Richmond Muimo

Histidine phosphorylation of annexin I in airway epithelia.

Although [Cl−] i regulates many cellular functions including cell secretion, the mechanisms governing these actions are not known. We have previously shown that the apical membrane of airway epithelium contains a 37-kDa phosphoprotein (p37) whose phosphorylation is regulated by chloride concentration. Using metal affinity (chelating Fe3+-Sepharose) and anion exchange (POROS HQ 20) chromatography, we have purified p37 from ovine tracheal epithelia to electrophoretic homogeneity. Sequence analysis and immunoprecipitation using monoclonal and specific polyclonal antibodies identified p37 as annexin I, a member of a family of Ca2+-dependent phospholipid-binding proteins. Phosphate on [32P]annexin I, phosphorylated using both [γ-32P]ATP and [γ-32P]GTP, was labile under acidic but not alkaline conditions. Phosphoamino acid analysis showed the presence of phosphohistidine. The site of phosphorylation was localized to a carboxyl-terminal fragment of annexin I. Our data suggest that cAMP and AMP (but not cGMP) may regulate annexin I histidine phosphorylation. We propose a role for annexin I in an intracellular signaling system involving histidine phosphorylation.

Making human nasal cilia beat in the cold: a real time assay for cell signalling.

Human nasal epithelium must adapt to cold climates, and yet, in vitro, human ciliary beat frequency (CBF) is zero at 4 degrees C. Similarly, hibernating mammals do not die of pneumonia despite a core body temperature as low as 6 degrees C, implying that cilia continue to beat. Here, we show that protein kinase C (PKC) and Ca(2+)/calmodulin-dependent kinase II (CaMKII) regulate the profile of human nasal CBF in response to rising temperature from 4 degrees C. Onset of ciliary beat was at 10 degrees C in Medium 199, 7 degrees C in the presence of the PKC activator phorbol 12-myristate 13-acetate (PMA), the calcium ionophore ionomycin, or the CAMKII blocker myristoylated autocamtide-2 related inhibitory peptide (MACI), and 6 degrees C for the myristoylated peptide PKC inhibitor EGF-R Fragment 651-658 (MyrPKCI). During cell warming to 32 degrees C, the thermal profile was sigmoid in all solutions except those containing MACI+PMA. Surprisingly, cilia continued to beat despite 4 degrees C and were significantly more responsive to rising temperature with either MACI+PMA, or MACI+MyrPKCI. Our data suggest that CaMKII and PKC regulate the thermal slope and profile of CBF in vitro, and that when these protein kinases are manipulated, cilia can continue to beat despite hypothermia. These findings may relate to adaptive responses to cold climates.

N-Acetylation of serotonin, octopamine and dopamine by adult Brugia pahangi.

The metabolism of biogenic amines by the filarial worm, Brugia pahangi, was investigated by incubating cut worms with radio-labelled amine substrates. Two-dimensional thin-layer chromatography and analysis on two high-performance liquid chromatography systems showed that [14C]5-hydroxytryptamine was metabolised to a less polar compound that was identified as N-acetyl 5-hydroxytryptamine. N-Acetyloctopamine and N-acetyldopamine were also formed when cut B. pahangi were incubated with [14C]octopamine and [3H]dopamine, respectively. N-Acetyltransferase activity towards 5-hydroxytryptamine was readily detected in nematode homogenates. This enzyme was localised in a 50,000 x g supernatant and required the addition of the co-substrate, acetyl CoA, for activity. No evidence was obtained for the involvement of monoamine oxidases in the metabolism of 5-HT in these filarial worms.

Na+ and K+ regulate the phosphorylation state of nucleoside diphosphate kinase in human airway epithelium

We describe how cations, in the presence of ATP, regulate the phosphorylated form of 19- and 21-kDa nucleoside diphosphate kinase (NDPK; EC 2.7.4.6 ), a kinase controlling K+ channels, G proteins, cell secretion, cellular energy production, and UTP synthesis. In apically enriched human nasal epithelial membranes, 10 mM Na+ inhibits phosphorylation of NDPK relative to other cations. Dose response showed that, whereas K+ induces a fourfold greater phosphate incorporation (EC50 10 mM), Na+ is inhibitory (EC50 10 mM) compared with respective buffer controls. Cation discrimination is nucleotide selective (not seen with [γ-32P]GTP) and NDPK specific (not seen with p37h, a previously characterized Cl--sensitive phosphoprotein). Na+ does not exert an inhibitory effect on NDPK phosphorylation directly but is likely to act via an okadaic acid-insensitive phosphatase. We speculate that the ability of NDPK to discriminate between physiologically relevant cation concentrations provides a novel example of cross talk within the apical membrane.

