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One of the major features of cancer is Otto Warburg's observation that many tumors have increased extracellular acidification compared to healthy tissues. Since Warburg's observation, the importance of extracellular acidification in cancer is now considered a hallmark of cancer. Human MAP3K4 functions upstream of the p38 and JNK mitogen activated protein kinases (MAPKs). Additionally, MAP3K4 is required for cell migration and extracellular acidification of breast cancer cells in response to HER2/HER3 signaling. Here, we demonstrate that GIT1 interacts with MAP3K4 by immunoprecipitation, while cellular lactate production and the capacity of MCF-7 cells for anchorage independent growth in soft agar were dependent on GIT1. Additionally, we show that activation of HER2/HER3 signaling leads to reduced expression of lactate receptor (GPR81) mRNA and that both, GIT1 and MAP3K4, are necessary for constitutive expression of GPR81 mRNA. Our study suggests that targeting downstream proteins in the HER2/HER3-induced extracellular lactate signaling pathway may be a way to inhibit the Warburg Effect to disrupt tumor growth.

Nonalcoholic fatty liver disease (NAFLD) is a globally widespread disease of increasing clinical significance. The pathological progression of the disease from simple steatosis to nonalcoholic steatohepatitis (NASH) has been well defined, however, the contribution of altered branched chain amino acid metabolomic profiles to the progression of NAFLD is not known. The three BCAAs: leucine, isoleucine and valine are known to mediate activation of several important hepatic metabolic signaling pathways ranging from insulin signaling to glucose regulation. The purpose of this study is to profile changes in hepatic BCAA metabolite levels with transcriptomic changes in the progression of human NAFLD to discover novel mechanisms of disease progression. Metabolomic and transcriptomic data sets representing the spectrum of human NAFLD (normal, steatosis, NASH fatty, and NASH not fatty livers) were utilized for this study. During the transition from steatosis to NASH, increases in the levels of leucine (127% of normal), isoleucine (139%), and valine (147%) were observed. Carnitine metabolites also exhibited significantly elevated profiles in NASH fatty and NASH not fatty samples and included propionyl, hexanoyl, lauryl, acetyl and butyryl carnitine. Amino acid and BCAA metabolism gene sets were significantly enriched among downregulated genes during NASH. These cumulative alterations in BCAA metabolite and amino acid metabolism gene profiles represent adaptive physiological responses to disease-induced hepatic stress in NASH patients.

Human MAP3K4 (MTK1) functions upstream of mitogen activated protein kinases (MAPKs). In this study we show MTK1 is required for human epidermal growth factor receptor 2/3 (HER2/HER3)-heregulin beta1 (HRG) induced cell migration in MCF-7 breast cancer cells. We demonstrate that HRG stimulation leads to association of MTK1 with activated HER3 in MCF-7 and T-47D breast cancer cells. Activated HER3 association with MTK1 is dependent on HER2 activation and is decreased by pre-treatment with the HER2 inhibitor, lapatinib. Moreover, we also identify the actin interacting region (AIR) on MTK1. Disruption of actin cytoskeletal polymerization with cytochalasin D inhibited HRG induced MTK1/HER3 association. Additionally, HRG stimulation leads to extracellular acidification that is independent of cellular proliferation. HRG induced extracellular acidification is significantly inhibited when MTK1 is knocked down in MCF-7 cells. Similarly, pre-treatment with lapatinib significantly decreased HRG induced extracellular acidification. Extracellular acidification is linked with cancer cell migration. We performed scratch assays that show HRG induced cell migration in MCF-7 cells. Knockdown of MTK1 significantly inhibited HRG induced cell migration. Furthermore, pre-treatment with lapatinib also significantly decreased cell migration. Cell migration is required for cancer cell metastasis, which is the major cause of cancer patient mortality. We identify MTK1 in the HER2/HER3-HRG mediated extracellular acidification and cell migration pathway in breast cancer cells., (© 2013 Elsevier Inc. All rights reserved.)

C-reactive protein (CRP) is an acute phase protein in humans. Elevated levels of CRP are produced in response to inflammatory cytokines and are associated with atherosclerosis, hypertension, cardiovascular disease and insulin resistance. Exposure to inorganic arsenic, a common environmental toxicant, also produces cardiovascular disorders, namely atherosclerosis and is associated with insulin-resistance. Inorganic arsenic has been shown to contribute to cardiac toxicities through production of reactive oxygen species (ROS) that result in the activation of NFκB. In this study we show that exposure of the hepatic cell line, HepG2, to environmentally relevant levels of arsenite (0.13 to 2 μM) results in elevated CRP expression and secretion. ROS analysis of the samples showed that a minimal amount of ROS are produced by HepG2 cells in response to these concentrations of arsenic. In addition, treatment of FvB mice with 100 ppb sodium arsenite in the drinking water for 6 months starting at weaning age resulted in dramatically higher levels of CRP in both the liver and inner medullary region of the kidney. Further, mouse Inner Medullary Collecting Duct cells (mIMCD-4), a mouse kidney cell line, were stimulated with 10 ng/ml CRP which resulted in activation of NFκB. Pretreatment with 10 nM Y27632, a known Rho-kinase inhibitor, prior to CRP exposure attenuated NFκB activation. These data suggest that arsenic causes the expression and secretion of CRP and that CRP activates NFκB through activation of the Rho-kinase pathway, thereby providing a novel pathway by which arsenic can contribute to metabolic syndrome and cardiovascular disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In the developing heart, the epicardium is a major source of progenitor cells that contribute to the formation of the coronary vessel system. These epicardial progenitors give rise to the different cellular components of the coronary vasculature by undergoing a number of morphological and physiological changes collectively known as epithelial to mesenchymal transformation (EMT). However, the specific signaling mechanisms that regulate epicardial EMT are yet to be delineated. In this study we investigated the role of TGFβ2 and hyaluronan (HA) during epicardial EMT and how signals from these two molecules are integrated during this important process. Here we show that TGFβ2 induces MEKK3 activation, which in turn promotes ERK1/2 and ERK5 phosphorylation. TGFβ2 also increases Has2 expression and subsequent HA production. Nevertheless, inhibition of MEKK3 kinase activity, silencing of ERK5 or pharmacological disruption of ERK1/2 activation significantly abrogates this response. Thus, TGFβ2 promotes Has2 expression and HA production through a MEKK3/ERK1/2/5-dependent cascade. Furthermore, TGFβ2 is able to induce epicardial cell invasion and differentiation but not proliferation. However, inhibition of MEKK3-dependent pathways, degradation of HA by hyaluronidases or blockade of CD44, significantly impairs the biological response to TGFβ2. Taken together, these findings demonstrate that TGFβ2 activation of MEKK3/ERK1/2/5 signaling modulates Has2 expression and HA production leading to the induction of EMT events. This is an important and novel mechanism showing how TGFβ2 and HA signals are integrated to regulate changes in epicardial cell behavior., (Published by Elsevier Inc.)

