Activated Src kinase may contribute to the progression and spread of breast cancers and recent in vitro evidence suggests a role for Src in acquired endocrine resistance. The purpose of this study ...was to investigate whether modulation of Src activity in endocrine-sensitive and endocrine-resistant breast cancer cells directly affected their phenotype and sensitivity to 4-hydroxytamoxifen (tamoxifen) and to determine whether Src activity in breast cancer tissue affected patient outcome. Expression of constitutively active Src in ER-positive, endocrine-sensitive MCF7 breast cancer cells resulted in the development of an aggressive phenotype, akin to that previously observed in cell models of tamoxifen resistance, and, significantly, attenuated their response to tamoxifen. Conversely, expression of dominant negative-Src in tamoxifen-resistant MCF7 cells re-sensitised them to tamoxifen. An exploratory immunohistochemical study of an archival primary breast tumour series (n = 75) with parallel clinicopathological data and in normal breast tissues (n = 19) revealed higher levels of activated Src in the cytoplasm (p < 0.01) and lower levels of nuclear Src (p < 0.01) in tumour tissue compared with normal tissue. Whereas elevated levels of activated-Src in the cytoplasm of tumours was significantly associated with reduced survival in ER+ patients (p = 0.031), elevated levels of activated Src within the nucleus appeared to associate with an improved hormonal response. Together these data are further suggestive of a role for Src in breast cancer where it may alter response to endocrine therapy.
We have recently reported that angiotensin II (Ang II)-induced mitogen-activated protein kinase (MAPK) activation is mainly mediated by Ca2+-dependent activation of a protein tyrosine kinase through ...Gq-coupled Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMC). In the present study, we found Ang II rapidly induced the tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and its association with Shc and Grb2. These reactions were inhibited by the EGF receptor kinase inhibitor, AG1478. The Ang II-induced phosphorylation of the EGF receptor was mimicked by a Ca2+ionophore and completely inhibited by an intracellular Ca2+chelator. Thus, AG1478 abolished the MAPK activation induced by Ang II, a Ca2+ ionophore as well as EGF but not by a phorbol ester or platelet-derived growth factor-BB in the VSMC. Moreover, Ang II induced association of EGF receptor with catalytically active c-Src. This reaction was not affected by AG1478. These data indicate that Ang II induces Ca2+-dependent transactivation of the EGF receptor which serves as a scaffold for pre-activated c-Src and for downstream adaptors, leading to MAPK activation in VSMC.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote
activation of the p42/p44 extracellular signal-regulated protein ...kinases (ERKs) in granulosa cells. Results show that the
ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L - type Ca 2+ channels are already partially activated in vehicle-treated cells and that FSH does not further activate them. This tonic
stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates
with ERK in vehicle-treated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation.
FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition
and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with
this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide
Myr-PKI as well as by inhibitors of MEK, Src, a Ca 2+ channel blocker, and chelation of extracellular Ca 2+ . These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by
a 100-kDa phosphotyrosine phosphatase.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Fluid shear stress (flow) modulates endothelial cell function via specific intracellular signaling events. Previously we showed that flow activated ERK1/2 in an integrin-dependent manner (Takahashi, ...M., and Berk, B. C. (1996) J. Clin. Invest. 98, 2623-2631). p130 Crk-associated substrate (Cas), a putative c-Src substrate, was originally identified as a highly phosphorylated protein that is localized to focal adhesions and acts as an adapter protein. Recent reports have shown that Cas is important in cardiovascular development and actin filament assembly. Flow (shear stress = 12 dynes/cm(2)) stimulated Cas tyrosine phosphorylation within 1 min in human umbilical vein endothelial cells. Phosphorylation peaked at 5 min (3.5 +/- 0.7-fold) and was sustained to 20 min. Tyrosine phosphorylation of Cas was functionally important because flow stimulated association of Cas with Crk in a time- and force-dependent manner. Flow-mediated activation of c-Src, phosphorylation of Cas, and association of Cas with Crk were all inhibited by calcium chelation and pretreatment with the Src family-specific tyrosine kinase inhibitor PP1. To determine the role of c-Src in flow-stimulated phosphorylation of Cas, we transduced cells with adenovirus encoding kinase-inactive Src. Expression of kinase-inactive Src prevented flow-induced Cas tyrosine phosphorylation but not ERK1/2 activation. Calcium-dependent activation of c-Src and tyrosine phosphorylation of Cas defines a new flow-stimulated signal pathway, different from ERK1/2 activation. This pathway may be involved in focal adhesion remodeling and actin filament assembly.