[Elements of cytoskeleton and pathogenesis of allergic reactions. 1. Elements of cytoskeleton and their functions (a literature review)].
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Animals↗
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[Elements of the cytoskeleton and pathogenesis of allergic reactions. 2. The cytoskeleton and its involvement in the pathogenesis of allergic reactions (literature review)].
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Animals↗
[Regulation of cytoskeleton mediated by cytocalbin (cytoskeleton-related calmodulin-binding protein)].
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Actins↗
Incorporation of Rap 1b into the platelet cytoskeleton is dependent on thrombin activation and extracellular calcium.
Rap 1b is a 22-kDa low molecular mass GTP-binding protein which is both a member of the Ras superfamily and a substrate for cAMP-dependent protein kinase. Recently, evidence has been presented to show that Rap 1b is incorporated into the detergent-extracted cytoskeleton of platelets during thrombin-induced activation. The aims of this study were to compare the incorporation of Rap 1b into the detergent-extracted cytoskeleton after activation with different agonists, to examine the role of extracellular calcium on the incorporation of Rap 1b into the cytoskeleton, to investigate the relationship between the association of Rap 1b and other proteins with the cytoskeleton, and to determine the effect of phosphorylation of Rap 1b incorporation into the cytoskeleton. Platelets were activated with thrombin, A23187, phorbol myristate acetate, ADP, epinephrine, and collagen in the presence and absence of calcium. The time dependence of Rap 1b incorporation into the detergent-extracted cytoskeleton was then measured. When platelets were activated by thrombin in the presence of extracellular calcium, conditions which permit aggregation, incorporation of Rap 1b into the detergent-extracted cytoskeleton was biphasic. Approximately 20% of the total cellular Rap 1b incorporated into the cytoskeleton within seconds and was followed by a slower second phase of incorporation. In contrast, when platelets were activated by thrombin in the absence of calcium, conditions which inhibit aggregation, or by the other agents in the presence or absence of calcium, only the initial phase of Rap 1b incorporation into the cytoskeleton was measured. The incorporation of Rap 1b paralleled the incorporation of membrane glycoproteins (GP) IIb/IIIa and PECAM-1, but not the incorporation of pp60c-src. The GTPase-activating protein for Ras (Ras-GAP) did not associate with the detergent-extracted cytoskeleton. Two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis of the total cellular and cytoskeletal Rap 1b showed that unphosphorylated as well as phosphorylated isoforms of Rap 1b were incorporated into the cytoskeleton in the same molar ratio as was present in the intact cell. Furthermore, the rates of incorporation of phosphorylated and unphosphorylated Rap 1b into the cytoskeleton were similar. These experiments show that Rap 1b can regulate events that take place within seconds after activation, such as the initial formation of the cytoskeleton, as well as longer term changes in the cytoskeleton that occur in response to thrombin-induced aggregation. Furthermore, phosphorylation could modulate the (unknown) functions of Rap 1b as a component of the cytoskeleton.
Blood Platelets↗
Interaction between phosphatidylserine and the isolated cytoskeleton of human blood platelets.
Binding experiments were performed to demonstrate a direct interaction between cytoskeletons from human blood platelets and phosphatidylserine. A centrifugation technique using radiolabeled phosphatidylserine-vesicles and Triton X-100 insoluble residues from unstimulated human platelets was used to assess the binding. Interaction between cytoskeleton and phospholipid is demonstrated to be specific for phosphatidylserine. No binding was observed for phosphatidylcholine. The binding of phosphatidylserine was saturable and dependent on the concentration of cytoskeleton used. The interaction between phosphatidylserine and the cytoskeleton appeared to be completely reversible. The existence of a reversible and specific interaction between phosphatidylserine and the cytoskeleton of unstimulated platelets would suggest a role for the cytoskeleton in the maintenance of the asymmetric distribution of this lipid in the plasma membrane. We have previously shown (Comfurius et al. (1985) Biochim. Biophys. Acta 815, 143-148) that in activated platelets a strong correlation exists between degradation of platelet cytoskeletal proteins by the endogenous calcium-dependent proteinase (calpain) and exposure of phosphatidylserine at their outer surface. Nevertheless, hydrolysis of the isolated cytoskeleton by calpain did not result in a change in the parameters of the binding between phosphatidylserine and cytoskeleton. Also, sulfhydryl oxidation of the cytoskeleton by diamide did not affect its binding properties for phosphatidylserine, in spite of the fact that diamide treatment of platelets results in exposure of phosphatidylserine at the outer surface. Exposition of phosphatidylserine upon activation of platelets cannot be directly ascribed to a change in affinity or number of binding sites of the modified cytoskeleton as measured in model systems. However, it cannot be excluded that topological rearrangements of the cytoskeleton as occur within the cell during platelet activation lead to a decreased contact between cytoskeleton and lipid, irrespective of the binding parameters.
