Progesterone receptor activation of extranuclear signaling pathways in regulating p53 expression in vascular endothelial cells

We previously showed that progesterone (P4) inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) through a p53-dependent pathway. Now we investigated further the molecular mechanism underlying the hormone activity. In cultured HUVECs, P4 increased the protein levels of phosphorylated Src (p-Src), Raf-1, and ERK. The levels of p-Src and p-Src-progesterone receptor complex in HUVECs were increased by P4 treatment. These effects were blocked by pretreatment with a progesterone receptor antagonist, RU486. The P4-induced increase in p53 transactivity was abolished by pretreatment with Src kinase inhibitors. Moreover, administration with cSrc antisense oligonucleotide prevented the P4-induced increases of the levels of p53 mRNA and protein. These data suggest that P4-induced up-regulation of p53 might be mediated through activation of cSrc. Pretreatment with Src kinase inhibitors also prevented P4-induced membrane translocation of Kras and increases of the protein levels of phosphorylated Raf and phosphorylated ERK. Transfection with dominant-negative ERK2 prevented the P4-induced increases of protein level and promoter activity of p53 and a decrease of thymidine incorporation. P4 also increased nuclear factor-κB (NF-κB) nuclear translocation and NF-κB binding onto the p53 promoter. These effects were abolished by pretreatment with ERK inhibitors. The P4-induced up-regulation of the p53 promoter activity was prevented by preadministration with dominant-negative ERK2 or NF-κB inhibitors. Taken together, our data suggest that the cSrc/Kras/Raf-1/ERK2/NF-κB signaling pathway contributes to the P4-induced up-regulation of p53 in HUVECs. These findings highlight progesterone receptor activation of extranuclear signaling pathways in regulating p53 and cell cycle progression in HUVECs.     

Aspirin and salicylates inhibit the IL-4- and IL-13-induced activation of STAT6.

Allergic diseases, including asthma, represent a major threat to human health. Over the three last decades, their incidence has risen in western countries. Aspirin treatment has been shown to improve allergic diseases, especially asthma, and the decreased use of aspirin has been hypothesized to contribute to the increase in childhood asthma. Because salicylate compounds suppress a number of enzymatic activities, and signaling through IL-4R participates in the development of allergic responses, we tested the effect of salicylates on IL-4 signal transduction. We found that treatment of cell lines and primary cells with aspirin and salicylates, but not acetaminophen, inhibited the activation of STAT6 by IL-4 and IL-13. This effect correlated with the inhibition of IL-4-induced CD23 expression. Although salicylates inhibited the in vivo activation of Janus kinases, their kinase activity was not affected in vitro by salicylates, suggesting that other kinases were involved in IL-4-induced STAT6 activation. Furthermore, we found that an Src kinase was involved in STAT6 activation because 1) Src kinase activity was induced by IL-4, 2) Src kinase activity, but not Janus kinase, was inhibited by salicylates in vitro, 3) cells expressing viral Src had constitutive STAT6 phosphorylation, and 4) cells lacking Src showed low STAT6 phosphorylation in response to IL-4. Because STAT6 activation by IL-4 and IL-13 participates in the development of allergic diseases, our results provide a mechanism to explain the beneficial effects of aspirin and salicylate treatment of these diseases.     

Enhanced activity of a large conductance, calcium-sensitive K+ channel in the presence of Src tyrosine kinase

