Sodium hydrogen exchanger (NHE1) palmitoylation and potential functional regulation.

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 [3H]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 (pHi), 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 pHi 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.

RhoA Kinase (Rock) and p90 Ribosomal S6 Kinase (p90Rsk) phosphorylation of the sodium hydrogen exchanger (NHE1) is required for lysophosphatidic acid-induced transport, cytoskeletal organization and migration.

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.

Integrating standard operating procedures and industry notebook standards to evaluate students in laboratory courses.

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.

Inside out: targeting NHE1 as an intracellular and extracellular regulator of cancer progression.

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.

Bringing the excitement and motivation of research to students; Using inquiry and research-based learning in a year-long biochemistry laboratory: Part I-guided inquiry-purification and characterization of a fusion protein: Histidine tag, malate dehydrogenase, and green fluorescent protein.

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.

Bringing the excitement and motivation of research to students; Using inquiry and research-based learning in a year-long biochemistry laboratory : Part II-research-based laboratory-a semester-long research approach using malate dehydrogenase as a research model.

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.

Sodium hydrogen exchanger and phospholipase D are required for alpha1-adrenergic receptor stimulation of metalloproteinase-9 and cellular invasion in CCL39 fibroblasts.

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.

alpha(1)-Adrenergic receptor stimulation of cell motility requires phospholipase D-mediated extracellular signal-regulated kinase activation.

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.

Isolation and characterization of a 66-kDa protein from rat liver plasma membrane with RhoA-stimulated phospholipase D activity.

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.