Phosphorylation of keratin intermediate filaments by protein kinase C, by calmodulin-dependent protein kinase and by cAMP-dependent protein kinase.

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type II keratin 8 (molecular mass 55 kDa; basic type) in a 1:1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by cAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2+/calmodulin-dependent protein kinase II were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.     

Cyclic AMP-dependent phosphorylation of two size forms of alpha 1 subunits of L-type calcium channels in rat skeletal muscle cells

Skeletal muscle dihydropyridine-sensitive calcium channels are in vitro substrates for cAMP-dependent protein kinase. In the present work, alpha 1 subunits were isolated from cultured skeletal muscle cells by immunoprecipitation with a specific monoclonal antibody under conditions where proteolysis and dephosphorylation were prevented. Two forms of alpha 1 subunit, 200 and 160 kDa, were identified by back phosphorylation in vitro with cAMP-dependent protein kinase, specific immunoprecipitation, and phosphopeptide mapping. Treatment of cells with forskolin, isoproterenol, calcitonin gene-related peptide, or 8-bromo-cAMP to increase intracellular cAMP reduced 32P incorporation into all phosphopeptides in vitro by 60-80% indicating that increases in cAMP caused endogenous phosphorylation of all sites on both alpha 1(200) and alpha 1(160) to nearly maximal levels. The extents of basal and stimulated phosphorylation in vivo were estimated by back phosphorylation methods to be 35-40% and 83-86%, respectively. In muscle cells metabolically labeled with 32P, 3 mol of phosphate were incorporated into alpha 1 subunits. Forskolin stimulated 32P incorporation into alpha 1 subunits 1.6-fold. Taken together, our results show that skeletal muscle cells contain two forms of the alpha 1 subunit which both are basally phosphorylated on cAMP-dependent phosphorylation sites and are further phosphorylated in response to agents that increase intracellular cAMP.     

Phosphorylation modulates keratin structure.

Bovine hoof keratin was shown to be a substrate for cAMP-dependent protein kinase using [gamma-32P]ATP. Natural-abundance cross-polarization (CP) MAS 13C NMR was used to examine the effect of phosphorylation on keratin structure. When short contact times were used, phosphorylation was shown to increase the number of residues in the motionally restricted portions of the protein; i.e., a portion(s) of the protein became more rigid upon phosphorylation. Circular dichroism (CD) spectra showed a spectral shape characteristic of alpha helix for this keratin. Phosphorylation of the keratin by cAMP-dependent protein kinase resulted in a CD spectrum with the same shape but of greater apparent intensity. This may have been the result of an increase in the alpha-helical content of the protein. These data showed that the structure of keratin changed significantly upon phosphorylation by cAMP-dependent protein kinase. The region of the keratin molecule most likely to be altering its structure was the end of the molecule, which was involved in the formation of, and intracellular attachment of, intermediate filaments. Therefore, these data suggested that cAMP-dependent phosphorylation may produce significant changes in the intracellular organization of intermediate filaments. When the keratin was phosphorylated using cold ATP, magic-angle spinning (MAS) 31P nuclear magnetic resonance (NMR) revealed two resonances arising from the phosphorylation sites on the keratin. The more shielded resonance was shown to arise from cAMP-dependent protein kinase phosphorylation. Static 31P NMR measurements suggested that at least two classes of cAMP-dependent sites existed with the same isotropic 31P chemical shift; one was considerably motionally restricted with respect to the other.     

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.     

Integration of different keratins into the same filament system after microinjection of mRNA for epidermal keratins into kidney epithelial cells

We have isolated poly (A)+ RNA, highly enriched in keratin mRNA from bovine muzzle epidermis, and injected it into epithelial cells of a different type, i.e., cultured kidney epithelial cells of the same (MDBK) or taxonomically distant (PtK2) species. Both recipient cell lines contain keratin polypeptides that are different from those present in epidermal cells. Using keratin subtype-specific antibodies in immunofluorescence and immunoelectron microscopy, we show that foreign keratin mRNAs when injected into a different type of epithelial cell can recruit polyribosomes and are translated together with the keratin mRNAs of the host cell. Foreign epidermal keratins are excluded from vimentin filaments and other structures but readily coassemble with the endogenous keratins and appear to be integrated into the meshwork of the preexisting kidney-type keratin filaments. Our observations indicate that different sets of keratin polypeptides from the same or different species can coassemble in the living cell into a common filament system. Thus we have developed a procedure that allows experimental alteration of the intermediate filament cytoskeleton within living epithelial cells.     

Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependent protein kinase and the myosin light chain kinase

Microinjection of the catalytic subunit of cAMP-dependent protein kinase (A-kinase) into living fibroblasts or the treatment of these cells with agents that elevate the intracellular cAMP level caused marked alterations in cell morphology including a rounded phenotype and a complete loss of actin microfilament bundles. These effects were transient and fully reversible. Two-dimensional gel electrophoresis was used to analyze the changes in phosphoproteins from cells injected with A-kinase. These experiments showed that accompanying the disassembly of actin microfilaments, phosphorylation of myosin light chain kinase (MLCK) increased and concomitantly, the phosphorylation of myosin P- light chain decreased. Moreover, inhibiting MLCK activity via microinjection of affinity-purified antibodies specific to native MLCK caused a complete loss of microfilament bundle integrity and a decrease in myosin P-light chain phosphorylation, similar to that seen after injection of A-kinase. These data support the idea that A-kinase may regulate microfilament integrity through the phosphorylation and inhibition of MLCK activity in nonmuscle cells.     

Locus of inhibitory action of cAMP-dependent protein kinase in the antigen receptor-triggered cytotoxic T lymphocyte activation pathway

The mechanism of the cAMP involvement in regulation of cellular functions was studied here using a novel functional assay (antigen receptor-triggered exocytosis of granules) of cloned cytotoxic T lymphocytes (CTL). We suggest that cAMP-dependent protein kinase, protein kinase A, counteracts the protein kinase C and Ca2+-mediated stimulatory T-cell antigen receptor (TcR)-triggered biochemical pathway. This suggestion is supported by experimental results which satisfy criteria for protein kinase A involvement in cellular functions. Pretreatment of CTL with cholera toxin induces cAMP accumulation in CTL, partially inhibits TcR-triggered "lethal hit" delivery to the target cell, and almost completely blocks TcR-triggered exocytosis of granules from CTL. Other agents that raise the intracellular level of cAMP, including forskolin and isobutylmethylxanthine (IBMX) also inhibit TcR-triggered CTL activation. Involvement of cAMP-dependent protein kinase in an inhibitory pathway is suggested by the synergistic effects of cyclic nucleotide analogs 8-bromo-cAMP and N6-benzoyl-cAMP in inhibition of TcR-triggered exocytosis. Forskolin and IBMX inhibited TcR-triggered phosphoinositide turnover in CTL, suggesting that cAMP affected very early events in signal transduction that follow TcR cross-linking by a ligand. The ability of IBMX to inhibit CTL activation when the TcR cross-linking step was by-passed by the combination of phorbol myristate acetate and ionophore A23187 suggests that the locus of inhibitory effect of cAMP is at both the early and late stages of the TcR-triggered transmembrane signaling pathway.     

The involvement of cyclic adenosine monophosphate in the control of schistosome miracidium cilia

This study examined the possible involvement of cyclic adenosine monophosphate (cAMP) in the control of ciliary action of Schistosoma mansoni miracidia. Miracidia immobilized in hypertonic NaCl solution were treated with 3 compounds that are known to increase intracellular cAMP concentrations. Forskolin, at a concentration of 50 microM, induced 50.1% of the miracidia to swim in hypertonic solution. The corresponding values obtained for 3-isobutyl-1-methylxanthine (IBMX) at 1 mM and 8-bromo-cAMP at 10 mM were 42.2 and 50.4%, respectively. The motility-enhancing effect of these compounds was dose dependent. Nevertheless, the swimming speed of miracidia activated in this way was only 10% of that observed in artificial pond water (APW). Cholera toxin had no apparent effect on miracidia swimming in hypertonic NaCl solution. Likewise, swimming in APW treated with forskolin at 50 microM, IBMX at 1 mM, or 8-bromo-cAMP at 10 mM did not induce any apparent change in motility. Miracidia swimming in APW were then treated with 3 compounds that decrease the intracellular concentration of cAMP. MDL-12,330A, at a concentration of 250 microM, caused a dramatic decrease in swimming over a period of 1 hr. Likewise, SQ22536 and imidazole, at concentrations of 20 and 50 mM, respectively, caused 36.5 and 73.4% decreases in swimming under the same conditions. Finally, inhibitors of cAMP-dependent protein kinase, i.e., PKI(14-22)amide, H89, and H88, completely inhibited miracidia swimming in APW at concentrations of 25, 50, and 100 microM, respectively. These results suggest that cAMP and cAMP-dependent protein kinase are involved in osmosis-controlled ciliary motion of schistosome miracidia.     