Binding Sites↗
Structure and dynamics of the fenestrae-associated cytoskeleton of rat liver sinusoidal endothelial cells.
This article describes the cytoskeleton associated with fenestrae and sieve plates of rat liver sinusoidal endothelial cells. Fenestrae control the exchange between the blood and parenchymal cells. We present evidence indicating that several agents that change the fenestrae and sieve plates also cause changes in the cytoskeleton. Cultured liver endothelial cells (LECs) were slightly fixed and treated with cytoskeleton extraction buffer. Detergent-extracted whole mounts of cultured cells were prepared for either scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Extracted cells show an integral intricate cytoskeleton; sieve plates and fenestrae are delineated by cytoskeleton elements. Fenestrae are surrounded by a filamentous, fenestrae-associated cytoskeleton with a mean filament thickness of 16 nm. Sieve plates are surrounded and delineated by microtubuli, which form a network together with additional branching cytoskeletal elements. The addition of ethanol to cultured cells enlarged the diameter for these fenestrae-associated cytoskeleton rings by 5%, whereas serotonin treatment reduced the diameter by 20%. These observations indicate that the fenestrae-associated cytoskeleton probably changes the size of fenestrae after different treatments. After treatment with cytochalasin B the number of fenestrae increased. However, cytochalasin B did not change the structure of the fenestrae-associated cytoskeleton ring, but disperses the microtubuli. In conclusion, LECs have a cytoskeleton that defines and supports sieve plates and fenestrae. Fenestrae-associated cytoskeleton is a dynamic structure and plays a role in maintaining and regulating the size of fenestrae after different treatments. Therefore, the fenestrae-associated cytoskeleton controls the important hepatic function of endothelial filtration.
Animals↗
Role of isoprenoids in cytoskeleton integrity and albumin endocytosis by opossum kidney cells.
BACKGROUND: The actin cytoskeleton has been increasingly implicated in endocytic events that are involved in the reabsorption of filtered protein by the proximal tubule. Isoprenylated small G proteins have emerged as key regulators of the actin cytoskeleton. This study examines the role of isoprenoid intermediates in organization of the cytoskeleton, and the effect of modification of the cytoskeleton on albumin endocytosis. METHODS: The effect of lovastatin on cytoskeleton morphology of opossum kidney cells (OK cells) was determined by staining with fluorescent isothiocyanate (FITC)-phalloidin followed by examination using confocal microscopy. Quantitative effects on albumin binding and uptake were determined using a well-established method. RESULTS: Inhibition of isoprenoid synthesis by lovastatin led to morphological disruption of the cytoskeleton with a concentration-dependent decrease in albumin uptake into OK cells. Addition of mevalonate, but not cholesterol, ameliorated the effects of lovastatin on the cytoskeleton and albumin uptake. Selective inhibition of isoprenoid intermediates with a farnesyltransferase inhibitor suggests that geranylgeranylated proteins mediate cytoskeleton organization. CONCLUSION: These results confirm the importance of cytoskeleton integrity in albumin endocytosis in renal proximal tubules. While the present data suggest that synthesis of isoprenoids via the mevalonate pathway is a critical step in maintenance of the cytoskeleton, the role of individual small G proteins in control of the cytoskeleton and other endocytic events is yet to be defined.
Albumins↗
Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton.
Previous studies indicated a correlation between the formation of EDTA-resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA-resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA-resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet-fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.
Antibodies, Monoclonal↗
Human platelet cytoskeletons: specific content of glycolipids and phospholipids.
