Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.25 (MEKK1)
 1,856 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The serine/threonine kinase Raf-1 functions downstream of Rats in a signal transduction cascade which transmits mitogenic stimuli from the plasma membrane to the nucleus. Raf-1 integrates signals coming from extracellular factors and, in turn, activates its substrate, MEK kinase. MEK activates mitogen-activated protein kinase (MAPK), which phosphorylates other kinases as well as transcription factors. Raf-1 exists in a complex with HSP90 and other proteins. The benzoquinone ansamycin geldanamycin (GA) binds to HSP90 and disrupts the Raf-1-HSP90 multimolecular complex, leading to destabilization of Raf-1. In this study, we examined whether Raf-1 destabilization is sufficient to block the Raf-1-MEK-MAPK signalling pathway and whether GA specifically inactivates the Raf-1 component of this pathway. Using the model system of NIH 3T3 cells stimulated with phorbol 12-myristate 13-acetate (PMA), we show that GA does not affect the ability of protein kinase C alpha to be activated by phorbol esters, but it does block activation of MEK and MAPK. Further, GA does not decrease the activity of constitutively active MEK in transiently transfected cells. Finally, disruption of the Raf-1-MEK-MAPK signalling pathway by GA prevents both the PMA-induced proliferative response and PMA-induced activation of a MAPK-sensitive nuclear transcription factor. Thus, we demonstrate that interaction between HSP90 and Raf-1 is a sine qua non for Raf stability and function as a signal transducer and that the effects observed cannot be attributed to a general impairment of protein kinase function.
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PMID:Destabilization of Raf-1 by geldanamycin leads to disruption of the Raf-1-MEK-mitogen-activated protein kinase signalling pathway. 881 98

Activation of 44 and 42 kDa extracellular signal-regulated kinases (ERK)1/2 by angiotensin II (angII) plays an important role in vascular smooth muscle cell (VSMC) function. The dual specificity mitogen-actived protein (MAP) kinase/ERK kinase (MEK) activates ERK1/2 in response to angII, but the MEK activating kinases remain undefined. Raf is a candidate MEK kinase. However, a kinase other than Raf appears responsible for angII-mediated signal transduction because we showed previously that treatment with 1 microM phorbol 12, 13-dibutyrate (PDBU) for 24 h completely blocked Raf-Ras association in VSMC but did not inhibit activation of MEK and ERK1/2 by angII. We hypothesized that an atypical protein kinase C (PKC) isoform, which lacks a phorbol ester binding domain, mediated ERK1/2 activation by angII. Western blot analysis of rat aortic VSMC with PKC isoform-specific antibodies showed PKC-alpha, -beta1, -delta, -epsilon, and -zeta in relative abundance. All isoforms except PKC-zeta were down-regulated by 1 microM PDBU for 24 h suggesting that PKC-zeta was responsible for angII-mediated ERK1/2 activation. In response to angII, PKC-zeta associated with Ras as shown by co-precipitation of PKC-zeta with anti-H-Ras antibody. To characterize further the role of PKC-zeta, PKC-zeta protein was depleted specifically by transfection with antisense PKC-zeta oligonucleotides. Antisense PKC-zeta oligonucleotide treatment significantly decreased PKC-zeta protein expression (without effect on other PKC isoforms) and angII-mediated ERK1/2 activation in a concentration-dependent manner. In contrast, ERK1/2 activation by platelet-derived growth factor and phorbol ester was not significantly inhibited. These results demonstrate an important difference in signal transduction by angII compared with PDGF and phorbol ester in VSMC, and suggest a critical role for PKC-zeta and Ras in angII stimulation of ERK1/2.
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PMID:Protein kinase C-zeta mediates angiotensin II activation of ERK1/2 in vascular smooth muscle cells. 904 26

Protein kinase C (PKC) is a multigene family of enzymes consisting of at least 11 isoforms. It has been implicated in the induction of c-fos and other immediate response genes by various mitogens. The serum response element (SRE) in the c-fos promoter is necessary and sufficient for induction of transcription of c-fos by serum, growth factors, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). It forms a complex with the ternary complex factor (TCF) and with a dimer of the serum response factor (SRF). TCF is the target of several signal transduction pathways and SRF is the target of the rhoA pathway. In this study we generated dominant-negative and constitutively active mutants of PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta to determine the roles of individual isoforms of PKC in activation of the SRE. Transient-transfection assays with NIH 3T3 cells, using an SRE-driven luciferase reporter plasmid, indicated that PKC-alpha and PKC-epsilon, but not PKC-delta or PKC-zeta, mediate SRE activation. TPA-induced activation of the SRE was partially inhibited by dominant negative c-Raf, ERK1, or ERK2, and constitutively active mutants of PKC-alpha and PKC-epsilon activated the transactivation domain of Elk-1. TPA-induced activation of the SRE was also partially inhibited by a dominant-negative MEKK1. Furthermore, TPA treatment of serum-starved NIH 3T3 cells led to phosphorylation of SEK1, and constitutively active mutants of PKC-alpha and PKC-epsilon activated the transactivation domain of c-Jun, a major substrate of JNK. Constitutively active mutants of PKC-alpha and PKC-epsilon could also induce a mutant c-fos promoter which lacks the TCF binding site, and they also induce transactivation activity of the SRF. Furthermore, rhoA-mediated SRE activation was blocked by dominant negative mutants of PKC-alpha or PKC-epsilon. Taken together, these findings indicate that PKC-alpha and PKC-epsilon can enhance the activities of at least three signaling pathways that converge on the SRE: c-Raf-MEK1-ERK-TCF, MEKK1-SEK1-JNK-TCF, and rhoA-SRF. Thus, specific isoforms of PKC may play a role in integrating networks of signal transduction pathways that control gene expression.
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PMID:Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. 989 Oct 65

