Cyclic phosphorylation/dephosphorylation cascade in bovine brain coated vesicles

Coated vesicles are involved in transport of membrane proteins between several intracellular membrane-bound compartments. These vesicles possess a specific 50-kDa protein which is phosphorylated and dephosphorylated by a coated-vesicle-specific kinase and phosphatase. We studied this phosphorylation/dephosphorylation cascade system and show that the phosphorylation level of the 50-kDa protein is governed by the ATP/ADP ratio.     

Molecular mechanisms determining sperm motility initiation in two sparids (Sparus aurata and Lithognathus mormyrus)

Molecular mechanisms involved in sperm motility initiation in two sparids (Sparus aurata and Lithognathus mormyrus) have been studied. Our comparative study demonstrates that osmolality is the key signal in sperm motility activation in both species, whereas K(+) and Ca(2+) do not have any role. The straight-line velocity that resulted, however, was significantly different when measured in sperm activated with non-ionic and/or calcium-free solutions with respect to that measured in seawater-activated sperm. In both species, motility initiation depends on cAMP-dependent protein phosphorylation. The phosphorylation/dephosphorylation patterns that resulted in gilthead and striped sea bream were quite different. In gilthead sea bream, the phosphorylated proteins have molecular weights of 174, 147, 138, 70, and 9-15 kDa, whereas the dephosphorylated proteins have molecular weights of 76, 57, and 33 kDa. In striped sea bream, phosphorylation after sperm motility activation occurred on proteins of 174, 147, 103, 96, 61, 57, and 28 kDa, whereas only one protein of 70 kDa resulted from dephosphorylation. Matrix-assisted laser desorption ionization-time of flight analyses allowed identification of the following proteins: In gilthead sea bream, the 9-15 kDa proteins that were phosphorylated after motility activation include an A-kinase anchor protein (AKAP), an acetyl-coenzyme A synthetase, and a protein phosphatase inhibitor, and in striped sea bream, 103- and 61-kDa proteins that were phosphorylated after motility activation were identified as a phosphatase (myotubularin-related protein 1) and a kinase (DYRK3), respectively.     

Cell cycle tyrosine phosphorylation of p34cdc2 and a microtubule-associated protein kinase homolog in Xenopus oocytes and eggs.

We have examined the time course of protein tyrosine phosphorylation in the meiotic cell cycles of Xenopus laevis oocytes and the mitotic cell cycles of Xenopus eggs. We have identified two proteins that undergo marked changes in tyrosine phosphorylation during these processes: a 42-kDa protein related to mitogen-activated protein kinase or microtubule-associated protein-2 kinase (MAP kinase) and a 34-kDa protein identical or related to p34cdc2. p42 undergoes an abrupt increase in its tyrosine phosphorylation at the onset of meiosis 1 and remains tyrosine phosphorylated until 30 min after fertilization, at which point it is dephosphorylated. p42 also becomes tyrosine phosphorylated after microinjection of oocytes with partially purified M-phase-promoting factor, even in the presence of cycloheximide. These findings suggest that MAP kinase, previously implicated in the early responses of somatic cells to mitogens, is also activated at the onset of meiotic M phase and that MAP kinase can become tyrosine phosphorylated downstream from M-phase-promoting factor activation. We have also found that p34 goes through a cycle of tyrosine phosphorylation and dephosphorylation prior to meiosis 1 and mitosis 1 but is not detectable as a phosphotyrosyl protein during the 2nd through 12th mitotic cell cycles. It may be that the delay between assembly and activation of the cyclin-p34cdc2 complex that p34cdc2 tyrosine phosphorylation provides is not needed in cell cycles that lack G2 phases. Finally, an unidentified protein or group of proteins migrating at 100 to 116 kDa increase in tyrosine phosphorylation throughout maturation, are dephosphorylated or degraded within 10 min of fertilization, and appear to cycle between low-molecular-weight forms and high-molecular-weight forms during early embryogenesis.     