The cyclic nucleotide-dependent phosphorylation of aortic smooth muscle membrane proteins

Membrane proteins of Mr 240,000, 130,000, and 85,000 (GS-proteins) were rapidly and selectively phosphorylated in particulate fractions of rabbit aortic smooth muscle in the presence of [Mg-32P]ATP and low concentrations of cGMP (Ka = 0.01 microM) or cAMP (Ka = 0.2 microM). The effects of both cyclic nucleotides in this preparation were mediated entirely by an endogenous, membrane-bound form of cGMP-dependent protein kinase (G-kinase). The GS-proteins were also phosphorylated by the soluble form of G-kinase purified from bovine lung; this effect was most evident following removal of endogenous G-kinase from the membranes using Na2CO3 and high salt washes. The membrane-bound and cytosolic forms of G-kinase phosphorylated the Mr 130,000 GS-protein with the same specificity as determined by two-dimensional peptide mapping. Despite this functional homology between the two forms of G-kinase, only the particulate enzyme appears to play a role in phosphorylating the GS-proteins. Although little endogenous cAMP-dependent protein kinase (A-kinase) activity was detected in washed aortic smooth muscle membranes, the GS-proteins could be phosphorylated when purified A-kinase catalytic subunit was added to this preparation. Peptide mapping of the Mr 130,000 GS-protein indicated that A-kinase phosphorylated a subset of the same peptides labeled by the two forms of G-kinase. The endogenous A-kinase of rabbit aortic smooth muscle homogenates was also found to phosphorylate the GS-proteins. Since the intracellular concentrations of cGMP or cAMP can be selectively elevated by different stimuli, these results suggest several possible mechanisms by which the phosphorylation state of the GS-proteins may be regulated by cyclic nucleotides: activation of the membrane-bound G-kinase by cGMP or cAMP; and activation of cytosolic A-kinase by cAMP.     

Activation of p34cdc2 kinase by cyclin is negatively regulated by cyclic amp-dependent protein kinase in Xenopus oocytes.

Microinjection of a bacterially expressed stable delta 90 sea urchin cyclin B into Xenopus prophase oocytes, in absence or presence of cycloheximide, provokes the activation of histone H1 kinase and the tyrosine dephosphorylation of p34cdc2. Unexpectedly, when prophase oocytes are submitted to a treatment known to elevate the intracellular cAMP level (3-isobutyl-1-methylxanthine and cholera toxin), delta 90 cyclin has no effect and the oocytes remain blocked in prophase. This inhibition is reverted by the microinjection of the inhibitor of cAMP-dependent protein kinase. When delta 90 cyclin is microinjected into oocytes depleted of endogenous cyclins (cycloheximide-treated metaphase I) and in the presence of a high intracellular concentration of cAMP, p34cdc2 kinase is tyrosine rephosphorylated. Altogether, our results indicate that in Xenopus oocyte, cAMP-dependent protein kinase (A-kinase) controls the formation of the cyclin B/p34cdc2 complex which remains inactive and tyrosine phosphorylated.     

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.     

Dynamics of keratin assembly: exogenous type I keratin rapidly associates with type II keratin in vivo

Keratin intermediate filaments (IF) are obligate heteropolymers containing equal amounts of type I and type II keratin. We have previously shown that microinjected biotinylated type I keratin is rapidly incorporated into endogenous bundles of keratin IF (tonofilaments) of PtK2 cells. In this study we show that the earliest steps in the assembly of keratin subunits into tonofilaments involve the extremely rapid formation of discrete aggregates of microinjected keratin. These are seen as fluorescent spots containing both type I and type II keratins within 1 min post-injection as determined by double label immunofluorescence. These observations suggest that endogenous type II keratin subunits can be rapidly mobilized from their endogenous state to form complexes with the injected type I protein. Furthermore, confocal microscopy and immunogold electron microscopy suggest that the type I-type II keratin spots from in close association with the endogenous keratin IF network. When the biotinylated protein is injected at concentrations of 0.3-0.5 mg/ml, the organization of the endogenous network of tonofilaments remains undisturbed during incorporation into tonofilaments. However, microinjection of 1.5-2.0 mg/ml of biotinylated type I results in significant alterations in the organization and assembly state of the endogenous keratin IF network soon after microinjection. The results of this study are consistent with the existence of a state of equilibrium between keratin subunits and polymerized keratin IF in epithelial cells, and provide further proof that IF are dynamic elements of the cytoskeleton of mammalian cells.     

