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1.
Purification of and production of an antibody against a 63,000 Mr stimulus-sensitive phosphoprotein in Paramecium 总被引:4,自引:0,他引:4
In vivo labeling of Paramecium cells with 32Pi most heavily labels a minor 63-kDa protein that undergoes a rapid, Ca2+-dependent dephosphorylation when the cell is stimulated to release. This stimulus-sensitive phosphoprotein was isolated and purified to apparent homogeneity. A polyclonal affinity purified antibody made against the purified protein recognizes both the phosphorylated and dephosphorylated forms of the protein. The phosphorylated 63-kDa protein is found in the cytosolic fraction; it is slightly acidic with two isoelectric forms at pI 5.8 and 6.2 and probably exists as a monomeric 60-65-kDa polypeptide in the native state. The labeled phosphoamino acid of the protein is phosphoserine. The affinity purified antibody recognizes a third isoelectric form at pI 6.3 that appears unlabeled. The specificity of the antibody was confirmed by showing that it immunoprecipitates the correct protein, i.e. the stimulus-sensitive 63-kDa phosphoprotein. The availability of purified 63-kDa protein as well as an antibody against it will now allow molecular, biochemical, and immunocytochemical studies into the role of this protein in the mechanism of exocytosis. 相似文献
2.
At the onset of mitosis in higher eukaryotic cells, the nuclear envelope and its components including subunits of the nuclear pore complexes are disassembled, and these are reassembled toward the end of mitosis. We have studied the role of protein phosphorylation in this process, by investigating the phosphorylation status of a specific pore-associated protein during mitosis. Using a monoclonal antibody, mAb E2, earlier shown to inhibit nuclear protein import in rat fibroblast cells, we have identified a 58-kDa protein termed NPA58 that is partially associated with nuclear pores based on a high degree of coincident immunofluorescence in dual labeling experiments with mAb 414, a well-studied pore-complex-reactive antibody. NPA58 is specifically phosphorylated during mitosis and dephosphorylated upon release from metaphase arrest. Confocal microscopy analysis shows that NPA58 is dispersed in the cytoplasm early in mitosis when it is phosphorylated, while its relocalization in the reforming nuclear envelope during telophase temporally correlates with its dephosphorylation upon release from metaphase arrest. Our data provide in vivo evidence that the modifications mediated by phosphorylation and dephosphorylation are required for regulating the mitotic localization of a nuclear-pore-associated protein. 相似文献
3.
The cis-Golgi matrix protein GM130 is phosphorylated in mitosis on serine 25. Phosphorylation inhibits binding to p115, a vesicle-tethering protein, and has been implicated as an important step in the mitotic Golgi fragmentation process. We have generated an antibody that specifically recognizes GM130 phosphorylated on serine 25, and used this antibody to study the temporal regulation of phosphorylation in vivo. GM130 is phosphorylated in prophase as the Golgi complex starts to break down, and remains phosphorylated during further breakdown and partitioning of the Golgi fragments in metaphase and anaphase. In telophase, GM130 is dephosphorylated as the Golgi fragments start to reassemble. The timing of phosphorylation and dephosphorylation correlates with the dissociation and reassociation of p115 with Golgi membranes. GM130 phosphorylation and p115 dissociation appear specific to mitosis, since they are not induced by several drugs that trigger nonmitotic Golgi fragmentation. The phosphatase responsible for dephosphorylation of mitotic GM130 was identified as PP2A. The active species was identified as heterotrimeric phosphatase containing the Balpha regulatory subunit, suggesting a role for this isoform in the reassembly of mitotic Golgi membranes at the end of mitosis. 相似文献
4.