Casein kinase I phosphorylates the 25-kDa mRNA cap-binding protein

The 25-kDa mRNA cap-binding protein (eIF-4E) exists in both phosphorylated and dephosphorylated forms in eukaryotic cells. Phosphorylated eIF-4E appears to be preferentially associated with 48 S initiation complexes and with the 220-kDa subunit of eIF-4F. In addition, dephosphorylation of eIF-4E has been observed during heat shock and mitosis which are accompanied by decreased protein synthesis. However, the control of eIF-4E phosphorylation and its regulatory role remain poorly understood. Using eIF-4E as a substrate we have identified and purified from rabbit reticulocytes a protein kinase that phosphorylates eIF-4E in vitro. This enzyme phosphorylated eIF-4E on both serine and threonine residues with an apparent Km of 3.7 microM. The molecular mass of the enzyme and specificity for substrates other than eIF-4E suggested that this enzyme was a species of casein kinase I. This was confirmed by comparing the phosphopeptide map of the purified reticulocyte enzyme with that of rabbit skeletal muscle casein kinase I and by comparing phosphopeptide maps of eIF-4E phosphorylated in vitro by each enzyme. We conclude that casein kinase I phosphorylates eIF-4E in vitro and suggest that eIF-4E may be phosphorylated by casein kinase I in intact cells under some physiologic conditions.     

Phosphorylation Modulates Calpain-Mediated Proteolysis and Calmodulin Binding of the 200-kDa and 160-kDa Neurofilament Proteins

Abstract: The effects of enzymatic dephosphorylation on neurofilament interaction with two calcium-binding proteins, calpain and calmodulin, were examined. Dephosphorylation increased the rate and extent of 200-kDa neurofilament protein proteolysis by calpain. In contrast, dephosphorylation of the 160-kDa neurofilament protein did not alter the rate or extent of calpain proteolysis. However, the calpain-induced breakdown products of native and dephosphorylated 160-kDa neurofilament protein were different. Dephosphorylation did not change the proteolytic rate, extent, or breakdown products of the 68-kDa neurofilament protein. Calmodulin binding to the purified individual 160- and 200-kDa neurofilament proteins was increased following dephosphorylation. These results suggest that phosphorylation may regulate the metabolism and function of neurofilaments by modulating interactions with the calcium-activated proteins calpain and calmodulin.     

Dephosphorylation of inner arm 1 is associated with ciliary reversals in Tetrahymena thermophila

In many organisms, depolarizing stimuli cause an increase in intraciliary Ca2+, which results in reversal of ciliary beat direction and backward swimming. The mechanism by which an increase in intraciliary Ca2+ causes ciliary reversal is not known. Here we show that Tetrahymena cells treated with okadaic acid or cantharidin to inhibit protein phosphatases do not swim backwards in response to depolarizing stimuli. We also show that both okadaic acid and cantharidin inhibit backward swimming in reactivated, extracted cell models treated with Ca2+. In contrast, treatment of whole cells or extracted cell models with protein kinase inhibitors has no effect on backward swimming. These results suggest that a component of the axonemal machinery is dephosphorylated during ciliary reversal. The phosphorylation state of inner arm dynein 1 (I1) was determined before and after cells were exposed to depolarizing conditions that induce ciliary reversal. An I1 intermediate chain is phosphorylated in forward swimming cells but is dephosphorylated in cells treated with a depolarizing stimulus. Our results suggest that dephosphorylation of Tetrahymena inner arm dynein 1 may be an essential part of the mechanism of ciliary reversal in response to increased intraciliary Ca2+.     

Light-induced dephosphorylation of a 65-kDa protein in the cyanobacterium Synechocystis sp. PCC6803

We found that a 65-kDa protein (p65) of Synechocystis sp. PCC 6803 is dephosphorylated in a light-dependent manner. In darkness, p65 was specifically phosphorylated and then completely dephosphorylated within 2 min upon exposure to high-intensity light. The phosphorylation of p65 recurred after 8 hours incubated in the dark following light exposure. Green (540-560 nm) and red (660 nm) light dephosphorylated p65 efficiently, with the efficiency being greater with green light. These results suggest that p65 is a novel substrate involved in the quantity and quality of light-dependent dephosphorylation in cyanobacteria.     

