Identification of 36 kDa phosphoprotein in fibrous sheath of hamster spermatozoa

In our previous studies (Fujinoki et al., 2001, 2003), we reported that two types of 36 kDa proteins, designated 36K-A protein and 36K-B protein, obtained from hamster sperm flagella, are associated with motility activation and phosphorylated in a cAMP-dependent manner at serine residues. In the present experiments, we focused on the hamster (Mesocricetus auratus) 36K-A protein, which was analyzed by peptide mass finger printing and amino acid sequencing. The results suggest that 36K-A protein is a pyruvate dehydrogenase E1 component β subunit lacking the N-terminal 30 amino acids. Moreover, our results suggest that 36 K-A protein is localized in the fibrous sheath of the principal piece of hamster spermatazoa.     

Identification of the 58-kDa phosphoprotein associated with motility initiation of hamster spermatozoa

In our previous paper [M. Fujinoki et al. (2001) BIOMED: Res. 22, 45-58], we reported that the 58-kDa protein obtained from hamster sperm flagella was phosphorylated at serine residues in association with the start of motility. In the present experiments, we identified and localized the 58-kDa protein. The 58-kDa protein was assumed to exist in the acrosomal region domain of the sperm head and the whole sperm flagellum. In particular, a large amount of 58-kDa protein was localized in the equatorial segment of the acrosomal region domain of the sperm head and the middle piece of the sperm flagellum. In the next step, the 58-kDa protein was identified by peptide mass finger printing and LC-MS/MS analysis. The results suggested that the 58-kDa protein was ATP synthase H(+) transporting F1 beta, which is one of the mitochondrial components. Therefore, it is likely that the 58-kDa protein is associated with ATP production in the mitochondrial sheath in the middle piece of the sperm flagellum, and H(+) transport in the sperm head and the sperm flagellum except for the middle piece, since ATP synthase also acts as an H(+) pump.     

Regulation of microtubule sliding by a 36-kDa phosphoprotein in hamster sperm flagella

Cyclic AMP has been shown essential for activation of sperm motility. When immotile hamster caudal epididymal spermatozoa were suspended in a Ca2+-deficient solution, they showed a sluggish motility. Spermatozoa were demembranated and transferred to an ATP-containing reactivation solution. Demembranated spermatozoa did not exhibit reactivated flagellar movement unless cAMP was added. Conversely, when the immotile epididymal spermatozoa were suspended in a Ca2+-containing solution, they were immediately activated to display a vigorous motility; demembranated spermatozoa also exhibited reactivated flagellar movement in the reactivation solution without cAMP. Further investigation of microtubule sliding properties revealed that the effects of Ca2+ on live spermatozoa were identical with the effects of cAMP on demembranated spermatozoa both in microtubule sliding velocity and sliding disintegration pattern. Moreover, a 36-kDa flagellar protein was found to be phosphorylated in a cAMP-dependent manner and coupled to the motility activation. A polyclonal antibody against this protein was developed and showed specific immunolocalization and significant inhibitory effects on microtubule sliding disintegration. These results indicate that extracellular Ca2+ owes its effect to triggering intracellular cAMP production, and cAMP-dependent phosphorylation of a 36-kDa phosphoprotein activates hamster sperm motility through regulation of microtubule sliding properties.     

Involvement of tyrosine protein kinase in the initiation of flagellar movement in rainbow trout spermatozoa

Initiation of flagellar motility in spermatozoa of the rainbow trout, Salmo gairdneri, is closely related to phosphorylation of a protein of molecular mass 15 kDa (Morisawa, M., and Hayashi, H. (1986) Biomed. Res. 6, 181-184). We have been able to solubilize the protein and its kinase and then construct an assay system in vitro for the phosphorylation of the 15-kDa protein. In vitro, the protein was phosphorylated in a cAMP-dependent manner. The phosphorylation absolutely required the presence of Mg2+ ions. at millimolar concentrations, but not of Ca2+ ions. The amino acid residue which was phosphorylated in the 15-kDa protein was tyrosine. The 15-kDa protein was purified to near homogeneity by affinity chromatography on a column of adenosine nucleotides conjugated to Eupergit and ion-exchange chromatography on DEAE-cellulose. The effects of synthetic inhibitors of protein kinase on the phosphorylation of the 15-kDa protein were also studied.     