The lipid composition of platelet cytoskeletons was analyzed. Triton X-100 (0.5%) was used to prepare cytoskeletons from thrombin-treated platelets. The lipid/protein ratio of platelet cytoskeletons was 0.260 and the phospholipid/protein ratio was 0.177, which were comparable to the ratios present in platelets. However, there was a selective enrichment of platelet lipids in platelet cytoskeletons. Only 2 of the 5 major platelet phospholipids were detected. About 14% platelet sphingomyelin and 2% platelet phosphatidylcholine were present in platelet cytoskeletons. Only 1 of the 4 platelet neutral glycolipids, trihexosyl ceramide, was detected and was about 7% of that in intact platelets. Two percent of platelet hematoside, the predominant ganglioside in platelets, was found in cytoskeletons. Six percent of platelet cholesterol was present in platelet cytoskeletons, while no other neutral lipid could be detected. The study demonstrates that the lipid/protein ratio of platelet cytoskeletons is similar to that in platelets, but the composition of cytoskeleton lipids is specific and distinctly different from that in platelets. The selective glycolipid and phospholipid composition of cytoskeletons may be important for cytoskeleton and platelet function.
Blood Platelets↗
Association of fibronectin with the platelet cytoskeleton.
Triton-insoluble cytoskeletons prepared from either normal or thrombasthenic platelets were found to contain approximately 1.3 micrograms of fibronectin/10(9) platelets as measured by a radioimmunoassay. Total endogenous platelet fibronectin was quantitatively retained on the platelet cytoskeleton, whereas 70% of exogenously added fibronectin that bound the surface of thrombin-activated platelets was recovered with the Triton-insoluble cytoskeleton. The exogenously added fibronectin specifically bound platelets and cytoskeletons with the same affinity giving an apparent binding constant of 1.47 X 10(-7) M. The possibility that fibrin associated with the platelet cytoskeleton could serve as the fibronectin receptor was investigated by measuring the binding constant of fibronectin for polymerizing fibrin and by measuring the amount of fibronectin associated with cytoskeletons of thrombasthenic platelets which contain 4-fold less fibrin than controls. The binding constant of fibronectin for polymerizing fibrin was 14-fold lower than that for cytoskeletons and cytoskeletons prepared from thrombasthenic platelets contained approximately the same amount of fibronectin as controls. Therefore, it is unlikely that fibrin is the platelet fibronectin receptor. These results support the hypothesis that platelet fibronectin is released from platelet alpha granules upon thrombin stimulation and becomes bound to the platelet surface and cytoskeleton either directly or through some intermediate protein that spans the membrane and interacts both with fibronectin and the internal cell cytoskeleton.
Antigen-Antibody Complex↗
Aggregation-related association of lipid with the cytoskeleton of rabbit and human platelets prelabeled with [3H]palmitic acid. Similar effects of adenosine diphosphate- and thrombin-induced aggregation.
To investigate the association of lipid with the cytoskeleton of platelets during aggregation, rabbit and human platelets were isolated and labeled with [3H]palmitic acid; lipid extraction showed approximately 80% in phospholipid. Limited aggregation was induced with ADP or thrombin, and the cytoskeleton was isolated after lysis with 1% Triton X-100, 5 mM EGTA. Cytoskeleton from unactivated platelets had approximately 0.03% of the total label in the platelets, but after aggregation with ADP (2 microM) or thrombin (0.1 U/ml) for 20-30 s, 1.5-8% of the label was with the cytoskeleton. Fibrinogen enhanced aggregation and the association of label with the cytoskeleton; incorporation of label increased exponentially as aggregation proceeded, decreased exponentially during deaggregation, and appeared to be related to the number of sites of contact. Inhibitors that increase cyclic AMP inhibited aggregation and cytoskeletal labeling, but aspirin had no effect. Some experiments were done with DNase I and Ca2+ in the Triton X-100 lysis medium to cause actin depolymerization, under conditions in which the Ca2+-dependent protease activity was inhibited. This greatly reduced the association of label with the cytoskeleton at early time points, but when aggregation had proceeded further, a large proportion of the label was not dissociated by this treatment. These findings, electron microscopy, and the enrichment of the cytoskeleton of aggregated platelets with only some of the membrane proteins that were labeled by the 125I-lactoperoxidase method, indicated that with limited aggregation, the 3H-labeled lipid was mainly associated with the cytoskeleton and not with trapped membrane fragments resulting from incomplete lysis. Since the pattern of cytoskeleton labeling ([3H]palmitate) and the selective association of some membrane proteins with the cytoskeleton/lipid complex was the same with ADP and thrombin, the reactions must be dependent on aggregation and not on events associated with the release of granule contents.
