Intracellular Ca2+ regulates the phosphorylation and the dephosphorylation of ciliary proteins via the NO pathway

Abstract

The phosphorylation profile of ciliary proteins under basal conditions and after stimulation by extracellular ATP was investigated in intact tissue and in isolated cilia from porcine airway epithelium using anti-phosphoserine and anti-phosphothreonine specific antibodies. In intact tissue, several polypeptides were serine phosphorylated in the absence of any treatment (control conditions). After stimulation by extracellular ATP, changes in the phosphorylation pattern were detected on seven ciliary polypeptides. Serine phosphorylation was enhanced for three polypeptides (27, 37, and 44 kD), while serine phosphorylation was reduced for four polypeptides (35, 69, 100, and 130 kD). Raising intracellular Ca2+ with ionomycin induced identical changes in the protein phosphorylation profile. Inhibition of the NO pathway by inhibiting either NO synthase (NOS), guanylyl cyclase (GC), or cGMP-dependent protein kinase (PKG) abolished the changes in phosphorylation induced by ATP. The presence of PKG within the axoneme was demonstrated using a specific antibody. In addition, in isolated permeabilized cilia, submicromolar concentrations of cGMP induced protein phosphorylation. Taken together, these results suggest that the axoneme is an integral part of the intracellular NO pathway. The surprising observation that ciliary activation is accompanied by sustained dephosphorylation of ciliary proteins via NO pathway was not detected in isolated cilia, suggesting that the protein phosphatases were either lost or deactivated during the isolation procedure. This work reveals that any pharmacological manipulation that abolished phosphorylation and dephosphorylation also abolished the enhancement of ciliary beating. Thus, part or all of the phosphorylated polypeptides are likely directly involved in axonemal regulation of ciliary beating.

Figure 1.

Electron micrograph showing a highly enriched preparation of cilia isolated from porcine airway epithelium. Thin section of the ciliary pellet demonstrates the features of intact axonemes (nine outer doublet microtubules and an inner pair). Bar, 1 μm.

Figure 2.

Effect of extracellular ATP on [Ca²⁺]_i and CBF enhancement. Representative experiment of time course responses of [Ca²⁺]_i (A) and CBF enhancement (B) to 100 μM ATP in porcine tracheal tissue cultures. [Ca²⁺]_i and CBF were sampled from the same cell every second, but only each third data point is displayed for purposes of clarity. A representative of 10 similar experiments is shown.

Figure 3.

Extracellular ATP induces phosphorylation and dephosphorylation of ciliary polypeptides in intact epithelial tissue. (A) Typical pattern of phosphorylated polypeptides in cilia isolated from epithelial tissue exposure to extracellular ATP (100 μM) for 3 min (lane 2) or 5 min (lane 3), as compared with untreated control (lane 1). Western blot analysis using anti-phosphoserine antibody indicates an increase in phosphorylation of 27, 37, and 44 kD polypeptides, and a decrease in phosphorylation of 35, 69, 100, and 130 kD polypeptides. (B) Reprobing of the nitrocellulose membrane presented in A with anti–α-tubulin antibody demonstrates equal protein loading. Representative blot of at least 10 similar experiments is shown. (C) Time course of extracellular ATP-dependent ciliary polypeptide phosphorylation. (D) Time course of extracellular ATP-dependent ciliary polypeptide dephosphorylation. Data represents a quantitative analysis of the magnitude of protein phosphorylation (C) and dephosphorylation (D) induced by extracellular ATP (100 μM). The optical density of protein bands on Western blots was measured, as described under materials and methods. The data indicate similar time dependences for the phosphorylation (C) of three major polypeptides (44, 37, and 27 kD) and dephosphorylation (D) of three additional polypeptides (130, 100, and 69 kD), which can be seen in A. The maximal phosphorylation and dephosphorylation effects were obtained at 1–5 min. Each point is the mean ± SEM of 4–10 experiments.

Figure 4.

Ionomycin treatment induces phosphorylation and dephosphorylation of the same polypeptides that are modified by extracellular ATP. Typical pattern of phosphorylated polypeptides in cilia isolated from epithelial tissue after exposure to ionomycin (1 μM) for 0.5 min (lane 2) or 1 min (lane 3), as compared with untreated control (lane 1). Western blot analysis using anti-phosphoserine antibody indicates an increase in phosphorylation of 27, 37, and 44 kD polypeptides, and a decrease in phosphorylation of 35, 69, 100, and 130 kD polypeptides. A representative blot of eight similar experiments is shown.