In vitro phosphorylation of sea urchin sperm adenylate cyclase by cyclic adenosine monophosphate-dependent protein kinase

Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase.     

Phosphorylation of phospholipase C-gamma by cAMP-dependent protein kinase

The mechanism by which cAMP modulates the activity of phosphoinositide-specific phospholipase C (PLC) was studied. Elevation of cAMP inhibited both basal and norepinephrine-stimulated phosphoinositide breakdown in C6Bu1 cells which contain at least three PLC isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of C6Bu1 cells with cAMP-elevating agents (cholera toxin, isobutylmethylxanthine, forskolin, and 8-bromo-cAMP) increased serine phosphate in PLC-gamma, but the phosphate contents in PLC-beta and PLC-delta were not changed. In addition, cAMP-dependent protein kinase selectively phosphorylated purified PLC-gamma among the three isozymes and added a single phosphate at serine. The serine phosphorylation, nevertheless, did not affect the activity of PLC-gamma in vitro. We propose, therefore, that the phosphorylation of PLC-gamma by cAMP-dependent protein kinase alters its interaction with putative modulatory proteins and leads to its inhibition.     

Cyclic-AMP-dependent phosphorylation of voltage-sensitive sodium channels in primary cultures of rat brain neurons

We have studied cAMP-dependent phosphorylation of sodium channels in rat brain neurons maintained in primary culture. In back phosphorylation studies, cells were treated with drugs to increase intracellular cAMP and sodium channels were solubilized and isolated by immunoprecipitation. Surface and intracellular pools of sodium channels were isolated separately. Purified channels were then phosphorylated with [gamma-32P]ATP by the catalytic subunit of cAMP-dependent protein kinase to incorporate 32P into available cAMP-dependent phosphorylation sites. The amount of 32P incorporated in vitro is inversely proportional to the extent of endogenous phosphorylation. Incubation of cells with forskolin (0.1-100 microM), 8-Br-cAMP (0.1-10 mM), or isobutylmethylxanthine (0.01-1.0 mM) inhibited subsequent incorporation of 32P into isolated sodium channels by 70-80%, indicating that treatment of cells with these drugs had increased endogenous phosphorylation to nearly maximum levels. The phosphopeptides phosphorylated in vivo and in vitro were identical. To examine the magnitude of basal phosphorylation and the extent of stimulated phosphorylation, the amount of 32P incorporated into sodium channels from control and stimulated cells was compared to that from matched samples which had been dephosphorylated with calcineurin. Sodium channels from control cells incorporated approximately 2-fold more 32P after dephosphorylation, indicating that cAMP-dependent sites on the channel are at least 47% phosphorylated in the basal state. Sodium channels from forskolin-treated cells incorporated 7-8-fold more 32P after dephosphorylation, indicating that cAMP-dependent phosphorylation sites are 80-90% phosphorylated after stimulation. Cell surface and intracellular pools of sodium channels were phosphorylated similarly. In cells metabolically labeled with 32P, cell surface sodium channels incorporated 2.7 mol of phosphate/mol of channel. Forskolin stimulated 32P incorporation into sodium channels 1.3-fold, consistent with the results obtained by back phosphorylation. We conclude that the rat brain sodium channel is substantially phosphorylated in both the cell surface and intracellular pools in vivo in unstimulated rat brain neurons, and the extent of phosphorylation is increased to 80-90% of maximum phosphorylation by agents that elevate intracellular cAMP.     