Actin Cytoskeleton↗
Interaction of epidermal growth factor receptors with the cytoskeleton is related to receptor clustering.
Recently it has been established that cytoskeleton-associated epidermal growth factor (EGF) receptors are predominantly of the high-affinity class and that EGF induces a recruitment of low-affinity receptors to the cytoskeleton. The nature of this EGF-induced receptor-cytoskeleton interaction, however, is still unknown. Therefore, we have studied the association of mutated EGF receptors with the cytoskeleton. Receptor deletion mutants lacking almost all intracellular amino acid residues displayed no interaction with the cytoskeleton, demonstrating that the cytoplasmic receptor domain is involved in this interaction. Further analysis revealed that receptor-cytoskeleton interaction is independent of receptor kinase activity and the C-terminal 126 amino acid residues, which include the auto-phosphorylation sites. Furthermore, it is shown that the high-affinity receptor subclass is not essential for association of low-affinity receptors to the cytoskeleton. EGF receptor-cytoskeleton interaction was increased, however, by treatment with sphingomyelinase, an enzyme known to induce membrane protein clustering, indicating that EGF receptor clustering may cause the association to the cytoskeleton.
Animals↗
Induction of a physical linkage between integrins and the cytoskeleton depends on intracellular calcium in an epithelial cell line.
In most cases epithelial cells reveal a polarized distribution of integrin receptors in basolateral domains of the plasma membrane. To evaluate the functional state of integrin receptors in these restricted sites we were interested in the physical association of integrins with the cytoskeleton. Basically, we extracted cells with Triton X-100 to obtain detergent insoluble cytoskeleton fractions and used monoclonal antibodies for the detection of integrins linked to the cytoskeleton. We found that no permanent physical integrin-cytoskeleton associations exist in a confluent culture of the hepatocyte cell line mHepR1. However, incubation with anti-integrin antibodies and cross linking with a secondary antibody induced a physical linkage of beta1 as well as of different alpha subunits to the cytoskeleton. The association of integrins with the cytoskeleton was also inducible in suspended cells, which was detected in flow cytometric analyses and indicates that the formation of a physical integrin-cytoskeleton connection is independent of the localization of integrins, cell shape, and adhesion on a substrate. Using the Ca2+ chelators BAPTA-AM and EGTA, we found that intracellular calcium is a necessary prerequisite to induce a connection of integrins to the cytoskeleton. ATP or tauroursodeoxycholic acid, which provoke an intracellular calcium elevation, partly induced the formation of an integrin-cytoskeleton linkage. These results indicate the obvious role of intracellular calcium in integrin-dependent outside-in as well as inside-out signaling.
Adenosine Triphosphate↗
Characterization of immunoglobulin binding to isolated human erythrocyte membranes: evidence for selective, temperature-induced binding of naturally occurring autoantibodies to the cytoskeleton.
Human plasma contains naturally occurring autoantibodies to the predominant components of the erythrocyte membrane: band 3 and spectrin bands 1 and 2 of the cytoskeleton. The titer of cytoskeletal plasma autoantibodies increases in various hemolytic conditions, suggesting that opsonization of the cytoskeleton may play an important role in the clearance of hemolyzed (not senescent) erythrocytes from the circulation. In this study, we use Alexa Fluor 488 goat anti-human IgG conjugate (Molecular Probes, Eugene, OR, USA), to characterize plasma immunoglobulin binding to erythrocyte membranes from osmotically hemolyzed cells ('ghosts'). The results show that exposure of ghosts to plasma results in 4-fold more immunoglobulin binding to the cytoskeleton than is bound to the proteins contained within the lipid bilayer. Preincubation of the ghosts at 37 degrees C causes 8-fold more immunoglobulin binding to the cytoskeleton compared to bilayer proteins. This temperature-induced change resulted from selective immunoglobulin binding to the cytoskeleton, with no change in immunoglobulin binding to bilayer proteins. However, the rate of increase in cytoskeletal antigenicity at 37 degrees C did correlate with the rate of a conformational change in band 3, a transmembrane protein which serves as a major membrane attachment site for the cytoskeleton. The results of this study suggest that the cytoskeleton is the primary target in the opsonization of hemolyzed erythrocyte membranes by naturally occurring plasma autoantibodies. The conformational changes which occur in ghosts at 37 degrees C are associated with selective exposure of new immunoglobulin binding sites on the cytoskeleton, and with a change in the structure of band 3. We propose a model suggesting that opsonization of the cytoskeleton occurs prior to the decomposition of hemolyzed erythrocytes at 37 degrees C.
