Regulation of pigment organelle translocation. II. Participation of a cAMP-dependent protein kinase

In intact goldfish xanthophores, the phosphorylation of a pigment organelle (carotenoid droplet) protein, p57, appears to play an important role in adrenocorticotropin (ACTH)- or cAMP-induced pigment organelle dispersion while the dephosphorylation of this protein upon withdrawal of ACTH or cAMP is implicated in pigment aggregation. In this paper, we report the cAMP-dependent phosphorylation of this protein in cell-free extracts of xanthophores as determined by the incorporation of 32P from [gamma-32P]ATP. As is the case in intact cells, p57 is the predominant protein phosphorylated in the presence of cAMP. The cAMP-dependent protein kinase which phosphorylates p57 is not bound to the isolated organelles but is found in the soluble portion of the cell extracts. Hence, the phosphorylation of p57 requires the carotenoid droplets bearing the substrate, soluble extract containing the kinase, cAMP (half-maximal activation at 0.5 microM), and Mg2+ (optimal at 5 mM or higher). The presence of protein phosphatase(s) in these extracts was shown indirectly by the stimulation of phosphorylation by fluoride. The phosphorylation of p57 does not appear to require a cell-specific kinase as soluble extracts of goldfish dermal nonpigment cells also phosphorylate p57 associated with isolated carotenoid droplets. Furthermore, using a constant amount of carotenoid droplets, a linear relationship was demonstrated between the rate of p57 phosphorylation and the amount of extract present in the assays. These results suggest that p57 is phosphorylated directly by a cAMP-dependent protein kinase and that the activity of this enzyme is important in regulating the intracellular movement of the pigment organelles of the xanthophore.     

cAMP-independent and cAMP-dependent protein phosphorylations by isolated goldfish xanthophore cytoskeletons: evidence for the association of cytoskeleton with a carotenoid droplet protein

Triton-insoluble cytoskeleton of nonpigment cells has bound protein kinase that phosphorylates, with or without added cAMP, tubulins and the intermediate filament proteins p60, p56, p53, and p45a to give multiple charge variants. In the absence of 8-Br-cAMP, Triton-insoluble cytoskeletons from xanthophores also phosphorylate p60, p56, and p45a, but not p53; tubulin phosphorylation may also be reduced. In the presence of 8-Br-cAMP, p53, as well as several other peptides, are phosphorylated. One of these latter peptides was identified as the carotenoid droplet (pigment organelle) protein p57, whose phosphorylation and dephosphorylation precede pigment dispersion and aggregation respectively (Lynch et al.: J. Biol. Chem. 261:4204-4211, 1986). The amount of pp57 produced depends on the state of pigment distribution in the xanthophores used to prepare the cytoskeletons for labeling. With cytoskeletons from xanthophores with aggregated pigment, pp57 is a major labeled phosphoprotein seen in two-dimensional gels. With cytoskeletons prepared from xanthophores with dispersed pigment, the yield of labeled pp57 is greatly reduced (by at least 90%). Together with earlier results, we propose that, in the aggregated state, p57 serves to bind carotenoid droplets to the cytoskeletons, most likely the microtubules. The significance of other cAMP-dependent phosphorylation reactions is unknown but may be related to cAMP-induced cytoskeleton rearrangement in intact xanthophores.     

Protein phosphorylation and the two stages of pigment organelle dispersion in permeabilized xanthophores: organelle protein phosphorylation alone supports only the first stage

We reported previously that, in cultured goldfish xanthophores, dispersion of aggregated carotenoid droplets (CDs) requires the specific phosphorylation of the CD protein p57 by a cAMP-dependent protein kinase and the presence of cytosol. We report here that, in permeabilized cells, the addition of the catalytic subunit of cAMP-dependent protein kinase and ATP phosphorylates p57 and converts the CDs from an immobile to a mobile state (first stage of CD dispersion). However, the CDs are restricted to the vicinity of the original site of the CD aggregate and do not actually disperse (second stage of CD dispersion) unless cytosol is also added. We propose that this process may be related to aspects of secretory processes.     

