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Malate dehydrogenases (MDHs) have been extensively studied since the 1960s due to their key roles in carbon metabolism and pathways such as redox balance and lipid synthesis. Recently, there has been renewed interest in these enzymes with the discovery of their role in the metabolic changes that occur during cancer and a widespread community of undergraduate teaching laboratories addressing MDH research questions, the Malate Dehydrogenase CUREs Community (MCC). This special issue describes different facets of MDH, including its physiological role, its structure-function relationships, its regulation through post-translational modifications, and perspectives on its evolutionary history. There are two human isoforms: a cytoplasmic isoform that carries out formation of NAD+ for glycolysis, and a mitochondrial isoform that plays a major role in the citric acid cycle. Although the sequences of these two isoforms vary, the structures of the enzymes are similar, and studies suggest that each isoform may form complexes with other enzymes in common pathways. Experimental and theoretical advances have helped to characterize the post-translational modifications of MDH, allowing us to ask more complex questions involving the regulation of the enzyme and substrate promiscuity in the context of cancer. Additionally, there are many unresolved questions on the role of malate dehydrogenase in other organisms, especially in parasites. The review articles in this issue seek to shed light on the latest advances in our understanding of MDH and highlight areas for future studies., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

In this review, we examine the protein-protein interactions of cytosolic malate dehydrogenase (MDH), an under-studied area in cellular metabolism. We provide a comprehensive overview of MDH involvement in metabolism, especially its interactions with metabolic partners and dynamics of changing metabolism. We present an analysis of the biophysical nature of these interactions and the current methods used to study them. Our review includes an assessment of computational docking studies, which offer initial hypotheses about potential MDH interaction partners. Furthermore, we provide a summary of the sparse yet insightful experimental evidence available, establishing a foundation for future research. By integrating biophysical analysis and methodological advancements, this paper aims to illuminate the intricate network of interactions involving cytosolic MDH and their metabolic implications. This work not only contributes to our understanding of MDH's role in metabolism but also highlights the potential impact of these interactions in metabolic disorders., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Malate dehydrogenase (MDH) is a key enzyme in mammalian metabolic pathways in cytosolic and mitochondrial compartments. Regulation of MDH through phosphorylation remains an underexplored area. In this review we consolidate evidence supporting the potential role of phosphorylation in modulating the function of mammalian MDH. Parallels are drawn with the phosphorylation of lactate dehydrogenase, a homologous enzyme, to reveal its regulatory significance and to suggest a similar regulatory strategy for MDH. Comprehensive mining of phosphorylation databases, provides substantial experimental (primarily mass spectrometry) evidence of MDH phosphorylation in mammalian cells. Experimentally identified phosphorylation sites are overlaid with MDH's functional domains, offering perspective on how these modifications could influence enzyme activity. Preliminary results are presented from phosphomimetic mutations (serine/threonine residues changed to aspartate) generated in recombinant MDH proteins serving as a proof of concept for the regulatory impact of phosphorylation. We also examine and highlight several approaches to probe the structural and cellular impact of phosphorylation. This review highlights the need to explore the dynamic nature of MDH phosphorylation and calls for identifying the responsible kinases and the physiological conditions underpinning this modification. The synthesis of current evidence and experimental data aims to provide insights for future research on understanding MDH regulation, offering new avenues for therapeutic interventions in metabolic disorders and cancer., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Malate dehydrogenase (MDH) is pivotal in mammalian tissue metabolism, participating in various pathways beyond its classical roles and highlighting its adaptability to cellular demands. This enzyme is involved in maintaining redox balance, lipid synthesis, and glutamine metabolism and supports rapidly proliferating cells' energetic and biosynthetic needs. The involvement of MDH in glutamine metabolism underlines its significance in cell physiology. In contrast, its contribution to lipid metabolism highlights its role in essential biosynthetic processes necessary for cell maintenance and proliferation. The enzyme's regulatory mechanisms, such as post-translational modifications, underscore its complexity and importance in metabolic regulation, positioning MDH as a potential target in metabolic dysregulation. Furthermore, the association of MDH with various pathologies, including cancer and neurological disorders, suggests its involvement in disease progression. The overexpression of MDH isoforms MDH1 and MDH2 in cancers like breast, prostate, and pancreatic ductal adenocarcinoma, alongside structural modifications, implies their critical role in the metabolic adaptation of tumor cells. Additionally, mutations in MDH2 linked to pheochromocytomas, paragangliomas, and other metabolic diseases emphasize MDH's role in metabolic homeostasis. This review spotlights MDH's potential as a biomarker and therapeutic target, advocating for further research into its multifunctional roles and regulatory mechanisms in health and disease., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Course-based undergraduate research experiences (CUREs) are laboratory courses that integrate broadly relevant problems, discovery, use of the scientific process, collaboration, and iteration to provide more students with research experiences than is possible in individually mentored faculty laboratories. Members of the national Malate dehydrogenase CUREs Community (MCC) investigated the differences in student impacts between traditional laboratory courses (control), a short module CURE within traditional laboratory courses (mCURE), and CUREs lasting the entire course (cCURE). The sample included approximately 1,500 students taught by 22 faculty at 19 institutions. We investigated course structures for elements of a CURE and student outcomes including student knowledge, student learning, student attitudes, interest in future research, overall experience, future GPA, and retention in STEM. We also disaggregated the data to investigate whether underrepresented minority (URM) outcomes were different from White and Asian students. We found that the less time students spent in the CURE the less the course was reported to contain experiences indicative of a CURE. The cCURE imparted the largest impacts for experimental design, career interests, and plans to conduct future research, while the remaining outcomes were similar between the three conditions. The mCURE student outcomes were similar to control courses for most outcomes measured in this study. However, for experimental design, the mCURE was not significantly different than either the control or cCURE. Comparing URM and White/Asian student outcomes indicated no difference for condition, except for interest in future research. Notably, the URM students in the mCURE condition had significantly higher interest in conducting research in the future than White/Asian students., Competing Interests: This work was supported by a grant for the National Science Foundations. NSF-1726932 EHR-IUSE, Principal Investigator J. Ellis Bell, Co-Investigators Jessica K. Bell and Joseph J. Provost. None of the authors have a financial, personal, or professional conflict of interest related to this work. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 DeChenne-Peters et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The implementation of course-based undergraduate research experiences (CUREs) has made it possible to expose large undergraduate populations to research experiences. For these research experiences to be authentic, they should reflect the increasingly collaborative nature of research. While some CUREs have expanded, involving multiple schools across the nation, it is still unclear how a structured extramural collaboration between students and faculty from an outside institution affects student outcomes. In this study, we established three cohorts of students: 1) no-CURE, 2) single-institution CURE (CURE), and 3) external collaborative CURE (ec-CURE), and assessed academic and attitudinal outcomes. The ec-CURE differs from a regular CURE in that students work with faculty member from an external institution to refine their hypotheses and discuss their data. The sharing of ideas, data, and materials with an external faculty member allowed students to experience a level of collaboration not typically found in an undergraduate setting. Students in the ec-CURE had the greatest gains in experimental design; self-reported course benefits; scientific skills; and science, technology, engineering, and mathematics (STEM) importance. Importantly this study occurred in a diverse community of STEM disciplinary faculty from 2- and 4-year institutions, illustrating that exposing students to structured external collaboration is both feasible and beneficial to student learning.