Barry AN Otoikhian A Bhatt S Shinde U Tsivkovskii R Blackburn NJ Lutsenko S 《The Journal of biological chemistry》2011,286(30):26585-26594
The copper-transporting ATPase ATP7A has an essential role in human physiology. ATP7A transfers the copper cofactor to metalloenzymes within the secretory pathway; inactivation of ATP7A results in an untreatable neurodegenerative disorder, Menkes disease. Presently, the mechanism of ATP7A-mediated copper release into the secretory pathway is not understood. We demonstrate that the characteristic His/Met-rich segment Met(672)-Pro(707) (HM-loop) that connects the first two transmembrane segments of ATP7A is important for copper release. Mutations within this loop do not prevent the ability of ATP7A to form a phosphorylated intermediate during ATP hydrolysis but inhibit subsequent dephosphorylation, a step associated with copper release. The HM-loop inserted into a scaffold protein forms two structurally distinct binding sites and coordinates copper in a mixed His-Met environment with an ~2:1 stoichiometry. Binding of either copper or silver, a Cu(I) analog, induces structural changes in the loop. Mutations of 4 Met residues to Ile or two His-His pairs to Ala-Gly decrease affinity for copper. Altogether, the data suggest a two-step process, where copper released from the transport sites binds to the first His(Met)(2) site, triggering a structural change and binding to a second 2-coordinate His-His or His-Met site. We also show that copper binding within the HM-loop stabilizes Cu(I) and protects it from oxidation, which may further aid the transfer of copper from ATP7A to acceptor proteins. The mechanism of copper entry into the secretory pathway is discussed. 相似文献
5.
Phosphorylation of tyrosine 992, 1068, and 1086 is required for conformational change of the human epidermal growth factor receptor c-terminal tail
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We reported previously that a conformation-specific antibody, Ab P2, to a 16-amino acid peptide (Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg) of the cytoplasmic domain of the beta-type platelet-derived growth factor receptor also recognizes the epidermal growth factor (EGF) receptor. Although the antibody is not directed to phosphotyrosine, it recognizes in immunoprecipitation the activated and hence phosphorylated form of both receptors. In P2 peptide, there are two tripeptide sequences, Asp-Glu-Glu and Tyr-Gln-Gln, that are also present in the EGF receptor. Our present studies using either EGF receptor C-terminal deletion mutants or point mutations (Tyr-->Phe) and our previous studies on antibody inhibition by P2-derived peptides suggest that Gln-Gln in combination with Asp-Glu-Glu forms a high-affinity complex with Ab P2 and that such complex formation is dependent on tyrosine phosphorylation. Of the five phosphate acceptor sites in the EGF receptor, clustered in the extreme C-terminal tail, phosphorylation of three tyrosine residues (992, 1068, and 1086) located between Asp-Glu-Glu and Gln-Gln is necessary for Ab P2 binding. In contrast, the acceptor sites Tyr 1173 and 1148 play no role in the conformation change. Asp-Glu-Glu and Gln-Gln are located 169 amino acids apart, and it is highly likely that the interactions among three negatively charged phosphotyrosine residues in the receptor C terminus may result in the bending of the peptide chain in such a way that these two peptides come close to each other to form an antibody-binding site. Such a possibility is also supported by our finding that receptor dephosphorylation results in complete loss of Ab P2-binding activity. In conclusion, we have identified a domain within the cytoplasmic part of the EGF receptor whose conformation is altered by receptor phosphorylation; furthermore, we have identified the tyrosine residues that positively regulate this conformation. 相似文献
6.
Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets 总被引:1,自引:0,他引:1
The focal adhesion protein VASP, a possible link between signal transduction pathways and the microfilament system, is phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro and in intact cells. Here, the analysis of VASP dephosphorylation by the serine/threonine protein phosphatases (PP) PP1, PP2A, PP2B and PP2C in vitro is reported. The phosphatases differed in their selectivity with respect to the dephosphorylation of individual VASP phosphorylation sites. Incubation of human platelets with okadaic acid, a potent inhibitor of PP1 and PP2A, caused the accumulation of phosphorylated VASP indicating that the phosphorylation status of VASP in intact cells is regulated to a major extent by serine/ threonine protein phosphatases. Furthermore, the accumulation of phosphorylated cAMP-dependent protein kinase substrate(s) appears to account for inhibitory effects of okadaic acid on platelet function. 相似文献
7.