The 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae are regulated differentially by phosphorylation via cAMP-dependent protein kinase.

Evidence is presented that demonstrated that the 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae (Morlock, K. R., McLaughlin, J. J., Lin, Y.-P., and Carman, G. M. (1991) J. Biol. Chem. 266, 3586-3593) were regulated differentially by phosphorylation. Purified 45-kDa phosphatidate phosphatase was phosphorylated by cAMP-dependent protein kinase whereas purified 104-kDa phosphatidate phosphatase was not phosphorylated. cAMP-dependent protein kinase catalyzed the phosphorylation of pure 45-kDa phosphatidate phosphatase at a serine residue which resulted in a stimulation (2.4-fold) of phosphatidate phosphatase activity. Alkaline phosphatase catalyzed the dephosphorylation of pure 45-kDa phosphatidate phosphatase which resulted in an inhibition (1.3-fold) of phosphatidate phosphatase activity. Results of studies using mutants (bcy1 and cyr1) defective in cAMP-dependent protein kinase activity corroborated the results of the phosphorylation studies using pure preparations of phosphatidate phosphatase. The 45-kDa phosphatidate phosphatase phosphorylated in vitro and in vivo had phosphopeptides in common. The activation of the GAL10-RAS2val19 allele in mutant cells resulted in an increase in the synthesis of diacylglycerols and triacylglycerols. These results were consistent with the phosphorylation and activation of 45-kDa phosphatidate phosphatase by cAMP-dependent protein kinase in vivo.     

Protein phosphatase 2C is involved in the cAMP-dependent ciliary control in Paramecium caudatum

Forward swimming of the Triton-extracted model of Paramecium is stimulated by cAMP. Backward swimming of the model induced by Ca(2+) is depressed by cAMP. Cyclic AMP and Ca(2+) act antagonistically in setting the direction of the ciliary beat. Some ciliary axonemal proteins from Paramecium caudatum are phosphorylated in a cAMP-dependent manner. In the presence of cAMP, axonemal 29- and 65-kDa polypeptides were phosphorylated by endogenous A-kinase in vitro. These phosphoproteins, however, were not dephosphorylated after in vitro phosphorylation, presumably because of the low endogenous phosphoprotein phosphatase activity associated with isolated axonemes. We purified the protein phosphatase that specifically dephosphorylated the 29- and 65-kDa phosphoproteins from Paramecium caudatum. The molecular weight of the protein phosphatase was 33 kDa. The protein phosphatase had common characteristics as protein phosphatase 2C (PP2C). The characteristics of the protein phosphatase were the same as those of the PP2C from Paramecium tetraurelia (PtPP2C) [Grothe et al., 1998: J. Biol. Chem. 273:19167-19172]. We concluded that the phosphoprotein phosphatase is the PP2C from Paramecium caudatum (PcPP2C). The PcPP2C markedly accelerated the backward swimming of the Triton-extracted model in the presence of Ca(2+). On the other hand, the PcPP2C slightly depressed the forward swimming speed. This indicates that the PP2C plays a role in the cAMP-dependent regulation of ciliary movement in Paramecium caudatum through dephosphorylation of 29- and/or 65-kDa regulatory phosphoproteins by terminating the action of cAMP.     

DNA-mediated transfer of cAMP resistance in CHO cells

Chinese hamster ovary (CHO) strain 10215 carries a dominant mutation which confers resistant to cAMP by virtue of an altered catalytic subunit of the cAMP-dependent protein kinase (Evain et al., 1979). This mutation was transferred to wild-type CHO cells by DNA-mediated gene transfer. Based on the absence of cAMP growth inhibition, seven transformant colonies were isolated. One of these, 11586, was studied in detail. This transformant showed the same phenotype as the mutant, including resistance to the morphological changes and growth inhibitory effects of 1 mM 8-Br-cAMP, reduced total cAMP dependent protein kinase activity and lowered sensitivity of the kinase to cAMP activation. When the cAMP-dependent protein kinase was fractionated on a DEAE-cellulose column, the transformant was lacking in type II cAMP dependent protein activity, to the same degree as the mutant. The transformant and mutant, but not wild-type cells, also failed to phosphorylate a 52,000-dalton protein in a cAMP-dependent manner. These characteristics support the conclusion that the gene for the mutant cAMP-dependent protein kinase has been transferred. The ability to transfer this gene by DNA-mediated transfer suggests that this methodology may be useful for the molecular isolation of the gene encoding the catalytic subunit of cAMP-dependent protein kinase.     

