Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

As transmembrane heterodimers, integrins bind to both extracellular ligands and intracellular proteins. We are currently investigating the interaction between integrins and the intracellular protein calreticulin. A prostatic carcinoma cell line (PC-3) was used to demonstrate that calreticulin can be found in the alpha3 immunoprecipitates of cells plated on collagen type IV, but not when plated on vitronectin. Conversely, alphav immunoprecipitates contained calreticulin only when cells were plated on vitronectin, i. e. not when plated on collagen IV. The interactions between these integrins and calreticulin were independent of actin cytoskeleton assembly and were transient, being maximal approx. 10-30 min after the cells came into contact with the substrates prior to complete cell spreading and formation of firm adhesive contacts. We demonstrate that okadaic acid, an inhibitor of intracellular serine/threonine protein phosphatases, inhibited the alpha3beta1-mediated adhesion of PC-3 cells to collagen IV and the alpha2beta1-mediated attachment of Jurkat cells to collagen I. This inhibition by okadaic acid was accompanied by inhibition of the ligand-specific interaction of calreticulin with the respective integrins in the two cell types. Additionally, we found that pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) resulted in prolongation of the calreticulin-integrin interaction, and enhancement of PC-3 cell attachment to collagen IV. We conclude that calreticulin interacts transiently with integrins during cell attachment and spreading. This interaction depends on receptor occupation, is ligand-specific, and can be modulated by protein phosphatase and MEK activity.
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PMID:Ligand-specific, transient interaction between integrins and calreticulin during cell adhesion to extracellular matrix proteins is dependent upon phosphorylation/dephosphorylation events. 1022 57

The mechanism of Taxol-induced apoptosis was investigated in MCF-7 human breast carcinoma cells. Taxol-induced apoptosis was associated with phosphorylation of both c-Raf-1 and Bcl-2 and activation of ERK and JNK MAP kinases. The serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) effectively blocked apoptosis, but N-p-tosyl-L-lysine chloromethyl ketone (TLCK), another serine protease inhibitor, was without effect. TPCK treatment also prevented phosphorylation of c-Raf-1 and Bcl-2 in response to Taxol treatment. The serine protease inhibitor did not alter JNK activity, but it enhanced Taxol-induced activation of ERK1/2. Treatment of cells with the inhibitor of MEK activation, PD98059, prevented Taxol-induced ERK activation both in the presence and absence of TPCK, but did not influence survival of either Taxol- or Taxol plus TPCK-treated cells. In addition, PD98059 had no effect on c-Raf-1 or Bcl-2 phosphorylation. Thus, while the Taxol-induced phosphorylations of c-Raf-1 and Bcl-2 proteins appear to be coupled, these events can be disassociated from ERK1/2 activation. In summary, these findings suggest that phosphorylation of c-Raf-1 and Bcl-2, but not ERK1/2, are important signaling events in Taxol-induced apoptosis of MCF-7 breast cancer cells and that a TPCK inhibitable protease(s) is required for these processes.
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PMID:Serine protease inhibitor TPCK prevents Taxol-induced cell death and blocks c-Raf-1 and Bcl-2 phosphorylation in human breast carcinoma cells. 1037 21

Intracellular signaling pathways that mediate survival of prostate carcinoma (PCa) cells are poorly understood. We examined the potential role of the phosphatidylinositol 3' kinase (PI3K) pathway as a mediator of cell survival in LNCaP human PCa cells, which express a variety of properties characteristic of human prostate cancer. LNCaP cell cultures rapidly became apoptotic when treated with the specific PI3K inhibitors, wortmannin and LY294002. In contrast, apoptosis was not induced when the cells were treated with: (a) rapamycin, an inhibitor of the ribosomal S6 kinase pp70S6K, which acts downstream of PI3K; (b) PD98059, a specific inhibitor of the extracellular signal-regulated kinase/mitogen-activated protein kinase (Erk/MAPK) kinase (MEK); or (c) the antiandrogen, Casodex; or when the cells were cultured under androgen-depleted conditions. Apoptosis induced by PI3K inhibition was attenuated by: (a) dihydrotestosterone; or (b) the ErbB1 activating ligands [epidermal growth factor (EGF), transforming growth factor alpha, or heparin-binding EGF-like growth factor]. In response to ErbB1 activation by ligand, the p85 regulatory subunit of PI3K associated specifically with ErbB3 but not detectably with ErbB1. The anti-apoptotic effect of ErbB1 activation was significantly reduced when cells were treated simultaneously with wortmannin and PD98059. These data indicate that survival signals can be evoked in LNCaP cells by several distinct pathways and can be triggered by nuclear and cell-surface receptors. Constitutive signaling through the PI3K pathway is required to prevent cell death in LNCaP, whereas activation of the Erk/MAPK and androgen response pathways is not obligatory for cell survival. These results also show that survival signals, as distinguished from mitogenic signals, can be evoked in PCa cells by ErbB1 ligands known to be synthesized within the human prostate.
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PMID:The phosphatidylinositol 3'-kinase pathway is a dominant growth factor-activated cell survival pathway in LNCaP human prostate carcinoma cells. 1038 51