Arsenic has been a recognized contaminant and toxicant, as well as a medicinal compound throughout human history. Populations throughout the world are exposed to arsenic and these exposures have been associated with a number of human cancers. Not much is known about the role of arsenic as a human carcinogen and more recently its role in non-cancerous diseases, such as cardiovascular disease, hypertension and diabetes mellitus have been uncovered. The health effects associated with arsenic are numerous and the association between arsenic exposure and human disease has intensified the search for molecular mechanisms that describe the biological activity of arsenic in humans and leads to the aforementioned disease states. Arsenic poses a human health risk due in part to the regulation of cellular signal transduction pathways and over the last few decades, some cellular mechanisms that account for arsenic toxicity, as well as, signal transduction pathways have been discovered. However, given the ubiquitous nature of arsenic in the environment, making sense of all the data remains a challenge. This review will focus on our knowledge of signal transduction pathways that are regulated by arsenic.

Congenital heart defects occur at a rate of 5% and are the most prevalent birth defects. A better understanding of the complex signaling networks regulating heart development is necessary to improve repair strategies for congenital heart defects. The mitogen-activated protein 3 kinase (MEKK3) is important to early embryogenesis, but developmental processes affected by MEKK3 during heart morphogenesis have not been fully examined. We identify MEKK3 as a critical signaling molecule during endocardial cushion development. We report the detection of MEKK3 transcripts to embryonic hearts before, during, and after cardiac cushion cells have executed epithelial-to-mesenchymal transition (EMT). MEKK3 is observed to endocardial cells of the cardiac cushions with a diminishing gradient of expression into the cushions. These observations suggest that MEKK3 may function during production of cushion mesenchyme as required for valvular development and septation of the heart. We used a kinase inactive form of MEKK3 (MEKK3(KI)) in an in vitro assay that recapitulates in vivo EMT and show that MEKK3(KI) attenuates mesenchyme formation. Conversely, constitutively active MEKK3 (ca-MEKK3) triggers mesenchyme production in ventricular endocardium, a tissue that does not normally undergo EMT. MEKK3-driven mesenchyme production is further substantiated by increased expression of EMT-relevant genes, including TGFbeta(2), Has2, and periostin. Furthermore, we show that MEKK3 stimulates EMT via a TGFbeta(2)-dependent mechanism. Thus, the activity of MEKK3 is sufficient for developmental EMT in the heart. This knowledge provides a basis to understand how MEKK3 integrates signaling cascades activating endocardial cushion EMT.

The cytoplasmic serine/threonine kinases transduce extracellular signals into regulatory events that impact cellular responses. The induction of one kinase triggers the activation of several downstream kinases, leading to the regulation of transcription factors to affect gene function. This arrangement allows for the kinase cascade to be amplified, and integrated according to the cellular context. An upstream mitogen or growth factor signal initiates a module of three kinases: a mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK; e.g., Raf) that phosphorylates and activates a MAP kinase kinase (MAPKK; e.g., MEK) and finally activation of MAP kinase (MAPK; e.g., ERK). Thus, this MAP3K-MAP2K-MAPK module represents critical effectors that regulate extracellular stimuli into cellular responses, such as differentiation, proliferation, and apoptosis all of which function during development. There are 21 characterized MAP3Ks that activate known MAP2Ks, and they function in many aspects of developmental biology. This review summarizes known transduction routes linked to each MAP3K and highlights mouse models that provide clues to their physiological functions. This perspective reveals that some of these MAP3K effectors may have redundant functions, and also serve as unique nexus depending on the context of the signaling pathway., (Copyright (c) 2008 Wiley-Liss, Inc.)

Chronic ingestion of arsenic is associated with increased incidence of respiratory and cardiovascular diseases. To investigate the role of arsenic in early events in vascular pathology, C57BL/6 mice ingested drinking water with or without 50 ppb sodium arsenite (AsIII) for four, five, or eight weeks. At five and eight weeks, RNA from the lungs of control and AsIII-exposed animals was processed for microarray. Sixty-five genes were significantly and differentially expressed. Differential expression of extracellular matrix (ECM) gene transcripts was particularly compelling, as 91% of genes in this category, including elastin and collagen, were significantly decreased. In additional experiments, real-time RT-PCR showed an AsIII-induced decrease in many of these ECM gene transcripts in the heart and NIH3T3 fibroblast cells. Histological stains for collagen and elastin show a distinct disruption in the ECM surrounding small arteries in the heart and lung of AsIII-exposed mice. Immunohistochemical detection of alpha-smooth muscle actin in blood vessel walls was decreased in the AsIII-exposed animals. These data reveal a functional link between AsIII exposure and disruption in the vascular ECM. These AsIII-induced early pathological events may predispose humans to respiratory and cardiovascular diseases linked to chronic low-dose AsIII exposure.

Chronic exposure to arsenic has been linked to tumorigenesis, cardiovascular disease, hypertension, atherosclerosis, and peripheral vascular disease; however, the molecular mechanisms underlying its pathological effects remain elusive. In this study, we investigated arsenic-induced alteration of focal adhesion protein complexes in normal, primary vascular smooth muscle cells. We demonstrate that exposure to environmentally relevant concentrations of arsenic (50 ppb As(3+)) can alter focal adhesion protein co-association leading to activation of downstream pathways. Co-associated proteins were identified and quantitated via co-immunoprecipitation, SDS-PAGE, and Western blot analysis followed by scanning densitometry. Activation of MAPK pathways in total cell lysates was evaluated using phosphor-specific antibodies. In our model, arsenic treatment caused a sustained increase in FAK-src association and activation, and induced the formation of unique signaling complexes (beginning after 3-hour As(3+) exposure and continuing throughout the 12-hour time course studied). The effects of these alterations were manifested as chronic stimulation of downstream PAK, ERK and JNK pathways. Past studies have demonstrated that these pathways are involved in cellular survival, growth, proliferation, and migration in VSMCs.