Large conductance, calcium-sensitive K(+) channels (BK(Ca) channels) contribute to the control of membrane potential in a variety of tissues, including smooth muscle, where they act as the target effector for intracellular "calcium sparks" and the endothelium-derived vasodilator nitric oxide. Various signal transduction pathways, including protein phosphorylation can regulate the activity of BK(Ca) channels, along with many other membrane ion channels. In our study, we have examined the regulation of BK(Ca) channels by the cellular Src gene product (cSrc), a soluble tyrosine kinase that has been implicated in the regulation of both voltage- and ligand-gated ion channels. Using a heterologous expression system, we observed that co-expression of murine BK(Ca) channel and the human cSrc tyrosine kinase in HEK 293 cells led to a calcium-sensitive enhancement of BK(Ca) channel activity in excised membrane patches. In contrast, co-expression with a catalytically inactive cSrc mutant produced no change in BK(Ca) channel activity, demonstrating the requirement for a functional cSrc molecule. Furthermore, we observed that BK(Ca) channels underwent direct tyrosine phosphorylation in cells co-transfected with BK(Ca) channels and active cSrc but not in cells co-transfected with the kinase inactive form of the enzyme. A single Tyr to Phe substitution in the C-terminal half of the channel largely prevented this observed phosphorylation. Given that cSrc may become activated by receptor tyrosine kinases or G-protein-coupled receptors, these findings suggest that cSrc-dependent tyrosine phosphorylation of BK(Ca) channels in situ may represent a novel regulatory mechanism for altering membrane potential and calcium entry.     

The Differential Expression of EphB2 and EphB4 Receptor Kinases in Normal Bladder and in Transitional Cell Carcinoma of the Bladder

Effective treatment of transitional cell carcinoma (TCC) of the bladder requires early diagnosis. Identifying novel molecular markers in TCC would guide the development of diagnostic and therapeutic targets. Ephrins mediate signals via tyrosine kinase activity that modulates diverse physiologic and developmental processes, and ephrins are increasingly implicated in carcinogenesis. The aim of our study was to examine the differential regulation of EphB4 and EphB2 in normal bladder and in TCC of the bladder in 40 patients undergoing radical cystectomy for curative intent. Immunostaining and Western blotting revealed that normal urothelium expresses EphB2 (20 of 24 cases, 83% of the time) not EphB4 (0 of 24 cases, 0%). In sharp contrast, TCC specimens show loss of EphB2 expression (0 of 34 cases, 0%) and gain of EphB4 expression (32 of 34, 94%). Furthermore, EphB4 signal strength statistically correlated with higher tumor stage, and trended toward the presence of carcinoma in situ (CIS). These results are confirmed by analysis of normal urothelial and tumor cell lines. EphB2 is not a survival factor in normal urothelium, while EphB4 is a survival factor in TCC. Treatment of bladder tumor xenograft with an EphB4 inhibitor sEphB4-HSA leads to 62% tumor regression and complete remission when combined with Bevacizumab. Furthermore, tissue analysis revealed that sEphB4-HSA led to increased apoptosis, decreased proliferation, and reduced vessel density, implicating direct tumor cell targeting as well as anti-angiogenesis effect. In summary loss of EphB2 and gain of EphB4 expression represents an inflection point in the development, growth and possibly progression of TCC. Therapeutic compounds targeting EphB4 have potential for diagnosing and treating TCC.     

Down-regulation of the tumor suppressor gene C-terminal Src kinase: an early event during premalignant colonic epithelial hyperproliferation

Hyperproliferation of the premalignant epithelium is critical for colonic carcinogenesis; however the mechanisms remain largely unexplored. We report herein that prior to occurrence of neoplastic lesions in the azoxymethane-rat model of colon carcinogenesis; the tumor suppressor gene C-terminal Src kinase (Csk) was down-regulated with a concomitant increase in Src activity. Furthermore, pharmacological or genetic (RNA interference) inhibition of Csk resulted in increased proliferation in colon cancer cell lines through the mitogen-activated protein kinase dependent pathway. Thus, we demonstrate, for the first time, that Csk suppression is an important early event in colorectal cancer pathogenesis.     