Integrating research into the classroom environment is an influential pedagogical tool to support student learning, increase retention of STEM students, and help students identify as scientists. The evolution of course-based undergraduate research experiences (CUREs) has grown from individual faculty incorporating their research in the teaching laboratory into well-supported systems to sustain faculty engagement in CUREs. To support the growth of protein-centric biochemistry-related CUREs, we cultivated a community of enthusiastic faculty to develop and adopt malate dehydrogenase (MDH) as a CURE focal point. The MDH CURE Community has grown into a vibrant and exciting group of over 28 faculty from various institutions, including community colleges, minority-serving institutions, undergraduate institutions, and research-intensive institutions in just 4 years. This collective has also addressed important pedagogical questions on the impact of CURE collaboration and the length of the CURE experience in community colleges, undergraduate institutions, and research-intensive institutions. This work provided evidence that modular or partial-semester CUREs also support student outcomes, especially the positive impact it had on underrepresented students. We are currently focused on expanding the MDH CURE Community network by generating more teaching and research materials, creating regional hubs for local interaction and increasing mentoring capacity, and offering mentoring and professional development opportunities for new faculty adopters., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

Aims: Determine the effect of palmitoylation on the sodium hydrogen exchanger isoform 1 (NHE1), a member of the SLC9 family., Main Methods: NHE1 expressed in native rat tissues or in heterologous cells was assessed for palmitoylation by acyl-biotinyl exchange (ABE) and metabolic labeling with [ 3 H]palmitate. Cellular palmitoylation was inhibited using 2-bromopalmitate (2BP) followed by determination of NHE1 palmitoylation status, intracellular pH, stress fiber formation, and cell migration. In addition, NHE1 was activated with LPA treatment followed by determination of NHE1 palmitoylation status and LPA-induced change in intracellular pH was determined in the presence and absence of preincubation with 2BP., Key Findings: In this study we demonstrate for the first time that NHE1 is palmitoylated in both cells and rat tissue, and that processes controlled by NHE1 including intracellular pH (pH i ), stress fiber formation, and cell migration, are regulated in concert with NHE1 palmitoylation status. Importantly, LPA stimulates NHE1 palmitoylation, and 2BP pretreatment dampens LPA-induced increased pH i which is dependent on the presence of NHE1., Significance: Palmitoylation is a reversible lipid modification that regulates an array of critical protein functions including activity, trafficking, membrane microlocalization and protein-protein interactions. Our results suggest that palmitoylation of NHE1 and other control/signaling proteins play a major role in NHE1 regulation that could significantly impact multiple critical cellular functions., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The COVID-19 pandemic has challenged undergraduate instructors and students in an unprecedented manner. Each has needed to find creative ways to continue the engaged teaching and learning process in an environment defined by physical separation and emotional anxiety and uncertainty. As a potential tool to meet this challenge, we developed a set of curricular materials that combined our respective life science teaching interests with the real-time scientific problem of the COVID-19 pandemic in progress. Discrete modules were designed that are engaging to students, implement active learning-based coursework in a variety of institutional and learning settings, and can be used either in person or remotely. The resulting interdisciplinary curriculum, dubbed "COVID-360," enables instructors to select from a menu of curricular options that best fit their course content, desired activities, and mode of class delivery. Here we describe how we devised the COVID-360 curriculum and how it represents our efforts to creatively and effectively respond to the instructional needs of diverse students in the face of an ongoing instructional crisis., (©2021 Author(s). Published by the American Society for Microbiology.)