Epidemiological studies link arsenic exposure to increased risks of cancers of the skin, kidney, lung, bladder and liver. Additionally, a variety of non-cancerous conditions such as diabetes mellitus, hypertension, and cardiovascular disease have been associated with chronic ingestion of low levels of arsenic. However, the biological and molecular mechanisms by which arsenic exerts its effects remain elusive. Here we report increased renal hexokinase II (HKII) expression in response to arsenic exposure both in vivo and in vitro. In our model, HKII was up-regulated in the renal glomeruli of mice exposed to low levels of arsenic (10 ppb or 50 ppb) via their drinking water for up to 21 days. Additionally, a similar effect was observed in cultured renal mesangial cells exposed to arsenic. This correlation between our in vivo and in vitro data provides further evidence for a direct link between altered renal HKII expression and arsenic exposure. Thus, our data suggest that alterations in renal HKII expression may be involved in arsenic-induced pathological conditions involving the kidney. More importantly, these results were obtained using environmentally relevant arsenic concentrations.

Congenital heart malformations occur at a rate of one per one hundred births and are considered the most frequent birth defects. This high incidence of cardiac defects underscores the complex developmental processes required to form the first functioning organ in mammals. The molecular cues which govern heart development are poorly defined and require an improved understanding in order to advance repair strategies for heart defects. The cytoplasmic MAP kinase kinase kinase, MEKK4, is a critical effector in cellular stress responses; however, the function of MEKK4 during embryonic development and cardiogenesis is not well understood. We have identified MEKK4 as a critical signaling molecule during cardiovascular development. We report the detection of MEKK4 transcripts to early myocardium, endocardium and to cardiac cushion cells that have executed epithelial to mesenchymal transformation (EMT). These observations suggest that MEKK4 may function during production of the cushion mesenchyme as required to create valves and the septated heart. We used a kinase inactive form of MEKK4(MEKK4(KI)) in an in vitro assay that recapitulates in vivo EMT, and show that MEKK4(KI) attenuates mesenchyme production. However, addition of a constitutively active MEKK4 into ventricular explants, a system that does not normally undergo EMT, is not able to cause mesenchymal cell outgrowth. Thus, the kinase activity of MEKK4 is essential, but not sufficient, to support developmental EMT. This knowledge provides a basis to understand how MEKK4 may integrate signaling cascades controlling heart development., ((c) 2006 Wiley-Liss, Inc.)

MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IkappaB kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3-dependent activation of an NF-kappaB reporter gene as well as ERK, JNK, and p38 MAP kinases correlated with a requirement for serine at position 526. Constitutively active mutants of MEKK3, consisting of S526D and S526E, were capable of activating a NF-kappaB luciferase reporter gene as well as ERK and MEK, suggesting that a negative charge at Ser526 was necessary for MEKK3 activity and implicating Ser526 as a phosphorylation site. An antibody was developed that specifically recognized phospho-Ser526 of MEKK3 but did not recognize the S526A point mutant. The catalytically inactive (K391M) mutant of MEKK3 was not phosphorylated at Ser526, indicating that phosphorylation of Ser526 occurs via autophosphorylation. Endogenous MEKK3 was phosphorylated on Ser526 in response to osmotic stress. In addition, phosphorylation of Ser526 was required for MKK6 phosphorylation in vitro, whereas dephosphorylation of Ser526 was mediated by protein phosphatase 2A and sensitive to okadaic acid and sodium fluoride. Finally, the association between MEKK3 and 14-3-3 was dependent on Ser526 and prevented dephosphorylation of Ser526. In summary, Ser526 of MEKK3 is an autophosphorylation site within the T-loop that is regulated by PP2A and 14-3-3 proteins.

Skeletal disorders and neural tube closure defects represent clinically significant human malformations. The signaling networks regulating normal skeletal patterning and neurulation are largely unknown. Targeted mutation of the active site lysine of MEK kinase 4 (MEKK4) produces a kinase-inactive MEKK4 protein (MEKK4(K1361R)). Embryos homozygous for this mutation die at birth as a result of skeletal malformations and neural tube defects. Hindbrains of exencephalic MEKK4(K1361R) embryos show a striking increase in neuroepithelial cell apoptosis and a dramatic loss of phosphorylation of MKK3 and -6, mitogen-activated protein kinase kinases (MKKs) regulated by MEKK4 in the p38 pathway. Phosphorylation of MAPK-activated protein kinase 2, a p38 substrate, is also inhibited, demonstrating a loss of p38 activity in MEKK4(K1361R) embryos. In contrast, the MEK1/2-extracellular signal-regulated kinase 1 (ERK1)/ERK2 and MKK4-Jun N-terminal protein kinase pathways were unaffected. The p38 pathway has been shown to regulate the phosphorylation and expression of the small heat shock protein HSP27. Compared to the wild type, MEKK4(K1361R) fibroblasts showed significantly reduced phosphorylation of p38 and HSP27, with a corresponding heat shock-induced instability of the actin cytoskeleton. Together, these data demonstrate MEKK4 regulation of p38 and that substrates downstream of p38 control cellular homeostasis. The findings are the first demonstration that MEKK4-regulated p38 activity is critical for neurulation.

IFNgamma (interferon-gamma) binding to its cognate receptor results, through JAK (Janus kinase), in direct activation of receptor-bound STAT1 (signal transducer and activator of transcription 1), although there is evidence for additional activation of a MAPK (mitogen-activated protein kinase) pathway. In the present paper, we report IFNgamma-dependent activation of the MEKK4 (MAPK/extracellular-signal-regulated kinase kinase kinase 4) pathway in HaCaT human keratinocytes. MEKK4 is tyrosine-phosphorylated and the IFNgamma-dependent phosphorylation requires intracellular calcium. Calcium-dependent phosphorylation of MEKK4 is mediated by Pyk2. Moreover, MEKK4 and Pyk2 co-localize in an IFNgamma-dependent manner in the perinuclear region. Furthermore, the calcium-binding protein, annexin II, and the calcium-regulated kinase, Pyk2, co-immunoprecipitate with MEKK4 after treatment with IFNgamma. Immunofluorescence imaging of HaCaT cells shows an IFNgamma-dependent co-localization of annexin II with Pyk2 in the perinuclear region, suggesting that annexin II mediates the calcium-dependent regulation of Pyk2. Tyrosine phosphorylation of MEKK4 correlates with its activity to phosphorylate MKK6 (MAPK kinase 6) in vitro and subsequent p38 MAPK activation in an IFNgamma-dependent manner. Additional studies demonstrate that the SH2 (Src homology 2)-domain-containing tyrosine phosphatase SHP2 co-immunoprecipitates with MEKK4 in an IFNgamma-dependent manner and co-localizes with MEKK4 after IFNgamma stimulation in the perinuclear region in HaCaT cells. Furthermore, we provide evidence that SHP2 dephosphorylates MEKK4 and Pyk2, terminating the MEKK4-dependent branch of the IFNgamma signalling pathway.

