2+ channels in SHR aorta

The contractile response to acidosis in isolated aorta from spontaneously hypertensive rat (SHR) depends upon tyrosine phosphorylation of phosphatidylinositol 3 kinase (PI3-kinase) and Ca 2+ influx via voltage-dependent Ca 2+ channels (VDCC). In this study, verapamil, a VDCC inhibitor, was shown to markedly inhibit acidic pH-induced contraction, whereas the residual contraction in the presence of verapamil was unaffected by the PI3-kinase inhibitor, 2-(4-morpholinyl)-8-phenyl-4 H-1-benzopyran-4-one hydrochloride (LY-294002). Interestingly, the LY-294002-insensitive component of contraction was further inhibited by verapamil in the presence of LY-294002. Western blotting revealed that acidosis stimulated tyrosine phosphorylation of p85, which was abolished when tissues were pretreated with tyrphostin 23, a tyrosine kinase inhibitor, verapamil or EGTA. In fura-2-loaded aortic strips, acidosis induced a rise in intracellular Ca 2+ ([Ca 2+] i) that was partially inhibited by LY-294002. The residual increase in [Ca 2+] i caused by acidosis in the presence of LY-294002 was abolished by verapamil. These findings suggest that acidosis-induced Ca 2+ influx through VDCC is the upstream event leading to the tyrosine phosphorylation of PI3-kinase, which in turn contributes to the enhancement of Ca 2+ entry to some extent in SHR aorta.

Hypothalamic PI3K and MAPK differentially mediate regional sympathetic activation to insulin.

The action of insulin in the central nervous system produces sympathetic nervous system activation (also called sympathoactivation), although the neuronal intracellular mechanisms that mediate this are unclear. We hypothesized that PI3K and MAPK, the major pathways involved in insulin receptor signaling, mediate sympathetic nerve responses to insulin. Intracerebroventricular administration of insulin in rat increased multifiber sympathetic nerve activity to the hindlimb, brown adipose tissue (BAT), adrenal gland, and kidney. Ex vivo biochemical studies of mediobasal hypothalamic tissue revealed that insulin stimulated the association of insulin receptor substrate-1 with the p85alpha subunit of PI3K and also tyrosine phosphorylation of p42 and p44 subunits of MAPK in the hypothalamus. In order to determine whether PI3K and/or MAPK were involved in insulin-mediated sympathoactivation, we tested the effect of specific inhibitors of PI3K (LY294002 and wortmannin) and MAPK (PD98059 and U0126) on regional sympathetic responses to insulin. Interestingly, regional sympathoactivation to insulin was differentially affected by blockade of PI3K and MAPK. Inhibition of PI3K specifically blocked insulin-induced sympathoactivation to the hindlimb, while inhibition of MAPK specifically blocked insulin-induced sympathoactivation to BAT. Sympathoactivation to corticotrophin-releasing factor, however, was not affected by inhibition of PI3K and MAPK. These data demonstrate that PI3K and MAPK are specific and regionally selective mediators of the action of insulin on the sympathetic nervous system.

Hypothalamic PI3K and MAPK differentially mediate regional sympathetic activation to insulin

The action of insulin in the central nervous system produces sympathetic nervous system activation (also called sympathoactivation), although the neuronal intracellular mechanisms that mediate this are unclear. We hypothesized that PI3K and MAPK, the major pathways involved in insulin receptor signaling, mediate sympathetic nerve responses to insulin. Intracerebroventricular administration of insulin in rat increased multifiber sympathetic nerve activity to the hindlimb, brown adipose tissue (BAT), adrenal gland, and kidney. Ex vivo biochemical studies of mediobasal hypothalamic tissue revealed that insulin stimulated the association of insulin receptor substrate–1 with the p85α subunit of PI3K and also tyrosine phosphorylation of p42 and p44 subunits of MAPK in the hypothalamus. In order to determine whether PI3K and/or MAPK were involved in insulin-mediated sympathoactivation, we tested the effect of specific inhibitors of PI3K (LY294002 and wortmannin) and MAPK (PD98059 and U0126) on regional sympathetic responses to insulin. Interestingly, regional sympathoactivation to insulin was differentially affected by blockade of PI3K and MAPK. Inhibition of PI3K specifically blocked insulin-induced sympathoactivation to the hindlimb, while inhibition of MAPK specifically blocked insulin-induced sympathoactivation to BAT. Sympathoactivation to corticotrophin-releasing factor, however, was not affected by inhibition of PI3K and MAPK. These data demonstrate that PI3K and MAPK are specific and regionally selective mediators of the action of insulin on the sympathetic nervous system.