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0-10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90-240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor alpha receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor alpha (TGFalpha) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFalpha cleavage 120-180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFalpha. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFalpha. Neutralization of TGFalpha function by an anti-TGFalpha antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFalpha-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.
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PMID:Radiation-induced release of transforming growth factor alpha activates the epidermal growth factor receptor and mitogen-activated protein kinase pathway in carcinoma cells, leading to increased proliferation and protection from radiation-induced cell death. 1043 7

Exposure of MDA-MB-231 human mammary carcinoma cells to an ionizing radiation dose of 2 Gy results in immediate activation and Tyr phosphorylation of the epidermal growth factor receptor (EGFR). Doxycycline induced expression of a dominant negative EGFR-CD533 mutant, lacking the COOH-terminal 533 amino acids, in MDA-TR15-EGFR-CD533 cells was used to characterize intracellular signaling responses following irradiation. Within 10 min, radiation exposure caused an immediate, transient activation of mitogen activated protein kinase (MAPK) which was completely blocked by expression of EGFR-CD533. The same radiation treatment also induced an immediate activation of the c-Jun-NH2-terminal kinase 1 (JNK1) pathway that was followed by an extended rise in kinase activity after 30 min. Expression of EGFR-CD533 did not block the immediate JNK1 response but completely inhibited the later activation. Treatment of MDA-TR15-EGFR-CD533 cells with the MEK1/2 inhibitor, PD98059, resulted in approximately 70% inhibition of radiation-induced MAPK activity, and potentiated the radiation-induced increase of immediate JNK1 activation twofold. Inhibition of Ras farnesylation with a concomitant inhibition of Ras function completely blocked radiation-induced MAPK and JNK1 activation. Modulation of EGFR and MAPK functions also altered overall cellular responses of growth and apoptosis. Induction of EGFR-CD533 or treatment with PD98059 caused a 3-5-fold increase in radiation toxicity in a novel repeated radiation exposure growth assay by interfering with cell proliferation and potentiating apoptosis. In summary, this data demonstrates that both MAPK and JNK1 activation in response to radiation occur through EGFR-dependent and -independent mechanisms, and are mediated by signaling through Ras. Furthermore, we have demonstrated that radiation-induced activation of EGFR results in downstream activation of MAPK which may affect the radiosensitivity of carcinoma cells.
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PMID:Dominant negative EGFR-CD533 and inhibition of MAPK modify JNK1 activation and enhance radiation toxicity of human mammary carcinoma cells. 1046 23

Hyper-activation of mitogen-activated protein kinase (MAPK) has recently been reported in several human cancers and activation of MAPK in those cancers may be associated with carcinogenesis through aberrant cell proliferation. To understand the roles of the MAPK pathway in colorectal tumorigenesis, we examined the status of extracellular signal-regulated protein kinases (ERK1/2) in 21 colorectal tumour specimens and compared it with that of paired normals. The specific MAPK activities were two- to tenfold lower in 71% (15 out of 21 cases) of colorectal tumours compared to those in paired normals. The individual MAPK kinase (MEK) correlated with MAPK activities (P = 0.006). Reduction of the MAPK and MEK activities in colorectal tumours was also observed in adenomas. These results suggested that down-regulation of the MAPK cascade may be caused by early genetic event(s) and that it may be related to the loss of normal growth control. Although MAPK activities were down-regulated both in adenomas and carcinomas, activities of the MAPKs in carcinomas were higher than those of paired adenomas. These results suggested that MAPK activities may be increased in the adenoma-to-carcinoma sequence and that it may play a role in the tumour progression. Observation of the differential regulation of MAPK activities in colorectal tumorigeneis suggested roles for the MAPK pathway in both positive and negative controls of cell growth.
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PMID:Differential regulation of MAP kinase cascade in human colorectal tumorigenesis. 1058 70