Although it is known that MEKK4 regulates MKK6, and p38 MAP kinase, extracellular stimuli that activate the serine/threonine kinase, MEKK4, are unknown. The aim of this study was then to identify stimuli that regulate MEKK4. By using recombinant MEKK4, as bait to attract interacting proteins, the calcium binding protein, annexin II, was identified by mass spectrometry as interacting with MEKK4, suggesting that MEKK4 might be regulated by calcium. A calcium-dependent interaction between MEKK4 and annexin II was observed when MEKK4 was immunoprecipitated from rat aortic smooth muscle cells that were treated with angiotensin II. Additional studies using recombinant MEKK4 in a Far-Western immunoblot identified a protein of 120 kDa as interacting directly with MEKK4. Prior studies indicated that MEKK4 was phosphorylated on tyrosine in vivo, and in fact, Pyk2 interacts with MEKK4 in an angiotensin II dependent manner in rat aortic smooth muscle cells. Pyk2 phosphorylates MEKK4 in vitro and Pyk2-dependent phosphorylation further regulates MEKK4-dependent phosphorylation of MKK6. Finally, dominant-negative MEKK4 inhibits angiotensin II mediated transcription of a luciferase reporter construct containing the cyclooxygenase II promoter, demonstrating that MEKK4 functions in a calcium-dependent manner as a substrate for Pyk2 and regulates transcription of cyclooxygenase II.

The development of apoptosis resistance appears to be an important factor in colon carcinogenesis. To gain an understanding of the molecular pathways altered during the development of apoptosis resistance, we selected three cell lines for resistance to induction of apoptosis by deoxycholate, an important etiologic agent in colon cancer. We then evaluated gene expression levels for 825 proteins in these resistant lines, compared with a parallel control line not subject to selection. Eighty-two proteins were identified as either over-expressed or under-expressed in at least two of the resistant lines, compared with the control. Thirty-five of the 82 proteins (43%) proved to have a known role in apoptosis. Of these 35 proteins, 21 were over-expressed and 14 were under-expressed. Of those that were over-expressed 18 of 21 (86%) are anti-apoptotic in some circumstances, of those that were under-expressed 11 of 14 (79%) are pro-apoptotic in some circumstances. This finding suggests that apoptosis resistance during selection among cultured cells, and possibly in the colon during progression to cancer, may arise by constitutive over-expression of multiple anti-apoptotic proteins and under-expression of multiple pro-apoptotic proteins. The major functional groups in which altered expression levels were found are post-translational modification (19 proteins), cell structure (cytoskeleton, microtubule, actin, etc.) (17 proteins), regulatory processes (11 proteins) and DNA repair and cell cycle checkpoint mechanisms (10 proteins). Our findings, overall, bear on mechanisms by which apoptosis resistance arises during progression to colon cancer and suggest potential targets for cancer treatment. In addition, assays of normal-appearing mucosa of colon cancer patients, for over- or under-expression of genes found to be altered in our resistant cell lines, may allow identification of early biomarkers of colon cancer risk.

Tyrosine hydroxylase (TH) is regulated by the reversible phosphorylation of serines 8, 19, 31 and 40. Upon initiation of this study, serine 19 was unique due to its requirement of 14-3-3 binding after phosphorylation for optimal enzyme activity, although it has been more recently demonstrated that phosphorylated serine 40 also binds 14-3-3. To identify proteins that interact with TH following phosphorylation of serine 19, this amino acid was mutated to alanine and THS19A was used as bait in a yeast two-hybrid system. From this, mouse-derived cyclin-dependent kinase 11 (CDK11)p110 was identified as an interacting partner with THS19A. The interaction was confirmed using human CDK11p110 cDNA in a mammalian system. Previous research has demonstrated that casein kinase 2 (CK2) interacts with CDK11p110, and both were observed to phosphorylate TH in vitro. In addition, CDK11p110 overexpression was observed to inhibit the interaction between TH and 14-3-3. A mechanism contributing to disruption of the interaction between TH and 14-3-3 may be due to CK2 phosphorylation of specific 14-3-3 isoforms, i.e. 14-3-3 tau. Collectively, these results imply that CDK11p110 and CK2 negatively regulate TH catecholamine biosynthetic activity since phosphoserine 19 of TH requires 14-3-3 binding for optimal enzyme activity and a decreased rate of dephosphorylation.

Cyclin-dependent kinase (CDK)11(p110), formerly known as PITSLRE, is a serine/threonine kinase whose catalytic activity has been associated with transcription and RNA processing. To further evaluate the regulation of CDK11(p110) catalytic activity, interacting proteins were identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Following the immunoprecipitation of CDK11(p110) from COS-7 cells, the serine/threonine kinase CK2 was identified by LC-MS/MS. These results were extended through the observation that CDK11(p110) serves as a substrate for CK2 and the identification of a phosphorylation site on CDK11(p110) at Ser227 by LC-MS/MS. To obtain CDK11(p110) devoid of CK2, CDK11(p110) was expressed in High Five insect cells and secreted into the media due to the presence of a honeybee melittin signal sequence encoded at the amino-terminus of CDK11(p110). Recombinant CDK11(p110) was purified from the media and phosphorylation of histone H1 subsequently demonstrated. After demonstrating retention of CDK11(p110) kinase activity, it was evaluated for activity on the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II), but only CK2 was found to phosphorylate the CTD.