Apert (Ap) syndrome is a craniofacial malformation characterized by premature fusion of cranial sutures (craniosynostosis). We previously showed that the Ser252Trp fibroblast growth factor receptor 2 (FGFR-2) mutation in Ap syndrome increases osteoblast differentiation and subperiosteal bone matrix formation, leading to premature calvaria ossification. In this study, we used the emerging technology of complementary DNA (cDNA) microarray to identify genes that are involved in osteoblast abnormalities induced by the Ser252Trp FGFR-2 mutation. To identify the signaling pathways involved in this syndrome, we used radioactively labeled cDNAs derived from two sources of cellular messenger RNAs (mRNAs) for hybridization: control (Co) and mutant Ap immortalized osteoblastic cells. Among genes that were differentially expressed, protein kinase Ca (PKC-alpha), interleukin-1alpha (IL-1alpha), and the small guanosine-5'-triphosphatase (GTPase) RhoA were increased in FGFR-2 mutant Ap cells compared with Co cells. The validity of the hybridization array was confirmed by Northern blot analysis using mRNAs derived from different cultures. Furthermore, immunochemical and Western blot analyses showed that mutant Ap cells displayed increased PKC-alpha, IL-1alpha, and RhoA protein levels compared with Co cells. Treatment of Co and Ap cells with the PKC inhibitor calphostin C decreased IL-1alpha and RhoA mRNA and protein levels in Ap cells, indicating that PKC is upstream of IL-1alpha and RhoA. Moreover, SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK), and PD-98059, a specific inhibitor of MAPK kinase (MEKK), also reduced IL-1alpha and RhoA expression in Ap cells. These data show that the Ser252Trp FGFR-2 mutation in Ap syndrome induces constitutive overexpression of PKC-alpha, IL-1alpha, and small GTPase RhoA, suggesting a role for these effectors in osteoblast alterations induced by the mutation. The cDNA microarray technology appears to be a useful tool to gain information on abnormal gene expression and molecular pathways induced by genetic mutations in bone cells.
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PMID:Increased expression of protein kinase Calpha, interleukin-1alpha, and RhoA guanosine 5'-triphosphatase in osteoblasts expressing the Ser252Trp fibroblast growth factor 2 receptor Apert mutation: identification by analysis of complementary DNA microarray. 1131 98

c-Jun NH(2)-terminal kinase (JNK), a member of the MAPK family of protein kinases, is a stress-response kinase that is activated by proinflammatory cytokines and growth factors coupled to membrane receptors or through nonreceptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (ROI). Ischemia followed by reperfusion or hypoxia with reoxygenation represents a condition of high oxidative stress where JNK activation is associated with elevated ROI. We recently demonstrated that the activation of JNK by this condition is initiated by ROI generated by mitochondrial electron transport and involves sequential activation of the proline-rich kinase 2 and the small GTP-binding factors Rac-1 and Cdc42. Here we present evidence that protein kinase C (PKC) and transforming growth factor-beta-activated kinase-1 (TAK-1) are also components of this pathway. Inhibition of PKC with the broad-range inhibitor calphostin C, the PKC-alpha/beta-selective inhibitor Go9367, or adenovirus-expressing dominant-negative PKC-alpha blocked the phosphorylation of proline-rich kinase 2 and JNK. Reoxygenation activated the mitogen-activated protein kinase kinase kinase, TAK-1, and promoted the formation of a complex containing Rac-1, TAK-1, and JNK but not apoptosis-stimulating kinase-1 or p21-activated kinase-1, which was detected within the first 10 min of reoxygenation. These results identify two new components, PKC and TAK-1, that have not been previously described in this signaling pathway.
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PMID:PKC-alpha and TAK-1 are intermediates in the activation of c-Jun NH2-terminal kinase by hypoxia-reoxygenation. 1720 6

Previous studies suggest that the novel protein kinase C (PKC) isoforms initiate a mitogen-activated protein kinase (MAPK) signaling cascade that regulates keratinocyte differentiation. However, assigning these functions has relied on treatment with pharmacologic inhibitors and/or manipulating kinase function using overexpression of wild-type or dominant-negative kinases. As these methods are not highly specific, an obligatory regulatory role for individual kinases has not been assigned. In this study, we use small interfering RNA knockdown to study the role of individual PKC isoforms as regulators of keratinocyte differentiation induced by the potent differentiating stimulus, 12-O-tetradecanoylphorbol-13-acetate (TPA). PKC-delta knockdown reduces TPA-activated involucrin promoter activity, nuclear activator protein-1 factor accumulation and binding to DNA, and cell morphology change. Knockdown of PKC downstream targets, including MEKK-1, MEK-6, MEK-3, or p38-delta, indicates that these kinases are required for these responses. Additional studies indicate that knockdown of PKC-eta inhibits TPA-dependent involucrin promoter activation. In contrast, knockdown of PKC-alpha (a classical PKC isoform) or PKC-epsilon (a novel isoform) does not inhibit these TPA-dependent responses. Further studies indicate that PKC-delta is required for calcium and green tea polyphenol-dependent regulation of end responses. These findings are informative as they suggest an essential role for selected PKC and MAPK cascade enzymes in mediating a range of end responses to a range of differentiation stimuli in keratinocytes.
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PMID:PKC-delta and -eta, MEKK-1, MEK-6, MEK-3, and p38-delta are essential mediators of the response of normal human epidermal keratinocytes to differentiating agents. 2063 51