Suppression of cofilin phosphorylation in insulin-stimulated ruffling membrane formation in KB cells
Various cellular events such as cell motility and division are directed by the actin cytoskeleton under the control of its regulatory system. Cofilin is a low molecular weight actin-modulating protein that severs and depolymerizes F-actin and is shown to enhance actin filament dynamics. The activity of cofilin is negatively regulated by phosphorylation at Ser-3. In human epidermoid carcinoma KB cells, insulin treatment induces characteristic ruffling membranes, and it was reported that LIMK1, a cofilin kinase, was activated in these cells treated with insulin. Since cofilin is a key protein responsible for establishing the rapid turnover of actin filaments, it appears to be contradictory that cofilin is phosphorylated (inactivated) by a stimulus that is known to induce the highly dynamic actin structure, ruffling membranes. Therefore, we examined the phosphorylation state of endogenous cofilin in KB cells treated with insulin. The dephosphorylated form of cofilin increased with insulin treatment, as analyzed by nonequilibrium pH gradient gel electrophoresis (NEpHGE)-immunoblotting. Cell labeling with (32)P orthophosphate indicated that cofilin was being continuously phosphorylated and dephosphorylated, and that the apparent insulin-induced dephosphorylation was due to suppression of continuous phosphorylation and not to enhanced dephosphorylation. Further, we examined the localization of the phosphorylated form of cofilin using phospho-specific antibody raised against phosphorylated cofilin. Surprisingly, phosphorylated cofilin was concentrated in the ruffling membranes induced by insulin. These results suggest that the examination of the kinetics and spatial regulation of phosphorylation is critical for the elucidation of the role of cofilin and upstream kinases in actin reorganization. 相似文献
8.
A kinetic analysis of the effects of inhibitor-1 and inhibitor-2 on the activity of protein phosphatase-1 总被引:1,自引:0,他引:1
The steady-state interaction between protein phosphatase-1 and its two inhibitor proteins was studied in vitro at low enzyme concentrations where the assumptions of the Michaelis-Menten equation appeared to be valid. Under these conditions, and in the absence of divalent cations, inhibitor-1 behaved as a mixed inhibitor using phosphorylase alpha as a substrate, whereas inhibitor-2 was a competitive inhibitor. The results demonstrate that inhibitor-1 and inhibitor-2 do not interact with protein phosphatase-1 in an identical manner. Inhibitor-1 was only a substrate for protein phosphatase-1 in the presence of Mn2+, and its dephosphorylation was inhibited competitively by inhibitor-2 (Kis = 8 nM). Inhibitor-1 did not inhibit its own dephosphorylation in the presence of Mn2+. Its Km as a substrate (190 nM) was very much higher than its Ki as an inhibitor (1.5-7.5 nM). The results are consistent with a model in which a single binding site for inhibitor-1 is present on protein phosphatase-1, distinct from the binding site for phosphorylase alpha. It is envisaged that the binding of inhibitor-1 to this site not only inhibits the dephosphorylation of other substrates but permits access of its phosphothreonine to the same catalytic group(s) responsible for the dephosphorylation of other substrates. G-substrate, a protein phosphorylated exclusively on threonine residues, did not inhibit the dephosphorylation of phosphorylase alpha and its dephosphorylation was potently inhibited by inhibitor-1 or inhibitor-2. The role of the phosphothreonine residue in inhibitor-1 is discussed in the light of these results. 相似文献
9.
We examined the dephosphorylation of p36, a protein ofD. discoideum that has previously been shown to be phosphorylated in a GDP-dependent manner (Anschutzet al., 1989). Specific dephosphorylation of p36 was found to occur in cell preparations but the activity responsible was strongly dependent upon the concentration of proteins in those extracts. When preparations were diluted, this activity was no longert detectable and the radiolabeled phosphate incorporated into p36 was stable. In contrast, p36 phosphorylation was seemingly unaffected by this treatment. Under the conditions where endogenous dephosphorylating activity was not detectable, the addition of GDP to the reaction resulted in substantial dephosphorylation of p36. The stimulation of this dephosphorylation process occurred at concentrations of GDP that were distinct from those that led to an increased p36 phosphorylation due to the previously reported stimulation of p36 protein kinase activity. Characterization of the dephosphorylation of p36 indicates that the same enzyme is responsible for the endogenous and GDP-stimulated activities. Additionally, these activities are identical when assayed with p36 that had been phosphortylated with ATP or GTP. In contrast to p36 kinase activity, the dephosphorylation of p36 did not display any developmental changes with respect to its regulatory features. 相似文献
10.
We examined the dephosphorylation of p36, a protein ofD. discoideum that has previously been shown to be phosphorylated in a GDP-dependent manner (Anschutzet al., 1989). Specific dephosphorylation of p36 was found to occur in cell preparations but the activity responsible was strongly dependent upon the concentration of proteins in those extracts. When preparations were diluted, this activity was no longert detectable and the radiolabeled phosphate incorporated into p36 was stable. In contrast, p36 phosphorylation was seemingly unaffected by this treatment. Under the conditions where endogenous dephosphorylating activity was not detectable, the addition of GDP to the reaction resulted in substantial dephosphorylation of p36. The stimulation of this dephosphorylation process occurred at concentrations of GDP that were distinct from those that led to an increased p36 phosphorylation due to the previously reported stimulation of p36 protein kinase activity. Characterization of the dephosphorylation of p36 indicates that the same enzyme is responsible for the endogenous and GDP-stimulated activities. Additionally, these activities are identical when assayed with p36 that had been phosphortylated with ATP or GTP. In contrast to p36 kinase activity, the dephosphorylation of p36 did not display any developmental changes with respect to its regulatory features. 相似文献
11.
