RCC2 is a novel p53 target in suppressing metastasis.

RCC2 (also known as TD60) is a highly conserved protein involved in prognosis in colorectal cancer. However, its relationship with tumor development is less understood. Here we demonstrate a signaling pathway defining regulation of RCC2 and its functions in tumor progression. We report that p53 is a transcriptional regulator of RCC2 that acts through its binding to a palindromic motif in the RCC2 promoter. RCC2 physically interacts and deactivates a small GTPase Rac1 that is known to be involved in metastasis. We solved a high-resolution crystal structure of RCC2 and revealed one RCC1-like domain with a unique ß-hairpin that is requisite for RCC2 interaction with Rac1. p53 or RCC2 deficiency leads to activation of Rac1 and deterioration of extracellular matrix sensing (haptotaxis) of surface-bound gradients. Ectopic expression of RCC2 restores directional migration in p53-null cells. Our results demonstrate that p53 and RCC2 signaling is important for regulation of cell migration and suppression of metastasis. We propose that the p53/RCC2/Rac1 axis is a potential target for cancer therapy.

Effects of Ambient Temperature on Growth Performance, Blood Metabolites, and Immune Cell Populations in Korean Cattle Steers.

Exposure to cold may affect growth performance in accordance with the metabolic and immunological activities of animals. We evaluated whether ambient temperature affects growth performance, blood metabolites, and immune cell populations in Korean cattle. Eighteen Korean cattle steers with a mean age of 10 months and a mean weight of 277 kg were used. All steers were fed a growing stage-concentrate diet at a rate of 1.5% of body weight and Timothy hay ad libitum for 8 weeks. Experimental period 1 (P1) was for four weeks from March 7 to April 3 and period 2 (P2) was four weeks from April 4 to May 1. Mean (8.7°C) and minimum (1.0°C) indoor ambient temperatures during P1 were lower (p<0.001) than those (13.0°C and 6.2°C, respectively) during P2. Daily dry matter feed intake in both the concentrate diet and forage groups was higher (p<0.001) during P2 than P1. Average daily weight gain was higher (p<0.001) during P2 (1.38 kg/d) than P1 (1.13 kg/d). Feed efficiency during P2 was higher (p = 0.015) than P1. Blood was collected three times; on March 7, April 4, and May 2. Nonesterified fatty acids (NEFA) were higher on March 7 than April 4 and May 2. Blood cortisol, glucose, and triglyceride concentrations did not differ among months. Blood CD4+, CD8+, and CD4+CD25+ T cell percentages were higher, while CD8+CD25+ T cell percentage was lower, during the colder month of March than during May, suggesting that ambient temperature affects blood T cell populations. In conclusion, colder ambient temperature decreased growth and feed efficiency in Korean cattle steers. The higher circulating NEFA concentrations observed in March compared to April suggest that lipolysis may occur at colder ambient temperatures to generate heat and maintain body temperature, resulting in lower feed efficiency in March.

NAMPT suppresses glucose deprivation-induced oxidative stress by increasing NADPH levels in breast cancer.

Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme involved in NAD+ biosynthesis. Although NAMPT has emerged as a critical regulator of metabolic stress, the underlying mechanisms by which it regulates metabolic stress in cancer cells have not been completely elucidated. In this study, we determined that breast cancer cells expressing a high level of NAMPT were resistant to cell death induced by glucose depletion. Furthermore, NAMPT inhibition suppressed tumor growth in vivo in a xenograft model. Under glucose deprivation conditions, NAMPT inhibition was found to increase the mitochondrial reactive oxygen species (ROS) level, leading to cell death. This cell death was rescued by treatment with antioxidants or NAD+. Finally, we showed that NAMPT increased the pool of NAD+ that could be converted to NADPH through the pentose phosphate pathway and inhibited the depletion of reduced glutathione under glucose deprivation. Collectively, our results suggest a novel mechanism by which tumor cells protect themselves against glucose deprivation-induced oxidative stress by utilizing NAMPT to maintain NADPH levels.