We investigated the role of the cdk inhibitor protein p21(Cip-1/WAF1/MDA6) (p21) in the ability of MAPK pathway inhibition to enhance radiation-induced apoptosis in A431 squamous carcinoma cells. In carcinoma cells, ionizing radiation (2 Gy) caused both primary (0-10 min) and secondary (90-240 min) activations of the MAPK pathway. Radiation induced p21 protein expression in A431 cells within 6 h via secondary activation of the MAPK pathway. Within 6 h, radiation weakly enhanced the proportion of cells in G(1) that were p21 and MAPK dependent, whereas the elevation of cells present in G(2)/M at this time was independent of either p21 expression or MAPK inhibition. Inhibition of the MAPK pathway increased the proportion of irradiated cells in G(2)/M phase 24-48 h after irradiation and enhanced radiation-induced apoptosis. This correlated with elevated Cdc2 tyrosine 15 phosphorylation, decreased Cdc2 activity, and decreased Cdc25C protein levels. Caffeine treatment or removal of MEK1/2 inhibitors from cells 6 h after irradiation reduced the proportion of cells present in G(2)/M phase at 24 h and abolished the ability of MAPK inhibition to potentiate radiation-induced apoptosis. These data argue that MAPK signaling plays an important role in the progression/release of cells through G(2)/M phase after radiation exposure and that an impairment of this progression/release enhances radiation-induced apoptosis. Surprisingly, the ability of irradiation/MAPK inhibition to increase the proportion of cells in G(2)/M at 24 h was found to be dependent on basal p21 expression. Transient inhibition of basal p21 expression increased the control level of apoptosis as well as the abilities of both radiation and MEK1/2 inhibitors to cause apoptosis. In addition, loss of basal p21 expression significantly reduced the capacity of MAPK inhibition to potentiate radiation-induced apoptosis. Collectively, our data argue that MAPK signaling and p21 can regulate cell cycle checkpoint control in carcinoma cells at the G(1)/S transition shortly after exposure to radiation. In contrast, inhibition of MAPK increases the proportion of irradiated cells in G(2)/M, and basal expression of p21 is required to maintain this effect. Our data suggest that basal and radiation-stimulated p21 may play different roles in regulating cell cycle progression that affect cell survival after radiation exposure.
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PMID:Roles for basal and stimulated p21(Cip-1/WAF1/MDA6) expression and mitogen-activated protein kinase signaling in radiation-induced cell cycle checkpoint control in carcinoma cells. 1058 55

alpha(V)beta(3), a broadly distributed member of the integrin family of adhesion receptors, has been implicated in a variety of physiological and pathophysiological events, including control of bone density, angiogenesis, apoptosis, tumor growth, and metastasis. Recently, it has been shown that activation of alpha(V)beta(3), its transition from a low- to a high-affinity/avidity state, influences its recognition of certain ligands. Bone sialoprotein (BSP) is recognized as an important ligand for alpha(V)beta(3) in processes ranging from bone formation to the homing of metastatic tumor cells. Here, the influence of alpha(V)beta(3) activation on the adhesion and migration of relevant cells to BSP has been examined. Stimulation of lymphoblastoid, osteoblastoid, and human umbilical vein endothelial cells (HUVEC) with PMA or Mn(2+) markedly enhanced alpha(V)beta(3)-dependent adhesion to BSP. alpha(V)beta(3)-mediated migration of HUVEC or osteoblastic cells to BSP was substantially enhanced by stimulation, demonstrating that alpha(V)beta(3) activation enhances both adhesive and migratory responses. However, adhesion and/or migration of certain tumor cell lines, including M21 melanoma and MDA MB435 and SKBR3 breast carcinoma cell lines, to BSP was constitutively high and was not augmented by alpha(V)beta(3)-activating stimuli. Inhibitors of the intracellular signaling molecules, phosphatidylinositol 3-kinase with wortmannin, hsp90-dependent kinases with geldanamycin, and calpain with calpeptin, but not MAPKK with PD98059, reduced the high spontaneous adhesion and migration of the M21 cells to BSP, consistent with the constitutive activation of the receptor on these tumor cells. These results indicate that the activation state of alpha(V)beta(3) can regulate cell migration and adhesion to BSP and, by extension, to other ligands of this receptor. The constitutive activation of alpha(V)beta(3) on neoplastic cells may contribute to tumor growth and metastatic potential.
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PMID:Activation of integrin alpha(V)beta(3) regulates cell adhesion and migration to bone sialoprotein. 1064 Apr 28