Role of Binding and Nucleoside Diphosphate Kinase A in the Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator by AMP-activated Protein Kinase

Background: AMPK phosphorylates CFTR and inhibits PKA-stimulated CFTR channel gating by unclear mechanisms. Results: NDPK-A, AMPK, and CFTR exist in a membrane-associated complex. AMPK-CFTR binding and NDPK-A catalytic function are required for CFTR inhibition by AMPK. Conclusion: NDPK-A plays an integral role in the regulation of CFTR by AMPK. Significance: Targeting the AMPK-CFTR interaction and NDPK-A function could yield new therapeutic strategies for CF. Cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel mutations cause cystic fibrosis lung disease. A better understanding of CFTR regulatory mechanisms could suggest new therapeutic strategies. AMP-activated protein kinase (AMPK) binds to and phosphorylates CFTR, attenuating PKA-activated CFTR gating. However, the requirement for AMPK binding to CFTR and the potential role of other proteins in this regulation are unclear. We report that nucleoside diphosphate kinase A (NDPK-A) interacts with both AMPK and CFTR in overlay blots of airway epithelial cell lysates. Binding studies in Xenopus oocytes and transfected HEK-293 cells revealed that a CFTR peptide fragment that binds AMPK (CFTR-1420-57) disrupted the AMPK-CFTR interaction. Introduction of CFTR-1420-57 into human bronchial Calu-3 cells enhanced forskolin-stimulated whole cell conductance in patch clamp measurements. Similarly, injection of CFTR-1420-57 into Xenopus oocytes blocked the inhibition of cAMP-stimulated CFTR conductance by AMPK in two-electrode voltage clamp studies. AMPK also inhibited CFTR conductance with co-expression of WT NDPK-A in two-electrode voltage clamp studies, but co-expression of a catalytically inactive H118F mutant or various Ser-120 NDPK-A mutants prevented this inhibition. In vitro phosphorylation of WT NDPK-A was enhanced by purified active AMPK, but phosphorylation was prevented in H118F and phosphomimic Ser-120 NDPK-A mutants. AMPK does not appear to phosphorylate NDPK-A directly but rather promotes an NDPK-A autophosphorylation event that involves His-118 and Ser-120. Taken together, these results suggest that NDPK-A exists in a functional cellular complex with AMPK and CFTR in airway epithelia, and NDPK-A catalytic function is required for the AMPK-dependent regulation of CFTR.

Nucleoside diphosphate kinase A as a controller of AMP-kinase in airway epithelia

This review integrates recent understanding of a novel role for NDPK-A in two related directions: Firstly, its role in an airway epithelial cell when bound to the luminal (apical) membrane and secondly in the cytosol of many different cells (epithelial and non-epithelial) where an isoform-specific interaction occurs with a regulatory partner, AMPKα1. Thus NDPK-A is present in both a membrane and cytosolic environment but in the apical membrane, its roles are not understood in detail; preliminary data suggest that it co-localises with the cystic fibrosis protein (CFTR). In cytosol, we find that NDPK-A is coupled to the catalytic alpha1 isoform of the AMP-activated protein kinase (AMPKα subunit), which is part of a heterotrimeric protein complex that responds to cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. We find that ATP is located within this complex and ‘fed’ from NDPK to AMPK without ever ‘seeing’ bulk solution. Importantly, the reverse can also happen such that AMPK activity can be made to decline when NDPK-A ‘steals’ ATP from AMPK. Thus we propose a novel paradigm in NDPK-A function by suggesting that AMP-kinase can be regulated by NDPK-A, independently of AMP.

The Transient Receptor Potential Ion Channel TRPV6 Is Expressed at Low Levels in Osteoblasts and Has Little Role in Osteoblast Calcium Uptake