Purification of and production of an antibody against a 63,000 Mr stimulus-sensitive phosphoprotein in Paramecium

In vivo labeling of Paramecium cells with 32Pi most heavily labels a minor 63-kDa protein that undergoes a rapid, Ca2+-dependent dephosphorylation when the cell is stimulated to release. This stimulus-sensitive phosphoprotein was isolated and purified to apparent homogeneity. A polyclonal affinity purified antibody made against the purified protein recognizes both the phosphorylated and dephosphorylated forms of the protein. The phosphorylated 63-kDa protein is found in the cytosolic fraction; it is slightly acidic with two isoelectric forms at pI 5.8 and 6.2 and probably exists as a monomeric 60-65-kDa polypeptide in the native state. The labeled phosphoamino acid of the protein is phosphoserine. The affinity purified antibody recognizes a third isoelectric form at pI 6.3 that appears unlabeled. The specificity of the antibody was confirmed by showing that it immunoprecipitates the correct protein, i.e. the stimulus-sensitive 63-kDa phosphoprotein. The availability of purified 63-kDa protein as well as an antibody against it will now allow molecular, biochemical, and immunocytochemical studies into the role of this protein in the mechanism of exocytosis.     

Phosphorylation and dephosphorylation of human platelet surface proteins by an ecto-protein kinase/phosphatase system.

We have characterized a novel ecto-protein kinase activity and a novel ecto-protein phosphatase activity on the membrane surface of human platelets. Washed intact platelets, when incubated with [gamma-32P]ATP in Tyrode's buffer, showed the phosphorylation of a membrane surface protein migrating with an apparent molecular mass of 42 kDa on 5-15% SDS polyacrylamide gradient gels. The 42 kDa protein could be further resolved on 15% SDS gels into two proteins of 39 kDa and 42 kDa. In this gel system, it was found that the 39 kDa protein became rapidly phosphorylated and dephosphorylated, whereas the 42 kDa protein was phosphorylated and dephosphorylated at a much slower rate. NaF inhibited the dephosphorylation of these proteins indicating the involvement of an ecto-protein phosphatase. The platelet membrane ecto-protein kinase responsible for the phosphorylation of both of these proteins was identified as a serine kinase and showed dependency on divalent cations Mg2+ or Mn2+ ions. Ca2+ ions potentiated the Mg(2+)-dependent ecto-protein kinase activity. The ecto-protein kinase rapidly phosphorylated histone and casein added exogenously to the extracellular medium of intact platelets. Following activation of platelets by alpha-thrombin, the incorporation of [32P]phosphate from exogenously added [gamma-32P]ATP by endogenous protein substrates was reduced by 90%, suggesting a role of the ecto-protein kinase system in the regulation of platelet function. The results presented here demonstrate that both protein kinase and protein phosphatase activities reside on the membrane surface of human platelets. These activities are capable of rapidly phosphorylating and dephosphorylating specific surface platelet membrane proteins which may play important roles in early events of platelet activation and secretion.     

Cilia regeneration in starved tetrahymena: an inducible system for studying gene expression and organelle biogenesis.

Deciliated starved Tetrahymena recover motility with kinetics similar to those of growing cells and, like growing cells, require RNA and protein synthesis for regeneration. Comparisons of polysome profiles and electrophoretic analyses of newly synthesized proteins indicate, however, that the basal level of protein synthesis in starved cells is markedly lower than that in growing cells. This difference allows demonstration of changes in protein synthesis following deciliation of starved cells which cannot be detected (if they occur at all) in growing cells. Deciliation of starved cells induces a specific and orderly program of protein synthesis. The synthesis of an 80,000 dalton protein (deciliation-induced protein, DIP) begins shortly after deciliation, comprises 15% of the protein synthesized from 20-60 min, and declines around 60 min after deciliation, shortly after most cells have begun to regenerate cilia. The synthesis of a 55,000 dalton protein is also induced during regeneration and has been identified as tubulin using a well characterized antibody made to ciliary tubulin. Tubulin synthesis is undetectable during the first hour after deciliation even though 60-80% of the cells regain mobility and regenerate short but clearly visible cilia. Tubulin synthesis begins 60 min after deciliation and continues for 2 hr. At its peak, tubulin comprises 7-8% of the protein synthesized. The results of actinomycin D addition at different times after deciliation suggest that RNA required for DIP synthesis is synthesized early (0-30 min), while RNA required for tubulin is synthesized later and over a longer period (30-90 min). Thus deciliation of starved cells, an event occurring at the cell periphery, initiates a well defined and reproducible series of events culminating in cilia formation. This system should be useful in elucidating the molecular mechanisms regulating gene expression and organelle biogenesis in Tetrahymena.     