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Reactive oxygen species (ROS) activate members of the Src kinase and mitogen-activated protein kinase superfamily, including big mitogen-activated protein kinase 1 (BMK1) and extracellular ...signal-regulated kinases (ERK1/2). A potentially important downstream effector of ERK1/2 is p90 ribosomal S6 kinase (p90RSK), which plays an important role in cell growth through the activation of several transcription factors, as well as the Na/H exchanger. Previously, we showed that Src regulates BMK1 via a redox-sensitive signaling pathway. Because ROS are generated during ischemia and reperfusion after ischemia, we assessed the effects of these stimuli (H2O2, ischemia, and reperfusion) in the activation of ERK1/2, p90RSK, Src, and BMK1 in perfused guinea pig hearts. H2O2 (100 μmol/L) significantly activated all kinases. Ischemia alone stimulated p90RSK, Src, and BMK1 but not ERK1/2. These results suggest that p90RSK activation through ischemia occurs via a pathway other than ERK1/2. A role of Src in ischemia-mediated BMK1 activation was demonstrated through inhibition with the Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo3,4-dpyrimidine. Reperfusion after ischemia stimulated both p90RSK and ERK1/2. In contrast, although ROS increase during reperfusion after ischemia, the activities of both BMK1 and its upstream regulator, Src, were markedly attenuated by reperfusion after ischemia. The activation of C-terminal Src kinase during ischemia but not during reperfusion suggests that the attenuation of Src and BMK1 activity by reperfusion was not regulated by C-terminal Src kinase activity. The antioxidant N-2-mercaptopropionylglycine completely inhibited ERK1/2 and p90RSK activation by reperfusion but only partially inhibited ischemia-induced Src and BMK1 activation. The present study is the first to show the coregulation of Src and BMK1 by reperfusion after ischemia, which we propose to occur via a novel, ROS-independent pathway.
Recent studies suggest that ischemia activates Src and members of the mitogen-activated protein (MAP) kinase superfamily and their downstream effectors, including big MAP kinase 1 (BMK1) and p90 ...ribosomal S6 kinase (p90RSK). It has also been reported that adenosine is released during ischemia and involved in triggering the protective mechanism of ischemic preconditioning. To assess the roles of Src and adenosine in ischemia-induced MAP kinases activation, we utilized the Src inhibitor PP2 (4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo3,4-dpyrimidine) and the adenosine receptor antagonist 8-(p-sulfophenyl) theophylline (SPT) in perfused guinea pig hearts. PP2 (1 μm) inhibited ischemia-induced Src, BMK1 and JNK activation but not JAK2 and p38 activation. SPT inhibited ischemia-mediated p38 and JNK activation. These results demonstrate that Src family kinase and adenosine regulate MAP kinases by parallel pathways. Preconditioning significantly improved both recovery of developed pressure and dp/dt in isolated guinea pig hearts. Since the protective effect of preconditioning was blocked by PP2 (1 μ m) and SPT (50 μ m), we next investigated the regulation of Src, MAP kinases and p90RSK during preconditioning. The activity and time course of ERK1/2 was not changed, but p90RSK activation by reperfusion was completely inhibited by preconditioning. In contrast, the activation by ischemia of Src, BMK1, p38 and JNK was significantly faster in preconditioned hearts. Maximal BMK1 activation by ischemia was also significantly enhanced by preconditioning. These data suggest important roles for Src family kinases and adenosine in mediating preconditioning, and suggest specific roles for individual MAP kinases in preconditioning.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Phosphorylation and dephosphorylation of Tyr-530 in human c-Src (Tyr-527 in avian c-Src) is critical in regulating c-Src kinase activity. So far, it has not been possible to distinguish the active ...and inactive forms in vivo. We now report a new monoclonal antibody that selectively recognizes the active form of c-Src. This antibody, termed clone 28, recognized a region adjacent to Tyr-530 (Q529YQP32) in the C-terminal regulatory domain of c-Src, and its binding was hindered by phosphorylation of this tyrosine as determined by peptide competition assay. Combined immunoprecipitation/Western blotting revealed that clone 28 reacted with a 60-kDa protein that was precipitated by mAb 327, a well known monoclonal antibody against v-Src and c-Src. Cyanogen bromide cleavage and two-dimensional tryptic maps confirmed that clone 28 was specific for the active form (Tyr-530 not phosphorylated), whereas mAb 327 recognized the inactive form (Tyr-530 phosphorylated) as well as the active form. Clone 28 selectively immunoprecipitated the active form and augmented its kinase activity. Preabsorption experiments revealed that clone 28 could not completely immunoprecipitate the mAb 327 binding 60-kDa protein in either an in vitro or an in vivo phosphorylation system. These observations, taken together, strongly suggest the existence of multiple forms of c-Src as proposed by Cooper and Howell(1993) (Cooper, J. A., and Howell, B.(1993) Cell 73, 1051-1054).