Endoglin Controls Cell Migration and Composition of Focal Adhesions: FUNCTION OF THE CYTOSOLIC DOMAIN

Mutations in the human endoglin gene result in hereditary hemorrhagic telangiectasia type 1, a vascular disorder characterized by multisystemic vascular dysplasia, arteriovenous malformations, and focal dilatation of postcapillary venules. Previous studies have implicated endoglin in the inhibition of cell migration in vivo and in vitro. In the course of studies to address the relationship of the conserved cytosolic domain to endoglin function, we identified zyxin, a LIM domain protein that is concentrated at focal adhesions, as an interactor with endoglin in human umbilical vein vascular endothelial cells. This interaction is localized within the 47-amino acid carboxyl-terminal cytosolic domain of endoglin, and maps within zyxin residues 326-572. The endoglin-zyxin interaction was found to be largely mediated by the third LIM domain of zyxin, and is specific for endoglin because the homologous cytosolic domain of the transforming growth factor-beta type III receptor, betaglycan, fails to interact with zyxin. Expression of endoglin is associated with reduction of zyxin, as well as its interacting proteins p130(cas) and CrkII, from a focal adhesion protein fraction, and this reduction is correlated with inhibition of cell migration. We also show that endoglin-dependent: (i) inhibition of cell migration, (ii) reduction of focal adhesion-associated p130(cas)/CrkII protein levels, (iii) tyrosine phosphorylation of p130(cas), and (iv) focal adhesion-associated endoglin levels are mediated by the cytosolic domain of endoglin. These results suggest a novel mechanism of endoglin function involving its interaction with LIM domain-containing proteins, and associated adapter proteins, affecting sites of focal adhesion.

Interactive effects of insulin-like growth factor-1 and β-estradiol on endothelial nitric oxide synthase activity in rat aortic endothelial cells

Insulin-like growth factor-1 (IGF-1) and β-estradiol (E2) have vasodilatory effects, in part, through stimulation of vascular nitric oxide (NO) production. However, their interactive effects on endothelial nitric oxide synthase (eNOS) and NO production have not been previously studied in endothelial cells (EC). Employing rat aortic EC (RAEC), the effects of acute (20 and 30 minutes) and prolonged (4 hours) stimulation with 100 nmol/L IGF-1 and 1 nmol/L E2 (alone or in combination) were assessed with respect to protein levels and enzymatic activities for phosphatidyl inositol 3-kinase (PI3K) and serine/threonine kinase Akt (Akt), enzymes involved in eNOS activation. Exposure to IGF-1 for 30 minutes or E2 for 20 minutes increased insulin receptor substrate-1 (IRS-1) association with the regulatory (p85) subunit of PI3K, enhanced tyrosine phosphorylation of p85, and increased PI3K activity. Combined treatment had a greater effect on p85 phosphorylation and PI3K activity then either agonist alone. Moreover, IGF-1 and E2 enhanced Akt Ser473 phosphorylation, with the effect of IGF-1 being much greater. Acute expose to both E2 (20 minutes) and IGF-1 (30 minutes) were associated with an increase in eNOS activity. Prolonged exposure (4 hours) to either IGF-1 or E2 increased expression of the p85 subunit as well as eNOS activity. Pretreatment with PI3K antagonist wortmannin (WT) prevented this increase in eNOS activity. The results suggest that IGF-1 and E2 may interact through PI3K/Akt-related pathways to increase eNOS activity. Copyright 2003 Elsevier, Inc. All rights.

IL-1 regulates cytoskeletal organization in osteoclasts via TNF receptor-associated factor 6/c-Src complex.