Approaches to learning and teaching have been undergoing massive changes. Technology has enabled many innovations while other methods have embedded authentic research approaches or looked to other disciplines. The tools in education session of the conference looked at tools being used to teach biochemistry and molecular biology ranging from online platforms, authentic research experiences to the use of music., (© 2020 International Union of Biochemistry and Molecular Biology.)

Accreditation of academic programs and recognition of student degrees provide academic institutions a measure of a set of community agreed upon standards. These can aid pedagogical change, support faculty to successfully engage students in their discipline and to provide a mechanism to maintain standards. Several professional scientific societies from engineering, chemistry, and biochemistry and molecular biology have developed standards by which departments can be recognized for accreditation. As one of the members of the American Society for Biochemistry and Molecular Biology who helped develop the accreditation and standardized exams and a committee member of the American Chemical Society's Committee on Professional Training I will present the evolution of the accreditation process, discuss the benefits and challenges with being an accreditation. How these programs serve their communities and at times can hinder or be used to support potential creativity and teaching pedagogies will also be discussed., (© 2020 International Union of Biochemistry and Molecular Biology.)

Maintaining intracellular pH is crucial for preserving healthy cellular behavior and, when dysregulated, results in increased proliferation, migration, and invasion. The Na + /H + exchanger isoform 1 is a highly regulated transmembrane antiporter that maintains pH homeostasis by exporting protons in response to intra- and extracellular signals. Activation of Na + /H + exchanger isoform 1 is exquisitely regulated by the extracellular environment and protein cofactors, including calcineurin B homologous proteins 1 and 2. While Na + /H + exchanger isoform 1 and calcineurin B homologous protein 1 are ubiquitously expressed, calcineurin B homologous protein 2 shows tissue-specific expression and upregulation in a variety of cancer cells. In addition, calcineurin B homologous protein 2 expression is modulated by tumorigenic extracellular conditions like low nutrients. To understand the role of calcineurin B homologous protein 2 in tumorigenesis and survival in lung cancer, we surveyed existing databases and formed a comprehensive report of Na + /H + exchanger isoform 1, calcineurin B homologous protein 1, and calcineurin B homologous protein 2 expression in diseased and non-diseased tissues. We show that calcineurin B homologous protein 2 is upregulated during oncogenesis in many adeno and squamous carcinomas. To understand the functional role of calcineurin B homologous protein 2 upregulation, we evaluated the effect of Na + /H + exchanger isoform 1 and calcineurin B homologous protein 2 depletion on cellular function during cancer progression in situ. Here, we show that calcineurin B homologous protein 2 functions through Na + /H + exchanger isoform 1 to effect cell proliferation, cell migration, steady-state pH i , and anchorage-independent tumor growth. Finally, we present evidence that calcineurin B homologous protein 2 depletion in vivo has potential to reduce tumor burden in a xenograft model. Together, these data support the tumor-promoting potential of aberrant calcineurin B homologous protein 2 expression and position calcineurin B homologous protein 2 as a potential therapeutic target for the treatment of non-small cell lung cancer.

The American Society for Biochemistry and Molecular Biology (ASBMB) began an accreditation program in 2013. The criteria for accreditation of undergraduate programs include sufficient infrastructure - number and expertise of faculty, physical space and equipment, support for faculty and students - and incorporation of core concepts in the curriculum - structure and function of biomolecules; information storage; energy transfer; and quantitative skills. Students in accredited programs are able to have their degrees ASBMB certified by taking an exam focused on knowledge or skills across the four core concept areas. Members of the accreditation committees administered a survey to key stakeholders in the BMB community: undergraduate programs, both those that have applied for accreditation and those that have not; alumni/ae of accredited programs; graduate and professional programs; and employers. The goals of the study were to gauge the success of the program and determine necessary areas of improvement. The results indicate that the major benefits of applying for accreditation are the impetus to gather data and analysis not generally collected, and access to assessment data via the exam. However, stakeholders outside of the undergraduate community showed little awareness of the accreditation program. Additionally, the application process itself was seen to be very time consuming. This feedback will be used to improve the process and engage in further outreach. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):464-471, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