Regulation of pigment organelle translocation. I. Phosphorylation of the organelle-associated protein p57

Treatment of goldfish xanthophores with adrenocorticotropin (ACTH) or cyclic AMP (cAMP) induces the centrifugal movement of their pigment organelles from the center of the cells. Using purified xanthophores pulse labeled with 32Pi, we have shown that the dispersion of the organelles is accompanied by the phosphorylation of a pair of polypeptides, termed p57. After fractionation on sucrose gradients, nearly all of the p57 is found associated with the pigment organelles. The phosphorylation induced by ACTH or cAMP apparently occurs at multiple sites on p57. The minimal effective doses of ACTH or cAMP required to induce full pigment dispersion also fully stimulate the phosphorylation of p57. Increased phosphorylation of p57 is detectable within a minute after stimulating the cells and appears to be near completion during the early phases of pigment dispersion. Upon withdrawal of ACTH, these events are reversed; the pigment organelles reaggregate toward the center of the cells and p57 is dephosphorylated. Again, dephosphorylation commences soon after ACTH is withdrawn and is complete before the organelles have completely reaggregated. These results suggest a novel mechanism for governing the movement of these organelles which acts on the organelles themselves through the phosphorylation and dephosphorylation of p57.     

Regulation of the distribution of carotenoid droplets in goldfish xanthophores and possible implication to secretory processes

In goldfish xanthophores, the formation of pigment aggregate requires: 1) that a pigment organelle (carotenoid droplet) protein p57 be in the unphosphorylated state; 2) that self-association of pigment organelles occur in a microtubule-independent manner; and 3) that pigment organelles via p57 associate with microtubules. In the fully aggregated state, the pigment organelles are completely stationary. Pigment dispersion is initiated by activation of a cAMP-dependent protein kinase, which phosphorylates p57 and allows pigment dispersion via an active process dependent on F-actin and a cytosolic factor. This factor is not an ATPase, and its function is unknown. However, its abundance in different tissues parallels secretory activity of the tissues, suggesting a similarity between secretion and pigment dispersion in xanthophores. The identity of the motor for pigment dispersion is unclear. Experimental results show that pigment organelles isolated from cells with dispersed pigment have associated actin and ATPase activity comparable to myosin ATPase. This ATPase is probably an organelle protein of relative molecular mass approximately 72,000, and unlikely to be an ion pump. Isolated pigment organelles without associated actin have 5x lower ATPase activity. Whether this organelle ATPase is the motor for pigment dispersion is under investigation. The process of pigment aggregation is poorly understood, with conflicting results for and against the involvement of intermediate filaments.     

Bidirectional pigment granule movements of melanophores are regulated by protein phosphorylation and dephosphorylation

Studies were conducted to investigate the molecular basis for bidirectional pigment granule transport in digitonin-lysed melanophores. Pigment granule dispersion, but not aggregation, required cAMP and resulted in the phosphorylation of a 57 kd polypeptide. cAMP-dependent protein kinase inhibitor prevented this phosphorylation as well as pigment dispersal. In contrast, both pigment aggregation and the concomitant dephosphorylation of the 57 kd polypeptide were blocked by phosphatase inhibitors. These data support a model in which pigment dispersion and aggregation require protein phosphorylation and dephosphorylation, respectively. Furthermore, studies using the ATP analog, ATP gamma S, suggest either that protein phosphorylation alone is sufficient for dispersion or that transport is mediated by a unique force-generating ATPase that can use ATP gamma S for hydrolyzable energy.     

Purification of anterogin, a protein factor necessary for the dispersion of carotenoid droplets in permeabilized xanthophores of goldfish

We reported previously that the dispersion of carotenoid droplets in permeabilized xanthophores requires cAMP, ATP, and a cytosolic factor present in several secretory tissues as well as in xanthophores. We have now purified this factor from beef liver to apparent/near homogeneity. It appears to be a heterodimer with Mr approximately 125,000. The purified factor has little or no ATPase activity, with or without the presence of actin. Nor does it stimulate the ATPase activity of carotenoid droplets. Its exact function in carotenoid droplet dispersion is thus unclear. Since dispersion of carotenoid droplets is an anterograde translocation, we propose the name anterogin for this protein. We also report that yeast cytosol has anterogin activity.     