Discovery of a BTK/MNK dual inhibitor for lymphoma and leukemia.

Bruton's tyrosine kinase (BTK) kinase is a member of the TEC kinase family and is a key regulator of the B-cell receptor (BCR)-mediated signaling pathway. It is important for B-cell maturation, proliferation, survival and metastasis. Pharmacological inhibition of BTK is clinically effective against a variety of B-cell malignances, such as mantle cell lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and activated B-cell-diffuse large B-cell lymphoma. MNK kinase is one of the key downstream regulators in the RAF-MEK-ERK signaling pathway and controls protein synthesis via regulating the activity of eIF4E. Inhibition of MNK activity has been observed to moderately inhibit the proliferation of AML cells. Through a structure-based drug-design approach, we have discovered a selective and potent BTK/MNK dual kinase inhibitor (QL-X-138), which exhibits covalent binding to BTK and noncovalent binding to MNK. Compared with the BTK kinase inhibitor (PCI-32765) and the MNK kinase inhibitor (cercosporamide), QL-X-138 enhanced the antiproliferative efficacies in vitro against a variety of B-cell cancer cell lines, as well as AML and CLL primary patient cells, which respond moderately to BTK inhibitor in vitro. The agent can effectively arrest the growth of lymphoma and leukemia cells at the G0-G1 stage and can induce strong apoptotic cell death. These primary results demonstrate that simultaneous inhibition of BTK and MNK kinase activity might be a new therapeutic strategy for B-cell malignances.

Immunotherapy for Lewis lung carcinoma utilizing dendritic cells infected with CK19 gene recombinant adenoviral vectors.

Dendritic cells (DCs) as 'professional' antigen-presenting cells (APCs) initiate and regulate immune responses to various antigens. DC-based vaccines have become a promising modality in cancer immunotherapy. Cytokeratin 19 (CK19) protein is expressed at high levels in lung cancer and many other tumor cells, suggesting CK19 as a potential tumor­specific target for cancer immune therapy. We constructed a recombinant adenoviral vector containing the CK19 gene (rAd-CK19). DCs transfected with rAd-CK19 were used to vaccinate C57BL/6 mice bearing xenografts derived from Lewis lung carcinoma (LLC) cells. The transfected DCs gave rise to potent CK19-specific cytotoxic T lymphocytes (CTLs) capable of lysing LLC cells. Mice immunized with the rAd­CK19-DCs exhibited significantly attenuated tumor growth (including tumor volume and weight) when compared to the tumor growth of mice immunized with rAd-c DCs or DCs during the 24-day observation period (P<0.05). The results revealed that the mice vaccinated with the rAd-CK19-DCs exhibited a potent protective and therapeutic antitumor immunity to LLC cells in the subcutaneous model along with an inhibitive effect on tumor growth compared to the mice vaccinated with the rAd-c DCs or DCs alone. The present study proposes a meaningful mode of action utilizing rAd-CK19 DCs in lung cancer immunotherapy.

Pyrosequencing Analysis of Subgingival Microbiota in Distinct Periodontal Conditions.

Subgingival microorganisms are potentially associated with periodontal diseases. However, changes in the subgingival microbiota during the progress of periodontal diseases are poorly understood. In this study, we analyzed bacterial communities in the subgingival paper point samples from 32 Korean individuals with no sign of disease, gingivitis, or periodontitis using 454 FLX Titanium pyrosequencing. A total of 256,113 reads representing 26 phyla, 433 genera, and 1,016 species were detected. Bacteroidetes, Fusobacteria, Synergistetes, and Spirochaetes were the abundant phyla in periodontitis subjects, whereas Firmicutes and Proteobacteria were identified as the dominant phyla in the gingivitis and healthy subjects, respectively. Although high levels of Porphyromonas, Fusobacterium, Fretibacterium, Rothia, Filifactor, and Treponema genera were observed in the periodontitis subjects, Streptococcus, Capnocytophaga, Leptotrichia, and Haemophilus genera were found at high frequency in the gingivitis subjects. Species including Porphyromonas gingivalis, Fusobacterium nucleatum, and Fretibacterium fastidiosum were significantly increased in periodontitis subjects. On the other hand, Streptococcus pseudopneumoniae, Haemophilus parainfluenzae, and Leptotrichia hongkongensis were preferentially observed in the gingivitis subjects. Intriguingly, the halophile Halomonas hamiltonii was revealed as a predominant species in the healthy subjects. Based on Fast UniFrac analysis, distinctive bacterial clusters were classified for the healthy, gingivitis, and periodontitis state. The current findings might be useful for understanding the pathogenesis, diagnosis, and treatment of periodontal diseases.