Autoantibodies↗
Thrombin and thrombin receptor agonist peptide induce tyrosine phosphorylation and tyrosine kinases in the platelet cytoskeleton. Translocation of pp60c-src and integrin alpha IIb beta 3 (glycoprotein IIb/IIIa) is not required for aggregation, but is dependent on formation of large aggregate structures.
The maximal aggregation of platelets induced by alpha-thrombin or by the receptor agonist peptide thrombin-(42-47)-peptide (TRP42/47) rapidly increased the pp60c-src associated with the cytoskeleton fraction. There was good correlation between the tyrosine kinase activity and the mass of pp60c-src. Tyrosine kinase activity associated with the cytoskeleton phosphorylated several endogenous cytoskeleton-associated proteins, as revealed by immunoblotting with anti-phosphotyrosine antibody following incubation with ATP in vitro. However, with the exception of pp60c-src, few phosphotyrosine-containing proteins were retained in the cytoskeleton in intact platelets when compared with total platelet lysates. Translocation of pp60c-src to the cytoskeleton induced by alpha-thrombin and TRP42/47 is dependent on glycoprotein IIb/IIIa (GPIIb/IIIa)-fibrinogen-mediated aggregation, but does not occur when ristocetin/von Willebrand factor produces GPIb-mediated platelet aggregation. The translocation of GPIIb/IIIa and pp60c-src to the cytoskeleton is not necessary for aggregation, as it is not seen when clearly visible small to moderate-sized aggregates are initially formed after exposure to thrombin. The linkage of these proteins to the cytoskeleton occurs only after later extensive formation of large aggregates. Translocation of GPIIa/IIIa to the cytoskeleton is not sufficient for the cytoskeletal association of pp60c-src, as the former occurs independently in platelets stimulated with concanavalin A in the absence of aggregation. Linkage of the integrin GPIIb/IIIa and pp60c-src to the internal cytoskeleton structure, and the corresponding tyrosine phosphorylation of certain proteins upon formation of large aggregates, may be an example of mechanochemical transduction by integrin receptors and may represent a structure with the requisite tensile strength to stabilize large platelet aggregates against high shear stresses.
Amino Acid Sequence↗
The cytoskeleton and the regulation of gluconeogenesis: a hypothesis.
Previous data from our laboratory indicated a role for the cytoskeleton in the hormonal stimulation of gluconeogenesis. To gain further insight into the role of the cytoskeleton in the regulation of gluconeogenesis, we performed further experiments to examine the possibility that the cytoskeleton is involved in the glucagon-mediated changes in calcium distribution. Perfused livers or isolated liver cells were pretreated with either cytochalasin B or colchicine, agents that disrupt microfilaments and microtubules, respectively. Pretreatment with either agent significantly decreased the glucagon-evoked efflux of labeled calcium. Pretreatment with colchicine was more effective in blocking the influx of calcium into the cells than pretreatment with cytochalasin B. These drugs also prevented the characteristic increase in O(2) uptake, which is usually observed after glucagon administration. Thus, an intact cytoskeleton seems to be a prerequisite for the glucagon-evoked changes in calcium distribution to occur. These changes in calcium fluxes were shown previously to be an essential link in the chain of events leading to the metabolic effects of glucagon. Based on these results and on data obtained by others, a hypothesis is presented here. The hypothesis presumes that the cytoskeleton plays a crucial role in the regulation of gluconeogenesis. The hypothesis further assumes that the cytoskeleton influences gluconeogenesis in 3 ways: (1) by influencing the process of calcium signaling; (2) by changing the rate of enzymatic reactions through association and dissociation of enzymes with the cytoskeleton; and (3) by altering the position of intracellular organelles and the movements of molecules. Each of these points is discussed separately. It is known that the intracellular environment exists as a dense mesh in dynamic motion. According to our hypothesis, hormonal stimulation changes this environment by affecting the ionic composition in the cytosol and the structure of the cytoskeleton. Motion and conformational changes by the cytoskeleton play crucial regulatory functions, influencing metabolic processes.