Tyrosine Phosphorylation of A17 during Vaccinia Virus Infection: Involvement of the H1 Phosphatase and the F10 Kinase

Vaccinia virus encodes two protein kinases (B1 and F10) and a dual-specificity phosphatase (VH1), suggesting that phosphorylation and dephosphorylation of substrates on serine/threonine and tyrosine residues are important in regulating diverse aspects of the viral life cycle. Using a recombinant in which expression of the H1 phosphatase can be regulated experimentally (vindH1), we have previously demonstrated that repression of H1 leads to the maturation of noninfectious virions that contain several hyperphosphorylated substrates (K. Liu et al., J. Virol. 69:7823-7834). In this report, we demonstrate that among these is a 25-kDa protein that is phosphorylated on tyrosine residues in H1-deficient virions and can be dephosphorylated by recombinant H1. We demonstrate that the 25-kDa phosphoprotein represents the product of the A17 gene and that A17 is phosphorylated on serine, threonine, and tyrosine residues during infection. Detection of phosphotyrosine within A17 is abrogated when Tyr(203) (but not Tyr(3), Tyr(6), or Tyr(7)) is mutated to phenylalanine, suggesting strongly that this amino acid is the site of tyrosine phosphorylation. Phosphorylation of A17 fails to occur during nonpermissive infections performed with temperature-sensitive mutants defective in the F10 kinase. Our data suggest that this enzyme, which was initially characterized as a serine/threonine kinase, might in fact have dual specificity. This hypothesis is strengthened by the observation that Escherichia coli induced to express F10 contain multiple proteins which are recognized by antiphosphotyrosine antiserum. This study presents the first evidence for phosphotyrosine signaling during vaccinia virus infection and implicates the F10 kinase and the H1 phosphatase as the dual-specificity enzymes that direct this cycle of reversible phosphorylation.     

Phosphorylation of NPA58, a rat nuclear pore-associated protein, correlates with its mitotic distribution

At the onset of mitosis in higher eukaryotic cells, the nuclear envelope and its components including subunits of the nuclear pore complexes are disassembled, and these are reassembled toward the end of mitosis. We have studied the role of protein phosphorylation in this process, by investigating the phosphorylation status of a specific pore-associated protein during mitosis. Using a monoclonal antibody, mAb E2, earlier shown to inhibit nuclear protein import in rat fibroblast cells, we have identified a 58-kDa protein termed NPA58 that is partially associated with nuclear pores based on a high degree of coincident immunofluorescence in dual labeling experiments with mAb 414, a well-studied pore-complex-reactive antibody. NPA58 is specifically phosphorylated during mitosis and dephosphorylated upon release from metaphase arrest. Confocal microscopy analysis shows that NPA58 is dispersed in the cytoplasm early in mitosis when it is phosphorylated, while its relocalization in the reforming nuclear envelope during telophase temporally correlates with its dephosphorylation upon release from metaphase arrest. Our data provide in vivo evidence that the modifications mediated by phosphorylation and dephosphorylation are required for regulating the mitotic localization of a nuclear-pore-associated protein.     

Dephosphorylation of cofilin accompanies heat shock-induced nuclear accumulation of cofilin

Cofilin is a widely distributed 21-kDa actin-modulating protein that forms intranuclear actin/cofilin rods in cultured fibroblastic cells exposed to heat shock or 10% dimethyl sulfoxide. In this study, cofilin was shown to be phosphorylated on a serine residue in cultured rat fibroblastic 3Y1 cells. Two-dimensional gel electrophoresis revealed that about 50% of the cofilin was phosphorylated in 3Y1 cells at 37 degrees C. Exposure of the cells to heat shock at 43 degrees C induced dephosphorylation of cofilin. The dephosphorylation of cofilin was detected about 30 min after the temperature shift and was completed within 120 min. Moreover, treatment of cells with 10% dimethyl sulfoxide also caused the dephosphorylation of cofilin. However, incubation of the cells with an isotonic NaCl solution, which induced cytoplasmic actin/cofilin rods, did not induce dephosphorylation of cofilin. Other cellular stress agents such as 6% ethanol or 50 microM sodium arsenite, which caused some heat shock responses in cells, did not induce dephosphorylation of cofilin. Thus, cofilin dephosphorylation was closely correlated with its nuclear accumulation. Incubation of the enucleated 3Y1 cells at 43 degrees C still induced dephosphorylation of cofilin, suggesting that the dephosphorylation occurred mostly in the cytoplasm in intact cells. It is likely that cofilin is dephosphorylated in the cytoplasm prior to its nuclear accumulation.     