Differential profiles of salivary proteins with affinity to Streptococcus mutans lipoteichoic acid in caries-free and caries-positive human subjects.

Streptococcus mutans is a representative oral pathogen that causes dental caries and pulpal inflammation. Its lipoteichoic acid (Sm.LTA) is known to be an important cell-wall virulence factor involved in bacterial adhesion and induction of inflammation. Since Sm.LTA-binding proteins (Sm.LTA-BPs) might play an important role in pathogenesis and host immunity, we identified the Sm.LTA-BPs in the saliva of caries-free and caries-positive human subjects using Sm.LTA-conjugated beads and LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Sm.LTA was conjugated to N-hydroxysuccinimidyl-Sepharose(®) 4 Fast Flow beads (Sm.LTA-beads). Sm.LTA retained its biological properties during conjugation, as determined by the expression of nitric oxide and interferon-γ-inducible protein 10 in a murine macrophage cell line and activation of Toll-like receptor 2 (TLR2) in CHO/CD14/TLR2 cells. Sm.LTA-BPs were isolated from pooled saliva prepared from 10 caries-free or caries-positive human subjects each, electrophoresed to see their differential expression in each group, and further identified by high-resolution mass spectrometry. A total of 8 and 12 Sm.LTA-BPs were identified with statistical significance in the pooled saliva from the caries-free and caries-positive human subjects, respectively. Unique Sm.LTA-BPs found in caries-free saliva included histone H4, profilin-1 and neutrophil defensin-1, and those in caries-positive saliva included cystatin-C, cystatin-SN, cystatin-S, cystatin-D, lysozyme C, calmodulin-like protein 3 and ß-actin. The Sm.LTA-BPs found in both groups were hemoglobin subunits α and ß, prolactin-inducible protein, protein S100-A9, and SPLUNC2. Collectively, we identified Sm.LTA-BPs in the saliva of caries-free and caries-positive subjects, which exhibit differential protein profiles.

Alpha-amylase is a human salivary protein with affinity to lipopolysaccharide of Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans lipopolysaccharide (Aa.LPS) is a major virulence factor associated with aggressive periodontitis. Although the recognition of Aa.LPS is potentially initiated by salivary proteins in the oral cavity, Aa.LPS-binding proteins (Aa.LPS-BPs) in saliva are poorly characterized. The purpose of this study was to capture and identify Aa.LPS-BPs in human saliva using a LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Aa.LPS conjugated onto N-hydroxysuccinimidyl-Sepharose(®) 4 Fast Flow beads (Aa.LPS-beads) activated Toll-like receptor 4 and produced nitric oxide and Interferon gamma-inducible protein-10, implying that the conjugation process did not alter the biological properties of Aa.LPS. Aa.LPS-BPs were subsequently isolated from the nine human saliva samples from healthy individuals with the Aa.LPS-beads followed by identification with the mass spectrometry. Aa.LPS-BPs include α-amylase, serum albumin, cystatin, lysozyme C, submaxillary gland androgen-regulated protein 3B, immunoglobulin subunits, polymeric immunoglobulin receptor, deleted in malignant brain tumors 1, prolactin-inducible protein, lipocalin-1, and basic salivary proline-rich protein 2. Specific binding was validated using a pull-down assay with α-amylase which was captured at the highest frequency. Alpha-amylase demonstrated to interfere with the adherence and biofilm formation of A. actinomycetemcomitans. Even heat-inactivated α-amylase showed the interference to the same extent. Conclusively, we identified unique Aa.LPS-BPs that provide useful information to understand bacterial pathogenesis and host innate immunity in the oral cavity.