Src Kinase Regulation in Progressively Invasive Cancer

Metastatic progression is a multistep process that involves tumor growth and survival, motility and invasion, and subsequent proliferation in an inappropriate environment. The Src protein tyrosine kinase has been implicated in many of the biochemical pathways that drive these behaviors. Although Src itself is only rarely mutated in human tumors, its aberrant activity has been noted in various cancers and suggested to serve as a barometer of metastatic potential. With these features in mind, we examined Src kinase regulation at the structural, enzymatic, and expression levels as a function of progressively invasive prostate cancer cell lines. Surprisingly, both total Src content and kinase activity decrease with increasing cell line aggressiveness, an observation that appears to be inconsistent with the well-documented role of Src in the signaling pathways that drive growth and invasion. However, we do observe a direct correlation between Src kinase specific activity (total Src kinase activity/total Src content) and metastatic aggressiveness, possibly suggesting that in highly aggressive cell lines, key signaling enzymes are globally recruited to drive the cancerous phenotype. In addition, although the expected enhanced phosphorylation of Src at Tyr-416 (activation site) is present in the most aggressive prostate cancer cell lines, unexpectedly high phosphorylation levels at the Tyr-527 inhibitory site are observed as well. The latter, rather than representative of inhibited enzyme, is more indicative of primed Src responsive to local phosphorylated binding partners.     

Lck is involved in interleukin-2 induced proliferation but not cell survival in human T cells through a MAP kinase-independent pathway

The role of Lck in IL-2-induced proliferation and cell survival is still controversial. Here, we show that the Src family kinase inhibitor, PP1, reduced the IL-2-induced proliferation of human T cells significantly without inhibiting the anti-apoptotic effect of IL-2. As Lck is the only Src family kinase activated upon IL-2 stimulation in T cells, this indicates that Lck is involved in IL-2-induced proliferation but not survival. IL-2-induced MAP kinase activation was only slightly inhibited by PP1, suggesting that Lck is not essential for IL-2-induced MAP kinase activation in human T cells. We found that an IL-2-sensitive, human mycosis fungoides-derived tumor T cell line is Lck negative, and that the IL-2-induced MAP kinase activation is comparable to non-cancerous T cells, although a little delayed in kinetics. An Lck expressing clone was established by transfecting Lck into mycosis fungoides tumor T cells, but Lck had no influence on the delayed kinetics of MAP kinase activation, indicating that Lck is not essential for MAP kinase activation in mycosis fungoides tumor T cells or in non-cancerous T cells. Taken together, this indicates that Lck is involved in IL-2-induced proliferation, but not cell survival, through a pathway not involving MAP kinase.     

Activation of Src and transformation by an RPTPα splice mutant found in human tumours

Receptor protein tyrosine phosphatase α (RPTPα)-mediated Src activation is required for survival of tested human colon and oestrogen receptor-negative breast cancer cell lines. To explore whether mutated RPTPα participates in human carcinogenesis, we sequenced RPTPα cDNAs from five types of human tumours and found splice mutants in ～30% of colon, breast, and liver tumours. RPTPα245, a mutant expressed in all three tumour types, was studied further. Although it lacks any catalytic domain, RPTPα245 expression in the tumours correlated with Src tyrosine dephosphorylation, and its expression in rodent fibroblasts activated Src by a novel mechanism. This involved RPTPα245 binding to endogenous RPTPα (eRPTPα), which decreased eRPTPα-Grb2 binding and increased eRPTPα dephosphorylation of Src without increasing non-specific eRPTPα activity. RPTPα245-eRPTPα binding was blocked by Pro210 → Leu/Pro211 → Leu mutation, consistent with the involvement of the structural 'wedge' that contributes to eRPTPα homodimerization. RPTPα245-induced fibroblast transformation was blocked by either Src or eRPTPα RNAi, indicating that this required the dephosphorylation of Src by eRPTPα. The transformed cells were tumourigenic in nude mice, suggesting that RPTPα245-induced activation of Src in the human tumours may have contributed to carcinogenesis.     

Role of Src kinase in diperoxovanadate-mediated activation of phospholipase D in endothelial cells.