Role of the N-terminal region of the regulatory light chain in the dephosphorylation of myosin by myosin light chain phosphatase. 总被引:1,自引:0,他引:1
Myosin regulatory light chain (RLC) is phosphorylated at various sites at its N-terminal region, and heterotrimeric myosin light chain phosphatase (MLCP) has been assigned as a physiological phosphatase that dephosphorylates myosin in vivo. Specificity of MLCP toward the various phosphorylation sites of RLC was studied, as well as the role of the N-terminal region of RLC in the dephosphorylation of myosin by MLCP. MLCP dephosphorylated phosphoserine 19, phosphothreonine 18, and phosphothreonine 9 efficiently with almost identical rates, whereas it failed to dephosphorylate phosphorylated serine 1/serine 2. Deletion of the N-terminal seven amino acid residues of RLC markedly decreased the dephosphorylation rate of phosphoserine 19 of RLC incorporated in the myosin molecule, whereas this deletion did not significantly affect the dephosphorylation rate of isolated RLC. On the other hand, deletion of only four N-terminal amino acid residues showed no effect on dephosphorylation of phosphoserine 19 of incorporated RLC. The inhibition of dephosphorylation by deletion of the seven N-terminal residues was also found with the catalytic subunit of MLCP. Phosphorylation at serine 1/serine 2 and threonine 9 did not influence the dephosphorylation rate of serine 19 and threonine 18 by MLCP. These results suggest that the N-terminal region of RLC plays an important role in substrate recognition of MLCP. 相似文献
12.
ABSTRACT. We have used the anti-phosphoprotein antibody MPM-2 to examine changes in phosphorytation of cortical proteins during cilia regeneration in Tetrahymena thermophila . Although numerous cortical proteins are phosphorylated in both nondeciliated and deciliated cells, deciliation induces a dramatic increase in the phosphorylation of a 90-kDa cortical protein. The 90-kDa protein remained phosphorylated during cilia regeneration and then gradually became dephosphorylated. The 90-kDa protein was phosphorylated and dephosphorylated normally in Tetrahymena mutants that assemble short cilia, suggesting that achievement of full length is not the signal that triggers dephosphorylation of the 90-kDa protein. When initiation of cilia assembly is blocked, the 90-kDa protein becomes phosphorylated and remains phosphorylated for an extended period of time, suggesting that initiation of cilia elongation triggers eventual dephosphorylation of the 90-kDa protein, regardless of how long the cilia actually become. 相似文献
13.
Siegmund Fischer Maria Tortolero Jean-Pierre Piau Jean Delaunay Georges Schapira 《生物化学与生物物理学报:生物膜》1980,598(3):463-471
Human erythrocyte membranes contain a phosphoprotein phosphatase able to dephosphorylate membrane protein previously phosphorylated by the endogenous protein kinase.The level of dephosphorylation obtained after prolonged incubation is about one half of the phosphorylated residues.The characteristics of this enzyme are similar to those described for the cytoplasmic phosphoprotein phosphatase.In a membrane preparation the phosphorylation and dephosphorylation reactions can be repeated, at least twice, achieving similar levels of phosphate esterified or hydrolyzed.The coordination of these two enzyme systems might play a role in some of the functions attributed to the protein kinase system. 相似文献
14.