Anti-bacterial and anti-toxic immunity induced by a killed whole-cell-cholera toxin B subunit cholera vaccine is essential for protection against lethal bacterial infection in mouse pulmonary cholera model.

The lack of appropriate animal model for studying protective immunity has limited vaccine development against cholera. Here, we demonstrate a pulmonary cholera model conferred by intranasal administration of mice with live Vibrio cholerae. The bacterial components, but not cholera toxin, caused lethal and acute pneumonia by inducing massive inflammation. Intranasal immunization with Dukoral, comprising killed whole bacteria and recombinant cholera toxin B subunit (rCTB), developed both mucosal and systemic antibody responses with protection against the lethal challenge. Either rCTB-free Dukoral or rCTB alone partially protected the mice against the challenge. However, reconstitution of rCTB-free Dukoral with rCTB restored full protection. Parenteral immunization with Dukoral evoked strong systemic immunity without induction of mucosal immunity or protection from the challenge. These results suggest that both anti-bacterial and anti-toxic immunity are required for protection against V. cholerae-induced pneumonia, and this animal model is useful for pre-clinical evaluation of candidate cholera vaccines.

The effects of fermented soybean meal on immunophysiological and stress-related parameters in Holstein calves after weaning.

The present experiment was conducted to investigate the effects of partial substitution of soybean meal (SBM) with fermented SBM (FSBM) on immunophysiological and stress-related parameters in Holstein calves after weaning. Eighteen Holstein calves were randomly assigned to receive either SBM or FSBM (5% of SBM was replaced with FSBM) calf starter and calves were weaned at 42 d of age. It was noted that FSBM contained a lower content of trypsin inhibitor but higher crude protein, amino acids, and small-sized peptides than those of SBM. The group fed FSBM calf starter significantly increased body weight gain and intakes of both feed and milk, when compared with those fed SBM calf starter at 4 wk of age. Calves fed the FSBM calf starter had significantly lower fecal scores than those fed the SBM calf starter during both pre- and postweaning periods. Calves also had better health scores when fed the FSBM calf starter than those fed SBM during the preweaning period. Weaning challenge significantly increased proinflammatory cytokines, tumor necrosis factor α, IL-1ß, and IL-6 levels at 1d postweaning (DPW). The TNF-α and IL-6 levels of the SBM group were significantly higher compared with those of the FSBM group at 3 DPW. Acute phase proteins (serum amyloid A and haptoglobin) in the serum were increased after weaning. Concentrations of serum amyloid A and haptoglobin in calves fed FSBM calf starter were significantly lower than those fed the SBM calf starter at 3 and 5 DPW, respectively. The concentration of cortisol was significantly lower in the FSBM group than that of the SBM group at 3 DPW. Weaning stress did not cause drastic changes in the total serum immunoglobulin levels and composition of peripheral lymphocytes. Our results indicate that FSBM may not only improve growth performance, feed intake, and health conditions during the preweaning period, but also alleviate stress responses, which was indicated by reduced induction of stress hormone, proinflammatory cytokines, and acute phase proteins in Holstein calves after weaning.

Effects of hydrolyzed yeast supplementation in calf starter on immune responses to vaccine challenge in neonatal calves.