Phosphorylation of regulatory subunit of type I cyclic AMP-dependent protein kinase: biphasic effects of cyclic AMP in intact S49 mouse lymphoma cells

Intact S49 mouse lymphoma cells were used as a model system to study the effects of cyclic AMP (cAMP) and its analogs on the phosphorylation of regulatory (R) subunit of type I cAMP-dependent protein kinase. Phosphorylation of R subunit was negligible in mutants deficient in adenylate cyclase; low levels of cAMP analogs, however, stimulated R subunit phosphorylation in these cells to rates comparable to those in wild-type cells. In both wild-type and adenylate cyclase-deficient cells, R subunit phosphorylation was inhibited by a variety of N6-substituted derivatives of cAMP; C-8-substituted derivatives were generally poor inhibitors. Two derivatives that were inactive as kinase activators (N6-carbamoylmethyl-5'-AMP and 2'-deoxy-N6-monobutyryl-cAMP) were also ineffective as inhibitors of R subunit phosphorylation. Preferential inhibition by N6-modified cAMP analogs could not be ascribed simply to selectivity for the more aminoterminal (site I) of the two cAMP-binding sites in R subunit: Analog concentrations required for inhibition of R subunit phosphorylation were always higher than those required for activation of endogenous kinase; 8-piperidino-cAMP, a C-8-substituted derivative that is selective for cAMP-binding site I, was relatively ineffective as in inhibitor; and, although thresholds for activation of endogenous kinase by site I-selective analogs could be reduced markedly by coincubation with low levels of site II-selective analogs, no such synergism was observed for the inhibitory effect. The uncoupling of cyclic nucleotide effects on R subunit phosphorylation from activation of endogenous protein kinase suggests that, in intact cells, activation of cAMP-dependent protein kinase requires more than one and fewer than four molecules of cyclic nucleotide.     

Decreased phosphorylation of a low molecular weight protein by cGMP-dependent protein kinase in variant HL-60 cells resistant to nitric oxide- and cGMP-induced differentiation

We previously described the isolation of a variant subline of HL-60 cells that does not differentiate in response to nitric oxide (NO)-generating agents or to cGMP analogs [7]. The variant cells have normal guanylate cyclase activity and normal NO-induced increases in the intracellular cGMP concentration. We now show that the variant cells have normal cGMP-dependent protein kinase (G-kinase) activity, both by an in vitro and in vivo assay, and using two-dimensional gel electrophoresis we have identified six G-kinase substrates in the parental cells. Of these six proteins, we found considerably less phosphorylation of one of the proteins in the variant cells than in parental cells, both in vitro and in intact cells, and by ³⁵S-methionine/³⁵S-cysteine incorporation we found much less of this protein in the variant cells than in parental cells. The protein is a shared substrate of cAMP-dependent protein kinase (A-kinase); since cAMP analogs still induce differentiation of the variant cells, it appears that the NO/cGMP/G-kinase and cAMP/A-kinase signal transduction pathways share some but not all of the same target proteins in inducing differentiation of HL-60 cells.     

Phosphorylation of the carotenoid droplet protein p57 by the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase and the effect of fluoride

We have previously shown that the dispersion and aggregation of carotenoid droplets in goldfish xanthophores are regulated, respectively, by phosphorylation and dephosphorylation of a carotenoid droplet protein p57. There is a basal level of p57 phosphorylation of p57 in unstimulated cells, which is greatly stimulated by adrenocorticotropic hormone (ACTH) or cyclic adenosine monophosphate (cAMP) acting via cAMP-dependent protein kinase. We have also observed that, in permeabilized xanthophores, pigment dispersion can be induced when cAMP is replaced by fluoride. Since p57 has multiple phosphorylation sites, there is the question of whether all p57 phosphorylation is by cAMP-dependent protein kinase or whether phosphorylation by cAMP-independent protein kinase coupled with inhibition of phosphatase activity by fluoride can replace cAMP-dependent protein kinase and that the ability of fluoride to replace cAMP for pigment dispersion in permeabilized cells is probably due to activation of adenylcyclase. We also show that ACTH causes an approximately threefold increase in the level of cAMP in these cells.     

Vimentin and keratin intermediate filament systems in cultured PtK2 epithelial cells are interrelated.

Certain cultured epithelial cells contain separate vimentin and keratin-type intermediate filament networks. The intracellular injection of monoclonal antibodies directed against either vimentin or keratin filaments into PtK2 cultured epithelial cells specifically disrupted the organization of both filament types. Neither antibody had any effect when injected into cells which, while containing vimentin or keratin filaments, lacked the specific filament type which that antibody recognized. These experiments suggest that keratin and vimentin filament networks are associated in some way with one another.