Phosphorylation and dephosphorylation of proteins play an important role in the regulation of mitosis and meiosis. In our previous studies we have described mitosis-specific monoclonal antibody MPM-2 that recognizes a family of phosphopeptides in mitotic cells but not in interphase cells. These peptides are synthesized in S phase but modified by phosphorylation during G2/mitosis transition. The epitope for the MPM-2 is a phosphorylated site. In this study, we attempted to determine which amino acids are phosphorylated during the G2-mitosis (M) transition. We raised a polyclonal antibody against one of the antigens recognized by MPM-2, i.e. a protein of 55 kDa, that is present in interphase cells but modified by phosphorylation during mitosis. This antibody recognizes the p55 protein in both interphase and mitosis while it is recognized by the monoclonal antibody MPM-2 only in mitotic cells. Phosphoamino acid analysis of protein p55 from 32P-labeled S-phase and M-phase HeLa cell extracts after immunoprecipitation with anti-p55 antibodies revealed that threonine was extensively phosphorylated in p55 during G2-M but not in S phase, whereas serine was phosphorylated during both S and M phases. Tyrosine was not phosphorylated. Identical results were obtained when antigens recognized by MPM-2 were subjected to similar analysis. As cells completed mitosis and entered G1 phase phosphothreonine was completely dephosphorylated whereas phosphoserine was not. These results suggest that phosphorylation of threonine might be specific to some of the mitosis-related events. 相似文献
15.
16.
The association of eIF-2 with Met-tRNAi or eIF-2B alters the specificity of eIF-2 phosphatase 总被引:2,自引:0,他引:2
In unfractioned reticulocyte lysate, interaction of eukaryotic initiation factor 2 (eIF-2) with other components regulates the accessibility of phosphatases and kinases to phosphorylation sites on its alpha and beta subunits. Upon addition of eIF-2 phosphorylated on both alpha and beta subunits (eIF-2(alpha 32P, beta 32P) to lysate, the alpha subunit is rapidly dephosphorylated, but the beta subunit is not. In contrast, both sites are rapidly dephosphorylated by the purified phosphatase. The basis of this altered specificity appears to be the association of eIF-2 with other translational components rather than an alteration of the phosphatase. Formation of an eIF-2(alpha 32P,beta 32P) Met-tRNAi X GTP ternary complex prevents dephosphorylation of the beta subunit, but has no effect on the rate of alpha dephosphorylation. eIF-2B, a 280,000-dalton polypeptide complex required for GTP:GDP exchange, also protects the beta subunit phosphorylation site from the purified phosphatase. However, the dephosphorylation of eIF-2(alpha 32P) is inhibited by 75% while complexed with eIF-2B. The altered phosphatase specificity upon association of eIF-2 with eIF-2B also affects the access of protein kinases to these phosphorylation sites. In the eIF-2B X eIF-2 complex, the alpha subunit is phosphorylated at 30% the rate of free eIF-2. Under identical conditions, phosphorylation of eIF-2 beta can not be detected. These results illustrate the importance of substrate conformation and/or functional association with other components in determining the overall phosphorylation state allowed by alterations of kinase and phosphatase activities. 相似文献
17.
We examined the paradoxical inhibition and stimulation of calcineurin, the calcium-activated protein phosphatase, using the drug FK506 (tacrolimus) which acts as a complex together with its binding protein; the complex is designated here as FKC. We reproduced FKC inhibition with RIIp, a phosphorylated peptide substrate, and FKC stimulation with p-nitrophenylphosphate (pNPP) as substrate. The presence of RIIp in the pNPP assay caused inhibition. Yet, under these conditions, FKC still stimulated pNPP dephosphorylation to the same extent. The effects of Mn2+ were strikingly different for the two substrates when calcineurin was measured under otherwise identical conditions: Mn2+ stimulated pNPP dephosphorylation several fold, but only stimulated RIIp dephosphorylation by about 50%. When Pi was used as product inhibitor, FKC stimulation, but not calmodulin stimulation, was attenuated. We conclude that FKC enhances substrate binding to the enzyme. This would lead to inhibition with RIIp, known to bind calcineurin tightly, but stimulation with pNPP, known to bind calcineurin weakly. The result not only resolves the paradox but also elucidates the mechanism of action for this class of immunosuppressive drugs. 相似文献
18.