Background TRPV6 ion channels are key mediators of regulated transepithelial absorption of Ca2+ within the small intestine. Trpv6 -/- mice were reported to have lower bone density than wild-type littermates and significant disturbances in calcium homeostasis that suggested a role for TRPV6 in osteoblasts during bone formation and mineralization. TRPV6 and molecules related to transepithelial Ca2+ transport have been reported to be expressed at high levels in human and mouse osteoblasts. Results Transmembrane ion currents in whole cell patch clamped SaOS-2 osteoblasts did not show sensitivity to ruthenium red, an inhibitor of TRPV5/6 ion channels, and 45Ca uptake was not significantly affected by ruthenium red in either SaOS-2 (P = 0.77) or TE-85 (P = 0.69) osteoblastic cells. In contrast, ion currents and 45Ca uptake were both significantly affected in a human bronchial epithelial cell line known to express TRPV6. TRPV6 was expressed at lower levels in osteoblastic cells than has been reported in some literature. In SaOS-2 TRPV6 mRNA was below the assay detection limit; in TE-85 TRPV6 mRNA was detected at 6.90±1.9 × 10−5 relative to B2M. In contrast, TRPV6 was detected at 7.7±3.0 × 10−2 and 2.38±0.28 × 10−4 the level of B2M in human carcinoma-derived cell lines LNCaP and CaCO-2 respectively. In murine primary calvarial osteoblasts TRPV6 was detected at 3.80±0.24 × 10−5 relative to GAPDH, in contrast with 4.3±1.5 × 10−2 relative to GAPDH in murine duodenum. By immunohistochemistry, TRPV6 was expressed mainly in myleocytic cells of the murine bone marrow and was observed only at low levels in murine osteoblasts, osteocytes or growth plate cartilage. Conclusions TRPV6 is expressed only at low levels in osteoblasts and plays little functional role in osteoblastic calcium uptake.

Defective formation of PKA/CnA-dependent annexin 2–S100A10/CFTR complex in ΔF508 cystic fibrosis cells

Cystic fibrosis (CF) is characterised by impaired epithelial ion transport and is caused by mutations in the cystic fibrosis conductance regulator protein (CFTR), a cAMP/PKA and ATP-regulated chloride channel. We recently demonstrated a cAMP/PKA/calcineurin (CnA)-driven association between annexin 2 (anx 2), its cognate partner -S100A10 and cell surface CFTR. The complex is required for CFTR and outwardly rectifying chloride channel function in epithelia. Since the cAMP/PKA-induced Cl(-) current is absent in CF epithelia, we hypothesized that the anx 2-S100A10/CFTR complex may be defective in CFBE41o cells expressing the commonest F508del-CFTR (DeltaF-CFTR) mutation. Here, we demonstrate that, despite the presence of cell surface DeltaF-CFTR, cAMP/PKA fails to induce anx 2-S100A10/CFTR complex formation in CFBE41o- cells homozygous for F508del-CFTR. Mechanistically, PKA-dependent serine phosphorylation of CnA, CnA-anx 2 complex formation and CnA-dependent dephosphorylation of anx 2 are all defective in CFBE41o- cells. Immunohistochemical analysis confirms an abnormal cellular distribution of anx 2 in human and CF mouse epithelia. Thus, we demonstrate that cAMP/PKA/CnA signaling pathway is defective in CF cells and suggest that loss of anx 2-S100A10/CFTR complex formation may contribute to defective cAMP/PKA-dependent CFTR channel function.

Properties of an arylalkylamine N-acetyltransferase from the nematode, Ascaridia galli

1. An arylalkylamine N-acetyltransferase (NAT) of the parasitic nematode, Ascaridia galli was studied using either [14C]serotonin (5-HT) or [14C]octopamine (OA) as substrates and with acetyl-CoA as the donor of the acetate group. 2. The NAT activity towards 5-HT and OA co-eluted from a size-exclusion column and appeared to have an M(r) of around 30,000. The enzyme had apparent Km values of 540 +/- 100 microM (+/- SEM) and 33 +/- 4 microM (+/- SEM) for 5-HT and octopamine, respectively, when assayed in the presence of 1 mM acetyl-CoA. 3. High levels of NAT were found in the gonads of male and female worms and the muscle/body wall. 4. N-acetylation was strongly inhibited by Cu2+ but not by other divalent metal ions and the effect of a number of compounds including biogenic amines, formamidines, hydrazines, and beta-carbolines on the arylalkylamine N-acetyltransferase activity was studied.

4α-Phorbol negates the inhibitory effects of phorbol-12-myristate-13-acetate on human cilia and alters the phosphorylation of PKC

In medium 199, ciliary beat frequency (CBF) in human nasal epithelium declines to 60% of baseline by 2 h and 1 nM phorbol‐12‐myristate‐13‐acetate (PMA) doubles the rate of decline by activating protein kinase C (PKC). We find that a reported negative control for PMA, 4α‐phorbol (1 pM–1 nM)±1 nM PMA, not only maintains CBF at baseline, but arrests a pre‐existing PMA‐induced decline in CBF and alters the profile of multiple phosphorylated PKC species. Thus, 4α‐phorbol not only potently prevents PMA from inhibiting CBF but also has potent effects on the phosphorylation of PKC.