The cytokine tumor necrosis factor alpha (TNF-alpha) stimulates the NF-kappaB, SAPK/JNK, and p38 mitogen-activated protein (MAP) kinase pathways by recruiting RIP1 and TRAF2 proteins to the tumor necrosis factor receptor 1 (TNFR1). Genetic studies have revealed that RIP1 links the TNFR1 to the IkappaB kinase (IKK) complex, whereas TRAF2 couples the TNFR1 to the SAPK/JNK cascade. In transfection studies, RIP1 and TRAF2 stimulate p38 MAP kinase activation, and dominant-negative forms of RIP1 and TRAF2 inhibit TNF-alpha-induced p38 MAP kinase activation. We found TNF-alpha-induced p38 MAP kinase activation and interleukin-6 (IL-6) production impaired in rip1(-/-) murine embryonic fibroblasts (MEF) but unaffected in traf2(-/-) MEF. Yet, both rip1(-/-) and traf2(-/-) MEF exhibit a normal p38 MAP kinase response to inducers of osmotic shock or IL-1alpha. Thus, RIP1 is a specific mediator of the p38 MAP kinase response to TNF-alpha. These studies suggest that TNF-alpha-induced activation of p38 MAP kinase and SAPK/JNK pathways bifurcate at the level of RIP1 and TRAF2. Moreover, endogenous RIP1 associates with the MAP kinase kinase kinase (MAP3K) MEKK3 in TNF-alpha-treated cells, and decreased TNF-alpha-induced p38 MAP kinase activation is observed in Mekk3(-/-) cells. Taken together, these studies suggest a mechanism whereby RIP1 may mediate the p38 MAP kinase response to TNF-alpha, by recruiting the MAP3K MEKK3.

We identified Ran-binding protein (RanBPM) as an interacting partner of the caspase-processed C-terminal domain of cyclin-dependent kinase 11 (CDK11(p46)) by using the yeast two-hybrid system. CDK11(p110) protein kinases are members of the cyclin-dependent kinase superfamily. During staurosporine-, Fas-, and tumor necrosis factor alpha-induced apoptosis caspase-processed activated CDK11(p46) is generated from larger CDK11(p110) isoforms. CDK11(p46) promotes apoptosis when it is ectopically expressed in human cells. However, the mechanism of signal transduction through CDK11(p46) is still unclear. In this study, we demonstrate that CDK11(p46) directly interacts with RanBPM in vitro and in human cells. RanBPM contains a conserved SPRY (repeats in splA and Ryr) domain and is localized both in the nucleus and cytoplasm. The SPRY domain of RanBPM is responsible for the association between CDK11(p46) and RanBPM. Furthermore, we show that CDK11(46) phosphorylates RanBPM.

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34(cdc2)-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. However, substrates of CDK11 during apoptosis have not been identified. We used a yeast two-hybrid screening strategy and identified eukaryotic initiation factor 3 p47 protein (eIF3 p47) as an interacting partner of caspase-processed C-terminal kinase domain of CDK11 (CDK11(p46)). We demonstrate that the eIF3 p47 can interact with CDK11 in vitro and in vivo, and the interaction can be strengthened by stimulation of apoptosis. EIF3 p47 contains a Mov34/JAB domain and appears to interact with CDK11(p46) through this motif. We show in vitro that the caspase-processed CDK11(p46) can phosphorylate eIF3 p47 at a specific serine residue (Ser(46)) and that eIF3 p47 is phosphorylated in vivo during apoptosis. Purified recombinant CDK11(p46) inhibited translation of a reporter gene in vitro in a dose-dependent manner. In contrast, a kinase-defective mutant CDK11(p46M) did not inhibit translation of the reporter gene. Stable expression of CDK11(p46) in vivo inhibited the synthesis of a transfected luciferase reporter protein and overall cellular protein synthesis. These data provide insight into the cellular function of CDK11 during apoptosis.

Much effort has focused on the identification of MAPK cascades that are activated by the MEKK family of protein kinases. However, direct phosphorylation and regulation of the MEKK proteins has not been shown. To address this question, we have expressed recombinant (His)6FLAG.MEKK3 in Sf9 insect cells and tethered the purified protein to Ni-Sepharose so that we could precipitate interacting proteins and then identify such proteins by liquid chromatography and mass spectrometry (LC-MS). We identified 14-3-3 proteins as interacting with MEKK3, which suggested that (His)6FLAG.MEKK3 was phosphorylated on serine since 14-3-3 proteins are known to associate with phosphorylated proteins. We identified two phosphorylated amino acids at Ser166 and Ser337 of tryptic peptides derived from (His)6FLAG.MEKK3 by using LC-MS. Antibodies were developed that recognize the specific phosphorylated amino acid and with these antibodies, we demonstrate that various stimuli (tumor necrosis factor, arsenite, forskolin, and serum) promote phosphorylation of Ser166 and Ser337. However, neither of these phosphorylated amino acids is required for association with 14-3-3 protein or regulation of MEKK3-dependent ERK and JNK activity. Nonetheless, these results suggest that MEKK3 is a convergence point of multiple upstream signaling pathways.

Associated with the metastatic progression of epithelial tumors is the dynamic regulation of cadherins. Whereas E-cadherin is expressed in most epithelium and carcinomas, recent studies suggest that the up-regulation of other cadherin subtypes in carcinomas, such as N-cadherin, may function in cancer progression. We demonstrate that a signal transduction cascade links the N-cadherin.catenin adhesion complex to up-regulation of the anti-apoptotic protein Bcl-2. In suspension, aggregates of DU-145 cells, an E-cadherin expressing human prostate carcinoma line, survive loss of integrin-dependent adhesion by a different anti-apoptotic signaling pathway than the N-cadherin expressing lines PC3 and PC3N. N-cadherin intercellular adhesion mediates a 3.5-fold increase in Bcl-2 protein expression, whereas the level of the proapoptotic protein Bax remains constant. Only N-cadherin ligation in PC3 cells, which express both N-cadherin and E-cadherin, is sufficient to induce activation of Akt/protein kinase B. N-cadherin homophilic ligation initiates phosphatidylinositol 3-kinase-dependent activation of Akt resulting in Akt phosphorylation of Bad on serine 136. Following N-cadherin homophilic adhesion phosphatidylinositol 3-kinase was identified in immunoprecipitates of the N-cadherin.catenin complex. The recruitment of phosphatidylinositol 3-kinase to the adhesion complex is dependent on ligation of N-cadherin and an organized actin cytoskeleton because cytochalasin D blocks the recruitment. We propose that N-cadherin homophilic adhesion can initiate anti-apoptotic signaling, which enhances the Akt cell survival pathway in metastatic cancer.