The sodium hydrogen exchanger isoform one (NHE1) plays a critical role coordinating asymmetric events at the leading edge of migrating cells and is regulated by a number of phosphorylation events influencing both the ion transport and cytoskeletal anchoring required for directed migration. Lysophosphatidic acid (LPA) activation of RhoA kinase (Rock) and the Ras-ERK growth factor pathway induces cytoskeletal reorganization, activates NHE1 and induces an increase in cell motility. We report that both Rock I and II stoichiometrically phosphorylate NHE1 at threonine 653 in vitro using mass spectrometry and reconstituted kinase assays. In fibroblasts expressing NHE1 alanine mutants for either Rock (T653A) or ribosomal S6 kinase (Rsk; S703A) we show that each site is partially responsible for the LPA-induced increase in transport activity while NHE1 phosphorylation by either Rock or Rsk at their respective site is sufficient for LPA stimulated stress fiber formation and migration. Furthermore, mutation of either T653 or S703 leads to a higher basal pH level and a significantly higher proliferation rate. Our results identify the direct phosphorylation of NHE1 by Rock and suggest that both RhoA and Ras pathways mediate NHE1-dependent ion transport and migration in fibroblasts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

To enhance the preparedness of graduates from the Biochemistry and Biotechnology (BCBT) Major at Minnesota State University Moorhead for employment in the bioscience industry we have developed a new Industry certificate program. The BCBT Industry Certificate was developed to address specific skill sets that local, regional, and national industry experts identified as lacking in new B.S. and B.A. biochemistry graduates. The industry certificate addresses concerns related to working in a regulated industry such as Good Laboratory Practices, Good Manufacturing Practices, and working in a Quality System. In this article we specifically describe how we developed a validation course that uses Standard Operating Procedures to describe grading policy and laboratory notebook requirements in an effort to better prepare students to transition into industry careers., (© 2013 by The International Union of Biochemistry and Molecular Biology.)

The sodium hydrogen exchanger isoform one is a critical regulator of intracellular pH, serves as an anchor for the formation of cytoplasmic signaling complexes, and modulates cytoskeletal organization. There is a growing interest in the potential for sodium hydrogen exchanger isoform one as a therapeutic target against cancer. Sodium hydrogen exchanger isoform one transport drives formation of membrane protrusions essential for cell migration and contributes to the establishment of a tumor microenvironment that leads to the rearrangement of the extracellular matrix further supporting tumor progression. Here, we focus on the potential impact that an inexpensive, $100 genome would have in identifying prospective therapeutic targets to treat tumors based upon changes in gene expression and variation of sodium hydrogen exchanger isoform one regulators. In particular, we will focus on the ezrin, radixin, moesin family proteins, calcineurin B homologous proteins, Ras/Raf/MEK/ERK signaling, and phosphoinositide signaling as they relate to the regulation of sodium hydrogen exchanger isoform one in cancer progression., (© 2012 John Wiley & Sons A/S.)

BACKGROUND: Non-small cell lung cancers (NSLC) are aggressive cancers that are insensitive to chemotherapies and accounts for nearly 33% of all cancer deaths in the United States. Two hallmarks of cancer that allow cells to invade and metastasize are sustained proliferation and enhanced motility. In this study we investigate the relationship between urokinase plasminogen activator (uPA)/uPA receptor (uPAR) signaling and Na(+)/H(+) exchanger isoform 1 (NHE1) expression and activity. METHODS AND RESULTS: The addition of 10nM uPA increased the carcinogenic potential of three NSCLC cell lines, NCI-H358, NCI-H460, and NCI-H1299. This included an increase in the rate of cell proliferation 1.6 to 1.9 fold; an increase in the percentage of cells displaying stress fibers 3.05 to 3.17 fold; and an increase in anchorage-independent growth from 1.64 to 2.0 fold. In each of these cases the increase was blocked when the experiments were performed with NHE1 inhibited by 10 μM EIPA (ethylisopropyl amiloride). To further evaluate the role of uPA/uPAR and NHE1 in tumor progression we assessed signaling events using full-length uPA compared to the uPA amino terminal fragment (ATF). Comparing uPA and ATF signaling in H460 cells, we found that both uPA and ATF increased stress fiber formation approximately 2 fold, while uPA increased matrix metalloproteinase 9 (MMP9) activity 5.44 fold compared to 2.81 fold for ATF. To expand this signaling study, two new cell lines were generated, one with reduced NHE1 expression (H460 NHE1 K/D) and one with reduced uPAR expression (H460 uPAR K/D). Using the K/D cell lines we found that neither uPA nor ATF could stimulate stress fiber formation or MMP9 activity in cells with dramatically decreased NHE1 or uPAR expression. Finally, using in vivo tumor formation studies in athymic mice we found that when mice were injected with H460 cells 80% of mice formed tumors with an average volume of 390 mm(3). This was compared to 20% of H460 uPAR K/D injected mice forming tumors with an average volume of 15 mm(3) and 10% of H460 NHE1 K/D injected mice forming tumors with an average volume of 5 mm(3). CONCLUSION: Taken together, these data demonstrate that uPA/uPAR-mediated tumor progression and metastasis requires NHE1 in NSCLC cells and suggests a potential therapeutic approach to blocking cancer progression.