Figure 5.

Inhibition of NOS abolishes extracellular ATP-dependent phosphorylation (A) and dephosphorylation (B) of ciliary polypeptides in intact tissue. Epithelial tissue was preincubated for 10 min with or without 50 μM L-NAME followed by stimulation with extracellular ATP (100 μM) for 5 min. Cilia were then isolated and subjected to Western blot analysis, as described under materials and methods. Quantitative analysis of the magnitude of phosphorylation shows the complete elimination of phosphorylation and dephosphorylation induced by ATP (100 μM) in the presence of L-NAME (50 μM). Each column represents the mean ± SEM averaged over six experiments.

Figure 6.

Inhibition of GC or PKG prevents ATP-dependent polypeptide phosphorylation and dephosphorylation in intact tissue. Epithelial tissue was preincubated for 10 min in the absence (lanes 1 and 2) or in the presence of either 50 μM LY-83583 (GC inhibitor, lanes 3 and 4) or 1 μM KT-5823 (PKG inhibitor, lanes 5 and 6) followed by stimulation with extracellular ATP (100 μM) for 5 min (lanes 2, 4, and 6). Cilia were then isolated and subjected to Western blot analysis, as described under materials and methods. Application of LY-83583 did not alter the basal level of phosphorylation (lane 3), although preincubation with KT-5823 resulted in a moderate phosphorylation of 44-kD polypeptide (lane 5), which was not influenced by the ATP addition (compare lanes 5 and 6). A representative of seven similar experiments is shown.

Figure 7.

cGMP induces phosphorylation of ciliary polypeptides in cilia isolated from untreated tissue. (A) Isolated cilia were incubated with or without 1 μM cGMP for 3 min, as described under materials and methods, followed by Western blot analysis with anti-phosphoserine antibody. The data represent cGMP-stimulated phosphorylation (lane 2) as compared with control (lane 1). A representative of five similar experiments is shown. (B) Quantitative analysis of the magnitude of polypeptide phosphorylation induced by 0.1 μM cGMP (empty bars) and 1 μM cGMP (hatched bars). The data indicate that cGMP modifies the major polypeptides, which are phosphorylated by extracellular ATP or ionomycin in intact tissue experiments (Figs. 3 and 4). Each column represents the mean ± SEM averaged over five to seven experiments.

Figure 8.

The ciliary polypeptides found to be modified by extracellular ATP (A) and PKG (B) are predominantly located in the axoneme. (A) Phosphorylated polypeptide profiles of untreated cilia and its fractions separated after detergent treatment. Cilia were isolated from untreated epithelial tissue (lane 1), separated to detergent-soluble membrane (lane 2) and detergent-insoluble axonemal (lane 3) fractions, and subjected to Western blot analysis, as described under materials and methods. The data indicate that the major polypeptides found to be phosphorylated (27 and 44 kD) and dephosphorylated (35, 100, and 130 kD) after exposure to extracellular ATP (Fig. 3) are predominantly located in the axoneme, while the 69-kD polypeptide appears mainly in the membrane fraction. Note that the 37-kD polypeptide is not presented either in whole cilia or in the separated fractions. This is consistent with the above data (Fig. 3 A, lane 1; Fig. 4, lane 1; Fig. 6, lane 1), showing the absence of the 37-kD band under control conditions (in unstimulated cilia). Moreover, enrichment of the axonemal fraction in all polypeptides, as compared with whole cilia, can be seen. A representative of seven similar experiments is shown. (B) PKG is an integral axonemal protein. Reprobing of the nitrocellulose membrane presented in A with cGKIα antibody demonstrates the polypeptide of 76 kD typical for PKG isoform in both whole cilia and the axonemal fraction. An enrichment of the axonemal fraction in PKG, as compared with whole cilia, can be seen. A representative of three similar blots is shown.

Figure 9.

Molecular events underlying ATP-induced CBF enhancement via phosphorylation and dephosphorylation of ciliary proteins. See details in the text.