Arsenate and arsenite activate c-Jun N-terminal kinase (JNK), however, the mechanism by which this occurs is not known. By expressing inhibitory mutant small GTP-binding proteins, p21-activated kinase (PAK) and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinases (MEKKs), we have identified specific proteins that are involved in arsenate- and arsenite-mediated activation of JNK. We observe a distinct difference between arsenate and arsenite signaling, which demonstrates that arsenate and arsenite are capable of activating unique proteins. Both arsenate and arsenite activation of JNK requires Rac and Rho. Neither arsenate nor arsenite signaling was inhibited by a dominant-negative mutant of Cdc42 or Ras. Arsenite stimulation of JNK requires PAK, whereas arsenate-mediated activation of JNK was unaffected by inhibitory mutant PAK. Of the four MEKKs tested, only MEKK3 and MEKK4 are involved in arsenate-mediated activation of JNK. In contrast, arsenite-mediated JNK activation requires MEKK2, MEKK3 and MEKK4. These results better define the mechanisms by which arsenate and arsenite activate JNK and demonstrate differences in the regulation of signal transduction pathways by these inorganic arsenic species.

Oncogenesis is a complicated process involving signal transduction pathways that mediate many different physiological events. Typically, oncogenes cause unregulated cell growth and this phenotype has been attributed to the growth-stimulating activity of oncogenes such as ras and src. In recent years, much research effort has focused on proteins that function downstream of Ras, leading to the identification of the Ras/Raf/MAPK pathway, because activation of this pathway leads to cellular proliferation. Activated receptor tyrosine kinases (RTKs) also utilize this pathway to mediate their growth-stimulating effects. However, RTKs activate many other signaling proteins that are not involved in the cellular proliferation process, per se and we are learning that these pathways also contribute to the oncogenic process. In fact, RTKs and many of the proteins involved in RTK-dependent signal transduction can also function as oncogenes. For example, the catalytic subunit of phosphoinositide 3-kinase (P13-K) was recently identified as an oncogenic protein. The scope of pathways that are activated by oncogenic RTKs is expanding. Thus, not only do RTKs activate Ras-dependent pathways that drive proliferation, RTKs activate P13-K-dependent pathways which also contribute to the oncogenic mechanism. P13-K can initiate changes in gene transcription, cytoskeletal changes through beta-catenin, changes in cell motility through the tumor suppressor, adenomatous polyposis coli (APC), and phosphorylation of BAD, a protein involved in apoptotic and antiapoptotic signaling. There is also cross-talk between RTKs and the oncostatin cytokine receptor which may positively and negatively influence oncogenesis. For this review, we will focus on oncogenic RTKs and the network of cellular proteins that are activated by RTKs because multiple, divergent pathways are responsible for oncogenesis.

Activation of receptor tyrosine kinases is thought to involve ligand-induced dimerization, which promotes receptor transphosphorylation and thereby increases the receptor's phosphotransferase activity. We used two platelet-derived growth factor beta-receptor (beta-PDGFR) mutants to identify events that are required for full engagement (autophosphorylation and activation of the kinase activity) of the beta-PDGFR kinase. The F79/81 receptor (Tyr to Phe substitution at 579 and 581 in the juxtamembrane domain of the receptor) was capable of only very modest ligand-dependent autophosphorylation and also failed to associate with numerous SH2 domain-containing proteins. Furthermore, stimulation with platelet-derived growth factor (PDGF) did not increase the kinase activity of the F79/81 mutant toward exogenous substrates. However, the F79/81 receptor had basal kinase activity and could be artificially stimulated by incubation with ATP. Because the low kinase activity of the F857 mutant (Tyr to Phe substitution at 857 in the putative activation loop) could not be increased by incubation with ATP, failure to phosphorylate Tyr-857 may be the reason why the F79/81 mutant has low kinase activity. Surprisingly, the F857 mutant underwent efficient PDGF-dependent autophosphorylation. Thus the PDGF-dependent increase in the kinase activity of the receptor is not required for autophosphorylation. We conclude that full activation of the beta-PDGFR kinase requires at least two, apparently distinct events.

MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) kinases (MEKKs) regulate c-Jun N-terminal kinase and extracellular response kinase pathways. The 14-3-3zeta and 14-3-3epsilon isoforms were isolated in a two-hybrid screen for proteins interacting with the N-terminal regulatory domain of MEKK3. 14-3-3 proteins bound both the N-terminal regulatory and C-terminal kinase domains of MEKK3. The binding affinity of 14-3-3 for the MEKK3 N terminus was 90 nM, demonstrating a high affinity interaction. 14-3-3 proteins also interacted with MEKK1 and MEKK2, but not MEKK4. Endogenous 14-3-3 protein and MEKK1 and MEKK2 were similarly distributed in the cell, consistent with their in vitro interactions. MEKK1 and 14-3-3 proteins colocalized using two-color digital confocal immunofluorescence. Binding of 14-3-3 proteins mapped to the N-terminal 393 residues of 196-kDa MEKK1. Unlike MEKK2 and MEKK3, the C-terminal kinase domain of MEKK1 demonstrated little or no ability to interact with 14-3-3 proteins. MEKK1, but not MEKK2, -3 or -4, is a caspase-3 substrate that when cleaved releases the kinase domain from the N-terminal regulatory domain. Functionally, caspase-3 cleavage of MEKK1 releases the kinase domain from the N-terminal 14-3-3-binding region, demonstrating that caspases can selectively alter protein kinase interactions with regulatory proteins. With regard to MEKK1, -2 and -3, 14-3-3 proteins do not appear to directly influence activity, but rather function as "scaffolds" for protein-protein interactions.

Growth factor receptor tyrosine kinase (RTK)-activated signaling pathways are well established regulators of neuronal growth and development, but whether these signals provide mechanisms for acute modulation of neuronal activity is just beginning to be addressed. We show in pheochromocytoma (PC12) cells that acute application of ligands for both endogenous RTKs [trkA, basic FGF (bFGF) receptor, and epidermal growth factor (EGF) receptor] and ectopically expressed platelet-derived growth factor (PDGF) receptors rapidly inhibits whole-cell sodium channel currents, coincident with a hyperpolarizing shift in the voltage dependence of inactivation. Sodium channel inhibition by trkA and PDGF receptors is mutually occlusive, suggestive of a common signal transduction mechanism. Furthermore, specific inhibitors for trkA and PDGF RTK activities abrogate sodium channel inhibition in response to NGF and PDGF, respectively, showing that the intrinsic RTK activity of these receptors is necessary for sodium channel inhibition. Use of PDGF receptor mutants deficient for specific signaling activities demonstrated that this inhibition is dependent on RTK interaction with Src but not with other RTK-associated signaling molecules. Inhibition was also compromised in cells expressing dominant-negative Ras. These results suggest a possible mechanism for acute physiological actions of RTKs, and they indicate regulatory functions for Ras and Src that may complement the roles of these signaling proteins in long-term neuronal regulation.