Four gas-permeable wrapping films exhibiting different degrees of water permeability (ranging from 1.6 to 500 g/m(2) per d) were tested to study their effect on soft-mold (Camembert-type) cheese-ripening dynamics compared with unwrapped cheeses. Twenty-three-day trials were performed in 2 laboratory-size (18L) respiratory-ripening cells under controlled temperature (6 ± 0.5°C), relative humidity (75 ± 2%), and carbon dioxide content (0.5 to 1%). The films allowed for a high degree of respiratory activity; no limitation in gas permeability was observed. The wide range of water permeability of the films led to considerable differences in cheese water loss (from 0.5 to 12% on d 23, compared with 15% for unwrapped cheeses), which appeared to be a key factor in controlling cheese-ripening progress. A new relationship between 2 important cheese-ripening descriptors (increase of the cheese core pH and increase of the cheese's creamy underrind thickness) was shown in relation to the water permeability of the wrapping film. High water losses (more than 10 to 12% on d 23) also were observed for unwrapped cheeses, leading to Camembert cheeses that were too dry and poorly ripened. On the other hand, low water losses (from 0.5 to 1% on d 23) led to over-ripening in the cheese underrind, which became runny as a result. Finally, water losses from around 3 to 6% on d 23 led to good ripening dynamics and the best cheese quality. This level of water loss appeared to be ideal in terms of cheese-wrapping film design., (Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Research-based learning in a teaching environment is an effective way to help bring the excitement and experience of independent bench research to a large number of students. The program described here is the second of a two-semester biochemistry laboratory series. Here, students are empowered to design, execute and analyze their own experiments for the entire semester. This style of laboratory replaces a variety of shorter labs in favor of an in depth research-based learning experience. The concept is to allow students to function in independent research groups. The research projects are focused on a series of wild-type and mutant clones of malate dehydrogenase. A common research theme for the laboratory helps instructors administer the course and is key to delivering a research opportunity to a large number of students. The outcome of this research-based learning laboratory results in students who are much more confident and skilled in critical areas in biochemistry and molecular biology. Students with research experience have significantly higher confidence and motivation than those students without a previous research experience. We have also found that all students performed better in advanced courses and in the workplace., (Copyright © 2010 International Union of Biochemistry and Molecular Biology, Inc.)

A successful laboratory experience provides the foundation for student success, creating active participation in the learning process. Here, we describe a new approach that emphasizes research, inquiry and problem solving in a year-long biochemistry experience. The first semester centers on the purification, characterization, and analysis of a novel fusion protein within a guided research experience. Throughout the semester, students gradually acquire skills as they are allowed to work independently. A fusion protein, malate dehydrogenase-green fluorescent protein with a histidine affinity tag (MGH), is used throughout the semester. The fusion protein allows for a high throughput analysis and is stable for duration of the semester. Students start with the purification and analysis of the plasmid DNA and end with an enzymatic analysis of MGH. As students take ownership of their experiments and choose two different chromatographic resins, they make many choices throughout the semester. Skills, motivation, confidence levels, and attitudes were assessed before and after the semester. Students achieved high levels of critical biochemical laboratory skills and critical thinking while increasing their confidence and motivation for working in a biochemical research setting., (Copyright © 2010 International Union of Biochemistry and Molecular Biology, Inc.)

Matrix metalloproteinase 9 (MMP-9) plays a critical role in digesting the extracellular matrix and has a vital function in tumor metastasis and invasion; this protease activity is significantly increased in non-small cell lung cancers. The sodium hydrogen exchanger isoform 1 (NHE1) functions as a focal point for signal coordination and cytoskeletal reorganization. NHE1 is thought to play a central role in establishing signaling components at the leading edge of a migrating cell. Therefore, we studied the relationship between NHE1 and MMP-9 activity in Chinese hamster lung fibroblasts (CCL39) stimulated with phenylephrine (PE). We show that PE increases MMP-9 gelatinolytic activity in CCL39 cells. The inhibition of phospholipase D (PLD) signaling abrogated PE-induced MMP-9 activity. The role of PLD as an essential signaling intermediate was confirmed when the addition of permeable phosphatidic acid increased MMP-9 activity in the same cells. PE-induced invasion was increased 1.9-fold over controls and the PE response was lost when 1-butanol was used to block PLD signaling. Cells pre-treated with the NHE1 inhibitor, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) prior to PE addition resulted in a notable decrease in MMP-9 activation and cell invasion as compared to untreated PE-stimulated cells. CCL39 NHE1 null cells demonstrated no increase in MMP-9 protease activity or cell invasion in response to PE treatment. Reconstitution of NHE1 expression recovered the PE-induced activation of protease activity and cell invasion. MMP-9 processing was altered in cells expressing a proton transport defective NHE1 but retained the ability to respond to PE. Conversely, cells expressing an ezrin, radixin, moesin (ERM)-binding deficient NHE1 had a lower MMP-9 activity and the protease did not respond to PE addition. Parallel studies on NCI-H358 non-small cell lung cancer (NSCL) cells showed that PE stimulated both MMP-9 activity and cell invasion in an NHE1 dependent manner. This work describes for the first time a PE-induced relationship between NHE1 and MMP-9 and a new potential mechanism by which NHE1 could promote tumor formation and metastasis.