The cytosol as site of phosphorylation of the cyclic AMP-dependent protein kinase in adrenal steroidogenesis

The mitochondria, the microsomes and the cystosol have been described as possible sites of cAMP-dependent phosphorylation. However, there has been no direct demonstration of a cAMP-dependent kinase associated with the activation of the side-chain cleavage of cholesterol. We have investigated the site of action of the cAMP-dependent kinase using a sensitive cell-free assay. Cytosol derived from cells stimulated with ACTH or cAMP was capable of increasing progesterone synthesis in isolated mitochondria when combined with the microsomal fraction. Cytosol derived from cyclase or kinase of negative mutant cells did not. Cyclic AMP and cAMP-dependent protein kinase stimulated in vitro a cytosol derived from unstimulated adrenal cells. This cytosol was capable of stimulating progesterone synthesis in isolated mitochondria. Inhibitor of cAMP-dependent protein kinase abolished the effect of the cAMP. ACTH stimulation of cytosol factors is a rapid process observable with a half maximal stimulation at about 3 pM ACTH. The effect was also abolished by inhibitor of arachidonic acid release. The function of cytosolic phosphorylation is still unclear. The effect of inhibitors of arachidonic acid release, and the necessity for the microsomal compartment in order to stimulate mitochondrial steroidogenesis, suggest that the factor in the cytosol may play a role in arachidonic acid release.     

Partial characterization of a carotenoid droplet ATPase and its possible significance in carotenoid droplet dispersion in goldfish xanthophores

The dispersion of carotenoid droplets in permeabilized goldfish xanthophores is dependent on ATP, F-actin, and cytosol. We report here that the motor (ATPase, translocator) resides with the permeabilized cell remnants and not in the cytosol. We also report that the carotenoid droplets have an ATPase that is not conventional myosin, dynein, or an ion pump. Its activity appears to correlate with the actin content of the carotenoid droplet preparation. A carotenoid droplet protein of Mr 72,000 (p72) is shown to be labeled by irradiation with 8-azido-ATP with concomitant loss of ATPase activity of the carotenoid droplets. We propose that this protein may be the ATPase responsible for carotenoid droplet dispersion.     

Evidence for the involvement of cAMP-dependent protein kinase in the exocytosis of amylase from parotid acinar cells.

To determine the role of cAMP-dependent protein kinase in the exocytosis of amylase from rat parotid acini, I studied the effects of various cAMP analogues on amylase release and protein phosphorylation in saponin-permeabilized parotid cells. The dose-dependent responses of amylase release and protein phosphorylation evoked by cAMP, dibutyryl-cAMP, and 8-chlorophenylthio-cAMP were closely correlated. Furthermore, when the permeabilized cells were incubated with a combination of site-selective cAMP analogues, such as N6-benzoyl-cAMP plus 8-thiomethyl-cAMP (RII-directed) or N6-benzoyl-cAMP plus 8-aminohexylamino-cAMP (RI-directed), synergistic stimulation of amylase release was clearly observed. As reported previously [T. Takuma (1988) Biochem. J. 256, 867-871], however, the protein phosphorylation evoked by 8-chlorophenylthio-cAMP was markedly inhibited by H-8 without a significant decrease in amylase release. These results suggest that cAMP-dependent protein kinase is involved in the regulation of amylase exocytosis, although the role of its catalytic subunit is still uncertain.     

Hormonal activation of the cAMP-dependent protein kinases in AtT20 cells. Preferential activation of protein kinase I by corticotropin releasing factor, isoproterenol, and forskolin