The effects of hydrolyzed yeast supplementation on growth performance, health and immune-physiological parameters in neonatal calves challenged with vaccine were investigated. Twelve Holstein calves were started in the experiment at 2 ± 1 days of age and were studied for 35 days. Calves were randomly assigned to each of two dietary treatments, a control (CON) and hydrolyzed yeast (HY) group. The calves in the HY group received control calf starter supplemented with 0.2% HY. All calves were given calf starter ad libitum for 5 weeks starting in week 1. Calves were also given whole milk according to a step-down milking protocol. In order to induce immune responses, all calves were challenged with Hog cholera and Erysipelothrix insidiossa live vaccines by intramuscular injection at 3 weeks of age. Growth performance and feed intake were not affected by dietary treatment throughout the experimental period, except that the HY group had significantly higher (P < 0.05) milk intake than did the CON group at 3 weeks of age. Calves in the HY group showed significantly better (P < 0.05) fecal and health scores at 3 weeks compared to those in the control group. After vaccine challenge, neutropenia, lymphophilia and thrombocytopenia were observed in the CON group, but calves in the HY group did not show significant changes of leukocytes. The average concentration of serum haptoglobin in the HY group was significantly higher (P < 0.05) at 1 and 3 days post-vaccine challenge (DPVC) than that of CON group. Feeding HY supplemented calf starter resulted in a higher (P < 0.05) relative amount of bacterial and viral - specific IgA than in the CON group at 5 DPVC. Although the percentage of CD4+ T cells was significantly (P < 0.05) higher in the HY group than in the CON group at -2 DPVC, significant differences between groups after vaccine challenge was not observed during the experimental period. These results suggest that 0.2% HY supplementation in calf starter can improve the health status and immune-related serum protein production and affect blood cell composition in neonatal calves after vaccine challenge.

Changes of immunophysiological characteristics in neonatal calves experimentally challenged with mixture of live bacteria and virus.

The aim of the present study was to define efficient immunophysiological parameters in neonatal Holstein calves with an experimentally induced microbial infection. Calves (n = 15) were challenged with classical swine fever virus (LOM strain) and Erysipelothrix insidiosa live vaccine by intravenous injection at 3 wk of age except for control calves (n = 4). The level of total serum IgA was significantly increased at 14 and 19 d post-experimental challenge (DPEC) compared with that in calves at -2 DPEC. At 5 DPEC, relative amounts of bacterial- and viral-specific IgA increased significantly and were sustained until 26 DPEC. In the hematology assay, the neutrophil:lymphocyte ratio (%) in whole blood was significantly decreased at 14 DPEC because of a significant increase in lymphocytes and a coincident decrease in neutrophils. The percentages of CD4+ and CD25+ T cells were significantly decreased at 14 DPEC and returned to initial levels at 19 DPEC. It is intriguing to note that the level of serum lactoferrin was significantly decreased by the microbial challenge within 1 d. The concentration of haptoglobin was increased within 3 d and gradually decreased in calves after microbial challenge. Our results suggest that 1) bovine serum lactoferrin plays an important role in the innate immune response against microbial infection at an early stage and 2) experimentally induced microbial challenge using porcine live bacterial and viral vaccine in calves could be a good experimental model to evaluate the effect of diet or stress induced by environmental change on the immune responses against microbial infection.

Acute ophthalmoplegia (without ataxia) associated with anti-GQ1b antibody.

BACKGROUND: Anti-GQ1b antibody has been found in Miller Fisher syndrome (MFS), Guillain-Barré syndrome (GBS) with ophthalmoplegia, Bickerstaff brainstem encephalitis (BBE), and acute ophthalmoplegia without ataxia (AO). The aim of this study was to determine the clinical features of AO associated with anti-GQ1b antibody. METHODS: We retrospectively collected 34 patients with anti-GQ1b antibody syndrome. Of these patients, 31 patients had ophthalmoplegia. The patients with ophthalmoplegia were classified into MFS (n = 13), AO (n = 11), GBS with ophthalmoplegia (n = 6), and BBE (n = 1). We analyzed clinical features and patterns of external and internal ophthalmoplegia of AO, and neuro-ophthalmologic findings were compared with those of other anti-GQ1b syndromes with ophthalmoplegia. RESULTS: AO was observed in 11 (32.4%) of the 34 patients with anti-GQ1b antibody. External ophthalmoparesis was present in all the patients and included mixed horizontal-vertical (n = 7), pure horizontal (n = 3), and pure vertical gaze palsy (n = 1). Binocular involvement was common, but unilateral ophthalmoparesis was also observed in 27.3%. Other findings included ptosis (n = 5, 45.5%) and internal ophthalmoplegia (n = 6, 54.5%). Other anti-GQ1b antibody syndromes had prominent neurologic signs including ataxia, weakness, and facial palsy in addition to ophthalmoplegia. The patterns of neuro-ophthalmologic findings did not differ between AO and other anti-GQ1b antibody syndromes with ophthalmoplegia. CONCLUSIONS: Acute ophthalmoplegia (AO) commonly occurs in anti-GQ1b antibody syndrome and manifests as various combinations of external and internal ophthalmoplegia. Internal ophthalmoplegia is fairly common and unilateral involvement may occur in AO.