Phospholipase D is suspected to play a role in tumorigenesis, and the inhibition of phospholipase D has been associated with changes in several cellular events including invasion and migration. We report here that the specific alpha(1)-adrenergic receptor agonist, phenylepherine, signals to a growth factor pathway in a manner that requires phospholipase D activity in CCL39 fibroblasts. Phenylepherine increased extracellular signal-regulated kinase phosphorylation eightfold and promoted stress fiber formation threefold. Stress fiber formation was blocked when extracellular signal-regulated kinase activation was inhibited. Stimulation of CCL39 fibroblasts by phenylepherine increased the rate of wound healing fourfold in a wounding assay, while treatment with the MEK inhibitor, PD98059 reduced the closure of phenylepherine-induced wound healing to control levels. Addition of 1-butanol but not 2-butanol inhibited extracellular signal-regulated kinase activation by phenylepherine, presumably by blocking the formation of phosphatidic acid. Exogenously added cell permeable phosphatidic acid increased extracellular signal-regulated kinase activation in a time- and dose-dependent manner as well as stimulated the formation of stress fibers. 1-butanol also significantly inhibited the ability of phenylepherine to stimulate stress fiber formation and wound healing. Taken together, these results indicate a novel role for phospholipase D in the activation of the extracellular signal-regulated kinase growth factor pathway to stimulate early cellular events induced by phenylepherine.

The activation of the Na+-H+ exchanger 1 (NHE1) and extracellular-signal regulated kinase (ERK) phosphorylation in Chinese hamster lung fibroblasts (CCL39) was characterized in response to the specific alpha1-adrenergic agonist, phenylephrine (PE). Addition of 100 micromol PE/L increased the steady-state intracellular pH (pHi) by 0.16 +/- 0.03 pH units, as well as increasing the phosphorylation of ERK. The response of NHE1 to PE in CCL39 cells was determined by the use of specific antagonists. Use of 2 specific chemical inhibitors of phosphoinositide-specific phospholipase C (PLC) reduced the ability of PE to activate either the exchanger or ERK. Studies were conducted in PLCbeta-deficient cell lines derived from parental CCL39 cells. NHE1 activity in both mutant cell lines was increased in response to phorbal esters or lysophosphatidic acid, whereas the addition of PE only caused a minimal change in either pHi or ERK phosphorylation. These results, combined with reconstitution experiments with exogenously expressed PLCbeta1, PLCbeta2, or PLCbeta3, revealed that stimulation of NHE1 activity by PE in CCL39 cells is a PLCbeta1-coupled event. Furthermore, the data indicate that alpha1-adrenergic signaling of PLCbeta is upstream of ERK activation. These data demonstrate that PLCbeta1 is primarily involved in the activation of NHE1 in CCL39 fibroblasts.

The sodium hydrogen exchanger isoform 1 (NHE1) is present in nearly all cells. Regulation of proton flux via the exchanger is a permissive step in cell growth and tumorgenesis and is vital in control of cell volume. The regulation of NHE1 by growth factors involves the Ras-extracellular signal regulated kinase (ERK) pathway, however, the mechanism for G protein-coupled receptor (GPCR) activation of NHE1 is not well established. In this report, the relationship between GPCRs, ERK, and NHE1 in CCL39 cells is investigated. We give evidence that two agonists, the specific alpha(1)-adrenergic agonist, phenylephrine and the water-soluble lipid mitogen, lysophosphatidic acid (LPA) activate NHE1 in CCL39 cells. Activation of ERK by phenylephrine and LPA occurs in a dose- and time-dependent manner. Optimal ERK activation was observed at 10 min and displayed a maximum stimulation at 100 microM phenylephrine and 10 microM LPA. alpha(1)-Adrenergic stimulation also led to a rise in steady-state pH(i) of 0.16+/-0.02 pH units, and incubation with LPA induced a 0.43+/-0.06 pH unit increase in pH(i). Phenylephrine-induced activation of NHE1 transport and ERK activity was inhibited by pretreating the cells with the MEK inhibitor PD98059. While only half of the LPA activatable exchange activity was abolished by PD98059 and U0126. To further demonstrate the specificity of the phenylephrine and LPA regulation of NHE1 and ERK, CCL39 cells were transfected with a kinase inactive MEK. The data indicate that ERK activation is essential for phenylephrine stimulation of NHE1, and that ERK and RhoA are involved in LPA stimulation of NHE1 by more than one mechanism. In addition, evidence of the convergence of these two pathways is shown by the loss of NHE1 activity when both pathways are inhibited and by the partial additivity of the two agonists on ERK and NHE1 activity. These studies indicate a direct involvement of ERK in the alpha(1)-adrenergic activation of NHE1 and a significant role for both ERK and RhoA in LPA stimulation of NHE1 in CCL39 fibroblasts.