The hormonal regulation of adenylate cyclase, cAMP-dependent protein kinase activation, and adrenocorticotropic hormone (ACTH) secretion was studied in AtT20 mouse pituitary tumor cells. Corticotropin releasing factor (CRF) stimulated cAMP accumulation and ACTH release in these cells. Maximal ACTH release was seen with 30 nM CRF and was accompanied by a 2-fold rise in intracellular cAMP. When cells were incubated with both 30 nM CRF and 0.5 mM 3-methylisobutylxanthine (MIX) cAMP levels were increased 20-fold, however, ACTH release was not substantially increased beyond release seen with CRF alone. The activation profiles of cAMP-dependent protein kinases I and II were studied by measuring residual cAMP-dependent phosphotransferase activity associated with immunoprecipitated regulatory subunits of the kinases. Cells incubated with CRF in the absence of MIX showed concentration-dependent activation of protein kinase I which paralleled stimulation of ACTH release. Protein kinase II was minimally activated. When cells were exposed to CRF in the presence of 0.5 mM MIX there was still a preferential activation of protein kinase I, although 50% of the cytosolic protein kinase II was activated. Complete activation of both protein kinases I and II was seen when cells were incubated with 0.5 mM MIX and 10 microM forskolin. Under these conditions cAMP levels were elevated 80-fold. CRF, isoproterenol, and forskolin stimulated adenylate cyclase activity in isolated membranes prepared from AtT20 cells. CRF and isoproterenol stimulated cyclase activity up to 5-fold while forskolin stimulated cyclase activity up to 15-fold. Our data demonstrate that ACTH secretion from AtT20 cells is mediated by small changes in intracellular levels of cAMP and activation of only a small fraction of the total cytosolic cAMP-dependent protein kinase in these cells is required for maximal ACTH secretion.     

The roles of cAMP and cAMP-dependent protein kinase in the regulation of adrenocortical functions: analysis using DNA-mediated gene transfer

DNA-mediated gene transfer was used to evaluate the cause and effect relationship between mutations in cAMP-dependent protein kinase activity and cellular resistance of adrenocortical tumor cells to ACTH and cAMP. Protein kinase defective, Kin 8 adrenocortical tumor cells were transformed with genomic DNA from an ACTH- and cAMP-responsive adrenocortical cell line and screened for the recovery of morphological responses to the cAMP analog 8-bromo-cAMP (8BrcAMP). 8BrcAMP-responsive transformants were recovered with a frequency of approximately 0.5 per 10(3) transformation-competent cells. These transformants recovered the ability to round up in the presence of ACTH and were able to respond to both ACTH and 8BrcAMP with increased steroidogenesis. They also recovered cAMP-dependent protein kinase activity. The transformants, however, were unstable and concomitantly lost cAMP-dependent protein kinase activity and steroidogenic and morphological responses to ACTH and 8BrcAMP. These observations suggest that a single gene, probably the gene encoding the regulatory subunit of cAMP-dependent protein kinase, is responsible for the resistance of the Kin 8 mutant to ACTH and cAMP.     

Substrates for adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase in the rat pituitary gland

1. Substrates for cAMP-dependent protein kinase were investigated in anterior, intermediate, and neural lobes of the rat pituitary gland. In a cell-free assay system, cAMP increased phosphorylation of 17 K, 33 K, and 60 K macromolecules of the anterior lobe, 17 K, 33 K, 60 K, and 80 K macromolecules of the intermediate lobe, and 60 K, 80 K, and 85 K macromolecules of the neural lobe. 2. Other nucleotides were tested in the intermediate lobe; 8 Br-cAMP mimicked cAMP, cGMP was much less effective than cAMP or 8 Br-cAMP, and 5'-AMP showed no significant effect. The purified catalytic subunit of cAMP-dependent protein kinase evoked the same phosphorylation pattern as the endogenous kinase. 3. Maximum cAMP-dependent phosphorylation occurred at between 1 and 2 min of incubation; after 20 min, phosphorylation was reduced by 80%. This suggests the presence of phosphatase activity in the intermediate lobe. 4. When tested upon dispersed intermediate lobe cells permeabilized by high-voltage electrical discharges, cAMP increased phosphorylation of the 17 K and 33 K macromolecules.     