Targeted disruption of either c-Src or TNFR-associated factor 6 (TRAF6) in mice causes osteoclast dysfunction and an osteopetrotic phenotype, suggesting that both molecules play important roles in osteoclastic bone resorption. We previously demonstrated that IL-1 induces actin ring formation and osteoclast activation. In this study, we examined the relationship between IL-1/TRAF6-dependent and c-Src-mediated pathways in the activation of osteoclast-like cells (prefusion cells (pOCs); multinucleated cells) formed in the murine coculture system. In normal pOCs, IL-1 induces actin ring formation and tyrosine phosphorylation of p130(Cas), a known substrate of c-Src. However, in Src-deficient pOCs, p130(Cas) was not tyrosine phosphorylated following IL-1 treatment. In normal pOCs treated with IL-1, anti-TRAF6 Abs coprecipitate p130(Cas), protein tyrosine kinase 2, and c-Src. In Src-deficient pOCs, this molecular complex was not detected, suggesting that c-Src is required for formation of the TRAF6, p130(Cas), and protein tyrosine kinase 2 complex. Moreover, an immunocytochemical analysis revealed that in osteoclast-like multinucleated cells, IL-1 induced redistribution of TRAF6 to actin ring structures formed at the cell periphery, where TRAF6 also colocalized with c-Src. Taken together, these data suggest that IL-1 signals feed into the tyrosine kinase pathways through a TRAF6-Src molecular complex, which regulates the cytoskeletal reorganization essential for osteoclast activation.

Anchorage-independent phosphorylation of p130(Cas) protects lung adenocarcinoma cells from anoikis.

The regulation and function of the signaling adaptor protein p130(Cas) in tumor cell anchorage-independent survival, or anoikis resistance, were investigated in human lung adenocarcinoma cells. The tyrosine phosphorylation and function of p130(Cas) during cell detachment were analyzed in tumor cells and compared with that of normal epithelial cells. Cell detachment trigged rapid dephosphorylation of p130(Cas) in the nontumorigenic and anoikis-sensitive normal epithelial cells, but had no effect on the tyrosine phosphorylation of p130(Cas) in the anoikis-resistant lung adenocarcinoma cells. Further analysis revealed that the total tyrosine kinase activities associated with p130(Cas) in the lung tumor cells are anchorage-independent and are significantly higher than that in the normal cells, in which the p130(Cas)-associated tyrosine kinase activities are anchorage-dependent. Analysis of two known p130(Cas)-associated tyrosine kinases FAK and Src indicated that the regulation of tyrosine phosphorylation of FAK and Src are altered in the tumor cells. Inhibition of Src specifically abolished phosphorylation of p130(Cas) and induced anoikis. Furthermore, overexpression of dominant-negative forms of p130(Cas) also induced apoptosis. Taken together, these data suggest that p130(Cas) mediates a cell survival signal from cell-matrix interaction. Alterations in tumor cells that lead to constitutive phosphorylation of p130(Cas) can prevent cells from anoikis, hence contribute to tumor cell anchorage independence and metastasis.

EDTA inhibits collagen-induced ATP+ADP secretion and tyrosine phosphorylation in platelets independently of Mg 2+ chelation and decrease in pH

The effects of Mg 2+ and EDTA on collagen-induced platelet dense granule secretion and protein tyrosine phosphorylation were studied in the presence and absence of various inhibitors of autocrine agonists that are released from secretory granules. Addition of EDTA to gel-filtered platelets in Tyrode's solution caused a decrease in pH of 0.7-0.9 pH units, due to chelation of Mg 2+ , and a marked inhibition of cATP+ADP secretion induced by collagen (25 w g/ml). Lowering pH of the platelet suspension to the same extent by HCl had no effect on secretion which also was the same in the absence of exogenous Mg 2+ as in its presence. Similarly, secretion induced by collagen per se , i.e., isolated from autocrine agonists by inhibitors, which was about 50% less than with autocrine agonists, was not affected by the presence or absence of exogenous Mg 2+ . The level of tyrosine phosphorylation of several proteins in resting platelets was higher in the absence of Mg 2+ than in its presence, but the onset of collagen-induced phosphorylation and dephosphorylation was more rapid in the absence of Mg 2+ . Tyrosine phosphorylation of p38 was specifically enhanced in the presence of inhibitors of autocrine phosphorylation and even more enhanced in the absence of Mg 2+ . EDTA inhibited the protein phosphorylation to the same extent in the presence as in the absence of exogenous Mg 2+ . These results show that EDTA inhibits collagen-induced dense granule secretion neither through chelation of Mg 2+ nor lowering of pH, and thus probably through other effects on the platelet by the EDTA2- species that are reflected in protein tyrosine phosphorylation signalling.