Intracellular Ca2+ regulates the phosphorylation and the dephosphorylation of ciliary proteins via the NO pathway
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Gertsberg I Hellman V Fainshtein M Weil S Silberberg SD Danilenko M Priel Z 《The Journal of general physiology》2004,124(5):527-540
The phosphorylation profile of ciliary proteins under basal conditions and after stimulation by extracellular ATP was investigated in intact tissue and in isolated cilia from porcine airway epithelium using anti-phosphoserine and anti-phosphothreonine specific antibodies. In intact tissue, several polypeptides were serine phosphorylated in the absence of any treatment (control conditions). After stimulation by extracellular ATP, changes in the phosphorylation pattern were detected on seven ciliary polypeptides. Serine phosphorylation was enhanced for three polypeptides (27, 37, and 44 kD), while serine phosphorylation was reduced for four polypeptides (35, 69, 100, and 130 kD). Raising intracellular Ca2+ with ionomycin induced identical changes in the protein phosphorylation profile. Inhibition of the NO pathway by inhibiting either NO synthase (NOS), guanylyl cyclase (GC), or cGMP-dependent protein kinase (PKG) abolished the changes in phosphorylation induced by ATP. The presence of PKG within the axoneme was demonstrated using a specific antibody. In addition, in isolated permeabilized cilia, submicromolar concentrations of cGMP induced protein phosphorylation. Taken together, these results suggest that the axoneme is an integral part of the intracellular NO pathway. The surprising observation that ciliary activation is accompanied by sustained dephosphorylation of ciliary proteins via NO pathway was not detected in isolated cilia, suggesting that the protein phosphatases were either lost or deactivated during the isolation procedure. This work reveals that any pharmacological manipulation that abolished phosphorylation and dephosphorylation also abolished the enhancement of ciliary beating. Thus, part or all of the phosphorylated polypeptides are likely directly involved in axonemal regulation of ciliary beating. 相似文献
19.
Examination of ciliate Blepharisma japonicum whole cell lysates with an antibody against phosphoserine and in vivo labeling of cells with radioactive phosphate revealed that the photophobic response in the ciliate is accompanied by a rapid dephosphorylation of a 28 kDa protein and an enhanced phosphorylation of a 46 kDa protein. Analysis with antibodies raised against rat phosducin or human phosducin-like proteins, identified one major protein of a molecular weight of 28 kDa, and two protein bands of 40 kDa and 93 kDa. While the identified ciliate phosducin is phosphorylated in a light-dependent manner, both phosducin-like proteins exhibit no detectable dependence of phosphorylation upon illumination. An immunoprecipitation assay also showed that the ciliate phosducin is indeed phosphorylated on a serine residue and exists in a phosphorylated form in darkness and that its dephosphorylation occurs in light. Immunocytochemical experiments showed that protozoan phosducin and phosducin-like proteins are localized almost uniformly within the cytoplasm of cells adapted to darkness. Cell exposure to light caused a pronounced displacement of the cell phosducin to the vicinity of the plasma membrane; however, no translocation of phosducin-like proteins was observed upon cell illumination. The obtained results are the first demonstration of the presence and morphological localization of a possible phosducin and phosducin-like proteins in ciliate protists. Phosducin and phosducin-like proteins were found to bind and sequester the betagamma-subunits of G-proteins with implications for regulation of G-protein-mediated signaling pathways in various eukaryotic cells. The findings presented in this study suggest that the identified phosphoproteins in photosensitive Blepharisma japonicum may also participate in the regulation of the efficiency of sensory transduction, resulting in the motile photophobic response in this cell. 相似文献
20.
A monoclonal antibody was prepared against the regulatory subunit (RII) of rat liver type II cAMP-dependent protein kinase. Autophosphorylated and nonphosphorylated RII in extracts from rat liver or hepatocytes were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and quantified by immunoblot analysis with this antibody. Under basal conditions, 90% of hepatocyte RII was in the phosphorylated form. Incubating hepatocytes with 8-bromo-cAMP and a phosphodiesterase inhibitor resulted in activation of cAMP-dependent protein kinase and glycogenolysis but did not affect phospho RII levels. RII phosphorylation was also unaffected by the inclusion of sufficient insulin to cause a decrease in cAMP-dependent protein kinase activity and glycogenolysis. The results indicate that unlike other cell types, dissociation of rat hepatocyte type II cAMP-dependent protein kinase does not result in dephosphorylation of RII. The biochemical basis for the apparent lack of RII dephosphorylation in intact hepatocytes was examined by comparison with smooth muscle where RII is rapidly dephosphorylated. Rat liver extract contained 4-fold less RII and had an 80-fold lower rate of dephosphorylation of endogenous RII compared to bovine smooth muscle extract. The differences in the rates of RII dephosphorylation in tissue extracts were not observed using purified RII from either tissue. These data suggested that the slow rate of RII dephosphorylation in rat hepatocytes is due to a difference in the susceptibility of endogenous rat liver RII to dephosphorylation rather than a difference in phosphatase activity. 相似文献