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.     

cAMP-dependent protein phosphorylation of membrane proteins in the parotid gland, platelets and liver. Comparison of a 22-kDa phosphoprotein from rat parotid microsomes (protein III) with phosphoproteins of similar molecular size from platelet and liver membranes

Stimulation of secretion in exocrine secretory glands leads to the phosphorylation of a 22-kDa membrane protein (protein III) whose function is still unknown [Jahn et al. (1980) Eur. J. Biochem. 112, 345-352; Jahn & S?ling (1980) Proc. Natl Acad. Sci. USA 78, 6903-6906]. This report describes the comparison of this protein with phosphorylated membrane proteins of similar molecular mass in platelets and liver. Incubation of platelets with agents which raise the intracellular cAMP concentration results in the phosphorylation of a 22-kDa protein which is also phosphorylated in membrane preparations by endogenous kinases or by exogenous cAMP-dependent protein kinase. It is shown that this protein is distinct from protein III although both proteins have the same molecular mass and are substrates of cAMP-dependent protein kinase. In contrast to platelets, protein III could be demonstrated in liver microsomes. This indicates that the function of protein III is not exclusively linked to the stimulus-secretion coupling in exocrine cells.     

Phosphorylation of the 165-kDa dihydropyridine/phenylalkylamine receptor from skeletal muscle by protein kinase C

Dihydropyridine-sensitive Ca2+ channels exist in many different types of cells and are believed to be regulated by various protein phosphorylation and dephosphorylation reactions. The present study concerns the phosphorylation of a putative component of dihydropyridine-sensitive Ca2+ channels by the calcium and phospholipid-dependent protein kinase, protein kinase C. A skeletal muscle peptide of 165 kDa, which is known to contain receptors for dihydropyridines, phenylalkylamines, and other Ca2+ channel effectors, was found to be an efficient substrate for protein kinase C when the peptide was phosphorylated in its membrane-bound state. Protein kinase C incorporated 1.5-2.0 mol of phosphate/mol of peptide within 2 min into the 165-kDa peptide in incubations carried out at 37 degrees C. In contrast to the membrane-bound peptide, the purified 165-kDa peptide in detergent solution was phosphorylated to a markedly less extent than its membrane-bound counterpart; less than 0.1 mol of phosphate/mol of peptide was incorporated. Preincubation of the membranes with several types of drugs known to be Ca2+ channel activators or inhibitors had no specific effects on the rate and/or extent of phosphorylation of the 165-kDa peptide by protein kinase C. The phosphorylation of the membrane-bound 165-kDa peptide by protein kinase C was compared to that catalyzed by cAMP-dependent protein kinase and was found to be not additive. Prior phosphorylation of the 165-kDa peptide by cAMP-dependent protein kinase prevented subsequent phosphorylation of the peptide by protein kinase C. Phosphoamino acid analysis indicated that protein kinase C phosphorylated the 165-kDa peptide at both serine and threonine residues. Phosphopeptide mapping experiments showed that protein kinase C phosphorylated one unique site in the 165-kDa peptide, and, in addition, other sites that were phosphorylated by either cAMP-dependent protein kinase or a multifunctional Ca2+/calmodulin-dependent protein kinase. The results suggest that the 165-kDa dihydropyridine/phenylalkylamine receptor could serve as a physiological substrate of protein kinase C in intact cells. It is therefore possible that the regulation of dihydropyridine-sensitive Ca2+ channels by activators of protein kinase C may occur at the level of this peptide.     