We have shown earlier that oxidant-induced activation of phospholipase D (PLD) in vascular endothelial cells (ECs) is regulated by protein tyrosine kinases. To further understand the regulation of oxidant-induced PLD activation, we investigated the role of Src kinase. Treatment of bovine pulmonary artery ECs (BPAECs) with a model oxidant, diperoxovanadate (DPV), at 5 microM concentration, for 30 min, stimulated PLD activity (four- to eightfold), which was attenuated by tyrosine kinase inhibitors and by Src kinase-specific inhibitors PP-1 and PP-2, in a dose- and time-dependent fashion. Furthermore, BPAECs exposed to DPV (5 microM) for 2 min showed activation of Src kinase as observed by increased tyrosine phosphorylation and autophosphorylation in Src immunoprecipitates, which was attenuated by PP-2. Src immunoprecipitates of cell lysates from control BPAECs exhibited PLD activity in cell-free preparations, which was Arf- and Rho-sensitive and was enhanced at 2 min of DPV (5 microM) treatment. Also, Western blots of Src immunoprecipitates of control cells revealed the presence of PLD(1) and PLD(2), suggesting the association of PLD with Src kinase under basal conditions. However, exposure of cells to DPV (5 microM) for 2 min enhanced the association of PLD(2) but not PLD(1) with Src. Western blotting of immunoprecipitates of PLD(1) and PLD(2) isoforms of control BPAECs revealed the presence of Src under basal conditions and exposure of cells to DPV (5 microM) for 2 min enhanced the association of PLD(2) with Src in PLD(2) immunoprecipitates. Transient expression of a dominant negative mutant of Src in BPAECs attenuated DPV- but not TPA-induced PLD activation. In cell-free preparations, Src did not phosphorylate either PLD(1) or PLD(2) compared to protein kinase Calpha or p38 mitogen-activated protein kinase. These data show for the first time a direct association of Src with PLD in ECs and regulation of PLD in intact cells.     

Overexpression of annexin a1 induced by terephthalic acid calculi in rat bladder cancer

Prolonged cell proliferation in response to irritation by bladder calculi can evoke malignant transformation of the urothelium. However, the molecular mechanisms responsible for calculi-associated bladder carcinogenesis are unknown. We compared the protein expression pattern of rat bladder transitional cell carcinomas (TCCs) induced by terephthalic acid with that of normal bladder tissues using 2-DE. Comparative analysis of the respective spot patterns on 2-DE showed 146 spots that were markedly changed in TCC samples. Subsequently, 56 of the variant protein spots were identified by MALDI-TOF MS. Among them, overexpression of annexin a1 (ANNA1) in rat TCCs was confirmed by Western blotting and real-time RT-PCR analysis. Immunohistochemical staining revealed that ANNA1, usually a cytoplasmic protein in normal urothelium, was translocated to the nucleus in rat bladder cancer cells. In contrast to the animal studies, examination of human clinical specimens showed that ANNA1 expression was reduced in TCC compared to normal urothelium. The expression of ANNA1 was inversely related to the level of differentiation of TCC. Our data suggest that overexpression of ANNA1 is involved in bladder carcinogenesis induced by bladder calculi and that translocation of the protein may be partly responsible for the effect. ANNA1 may serve as a new marker of differentiation for the histopathological grading of human TCC.     

Distinct functions of Cdk5(Y15) phosphorylation and Cdk5 activity in stress fiber formation and organization

Previous studies have shown that Cdk5 promotes lens epithelial cell adhesion. Here we use a cell spreading assay to investigate the mechanism of this effect. As cells spread, forming matrix adhesions and stress fibers, Cdk5(Y15) phosphorylation and Cdk5 kinase activity increased. Cdk5(Y15) phosphorylation was inhibited by PP1, a Src family kinase inhibitor. To identify the PP1-sensitive kinase, we transfected cells with siRNA oligonucleotides for cSrc and related kinases. Only cSrc siRNA oligonucleotides inhibited Cdk5(Y15) phosphorylation. Cdk5(pY15) and its activator, p35, colocalized with actin in stress fibers. To examine Cdk5 function, we inhibited Cdk5 activity under conditions that also prevent phosphorylation at Y15: expression of kinase inactive mutations Cdk5(Y15F) and Cdk5(K33T), and siRNA suppression of Cdk5. Stress fiber formation was severely inhibited. To distinguish between a requirement for Cdk5 kinase activity and a possible adaptor role for Cdk5(pY15), we used two methods that inhibit kinase activity without inhibiting phosphorylation at Y15: pharmacological inhibition with olomoucine and expression of the kinase inactive mutation, Cdk5(D144N). Stress fiber organization was altered, but stress fiber formation was not blocked. These findings indicate that Cdk5(Y15) phosphorylation and Cdk5 activity have distinct functions required for stress fiber formation and organization, respectively.     