SRF is a nuclear repressor of Smad3-mediated TGF-beta signaling.

Serum response factor (SRF) is a widely expressed transcription factor involved in immediate-early and tissue-specific gene expression, cell proliferation and differentiation. We defined a new role of SRF as a nuclear repressor of the tumor growth factor beta1 (TGF-beta1) growth-inhibitory signal during cell proliferation. We show that SRF significantly inhibits the TGF-beta1/Smad-dependent transcription by associating with Smad3. SRF causes resistance to the TGF-beta1 cytostatic response by directly repressing the Smad transcriptional activity and Smad binding to DNA. Furthermore, we demonstrated that overexpression of SRF markedly decreases the level of Smad3 complex binding to the promoters of Smad3 target genes, p15(INK4b) and p21(Cip1). This leads to the inhibition of expression of TGF-beta1-responsive genes. SRF therefore acts as a nuclear repressor of Smad3-mediated TGF-beta1 signaling.

Intracranial ictal onset zone in nonlesional lateral temporal lobe epilepsy on scalp ictal EEG.

OBJECTIVE: To determine the ictal focus and the role of seizure characteristics, fluorodeoxyglucose (FDG) PET, and subtraction ictal SPECT in patients diagnosed as having nonlesional lateral temporal lobe epilepsy by long-term scalp video-EEG monitoring. METHODS: The authors studied 33 consecutive patients with nonlesional neocortical epilepsy who had a scalp ictal onset zone localized in the temporal lobe and good surgical outcome after focal neocortical resection. All patients were evaluated using intracranial recordings prior to resection. Semiology, FDG-PET, and ictal-interictal subtraction SPECT were used to verify the diagnostic role of these methods in the localization of epileptic foci. RESULTS: The ictal onset zones, confirmed by intracranial study, were the lateral temporal (22 patients), parietal (5), frontal (3), temporoparietal (2), and occipital (1) areas. FDG-PET analyzed by statistical parametric mapping correctly localized the epileptogenic lobe in 18 of 33 patients and subtraction ictal SPECT correctly localized it in 13 of 25 patients. However, in patients with extratemporal ictal onset zones, FDG-PET and ictal SPECT in combination correctly localized the epileptogenic lobe in only 3 of 11 cases. CONCLUSIONS: An extratemporal ictal onset zone was encountered in patients with nonlesional lateral temporal lobe seizures based on scalp video-EEG monitoring. FDG-PET and subtraction SPECT had localizing value in no more than half of patients.

The adenosine 2b receptor is recruited to the plasma membrane and associates with E3KARP and Ezrin upon agonist stimulation.

We have previously shown that adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5'-AMP. Acting through the adenosine A2b receptor (A2bR), the luminally derived adenosine induces vectorial chloride secretion and a polarized secretion of interleukin-6 to the intestinal lumen. Although some G protein-coupled receptors interact with anchoring or signaling molecules, not much is known in this critical area for the A2bR. We used the model intestinal epithelial cell line, T84, and Caco2-BBE cells stably transfected with GFP-A2b receptor to study the intestinal A2bR. The A2bR is present in both the apical and basolateral membranes of intestinal epithelia. Apical or basolateral stimulation of the A2bR induces recruitment of the receptor to the plasma membrane and caveolar fractions. The A2bR co-immunoprecipitates with E3KARP and ezrin upon agonist stimulation. Ezrin interacts with E3KARP and PKA and the interaction between ezrin and E3KARP is enhanced by agonist stimulation. Our data suggest that the A2bR is recruited to the plasma membrane upon apical or basolateral agonist stimulation and interacts with E3KARP and ezrin. We speculate that such an interaction may not only anchor the A2bR to the plasma membrane but may also function to stabilize the receptor in a signaling complex in the plasma membrane.