Actin-dependent carotenoid droplet dispersion in permeabilized cultured goldfish xanthophores

Organelle translocations are essential cellular processes. Although much progress has been made with regards to microtubule-dependent organelle translocations, little is known about actin-dependent organelle translocation(s) except cytoplasmic streaming in Nitella. On the other hand, there is indirect evidence that actin-dependent organelle translocation may be involved in secretion. We now present evidence that the dispersion of the pigment organelles carotenoid droplets in goldfish xanthophores is apparently actin dependent and that this process may be related to secretory processes. We show that, in digitonin-permeabilized goldfish xanthophores, the pigment organelles can be induced to disperse by a combination of cAMP, ATP, and xanthophore cytosol. This induced dispersion is inhibited by DNase I, phalloidin, or anti-actin, but not by anti-tubulin or anti-intermediate filament proteins, suggesting a dependence on F-actin. Since the dispersion of carotenoid droplets and secretion both involve outward translocation of organelles, we tested the possibility that cytosols of secretory tissues have similar activity. Such activity was indeed found in different tissues, apparently in parallel with the secretory activity of the tissues, suggesting that pigment dispersion in xanthophores and some secretory processes may share a common component.     

Genetic and biochemical evidence that trehalase is a substrate of cAMP-dependent protein kinase in yeast

In Saccharomyces cerevisiae, trehalase activity in crude extracts obtained from wild type cells was activated about 3-fold by preincubation with cAMP and ATP. The inactive trehalase fractionated by DEAE-Sephacel chromatography was activated by the addition of the cAMP-dependent protein kinase fraction from wild type cells in the presence of cAMP and ATP. Using the crude extract obtained from bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase, the stimulation of trehalase activity was observed in the absence of cAMP. The cAMP-dependent protein kinase of CYR3 mutant cells which had a high Ka value for cAMP in the phosphorylation reaction required a high cAMP concentration for activation of trehalase. Increased activation of partially purified inactive trehalase (Mr = 320,000) was observed to correlate with increased phosphorylation of a protein (Mr = 80,000) identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay results using various mutants altered in cAMP metabolism indicated that the activation and phosphorylation of inactive trehalase fractions depended on the cAMP concentration accumulated in mutant cells. Inactivation and dephosphorylation of active trehalase fractions were observed by treatment with alkaline phosphatase or crude cell extracts. The results indicated that the conversion of inactive form of trehalase to the active form is regulated by cAMP through cAMP-dependent protein kinase.     

cAMP-dependent phosphorylation and hexamethylene-bis-acetamide induced dephosphorylation of p19 in murine erythroleukemia cells

The objective of this study was to investigate cyclic-adenosinemonophosphate (cAMP)-dependent phosphorylation in murine erythroleukemia (MEL) cells and to identify either direct substrates of cAMP-dependent kinase or downstream effectors of cAMP dependent phosphorylation with a potential function in growth and differentiation. MEL-cells rendered deficient in cAMP-dependent protein kinase (A-kinase) activity by stable transfection with DNA encoding for either a mutant regulatory subunit or a specific peptide inhibitor of A-Kinase (PKI) are unable to differentiate normally in response to chemical inducers. We have identified by 2-D Western blotting 2 phosphorylated forms of p19, a highly conserved 18-19 kDa cytosolic protein that is frequently upregulated in transformed cells and undergoes phosphorylation in mammalian cells upon activation of several signal transduction pathways. The phosphorylation of the more acidic phosphorylated form is increased in a cAMP-dependent fashion and impaired in cells deficient in cAMP-dependent kinase (A-kinase). Treatment of MEL-cells with the chemical inducer of differentiation hexamethylene-bisacetamide (HMBA) led to dephosphoryation of this phosphoform. Our data are compatible with previous observations which imply that phosphorylation of Ser 38 in p19 by p34cdc2-kinase leads to a more basic phosphoform and simultaneous phosphorylation by mitogen-activated kinase of Ser 25 in response to protein kinase C and the cAMP- dependent kinase creates the more acidic species.     

