Histidine Phosphorylation of Annexin I in Airway Epithelia

Abstract

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 Fe 3؉ -Sepharose) and anion exchange (PO-ROS 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 Ca We have previously shown that [Cl Ϫ ]/[anions]/ATP/GTP differentially determine the profile of phosphorylation of a number of apical membrane proteins in airway epithelia (3, 4). We identified two of the phosphoproteins, a 19/21-kDa doublet that showed chloride concentration-dependent phosphorylation, as two isoforms of nucleoside-diphosphate kinase (3, 5). We also observed that phosphorylation of a 37-kDa protein (p37) from both human nasal and sheep tracheal epithelia was differentially modulated by nucleotides (GTP, ATP, and GDP), [Cl Ϫ ], and other anion species (3, 4). We found that GTP was the principal phosphate donor for p37 from both species; and in the presence of ATP/GDP, phosphorylation of p37 was enhanced 20-fold. However, this phosphorylation could not be inhibited by known serine/threonine and tyrosine protein kinase inhibitors, suggesting that phosphorylation was not due to the activity of these enzymes.In this study, our objective was to establish the identity of the ion-sensitive phosphoprotein p37 and to characterize its phosphorylated amino acid residue. By selectively enhancing phosphorylation of p37 (3) and by applying metal affinity chromatography, we show that p37 is identical to annexin I and provide evidence for novel annexin phosphorylation on histidine residues. Our data suggest that annexin is a component of an intracellular signaling system involving histidine phosphorylation, which is regulated by chloride concentration. EXPERIMENTAL PROCEDURESSample Preparation-A membrane fraction was prepared from ovine airway epithelia as described previously (3). Briefly, tracheal epithelia were scraped and dislodged into homogenization buffer (250 mM sucrose, 10 mM triethanolamine; one protease inhibitor tablet from Roche Molecular Biochemicals per 50 ml buffer). Pooled scrapings were homogenized and spun at 600 ϫ g for 15 min. The post-nuclear supernatant was re-spun at 100,000 ϫ g for 2 h. The pellet was resuspended in homogenization buffer and spun for 30 min at 16,000 ϫ g (this procedure was repeated three times). All procedures were conducted at 4°C. Use of lactate dehydrogenase as a cytosol marker showed that the pellet contained no cytosol (3). Aliquots of the cytosol and membrane pellet were stored in liquid nitrogen.Phosphorylation and Electronic Autoradiography-Phosphorylation and detection of phosphoproteins were performed as described previously (3). Briefl...

Identification and characterization of a mammalian 14‐kDa phosphohistidine phosphatase

“…Whether the small amounts of larger sized mRNA seen in the Northern blot in addition to the 0.6 kb mRNA mirrors alternative splicing remains to be studied. A putative simultaneous expression of the 14-kDa phosphohistidine phosphatase and an endosomal protein may be of interest in the light of the recently described histidine phosphorylation of annexin I from a membrane preparation of ovine tracheal epithelia [34]. Although the detailed subcellular location of the phosphorylated annexin I was not reported, and the effect of the 14-kDa phosphatase on phosphorylated annexin I is still unknown, it is worth noting that annexin I shows endosomal binding in live HeLa cells [35].…”

Advances in development of new tools for the study of phosphohistidine

“…86 Enrichment of pHis-containing proteins using IMAC has had some success but has its limitations. Muimo et al 55 enriched His phosphorylated Annexin A1 with Fe 3+ and Ca 2+ affinity columns, but the method was found to be inefficient. Napper et al 87 used Cu 2+ in what they describe as the selective enrichment of pHiscontaining HPr protein from E. coli.…”

“…For example, pHis has been shown to be present in heteromeric G proteins (GNB1), which are involved in G protein signalling, 15,47,48 KCa3.1 potassium channel, which is involved in ion conductance, 18,49 ATP-citrate lyase (ACLY), which is involved in cell metabolism, 9 histone H4, which is involved in chromatin biology, 21,22,28 transient receptor potentialvanilloid-5 (TRPV5), which regulates urinary Ca 2+ excretion, 17 and phosphoglycerate mutase 1 (PGAM1), which is involved in glycolysis. [50][51][52][53] Other mammalian pHis proteins include P-selectin, which has an important role in the function of blood platelets, 54 annexin A1 a multi-functional Ca 2+ -dependant phospholipid-binding protein found in airway epithelia cells, 55 thymidylate synthase, which catalyzes Nmethylenetetrahydrofolate assisted C(5)-methylation of dUMP required for DNA synthesis, 56 glucose-6-phosphatase involved in glucose homeostasis, 57,58 nicotinamide phosphotransferase (NAMTP) involved in reforming nicotinamide adenine dinucleotide (NAD + ) from nicotinamide 59 and prostatic acid phosphatase, which is found in high levels in prostate cancer cells. 60,61 However, the functions of many of these pHis proteins, and the specific pHis isomer involved, as well as corresponding kinases and phosphatases remain unknown.…”

Progress on Nme (NDP kinase/Nm23/Awd) gene family-related functions derived from animal model systems: studies on development, cardiovascular disease, and cancer metastasis exemplified

“…In recent years, increasing evidence has been presented that Nme2 can act as protein histidine kinase in mammals. The so far described few substrates of the Nme2, the channels K Ca 3.1 and TRPV5 (Cai et al 2014;Srivastava et al 2006b), the G protein β subunit (Cuello et al 2003), and annexin 1 (Muimo et al 2000) fall into at least two subgroups. The histidine phosphorylation of the channels follows the classical paradigm in which phosphorylation of a protein alters its confirmation and/or activity.…”