A 66-kDa molecular weight protein with phospholipase D activity was solubilized and partially purified from rat liver plasma membrane. The activity and regulation of this phospholipase D have been characterized. Immunoblot analyses indicated that the enzyme was distinct from hPLD1 and PLD2, but was recognized by an antibody to the 12 terminal amino acids of PLD1. PLD activity was stimulated by 1-100 microM Ca(2+) and Mg(2+) and displayed a pH optimum of 7.5. Activity was inhibited by both saturated and unsaturated fatty acids. This PLD was activated in an ATP-independent manner by the PKC isozymes alpha and betaII but not activated by other PKC isozymes. It was also stimulated by the small G-proteins RhoA and ARF. RhoA stimulated the greatest activation, followed by ARF and PKC(alpha). This enzyme was further activated in a synergistic manner when combinations of PKC(alpha) and RhoA or ARF were used. This enzyme displayed a greater response activation by RhoA than to activation by ARF. While a potential breakdown product of PLD1, activation by RhoA indicates that the PLD characterized here is distinct from the other PLDs cloned or isolated to date., (Copyright 2002 Elsevier Science (USA).)

ADP-ribosylation factors (ARFs) regulate coatomer assembly on the Golgi as well as recruitment of clathrin adapter proteins and are therefore involved in vesicle budding from the Golgi and vesicular transport. They are also regulators of phospholipase D (PLD) activity. Arfaptin 1 is an ARF binding protein that inhibits PLD activation, vesicular trafficking and secretion. In the present report, we show that arfaptin 1 interacts with 'high speed' membranes independently of ARF. However, addition of myristoylated ARF3 (myrARF3) increases the association of arfaptin 1 with the membranes, suggesting that arfaptin 1 and ARF form a complex on the Golgi. Utilizing several deletion mutants of arfaptin 1 it is shown that the association of arfaptin 1 with myrARF3 is mediated via two binding sites on arfaptin 1. These two domains are needed for arfaptin 1 inhibition of PLD activation by myrARF3 in vitro.

The regulation of phospholipase D cloned from rat brain (rPLD) was examined in vivo and in vitro. The enzyme was a shorter splice variant of human phospholipase D 1 (Hammond, S. M., Altshuller, Y. M. , Sung, T.-C., Rudge, S. M., Rose, K., Engebrecht, J. A., Morris, A. J., and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640-29643). Its expression in COS-7 cells led to increased phospholipase D (PLD) activity that was further stimulated by constitutively active V14RhoA. V14RhoA had no effect on the endogenous PLD of the COS-7 cells, but constitutively active L71ARF3 increased its activity. In contrast, L71ARF3 did not activate rPLD expressed in the cells. Addition of phorbol ester markedly increased the endogenous PLD activity of COS-7 cells, and there was a further increase in the cells expressing rPLD. In membranes from COS-7 cells expressing rPLD, addition of myristoylated ADP-ribosylation factor (ARF) and RhoA in vitro stimulated PLD activity. The effect of ARF was greater than that of RhoA, although the concentrations for half-maximal stimulation (0.08-0.2 microM) were similar. Membranes isolated from cells expressing rPLD plus L71ARF3 and/or V14RhoA also showed higher PLD activity but no synergism between the two G proteins. Addition of phorbol ester and protein kinase C alpha (PKCalpha) also stimulated PLD activity in membranes from COS-7 cells expressing rPLD, but it had no effect on the activity in control (vector) membranes and did not enhance the effects of constitutively active ARF or Rho. The stimulation by PKCalpha did not require ATP and was not increased by addition of this nucleotide. No synergism between ARF and Rho and between these and PKCalpha on PLD activity was observed when these were added to membranes from cells expressing rPLD. Oleate inhibited the PLD activity of membranes from both control and rPLD-expressing cells. In summary, these results indicate that in vitro, rPLD is stimulated by ARF, RhoA, and PKCalpha and inhibited by oleate. However, in intact COS-7 cells, ARF activates endogenous PLD but not rPLD, whereas the reverse is true for RhoA. In addition, the effects of phorbol ester are much greater in the intact cells. It is concluded that the regulation of rPLD in intact COS-7 cells differs significantly from that seen in vitro; possible reasons for this are discussed.

Platelet-derived growth factor (PDGF) stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptd InsP2) via phospholipase C-gamma1 (PLC-gamma1) in Swiss 3T3 cells. Treatment of cells with the protein kinase C (PKC) inhibitor Ro-31-8220 greatly decreased PDGF-induced tyrosine phosphorylation of PLC-gamma1, but paradoxically enhanced the production of inositol phosphates (InsPs). The inhibitor also caused an increase of PDGF receptor tyrosine phosphorylation at later times. The changes in phosphorylation of the receptor were correlated with alterations in PLC-gamma1 translocation to the particulate fraction. Thus, although activation of PLC-gamma1 was associated with phosphorylation of the receptor and translocation of the enzyme to the particulate fraction, it was dissociated from its tyrosine phosphorylation. A non-receptor-associated, cytosolic tyrosine kinase also was found to phosphorylate PLC-gamma1 in a PDGF-dependent manner, but was not inhibited by Ro-31-8220 in vitro. PKC depletion by phorbol ester treatment decreased the tyrosine phosphorylation of PLC-gamma1 induced by PDGF and slowed the translocation of PLC-gamma1, but Ro-31-8220 produced further effects. The effect of Ro-31-8220 to enhance the production of InsPs could not be attributed to inhibition of PKC since InsPs production with PDGF was decreased in PKC-depleted cells and a stimulatory effect of the inhibitor was still evident. Interestingly, Ro-31-8220 decreased the radioactivity in phosphatidylinositol and increased that in phosphatidylinositol 4-phosphate and PtdInsP2 in cells labeled with myo[3H]inositol. The increased synthesis of PtdInsP2 could contribute to the increased production of InsPs induced by Ro-31-8220. In summary, these results support the conclusion that the activation of PLC-gamma1 in response to PDGF requires autophosphorylation of the receptor and membrane association of PLC-gamma1, but not phosphorylation of the enzyme. Furthermore, the effects of Ro-31-8220 on the tyrosine phosphorylation and activity of PLC-gamma1, and on PtdInsP2 synthesis cannot be attributed to inhibition of PKC.