Using clone 28, we demonstrated a distinct localization of the active form of c-Src within cultured normal fibrobast cells. In liver tissue sections, we also examined the distribution of the active form in embryonic mice. Megakaryocytes were strongly stained, in contrast to completely negative immunoreactivity in hepatocytes, reticulocytes, and granulocytes. This result provides the first direct evidence that c-Src is highly activated in platelets.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In order to elucidate the function of c-Src in keratinocytes, we studied the intracellular distribution of its active and inactive form in cultured normal human keratinocyte, using anti-c-Src ...monoclonal antibody clone 28, which recognizes the active form of c-Src (dephosphorylated at COOH-terminal residue Tyr 530), and monoclonal antibody clone 327 which recognizes both active and inactive forms. Since c-Src has been suggested to be involved in the control of cell adhesion in other cells, we produced a dynamic condition of cell migration by cutting culture cell colonies into squares to form a mesh pattern with a blade (culture wound model). Before cutting, the active form was expressed in cells located only at the periphery of colonies or isolated migrating cells, and was associated with microtubules. Wounding the colony generated a dramatic and rapid activation of c-Src in a few rows of cells along the cut edges, which were made even at the middle of colony, resulting in the association of the active form with microtubules. This increase of the active form was also detected by immunoblotting of cell extracts. These reactions were inhibited by 1 mM sodium orthovanadate, a protein-tyrosine phosphatase inhibitor. ST 638, a potent Src family tyrosine kinase inhibitor, inhibited the migration of keratinocytes in the culture wound healing model. These results suggest that wounding the culture causes activation of c-Src in keratinocytes, and thus activated c-Src may play a role in the function of microtubules during cell migration, especially at an early stage of wound healing.
Acute lung injury (ALI) is a devastating syndrome characterized by diffuse alveolar damage, elevated airspace levels of pro-inflammatory cytokines, and flooding of the alveolar spaces with ...protein-rich edema fluid. Interleukin-1β (IL-1β) is one of the most biologically active cytokines in the distal airspaces of patients with ALI. IL-1β has been shown to increase lung epithelial and endothelial permeability. In this study, we hypothesized that IL-1β would decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we measured the effects of IL-1β on transepithelial current, resistance, and sodium transport in primary cultures of alveolar epithelial type II (ATII) cells. IL-1β significantly reduced the amiloride-sensitive fraction of the transepithelial current and sodium transport across rat ATII cell monolayers. Moreover, IL-1β decreased basal and dexamethasone-induced epithelial sodium channel α-subunit (αENaC) mRNA levels and total and cell-surface protein expression. The inhibitory effect of IL-1β on αENaC expression was mediated by the activation of p38 MAPK in both rat and human ATII cells and was independent of the activation of αvβ6 integrin and transforming growth factor-β. These results indicate that IL-1β may contribute to alveolar edema in ALI by reducing distal lung epithelial sodium absorption. This reduction in ion and water transport across the lung epithelium is in large part due to a decrease in αENaC expression through p38 MAPK-dependent inhibition of αENaC promoter activity and to an alteration in ENaC trafficking to the apical membrane of ATII cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Phorbol ester treatment of MCF-7 cells led to the tyrosine phosphorylation and activation of PKC delta. However, through Western blot analysis and in vitro immunecomplex kinase assays, we detected a ...differential localization of tyrosine-phosphorylated PKC delta and catalytically active PKC delta. Catalytically active PKC delta was concentrated in Triton X-100 solubilized-membrane fractions while tyrosine-phosphorylated PKC delta was localized to the cytosol fraction. Phorbol ester treatment of MCF-7 cells stimulated both the time-dependent in vivo association of Src with PKC delta, evidenced in Src immunoprecipitates by the co-immunoprecipitation of PKC delta, and activation of Src, evidenced in Src immunoprecipitates as an increase in reactivity with a Src antibody (clone 28) reactive only with active Src (dephosphorylated on residue 530) and in Src and PKC delta immunoprecipitates by an increase in Src kinase activity. While our data are consistent with reports in the literature showing the activator/stimulus-dependent tyrosine phosphorylation of PKC delta, our data show that the tyrosine phosphorylation of PKC delta is not essential for kinase activity. These results are the first to demonstrate an in vivo association between PKC delta and active Src in the absence of over-expression of either PKC delta or Src, and support the association of Src and PKC delta towards a physiological function.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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