Bi-directional regulation of dephosphorylation of cAMP-dependent phosphorylated proteins by cAMP and calcium in permeabilized rat heart cells

We studied the regulation of dephosphorylation of cAMP-dependent phosphorylated proteins of isolated, permeabilized (skinned) myocardial cells from adult rat. Staurosporine, a potent inhibitor of protein kinase, inhibited cAMP-dependent phosphorylation of phospholamban and troponin-I, the key proteins in the control of contraction and relaxation of the myocardial cells. Staurosporine antagonized the stimulatory action of cAMP on the spontaneous beating of the myocytes accompanied by dephosphorylation of phospholamban but not of troponin-I at pCa 7-8. In cold ATP dilution experiments with apparent stoppage of protein phosphorylation, dephosphorylation of phospholamban was accelerated both by Ca2+ and staurosporine but that of troponin-I took place only in the presence of Ca2+ ion (pCa less than 6.5). These phenomena suggest a bi-directional regulation of dephosphorylation of the key proteins by the intracellular messengers cAMP and Ca2+.     

The effect of retinoic acid on protein phosphorylation in mouse melanoma cells

Vitamin A inhibits growth and increases the activity of cAMP-dependent protein kinase in B16 mouse melanoma cells. In this report we show that retinoic acid (RA) treatment of intact cells alters their subsequent in vitro protein phosphorylation, but we could not demonstrate any changes in in vivo protein phosphorylation. A 48-h treatment with RA results in a concentration-dependent decrease of protein phosphorylation of a 95K molecular weight (MW) protein in both supernatant and particulate fractions. The phosphorylation of this protein does not appear to be regulated by cAMP. Proteins at 92K and 82K MW in the supernatant fraction are increased in phosphorylation. The former (but not the latter) is regulated by cAMP. In the particulate fraction a variety of proteins 12K-68K MW are increased in phosphorylation, as the cells are treated with increasing amounts of RA. The phosphorylation of most of these proteins is regulated by cAMP. Another inhibitor of B16 cell growth, melanocyte-stimulating hormone (MSH) also alters protein phosphorylation. At short incubation periods (1 h), this hormone stimulates phosphorylation of a number of proteins (17-40K MW), while in longer incubation periods (48 h) phosphorylation is inhibited. All of these phosphorylations appear to be regulated by cAMP. We attempted to repeat these observations using intact-cell phosphorylation with 32PO4. In two experiments we saw small changes in the phosphorylation of proteins. In most experiments, however, we could find no change in the phosphoproteins. Further experiments have led us to question the in vivo phosphorylation, since treatment of the cells with MSH, cholera toxin, or db-cAMP also did not affect intact-cell protein phosphorylation. We have previously documented that under these latter conditions cAMP levels are greatly elevated and cAMP-dependent protein kinase is activated. The in vitro phosphorylation results suggests that in RA-treated cells, kinase activities and/or protein substrate levels are changing. However, the physiological significance of the particular MW phosphoproteins changes we have described must await resolution of the in vivo phosphorylation data.     

Ultrastructural Immunogold Localization of Some Organelle-Transport Relevant Proteins in Wholemounted Permeabilized Nonextracted Goldfish Xanthophores

By whole-cell transmission electron microscopy (WCTEM), we recently demonstrated that carotenoid droplets are transported by elongating or retracting endoplasmic reticular cisternae in goldfish xanthophores. Here we report that permeabilized xanthophores demonstrate immunogold reactivity against several proteins involved in organelle translocation. The gold labeling against β-tubulin and the intermediate filament protein p45a were found on microtubules and intermediate filaments. Labeling with antiactin was found on nonidentifiable structures, on vesicles of unknown origin, occasional labeling on carotenoid droplets, and on occasional microfilaments. Immunoreactivity was demonstrated with anti-p57 on the carotenoid droplet surface, confirming previous results (Lynch et al., 1986a,b). Labeling with anti-PCD6 subunit (of the inositol trisphosphate/ryanodine receptor) was demonstrated on carotenoid droplets suggesting they possess calcium channels. Anti-MAP 1C (dynein) immunolabeling was generally seen on club-shaped structures in the cytomatrix and on carotenoid droplets. Finally, immunogold labeling with anti-MAP 2a + 2b was seen on a meshwork of microfilaments and intermediate filaments. Finally, this is the first report of a WCTEM technique for permeabilized cells that reveals immunoreactive elements, organelles, and cytomatrix components without the additional requirements of extraction or fracturing.