Differential regulation of basic protein phosphorylation by calcium phospholipid and cyclic-AMP-dependent protein kinases

Myelin basic protein, an 80-kilodalton (kDa) protein in rat oligodendrocytes, and an 80-kDa basic protein in neuroblastoma x neonatal Chinese hamster brain explant hybrids were phosphorylated extensively when the cells were treated with either phorbol esters (TPA) or diacylglycerols (e.g., oleyoyl-acetylglycerol). TPA-stimulated phosphorylation was inhibited by pre-incubation with 50 microM psychosine (galactosyl-sphingosine), confirming that it is mediated through the phospholipid-dependent protein kinase C (PK-C). Surprisingly, phosphorylation of these proteins was inhibited by incubation of cells with agents which result in activation of cyclic-AMP-dependent protein kinase (dibutyryl cyclic AMP or forskolin). In contrast, phosphorylation of other nonbasic proteins, for example, the oligodendrocyte-specific 2',3'-cyclic nucleotide phosphohydrolase, was stimulated under these conditions (Vartanian et al.: Proceedings of the National Academy of Sciences of the United States of America 85:939, 1988). The possible role of cyclic AMP in activating specific phosphatases or restricting the availability of diacylglycerol for PK-C activation is discussed.     

Hyperactivation of hamster sperm motility by temperature-dependent tyrosine phosphorylation of an 80-kDa protein.

Protein phosphorylation and dephosphorylation are believed to play key roles in regulation of sperm motility. Here we examine the effect of temperature on hamster sperm motility and protein tyrosine phosphorylation status. As in previous work, a decrease from 37 degrees C to 22 degrees C caused loss of hyperactivated motility. We now find that cooling also produces a dephosphorylation of several 48-80-kDa flagellar peptides. A return to 37 degrees C restored hyperactivation but resulted in rephosphorylation of only an 80-kDa protein. Conversely, hyperactivation and phosphorylation of the 80-kDa component were insensitive to incubation temperature for sperm incubated with the protein phosphatase inhibitor, calyculin A, or for sperm demembranated by detergent extraction. These results strongly indicate that the temperature-sensitive tyrosine phosphorylation status of an 80-kDa sperm flagellar peptide explains the sensitivity of hyperactivation to temperature.     

cAMP-dependent phosphorylation associated with activation of motility of Ciona sperm flagella

Demembranated spermatozoa of Ciona do not become motile when provided with MgATP, unless their motility is activated in vivo before demembranation or unless the demembranated spermatozoa are activated in vitro with cAMP or with the catalytic subunit of a cAMP-dependent protein kinase. CAMP causes a greater than fivefold enhancement of ³²P incorporation by demembranated spermatozoa. Analysis by one-dimensional PAGE and autoradiography shows several axonemal protein bands that become ³²P-labeled during in vitro activation with cAMP and identifies protein bands whose labeling is specifically reduced if motility of the spermatozoa is activated before demembranation, suggesting that these proteins also become phosphorylated during activation of motolity in vivo. These phosphorylated proteins appear to include dynein heavy-chain components, but axonemal tubulin is not phosphorylated. Partially phosphorylated spermatozoa can be activated by an increase in KCI concentration, which appears to dissociate one phosphorylated component from the axoneme.     

Multiple phosphorylation sites in the 165-kilodalton peptide associated with dihydropyridine-sensitive calcium channels

Evidence from electrophysiological and ion flux studies has established that dihydropyridine-sensitive calcium channels are subject to regulation by neurotransmitter-mediated phosphorylation and dephosphorylation reactions. In the present study, we have further characterized the phosphorylation by cAMP-dependent protein kinase and a multifunctional Ca/calmodulin-dependent protein kinase of the membrane-associated form of the 165-kDa polypeptide identified as the skeletal muscle dihydropyridine receptor. The initial rates of phosphorylation of the 165-kDa peptide by both protein kinases were found to be relatively good compared to the rates of phosphorylation of established substrates of the enzymes. Phosphorylation of the 165-kDa peptide by both protein kinases was additive. Prior phosphorylation by either one of the kinases alone did not preclude phosphorylation by the second kinase. The cAMP-dependent protein kinase phosphorylated the 165-kDa peptide preferentially at serine residues, although a small amount of phosphothreonine was also formed. In contrast, after phosphorylation of the 165-kDa peptide by the Ca/calmodulin-dependent protein kinase, slightly more phosphothreonine than phosphoserine was recovered. Phosphopeptide mapping indicated that the two kinases phosphorylated the peptide at distinct as well as similar sites. Notably, one major site phosphorylated by the cAMP-dependent protein kinase was not phosphorylated by the Ca/calmodulin-dependent protein kinase, while other sites were phosphorylated to a high degree by the Ca/calmodulin-dependent protein kinase, but to a much lesser degree by the cAMP-dependent protein kinase. The results show that the 165-kDa dihydropyridine receptor from skeletal muscle can be multiply phosphorylated at distinct sites by the cAMP- and Ca/calmodulin-dependent protein kinases. As the 165-kDa peptide may be the major functional unit of the dihydropyridine-sensitive Ca channel, the results suggest that the phosphorylation-dependent modulation of Ca channel activity by neurotransmitters may involve phosphorylation of the 165-kDa peptide at multiple sites.     