Animals↗
Stopped-flow measurement of cytoskeletal contraction: Dictyostelium myosin II is specifically required for contraction of amoeba cytoskeletons.
Cytoskeletons provide valuable information on the composition and organization of the cell's contractile machinery, and in many cases these cell models retain the ability to contract. To quantitate contraction rates, we developed a novel stopped-flow assay permitting simultaneous analysis of thousands of Dictyostelium cytoskeletons within milliseconds of mixing with Mg-ATP. Cytoskeletons were placed in one syringe of the stopped flow apparatus and the appropriate buffer was placed in the second syringe. Mixing with Mg-ATP caused an immediate increase in the absorbance at 310 nm. Rapid fixation of the cytoskeletons during the reaction confirmed that this change in absorbance was highly correlated with contraction of the cytoskeletons. This spectroscopic change was used to measure the effects of temperature, pH, ionic strength, and nucleotides on contraction rate. Treatment with high salt and ATP removed most of the myosin, some actin, and small amounts of minor proteins. These extracted cytoskeletons lost the ability to contract, but after the addition of purified Dictyostelium myosin they regained full function. In contrast, rabbit skeletal muscle myosin was unable to restore contractility, even though it bound to the extracted cytoskeletons. Cytoskeletons prepared from a myosin-null mutant did not contract. Upon the addition of purified ameba myosin, however, they became contractile. These results suggest that filamentous Dictyostelium myosin II is essential for contraction, and that the actin cytoskeleton and associated proteins retain their functional organization in the absence of myosin.
Adenosine Triphosphate↗
Adenosine 3',5'-monophosphate-dependent protein kinase associated with the cytoskeleton of adrenal tumor cells.
Preparations of cytoskeleton from Y-1 cells were found to phosphorylate various cytoskeletal proteins when incubated with [gamma-32P]ATP. When cAMP was added to the cytoskeleton, a rapid increase in phosphorylation of cytoskeletal protein was observed, and changes were seen in the phosphorylation of individual proteins; four additional proteins were phosphorylated (mol wt, 165,000, 92,000, 45,000, and 24,000) and three proteins were more intensely phosphorylated than without cAMP (mol wt, 125,000, 51,000, and 38,000). In addition, one protein (mol wt, 96,000) that was intensely phosphorylated without cAMP was not phosphorylated with the cyclic nucleotide, and a second (mol wt, 48,000) was less phosphorylated. The increased level of total phosphorylation returned to the unstimulated level within 10 min. The increased phosphorylation of proteins produced by cAMP was inhibited by protein kinase inhibitor. cAMP-dependent protein kinase activity was closely associated with the cytoskeleton, since it was not removed by Triton X-100 (1%, wt/vol), although some activity could be extracted with buffer containing high concentrations of salt. When the cytoskeleton of Y-1 cells was subjected to treatments that disrupt the cytoskeleton before the cells were extracted (cytochalasin B, colchicine, and sonication), no change was seen in cAMP-dependent protein kinase activity. However, cytochalasin B increased phosphorylation of two proteins that were not phosphorylated by cAMP-dependent kinase (mol wt, 63,000 and 43,000). Sonication of the cytoskeleton before addition of [gamma-32P]ATP caused a number of changes in cAMP-independent phosphorylation, but did not affect cAMP-dependent phosphorylation. cAMP-dependent phosphorylation required Mg2+ and was inhibited by Ca2+. It is concluded that the cytoskeleton of Y-1 cells contains bound cAMP-dependent protein kinase that phosphorylates certain cytoskeleton proteins. The cytoskeleton also contains one or more cAMP-independent kinase systems. It is suggested that the cAMP-dependent protein kinase described here may be important in the cytoskeletal responses to ACTH.
Adenosine Triphosphate↗