Denbinobin suppresses breast cancer metastasis through the inhibition of Src-mediated signaling pathways

Denbinobin (5-hydroxy-3,7-dimethoxy- 1,4-phenanthraquinone), a biologically active chemical isolated from Ephemerantha lonchophylla, has been demonstrated to display anti-cancer activity. Breast cancer is the leading cause of female mortality, and the high mortality is mainly attributable to metastasis. Src kinase activity is elevated in many human cancers, including breast cancer, and is often associated with aggressive disease. In the present study, we examined the anti-metastatic effects of denbinobin through decreasing Src kinase activity in human and mouse breast cancer cells. Denbinobin caused significant block of Src kinase activity in both human and mouse breast cancer cells. Moreover, phosphorylation of the signaling molecules focal adhesion kinase, Crk-associated substrate and paxillin downstream of Src was also inhibited by denbinobin. Furthermore, denbinobin inhibited the in vitro migration, invasion and in vivo metastasis of breast cancers in a mouse metastatic model. The denbinobin-treated group showed a significant reduction in tumor metastasis, orthrotopic tumor volume, and spleen enlargement compared to the control group. In addition, transfection of breast cancer cells with a plasmid coding for a constitutively active Src prevented the denbinobin-mediated phosphorylation of Src and downstream molecules and cell migration. Our findings provide evidences that denbinobin inhibits Src-mediated signaling pathways involved in controlling breast cancer migration and metastasis, suggesting that it has therapeutic potential in breast cancer treatment.     

Colon cancer cell adhesion in response to Src kinase activation and actin-cytoskeleton by non-laminar shear stress

Malignant cells shed from tumors during surgical resection or spontaneous metastasis experience physical forces such as shear stress and turbulence within the peritoneal cavity during irrigation, laparoscopic air insufflation, or surgical manipulation, and within the venous or lymphatic system. Since physical forces can activate intracellular signals that modulate the biology of various cell types in vitro, we hypothesized that shear stress and turbulence might increase colon cancer cell adhesion to extracellular matrix, potentiating metastatic implantation. Primary human malignant colon cancer cells isolated from resected tumors and SW620 were subjected to shear stress and turbulence by stirring cells in suspension at 600 rpm for 10 min. Shear stress for 10 min increased subsequent SW620 colon cancer cell adhesion by 40.0 +/- 3.0% (n = 3; P < 0.001) and primary cancer cells by 41.0 +/- 3.0% to collagen I when compared to control cells. In vitro kinase assay (1.5 +/- 0.13 fold) and Western analysis (1.34 +/- 0.04 fold) demonstrated a significant increase in Src kinase activity in cells exposed shear stress. Src kinase inhibitors PP1 (0.1 microM), PP2 (20 microM), and actin-cytoskeleton stabilizer phalloidin (10 microM) prevented the shear stress stimulated cell adhesion to collagen I. Furthermore, PP2 inhibited basal (50.0 +/- 2.8%) and prevented shear stress induced src activation but phalloidin pretreatment did not. These results raise the possibility that shear stress and turbulence may stimulate the adhesion of malignant cells shed from colon cancers by a mechanism that requires both actin-cytoskeletal reorganization an independent physical force activation of Src kinase. Blocking this pathway might reduce tumor metastasis during surgical resection.     

PP2 对人胆管癌细胞株QBC939 侵袭能力的影响

目的：探讨Src 激酶特异性抑制剂PP2 对人胆管癌QBC939 细胞侵袭能力的影响和机制。方法：通过Western Blotting 技术 检测PP2 对人胆管癌QBC939细胞中Src 激酶活化的影响;用Transwell 小室法观察PP2 对QBC939细胞的影响;用RT-PCR 和 Western Blotting 技术检测PP2对QBC939 细胞侵袭能力相关分子的作用。结果：实验组p-Src 蛋白表达水平明显低于对照组,差 异具有统计学意义（P<0.05）;实验组QBC939 细胞体外侵袭能力较对照组显著降低,差异具有统计学意义（P<0.05）;与对照组相 比,实验组E-cadherin 表达显著增强,CD44表达明显减弱,差异具有统计学意义（P<0.05）。结论：PP2 通过抑制Src 激酶活化,增 强E-cadherin 表达、减弱CD44 表达,抑制人胆管癌QBC939 细胞侵袭能力。     