The mechanisms governing neuronal differentiation, including the signals underlying the induction of voltage-dependent sodium (Na+) channel expression by neurotrophic factors, which occurs independent of Ras activity, are not well understood. Therefore, Na+ channel induction was analyzed in sublines of PC12 cells stably expressing platelet-derived growth factor (PDGF) beta receptors with mutations that eliminate activation of specific signalling molecules. Mutations eliminating activation of phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein (GAP), and Syp phosphatase failed to diminish the induction of type II Na+ channel alpha-subunit mRNA and functional Na+ channel expression by PDGF, as determined by RNase protection assays and whole-cell patch clamp recording. However, mutation of juxtamembrane tyrosines that bind members of the Src family of kinases upon receptor activation inhibited the induction of functional Na+ channels while leaving the induction of type II alpha-subunit mRNA intact. Mutation of juxtamembrane tyrosines in combination with mutations eliminating activation of PI3K, PLC gamma, GAP, and Syp abolished the induction of type II alpha-subunit mRNA, suggesting that at least partially redundant signaling mechanisms mediate this induction. The differential effects of the receptor mutations on Na+ channel expression did not reflect global changes in receptor signaling capabilities, as in all of the mutant receptors analyzed, the induction of c-fos and transin mRNAs still occurred. The results reveal an important role for the Src family in the induction of Na+ channel expression and highlight the multiplicity and combinatorial nature of the signaling mechanisms governing neuronal differentiation.

The PDGF beta-receptor in which the active-site lysine in the kinase domain has been mutated to arginine (K634R) tacks intrinsic kinase activity. When expressed in HepG2 cells, the kinase-inactive PDGF beta-receptor was tyrosine phosphorylated in response to PDGF-BB. Previously, HepG2 cells were thought to be devoid of PDGF alpha-receptor primarily due to lack of specific antibody which precluded detection of the PDGF alpha-receptor. In fact, these cells express low levels of PDGF alpha-receptor. In HepG2 cells that express the kinase-inactive PDGF beta-receptor, PDGF-BB activates the PDGF alpha-receptors to trans phosphorylate the kinase-inactive PDGF beta-receptor in an intermolecular fashion. As a result, stimulation of HepG2 cells that express the kinase-inactive receptor leads to activation of serine/threonine kinases of the MAP kinase cascade which include RAF-1, MEK-1 and p42 MAP kinase. In contrast, the kinase-inactive receptor does not activate any signaling pathways when it is expressed in PC12 cells which do not express the endogenous PDGF alpha-receptor. Thus, the kinase-inactive K634R PDGF beta-receptor is able to enhance PDGF-BB signaling in HepG2 cells that express the PDGF alpha-receptor.

Recently, we reported the synthesis and use of [32P]2-azido-NAD+ as a probe to study the structural organization of G-proteins. Pertussis toxin was used to 'tether' [32P]2-azido-ADP-ribose of [32P]2-azido-NAD+ to Cys347 of the alpha subunit of the G-protein Gt. Light activation of the azide moiety covalently cross-linked the domain containing Cys347 at the C-terminus of alpha t with neighbouring intra- and inter-molecular domains of holo-transducin. The radiolabel from [32P]2-azido-ADP-ribose was then transferred to the 'acceptor' domain by cleaving the thioglycosidic bond between Cys347 and [32P]2-azido-ADP- ribose with mercuric acetate. ADP-ribosylation followed by photocross-linking of holo-transducin indicated intramolecular interactions of the C-terminal domain with other alpha t domains and intermolecular interactions with holotransducin alpha and gamma subunits. The radiolabelled peptides, which were radiolabelled because of the transfer of the photoactive moiety, were identified by utilizing 2-(2'-nitrophenylsulphenyl)-3-methyl-3'- bromoindolenine ('BNPS-skatole') and CNBr. The results indicate that the C-terminus of alpha t interacts with both N-terminal and C-terminal domains within the alpha t molecular. Mapping the interacting sites between cross-linked alpha dimers and alpha trimers indicates that the C-terminal domain of alpha t is involved in the formation of alpha t homopolymers in solution. In addition, our studies place the beta gamma subunit in close proximity to Cys347 of alpha t, as indicated by the transfer of [32P]2-azido-ADP-ribose from Cys347 to the gamma subunit, which was further localized to the C-terminal half of gamma t. The studies presented here identify the C-terminal intra- and inter-molecular interactions of the alpha subunit of holo-transducin.

When expressed in PC12 cells, the platelet-derived growth factor beta receptor (beta PDGF-R) mediates cell differentiation. Mutational analysis of the beta PDGF-R indicated that persistent receptor stimulation of the Ras/Raf/mitogen-activated protein (MAP) kinase pathway alone was insufficient to sustain PC12 cell differentiation. PDGF receptor activation of signal pathways involving p60c-src or the persistent regulation of phospholipase C gamma was required for PC12 cell differentiation. beta PDGF-R regulation of phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, and the tyrosine phosphatase, Syp, was not required for PC12 cell differentiation. In contrast to overexpression of oncoproteins involved in regulating the MAP kinase pathway, growth factor receptor-mediated differentiation of PC12 cells requires the integration of other signals with the Ras/Raf/MAP kinase pathway.

The E5 protein of bovine papillomavirus type 1 binds to and activates the endogenous platelet-derived growth factor (PDGF) beta receptor in fibroblasts, resulting in cell transformation. We have developed a functional assay to test the ability of PDGF beta receptor mutants to mediate a mitogenic signal initiated by the E5 protein. Lymphoid Ba/F3 cells are strictly dependent on interleukin-3 for growth, but coexpression of the wild-type PDGF beta receptor and the E5 or v-sis-encoded protein generated a mitogenic signal which allowed Ba/F3-derived cells to proliferate in the absence of interleukin-3. In these cells, the E5 protein bound to and caused increased tyrosine phosphorylation of both the mature and the precursor forms of the wild-type PDGF beta receptor. The tyrosine kinase activity of the receptor was necessary for E5-induced receptor tyrosine phosphorylation and mitogenic activity but not for complex formation with the E5 protein. In contrast, the PDGF-binding domain of the receptor was not required for complex formation with the E5 protein, E5-induced tyrosine phosphorylation or mitogenic activity, demonstrating that E5-mediated receptor activation is ligand independent. Analysis of receptor mutants lacking various combinations of tyrosine phosphorylation sites revealed that the E5 and v-sis-encoded proteins display similar requirements for signaling and suggested that the wild-type PDGF beta receptor can generate multiple independent mitogenic signals. Importantly, these mutants dissociated two activities of the PDGF beta receptor tyrosine kinase, both of which are required for sustained mitogenic signaling: (i) receptor autophosphorylation and creation of binding sites for SH2 domain-containing proteins and (ii) phosphorylation of substrates other than the receptor itself.