The MgATP-dependent protein phosphatase and protein phosphatase 1 have identical substrate specificities

The MgATP-dependent phosphorylase phosphatase was found to have a broad substrate specificity. Its activity against all phosphoproteins tested was dependent upon preincubation with the activating factor FA and MgATP. The enzyme dephosphorylated and inactivated phosphorylase kinase and inhibitor 1, and dephosphorylated and activated glycogen synthase and acetyl-CoA carboxylase. Glycogen synthase was dephosphorylated at similar rates whether it had been phosphorylated by cyclic-AMP-dependent protein kinase, phosphorylase kinase or glycogen synthase kinase 3. The enzyme also catalysed the dephosphorylation of ATP citrate lyase, initiation factor eIF-2, and troponin I. The properties of the MgATP-dependent protein phosphatase from either dog liver or rabbit skeletal muscle showed a remarkable similarity to highly purified preparations of protein phosphatase 1 from rabbit skeletal muscle. The relative activities of the two enzymes against all phosphoproteins tested was very similar. Both enzymes dephosphorylated the beta-subunit of phosphorylase kinase 40-fold faster than the alpha-subunit, and both enzymes were inhibited by identical concentrations of the two proteins termed inhibitor 1 and inhibitor 2, which inhibit protein phosphatase 1 specifically. These results demonstrate that the MgATP-dependent protein phosphatase is a type-1 protein phosphatase, and is distinct from type-2 protein phosphatases which dephosphorylate the alpha-subunit of phosphorylase kinase and are unaffected by inhibitor 1 and inhibitor 2. The possibility that the MgATP-dependent protein phosphatase is an inactive form of protein phosphatase 1 and that both proteins share the same catalytic subunit is discussed.     

Phosphorylation of the myosin light chains and satellite proteins in detergent-skinned arterial smooth muscle

Isoelectric focusing of extracts prepared from detergent-skinned porcine carotid artery showed that contraction was associated with phosphorylation of the regulatory myosin light chains and two additional proteins of the same apparent molecular weight (20,000). These two proteins, previously described as satellites, did not appear to be artifactually derived from the phosphorylated light chains during electrophoresis. That is, each of the phosphorylated proteins migrated as separate and distinct proteins when subjected to a second cycle of isoelectric focusing. Moreover, relaxation of skinned fibers was associated with dephosphorylation of the light chains and both satellites. These findings suggest that the satellites may represent varients of the light chains per se, or another regulatory protein which is reversibly phosphorylated and dephosphorylated during contraction and relaxation of vascular smooth muscle.     

Quantitative proteomics indicate a strong correlation of mitotic phospho-/dephosphorylation with non-structured regions of substrates

Protein phosphorylation plays a critical role in the regulation and progression of mitosis. >40,000 phosphorylated residues and the associated kinases have been identified to date via proteomic analyses. Although some of these phosphosites are associated with regulation of either protein-protein interactions or the catalytic activity of the substrate protein, the roles of most mitotic phosphosites remain unclear. In this study, we examined structural properties of mitotic phosphosites and neighboring residues to understand the role of heavy phosphorylation in non-structured domains. Quantitative mass spectrometry analysis of mitosis-arrested and non-arrested HeLa cells revealed >4100 and > 2200 residues either significantly phosphorylated or dephosphorylated, respectively, at mitotic entry. The calculated disorder scores of amino acid sequences of neighboring individual phosphosites revealed that >70% of dephosphorylated phosphosites exist in disordered regions, whereas 50% of phosphorylated sites exist in non-structured domains. A clear inverse correlation was observed between probability of phosphorylation in non-structured domain and increment of phosphorylation in mitosis. These results indicate that at entry to mitosis, a significant number of phosphate groups are removed from non-structured domains and transferred to more-structured domains. Gene ontology term analysis revealed that mitosis-related proteins are heavily phosphorylated, whereas RNA-related proteins are both dephosphorylated and phosphorylated, suggesting that heavy phosphorylation/dephosphorylation in non-structured domains of RNA-binding proteins plays a role in dynamic rearrangement of RNA-containing organelles, as well as other intracellular environments.     