Tumor necrosis factor-alpha (TNF-alpha) inhibits growth of normal cervical keratinocytes but stimulates proliferation of human papillomavirus (HPV)-immortalized and cervical carcinoma-derived cell lines when mitogens such as epidermal growth factor (EGF) or serum are depleted. Current work identifies the mechanism of growth stimulation. TNF-alpha promoted cell cycle progression by increasing expression of HPV-16 E6/E7 RNAs and enhancing activity of cyclin-dependent kinase (cdk)2 and cdc2 after 3 d. Increased kinase activity was mediated by upregulation of cyclins A and B and decreases in cdk inhibitors p21(waf) and p27(kip). TNF-alpha stimulated these changes in part by increasing transcription and stabilization of RNA for amphiregulin, an EGF receptor ligand, and amphiregulin directly increased HPV-16 E6/E7 and cyclin A RNAs. To define which components of the EGF receptor signaling pathway were important, HPV-immortalized cells were transfected with activated or dominant negative mutants of Ha-ras, raf, or MAPKK. Expression of activated Ha-ras maintained HPV-16 and cyclin gene expression and promoted rapid growth in the absence of EGF. Furthermore, ras activation was necessary for TNF-alpha mitogenesis as transfection with a dominant negative ras mutant (Asn-17) strongly inhibited growth. Thus, activation of ras promotes expression of HPV-16 E6/E7 RNAs, induces cyclins A and B, and mediates growth stimulation of immortal keratinocytes by TNF-alpha. These studies define a pathway by which ras mutations, which occur in a subset of cervical cancers, may contribute to pathogenesis. Mol. Carcinog. 27:97-109, 2000. Published by Wiley-Liss, Inc.
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PMID:Tumor necrosis factor-alpha promotes human papillomavirus (HPV) E6/E7 RNA expression and cyclin-dependent kinase activity in HPV-immortalized keratinocytes by a ras-dependent pathway. 1065 2

Degenerate polymerase chain reaction against conserved kinase catalytic subdomains identified 15 tyrosine and serine-threonine kinases expressed in surgically removed prostatic carcinoma tissues, including six receptor kinases (PDGFBR, IGF1-R, VEGFR2, MET, RYK, and EPH-A1), six non-receptor kinases (ABL, JAK1, JAK2, TYK2, PLK-1, and EMK), and three novel kinases. Several of these kinases are oncogenic, and may function in the development of prostate cancer. One of the novel kinases is a new member of the sterile 20 (STE20) family of serine-threonine kinases which we have called prostate-derived STE20-like kinase (PSK) and characterized functionally. PSK encodes an open reading frame of 3705 nucleotides and contains an N-terminal kinase domain. Immunoprecipitated PSK phosphorylates myelin basic protein and transfected PSK stimulates MKK4 and MKK7 and activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway. Microinjection of PSK into cells results in localization of PSK to a vesicular compartment and causes a marked reduction in actin stress fibers. In contrast, C-terminally truncated PSK (1-349) did not localize to this compartment or induce a decrease in stress fibers demonstrating a requirement for the C terminus. Kinase-defective PSK (K57A) was unable to reduce stress fibers. PSK is the first member of the STE20 family lacking a Cdc42/Rac binding domain that has been shown to regulate both the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and the actin cytoskeleton.
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PMID:PSK, a novel STE20-like kinase derived from prostatic carcinoma that activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and regulates actin cytoskeletal organization. 1066 Jun


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