Flagellar motility requires the cAMP-dependent phosphorylation of a heat-stable NP-40-soluble 56 kd protein, axokinin

Using NP-40-treated dog sperm as a model, the stimulatory effect of cAMP upon reactivated flagellar motility has been shown to be dependent upon the cAMP-dependent phosphorylation of a heat-stable NP-40-soluble protein of 56 kd. Examination by two-dimensional polyacrylamide gel electrophoresis of NP-40 extract proteins phosphorylated with gamma-32P-ATP revealed a major cAMP-dependent phosphopeptide at 56 kd. This is the only cAMP-dependent phosphoprotein common to NP-40 extracts of all tissues that show cAMP-dependent stimulation of flagellar motility. These cells and tissues include sea urchin, dog, and human sperm, as well as dog trachea and retina. Moreover, this phosphoprotein is absent in nonstimulatory extracts from tissues such as skeletal muscle, brain, and liver. We conclude that the cAMP-dependent phosphorylation of the 56 kd peptide represents a major regulatory component of not only sperm but other types of axonemal motility as well.     

Phosphorylation of Endogenous Proteins by Adenosine 3':5'-Monophosphate-Dependent Protein Kinase in Mouse Neuroblastoma Cells

Abstract: Increased intracellular adenosine 3':5'-monophosphate (cAMP) levels and activation of cAMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) in vivo were correlated in mouse neuroblastoma cells grown in the presence of 1 mM-⁶ N.O ²-dibutyryl 3':5'-monophosphate (Bt₂cAMP). The time course for activation showed that cAMP-dependent protein kinases were activated by 30 min. A heat-stable inhibitor protein inhibited a majority of activated cAMP-dependent protein kinase. Activation of cAMP—dependent protein kinase caused additional phosphorylation of proteins when compared with untreated control cells, as demonstrated by endogenous phosphorylation of proteins in vitro using [γ-³²P]ATP and analysis by two—dimensional polyacrylamide gel electrophoresis. The phosphorylation data show selective phosphorylation of specific proteins by cAMP-independent and cAMP-dependent protein kinase. Among the proteins in the postmitochondrial supernatant fraction phosphorylated by cAMP-dependent protein kinases, two proteins with a molecular weight of 43,000 were heavily phosphorylated. It is suggested that phosphorylation of cellular proteins by cAMP-dependent protein kinases might be involved in the cAMP-modulated biochemical changes in neuroblastoma cells.     

Role of tyrosine phosphorylation of flagellar proteins in hamster sperm hyperactivation.

Despite extensive study of sperm motility, little is known of the mechanism of mammalian sperm hyperactivation. Here we describe a novel method for preparation of rodent sperm flagella and use it to show a correlation between tyrosine phosphorylation of flagellar proteins and hyperactivation of hamster sperm. When hyperactivation was produced by a 3.5-h incubation in a medium supporting capacitation, four major tyrosine-phosphorylated peptides of 90-, 80-, 62-, and 48-kDa mass were detected in flagellar extracts. Incubation with calyculin A, an inhibitor of protein phosphatases 1 and 2A, produced hyperactivation within 40 min but only a single 80-kDa phosphotyrosine-containing flagellar component. Conversely, incubation with inhibitors of either protein kinase A (H8) or protein tyrosine kinase (tyrphostin 47) prevented both hyperactivation and the production of tyrosine-phosphorylated flagellar peptides. These results indicate a strong correlation of hyperactivation with the tyrosine phosphorylation of sperm flagellar peptides, and they strongly implicate an 80-kDa component as a major mediator of the mechanism that produces hyperactivated motility of hamster sperm.     