Role of Hck in the pathogenesis of encephalomyocarditis virus-induced diabetes in mice

Soluble mediators such as interleukin-1beta, tumor necrosis factor alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) produced from activated macrophages play an important role in the destruction of pancreatic beta cells in mice infected with a low dose of the D variant of encephalomyocarditis (EMC-D) virus. The tyrosine kinase signaling pathway was shown to be involved in EMC-D virus-induced activation of macrophages. This investigation was initiated to determine whether the Src family of kinases plays a role in the activation of macrophages, subsequently resulting in the destruction of beta cells, in mice infected with a low dose of EMC-D virus. We examined the activation of p59/p56(Hck), p55(Fgr), and p56/p53(Lyn) in macrophages from DBA/2 mice infected with the virus. We found that p59/p56(Hck) showed a marked increase in both autophosphorylation and kinase activity at 48 h after infection, whereas p55(Fgr) and p56/p53(Lyn) did not. The p59/p56(Hck) activity was closely correlated with the tyrosine phosphorylation level of Vav. Treatment of EMC-D virus-infected mice with the Src kinase inhibitor, PP2, resulted in the inhibition of p59/p56(Hck) activity and almost complete inhibition of the production of TNF-alpha and iNOS in macrophages and the subsequent prevention of diabetes in mice. On the basis of these observations, we conclude that the Src kinase, p59/p56(Hck), plays an important role in the activation of macrophages and the subsequent production of TNF-alpha and nitric oxide, leading to the destruction of pancreatic beta cells, which results in the development of diabetes in mice infected with a low dose of EMC-D virus.     

Src kinase activity is essential for osteoclast function

Deletion of the c-src gene impairs osteoclast bone resorbing activity, causing osteopetrosis. Although it has been concluded that restoring only the Src adaptor function at least partly rescues the cell attachment and skeletal phenotypes, the contribution of Src kinase activity remains controversial. Src forms a complex with Pyk2 and Cbl after adhesion-induced stimulation of alpha(V)beta(3) integrin. To demonstrate the importance of the Pyk2-Src association in osteoclasts and to distinguish the contributions of the Src adaptor and kinase activities in cytoskeletal organization and osteoclast function, we expressed mutants of Src and Pyk2 in osteoclasts using adenovirus vectors. Eliminating the Src-binding site on Pyk2 (Pyk2(Y402F)) markedly inhibited bone resorption by osteoclast-like cells, whereas kinase-dead Pyk2 had little effect. Kinase-dead Src, unlike kinase-dead Pyk2, markedly inhibited the bone-resorbing activity of wild type osteoclasts and failed to significantly restore bone-resorbing activity to Src(-/-) osteoclast-like cells. Activation of Src kinase by overexpressing kinase-dead Csk failed to reverse the inhibitory effect of Pyk2(Y402F), suggesting that osteoclastic bone resorption requires both c-Src kinase activity and the targeting of Src kinase by Pyk2. Src-catalyzed phosphorylation of Cbl on Tyr-731 is reported to induce the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. Expressing the Cbl(Y731F) mutant in osteoclasts markedly reduced their bone resorbing activity, suggesting that phosphorylation of Cbl(Y731) and the subsequent recruitment and activation of phosphatidylinositol 3-kinase may be critical signaling events downstream of Src in osteoclasts.     