In this paper we demonstrate for the first time that human placenta contains a cytosolic phospholipase D (PLD) activity. This activity had a pH optimum of 7.0 and was stimulated by PIP2 and inhibited by oleate. Furthermore, cytosolic PLD was stimulated by 30 microM GTP gamma S (6-14-fold) and by the small G proteins 1 microM mArf3 (2-fold) and 0.37 nM RhoA (2-fold). This is the first report to show RhoA activation of a cytosolic PLD. The activation by mArf3 was maintained after partial purification on DEAE Sepharose of the enzyme. We have previously reported the existence of a membrane-bound PLD from human placenta, which is stimulated by PIP2, but not by oleate (Vinggaard, A. M. & Hansen, H. S. (1995) Biochim. Biophys. Acta 1258, 169-176). Here we show that oleic acid and alpha-linolenic acid both dose-dependently inhibited solubilized membrane PLD (65% inhibition at 4 mM), whereas stearic acid (4 mM) had no effect. Thus, the presence of double bonds in the fatty acid is important for the inhibitory effect. Furthermore, placental membrane PLD was activated by 30 microM GTP gamma S (4-fold) and by mArf3 (1 microM) and RhoA (0.37 nM) by a factor of 3 and 2, respectively. The solubilized membrane phospholipase D was partially purified to a basal specific activity of 25-37 nmol/min/mg. This preparation was devoid of endogenous RhoA and Arf and could not be stimulated by GTP gamma S. However, mArf3 (1 microM) still activated this partially purified membrane PLD, whereas RhoA (0.37 nM) was not able to activate this PLD fraction. In conclusion, our results suggest that the human placenta contains a PLD that is located both in the cytosol and the membranes, and that is activated by PIP2, mArf3 and RhoA but inhibited by oleate.

Phospholipase D (PLD) is regulated by many factors including the small G-proteins, RhoA and ADP-ribosylation factor (ARF). The present study examined the distribution of RhoA- and ARF-responsive PLD in membranes, microsomes and cytosol of rat tissues and in rat liver subcellular fractions. PLD was present in all tissue fractions examined and was stimulated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]), with the highes: specific activities being in lung, kidney and spleen. When myristoylated recombinant ARF (mARF) was added with GTP[S], the PLD activity was stimulated further, but the addition of RhoA was without effect. However, in extracts from crude membranes both mARF and RhoA enhanced the stimulation by GTP[S], with high specific activities of PLD being observed in all tissues except muscle. The response to mARF was usually greater than to RhoA, and the responses were additive, except for liver, which showed synergism. When the PLD activity of subcellular fractions of liver was examined, GTP[S] caused increases in all fractions except microsomes and mitochondria, which exhibited low activity. All fractions except mitochondria showed responses to RhoA and mARF, with the response to RhoA being greater in plasma membranes and that to mARF being greater in Golgi and nuclei. Western blotting showed that RhoA was located mainly in the cytosol and plasma membranes, whereas ARF was principally in the cytosol. These findings demonstrate the widespread occurrence of significant activity of both Rho- and ARF-responsive forms of PLD in membranes from all tissues except muscle, and the presence of both forms in liver subcellular fractions except mitochondria. The large variations in the relative responses of PLD to Rho and ARF observed in different tissues and fractions support the existence of different isoforms of the enzyme.

A procedure was developed for purifying the cytosolic isoforms of malate dehydrogenase, aspartate transaminase, enolase and nucleoside diphosphate kinase from a single preparation of rabbit liver. The procedure includes chromatography on reactive-dye, radial-flow columns, and elution of enzymes from columns by substrates, to obtain high yields in a minimal amount of time. The scheme avoids steps used in previous methods that are either difficult to execute in large-scale preparations, or alter the native forms of the enzymes. Examination of the purified enzymes by SDS-PAGE indicated that nearly homogeneous preparations had been obtained. The native molecular weight, subunit molecular weight, and isoelectric point(s) were determined for each enzyme. Although preparations of nucleoside diphosphate kinase purified from cytosol showed only a single band on SDS-PAGE, isoelectric focusing revealed the presence of multiple isoforms.