Cyclic AMP- and Ca2(+)-dependent protein kinases in Plasmodium falciparum

The cyclic AMP- and Ca2(+)-dependent protein kinase activities of Plasmodium falciparum were partially characterized after purification of parasites from host erythrocytes by N2 cavitation and Percoll gradient centrifugation. Proteins of molecular weights 80, 54, 51, and 31.5 kDa were phosphorylated in a cAMP-dependent manner in cytosolic extracts of isolated P. falciparum. Cytosolic extracts also contained cAMP-dependent histone II-A kinase activity with an average Vmax of 131.1 pmol/32P/min/mg protein and a Km for cAMP of 85nM. Upon photoaffinity labeling with [32P]-8-N3-cAMP, a 53-kDa protein was specifically labeled in parasite cytosol. A metabolically labeled protein of the same molecular weight was identified by cAMP-agarose affinity chromatography. The 53-kDa protein cochromatographed with cAMP-dependent histone II-A kinase activity on DEAE-cellulose, suggesting that it is the regulatory subunit of the kinase. Ca2(+)-dependent phosphorylation of proteins of molecular weights 195, 158, 51, 47.5, and 15 kDa was demonstrated in a membrane fraction from parasites free of the erythrocyte membrane. This activity was not stimulated by either calmodulin or phospholipid plus diacylglycerol and was absent from the membranes of uninfected erythrocytes. Of several exogenous substrates tested, none were found to be a substrate for this Ca2(+)-dependent kinase. Both cAMP- and Ca2(+)-dependent kinases phosphorylated serine and threonine residues.     

Inhibition by cyclic AMP of guanine nucleotide-induced activation of phosphoinositide-specific phospholipase C in human platelets

Phosphoinositide-specific phospholipase C (PLC) activity of human platelet membranes was activated by the nonhydrolyzable guanine nucleotide GTP gamma S. This activation did not occur in either membranes prepared from dibutyryl cyclic AMP-pretreated platelets (A-membranes) or those prepared from untreated cells and subsequently incubated with cyclic AMP (cAMP) (B-membranes). This cAMP-mediated inhibition was abolished in the presence of inhibitors of cAMP-dependent protein kinase (A-kinase), suggesting that the inhibition was due to phosphorylation of (a) protein component(s). No significant differences were observed in the basal PLC activity and the extent of pertussis toxin-catalyzed ADP-ribosylation among control membranes and the two types of phosphorylated membranes (A- and B-membranes). GTP-binding activities of Gs, Gi and GTP-binding proteins of lower molecular masses were not altered by the phosphorylation of the membranes. These findings suggest that a GTP-binding protein is involved in the GTP gamma S-mediated activation of PLC and that cAMP (plus A-kinase) inhibits this activation by phosphorylating a membrane protein (probably a 240-kDa protein), rather than the GTP-binding protein or PLC itself. It is likely that this phosphorylation uncouples the GTP-binding protein from PLC.     

Phosphorylation of a single subunit of the epithelial Na+ channel protein following vasopressin treatment of A6 cells

Arginine vasopressin (antidiuretic hormone, ADH) stimulation of sodium transport in high electrical resistance epithelia is accompanied by adenylate cyclase stimulation and cAMP accumulation. The hypothesis of direct phosphorylation of the purified amiloride-blockable epithelial Na+ channel protein by cAMP-dependent protein kinase A after ADH treatment of cultured cells was investigated in this study. Phosphate-depleted A6 cells (a cell line derived from toad kidney) were exposed to 32PO4(3-) in the absence or presence of basolateral ADH (100 milliunits/ml). After 20 min (the time needed for ADH to increase maximally Na+ transport), the Na+ channels were extracted from the cells and purified. At every stage of purification, only one subunit of the Na+ channel, namely, the 315-kDa subunit, was specifically phosphorylated as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography or scintillation counting. In addition, a polyclonal antibody raised against purified epithelial Na+ channel protein was able to immunoprecipitate the phosphorylated channel protein from a detergent-solubilized fraction of vasopressin-treated A6 cells. This same subunit was also specifically phosphorylated in vitro when the purified Na+ channel protein was incubated with gamma-[32P]ATP and the purified catalytic subunit of the cAMP-dependent protein kinase. Thus, only a single component, the 315-kDa subunit, of the Na+ channel protein complex (which is composed of six subunits) can be phosphorylated both in vivo and in vitro. This subunit is selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 2-3 mol of 32P/mol of protein.