PI3K activation is required for PMA-directed activation of cSrc by AFAP-110

Activation of PKC will induce the cSrc binding partner AFAP-110 to colocalize with and activate cSrc. The ability of AFAP-110 to colocalize with cSrc is contingent on the integrity of the amino-terminal pleckstrin homology (PH1) domain, while the ability to activate cSrc is dependent on the integrity of its SH3 binding motif, which engages the cSrc SH3 domain. The outcome of AFAP-110-directed cSrc activation is a change in actin filament integrity and the formation of podosomes. Here, we address what cellular signals promote AFAP-110 to colocalize with and activate cSrc, in response to PKC activation or PMA treatment. Because PH domain integrity in AFAP-110 is required for colocalization, and PH domains are known to interact with both protein and lipid binding partners, we sought to determine whether phosphatidylinositol 3-kinase (PI3K) activation played a role in PMA-induced colocalization between AFAP-110 and cSrc. We show that PMA treatment is able to direct activation of PI3K. Treatment of mouse embryo fibroblast with PI3K inhibitors blocked PMA-directed colocalization between AFAP-110 and cSrc and subsequent cSrc activation. PMA also was unable to induce colocalization or cSrc activation in cells that lacked the p85 and - regulatory subunits of PI3K. This signaling pathway was required for migration in a wound healing assay. Cells that were null for cSrc or the p85 regulatory subunits or expressed a dominant-negative AFAP-110 also displayed a reduction in migration. Thus PI3K activity is required for PMA-induced colocalization between AFAP-110 and cSrc and subsequent cSrc activation, and this signaling pathway promotes cell migration. phorbol 12-myristate 13-acetate; Src; protein kinase C; AFAP-110; phosphatidylinositol 3-kinase; pleckstrin homology domain     

Ligation of HLA class I molecules on endothelial cells induces phosphorylation of Src,paxillin, and focal adhesion kinase in an actin-dependent manner

The development of chronic rejection is the major limitation to long-term allograft survival. HLA class I Ags have been implicated to play a role in this process because ligation of class I molecules by anti-HLA Abs stimulates smooth muscle cell and endothelial cell proliferation. In this study, we show that ligation of HLA class I molecules on the surface of human aortic endothelial cells stimulates phosphorylation of Src, focal adhesion kinase, and paxillin. Signaling through class I stimulated Src phosphorylation and mediated fibroblast growth factor receptor (FGFR) translocation to the nucleus. In contrast, Src kinase activity was not involved in class I-mediated transfer of FGFR from cytoplasmic stores to the cell surface. Inhibition of Src protein kinase activity blocked HLA class I-stimulated tyrosine phosphorylation of paxillin and focal adhesion kinase. Furthermore, HLA class I-mediated phosphorylation of the focal adhesion proteins and FGFR expression was inhibited by cytochalasin D and latrunculin A, suggesting a role for the actin cytoskeleton in the signaling process. These findings indicate that anti-HLA Abs have the capacity to transduce activation signals in endothelial cells that may promote the development of chronic rejection.     

Phosphorylation of MNAR promotes estrogen activation of phosphatidylinositol 3-kinase

Estrogen actions are mediated by a complex interface of direct control of gene expression (the so-called "genomic action") and by regulation of cell signaling/phosphorylation cascades, referred to as the "nongenomic," or extranuclear, action. We have previously described the identification of MNAR (modulator of nongenomic action of estrogen receptor) as a novel scaffold protein that regulates estrogen receptor alpha (ERalpha) activation of cSrc. In this study, we have investigated the role of MNAR in 17beta-estradiol (E2)-induced activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Consistent with our previous results, a direct correlation was established between MNAR expression levels and E2-induced activation of PI3 and Akt kinases. Endogenous MNAR, ERalpha, cSrc, and p85, the regulatory subunit of PI3 kinase, interacted in MCF7 cells treated with E2. The interaction between p85 and MNAR required activation of cSrc and MNAR phosphorylation on Tyr 920. Consequently, the mutation of this tyrosine to alanine (Y920A) abrogated the interaction between MNAR and p85 and the E2-induced activation of the PI3K/Akt pathway, which was required for the E2-induced protection of MCF7 cells from apoptosis. Nonetheless, the Y920A mutant potentiated the E2-induced activation of the Src/MAPK pathway and MCF7 cell proliferation, as observed with the wild-type MNAR. These results provide new and important insights into the molecular mechanisms of E2-induced regulation of cell proliferation and apoptosis.