The phosphorylation state of an aurora-like kinase marks the length of growing flagella in Chlamydomonas

Flagella and cilia are structurally polarized organelles whose lengths are precisely defined, and alterations in length are related to several human disorders. Intraflagellar transport (IFT) and protein signaling molecules are implicated in specifying flagellar and ciliary length, but evidence has been lacking for a flagellum and cilium length sensor that could participate in active length control or establishment of structural polarity. Previously, we showed that the phosphorylation state of the aurora-like protein kinase CALK in Chlamydomonas is a marker of the absence of flagella. Here we show that CALK phosphorylation state is also a marker for flagellar length. CALK is phosphorylated in cells without flagella, and during flagellar assembly it becomes dephosphorylated. Dephosphorylation is not simply a consequence of initiation of flagellar assembly or of time after experimentally induced flagellar loss, but instead requires flagella to be assembled to a threshold length. Analysis of cells with flagella of varying lengths shows that the threshold length for CALK dephosphorylation is ~6 μm (half length). Studies with short and long flagellar mutants indicate that cells detect absolute rather than relative flagellar length. Our results demonstrate that cells possess a mechanism for translating flagellar length into a posttranslational modification of a known flagellar regulatory protein.     

Novel Regulatory Role of Phosphorylated Clathrin Light Chain β in Bovine Brain Coated Vesicles

The 50-kilodalton (kDa) assembly polypeptide of bovine brain clathrin coated vesicles (CCVs) is phosphorylated in a cyclic nucleotide- and Ca2+-independent manner and is dephosphorylated by a Mg2+-ATP-dependent CCV phosphatase. This report provides evidence for modulation of the phosphorylation reaction of the 50-kDa assembly polypeptide by phosphorylated clathrin light chain beta (pLC beta). In vitro, phosphorylated LC beta inhibits phosphorylation of the 50-kDa polypeptide in CCVs. Furthermore, incubation of previously phosphorylated 50-kDa polypeptide in CCVs with phosphorylated LC beta results in a rapid dephosphorylation of the 50-kDa assembly polypeptide. Both phenomena are time and concentration dependent. Monoclonal antibodies to LC beta prevent the modulatory effect of phosphorylated LC beta on the 50-kDa assembly polypeptide phosphorylation in CCVs. The results obtained indicate for the first time, to our knowledge, that phosphorylated LC beta has a modulatory role in CCVs. The data also suggest that phosphorylated LC beta promotes activation of a coated vesicle phosphatase.     

Calcium-dependent phosphorylation of a protein in the frog olfactory cilia

In the cilia of vertebrate olfactory sensory neurons, cytoplasmic Ca(2+) concentration increases in response to odorant stimulation, and this increase has been implicated to have important roles in the regulation of olfactory responses. Since protein phosphorylation is often a regulatory mechanism of biological reactions, we explored the effect of Ca(2+) on phosphorylation reactions in the frog olfactory cilia. First, we found that a 45-kDa phosphoprotein (p45) is predominantly phosphorylated in vitro in the isolated cilia in a Ca(2+)-dependent manner. However, later studies showed that the phosphorylation level of p45 is controlled by a dynamic equilibrium between phosphorylation and dephosphorylation. Although both activities are enhanced at high Ca(2+) concentrations (K(1/2) = approximately 2 microM in both reactions), the enhancement of dephosphorylation is relatively greater than that of phosphorylation. As a result, the steady phosphorylation level of p45 is lower at high than at low Ca(2+) concentration. The phosphorylation/dephosphorylation equilibrium was founed to involve protein kinases sensitive to zinc and heparin, and an unknown phosphatase(s). The present result suggests the presence of a novel Ca(2+)-signaling pathway that involves phosphorylation of p45 in the olfactory cilia.     

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

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.