Ca(2+)-dependent inhibition of Na+/H+ exchanger 3 (NHE3) requires an NHE3-E3KARP-alpha-actinin-4 complex for oligomerization and endocytosis.

Two PDZ domain-containing proteins, NHERF and E3KARP are necessary for cAMP-dependent inhibition of Na(+)/H(+) exchanger 3 (NHE3). In this study, we demonstrate a specific role of E3KARP, which is not duplicated by NHERF, in Ca(2+)-dependent inhibition of NHE3 activity. NHE3 activity is inhibited by elevation of intracellular Ca(2+) ([Ca(2+)](i)) in PS120 fibroblasts stably expressing E3KARP but not those expressing NHERF. In addition, this Ca(2+)-dependent inhibition requires Ca(2+)-dependent association between alpha-actinin-4 and E3KARP. NHE3 is indirectly connected to alpha-actinin-4 in a protein complex through Ca(2+)-dependent interaction between alpha-actinin-4 and E3KARP, which occurs through the actin-binding domain plus spectrin repeat domain of alpha-actinin-4. Elevation of [Ca(2+)](i) results in oligomerization and endocytosis of NHE3 as well as in inhibition of NHE3 activity. Overexpression of alpha-actinin-4 potentiates the inhibitory effect of ionomycin on NHE3 activity by accelerating the oligomerization and endocytosis of NHE3. In contrast, overexpression of the actin-binding domain plus spectrin repeat domain acts as a dominant-negative mutant and prevents the inhibitory effect of ionomycin on NHE3 activity as well as the oligomerization and internalization of NHE3. From these results, we propose that elevated Ca(2+) inhibits NHE3 activity through oligomerization and endocytosis of NHE3, which occurs via formation of an NHE3-E3KARP-alpha-actinin-4 complex.

Protein kinase C epsilon-dependent regulation of cystic fibrosis transmembrane regulator involves binding to a receptor for activated C kinase (RACK1) and RACK1 binding to Na+/H+ exchange regulatory factor.

Protein kinase C (PKC) regulation of cystic fibrosis transmembrane regulator (CFTR) chloride function has been demonstrated in several cell lines, including Calu-3 cells that express native, wild-type CFTR. We demonstrated previously that PKC epsilon was required for cAMP-dependent CFTR function. The goal of this study was to determine whether PKC epsilon interacts directly with CFTR. Using overlay assay, immunoprecipitation, pulldown and binding assays, we show that PKC epsilon does not bind to CFTR, but does bind to a receptor for activated C kinase (RACK1), a 37-kDa scaffold protein, and that RACK1 binds to Na(+)/H(+) exchange regulatory factor (NHERF1), a binding partner of CFTR. In vitro binding assays demonstrate dose-dependent binding of PKC epsilon to RACK1 which is inhibited by an 8-amino acid peptide based on the sequence of the sixth Trp-Asp repeat in RACK1 or by an 8-amino acid sequence in the V1 region of PKC epsilon, epsilon V1-2. A 4-amino acid sequence INAL (70-73) expressed in CFTR shares 50% homology to the RACK1 inhibitory peptide, but it does not bind PKC epsilon. NHERF1 and RACK1 bind in a dose-dependent manner. Immunofluorescence and confocal microscopy of RACK1 and CFTR revealed colocalization of the proteins to the apical and lateral regions of Calu-3 cells. The results indicate the RACK1 binds PKC epsilon and NHERF1, thus serving as a scaffold protein to anchor the enzyme in proximity to CFTR.