Growth factor receptor tyrosine kinase regulation of the sequential phosphorylation reactions leading to mitogen-activated protein (MAP) kinase activation in PC12 cells has been investigated. In response to epidermal growth factor, nerve growth factor, and platelet-derived growth factor, B-Raf and Raf-1 are activated, phosphorylate recombinant kinase-inactive MEK-1, and activate wild-type MEK-1. MEK-1 is the dual-specificity protein kinase that selectively phosphorylates MAP kinase on tyrosine and threonine, resulting in MAP kinase activation. B-Raf and Raf-1 are growth factor-regulated Raf family members which regulate MEK-1 and MAP kinase activity in PC12 cells. Protein kinase A activation in response to elevated cyclic AMP (cAMP) levels inhibited B-Raf and Raf-1 stimulation in response to growth factors. Ras.GTP loading in response to epidermal growth factor, nerve growth factor, or platelet-derived growth factor was unaffected by protein kinase A activation. Even though elevated cAMP levels inhibited Raf activation, the growth factor activation of MEK-1 and MAP kinase was unaffected in PC12 cells. The results demonstrate that tyrosine kinase receptor activation of MEK-1 and MAP kinase in PC12 cells is regulated by B-Raf and Raf-1, whose activation is inhibited by protein kinase A, and MEK activators, whose activation is independent of cAMP regulation.

Sequential protein kinase reactions involve the phosphorylation and activation of multiple kinases in a pathway. The growth factor receptor tyrosine kinase regulation of the mitogen-activated protein kinase (MAPK) pathway was defined in 1993. The MAPK pathway involves sequential protein kinase reactions. Notable advances were made in defining tyrosine kinase receptor regulation of Ras, and these discoveries were combined with the identification of Raf-1, a serine-threonine protein kinase in the MAPK pathway, as an effector for Ras GTP.

MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK-1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.

Mitogen-activated protein kinases (MAPKs) are rapidly phosphorylated and activated in response to a variety of extracellular stimuli in many different cell types. The kinases that activate MAPK, the MAPK/ERK Kinases (MEKs), are also activated by phosphorylation. We have studied the influence of specific oncogenes on the regulation of MEK activity in NIH3T3 and Rat1a fibroblasts. We show that a similar MEK activity phosphorylates and activates MAPK in both growth factor-stimulated (epidermal growth factor and thrombin) and oncogene (gip2, v-src, and v-raf)-transfected cells. Gip2 and v-Src activated MEK-1 in transfected Rat 1a cells, whereas v-Raf activated MEK-1 in transfected NIH3T3 cells. These cell-selective differences in MEK activation parallel constitutive MAPK activation in these cell lines. Stable expression of the v-ras oncogene resulted in little constitutive MEK activation in either cell line, even though both were highly transformed. The growth factor and oncoprotein regulated MEK activity co-fractionated by Mono S chromatography with the 45-kDa MEK-1 protein. We further demonstrate in NIH3T3 and Rat 1a cells that Raf-1 is activated, as measured by its ability to phosphorylate MEK-1, in response to epidermal growth factor but not thrombin. Thus, the regulatory network of protein kinases that activate MAPK converges at MEK but diverges with the kinases that phosphorylate and activate MEK.

Genetic and structural analysis of the alpha chain polypeptides of heterotrimeric G proteins defines functional domains for GTP/GDP binding, GTPase activity, effector activation, receptor contact and beta gamma subunit complex regulation. The conservation in sequence comprising the GDP/GTP binding and GTPase domains among G protein alpha subunits readily allows common mutations to be made for the design of mutant polypeptides that function as constitutive active or dominant negative alpha chains when expressed in different cell types. Organization of the effector activation, receptor and beta gamma contact domains is similar in the primary sequence of the different alpha subunit polypeptides relative to the GTP/GDP binding domain sequences. Mutation within common motifs of the different G protein alpha chain polypeptides have similar functional consequences. Thus, what has been learned with the Gs and Gi proteins and the regulation of adenylyl cyclase can be directly applied to the analysis of newly identified G proteins and their coupling to receptors and regulation of putative effector enzymes.

A radioactive and photoactivatable derivative of NAD+, 2-azido-[adenylate-32P]NAD+, has been synthesized and used with pertussis toxin to ADP-ribosylate Cys347 of the alpha subunit (alpha T) of GT, the retinal guanine nucleotide-binding protein. ADP-ribosylation of alpha T followed by light activation of the azide moiety of 2-azido-[adenylate-32P]ADP-ribose produced four crosslinked species involving the alpha and gamma subunits of the GT heterotrimer: an alpha trimer (alpha-alpha-alpha), and alpha-alpha-gamma crosslink, an alpha dimer (alpha-alpha), and an alpha-gamma crosslink. The alpha trimer, alpha-alpha-gamma complex, alpha dimer, and alpha-gamma complexes were immunoreactive with alpha T antibodies. The alpha-alpha-gamma and the alpha-gamma complexes were immunoreactive with antisera recognizing gamma subunits. No evidence was found for crosslinking of alpha T to beta T subunits. Hydrolysis of the thioglycosidic bond between Cys347 and 2-azido-[adenylate-32P]ADP-ribose using mercuric acetate resulted in the transfer of radiolabel from Cys347 of alpha T in the crosslinked oligomers to alpha monomers, indicative of intermolecular photocrosslinking, and to gamma monomers, indicative of either intermolecular crosslinked complexes (between heterotrimers) or intramolecular crosslinked complexes (within the heterotrimer). These results demonstrate that GT exists as an oligomer and that ADP-ribosylated Cys347, which is four residues from the alpha T-carboxyl terminus, is oriented toward and in close proximity to the gamma subunit.