cAMP-dependent protein kinase phosphorylates gap junction protein in lens cortex but not in lens nucleus

MP70 (a 70 kDa membrane protein) is a component of the gap junctions of the young fibre cells in the lens outer cortex. In the older fibres deeper in the mammalian lens (lens nucleus), MP70 is processed to MP38 by cleavage and removal of the carboxy terminal half. It is shown here that cortical MP70, and its derivative MP64, can be phosphorylated with cAMP-dependent protein kinase. In contrast, MP38 from the lens nucleus is not phosphorylated by the enzyme. Proteolytic processing and this lens region specific phosphorylation are relevant for the future development of functional assays for lens gap junctions.     

cAMP-dependent protein kinase of Manduca sexta phosphorylates but does not activate the fat body triglyceride lipase

cAMP-dependent-protein kinase (PKA) is a central player of the adipokinetic signal that controls the mobilization of stored lipids in the fat body. Previous studies showed that adipokinetic hormone (AKH) rapidly activates PKA from the fat body of Manduca sexta (Arrese et al. (J. Lipid. Res. 40(3): 556)). As a part of our investigation on lipolysis in insects, here we report the purification and characterization of the catalytic subunit of PKA from the fat body of M. sexta and its role in the direct activation of the TG lipase in vitro. PKA was purified to apparent homogeneity and the identity of the protein was confirmed by MALDI-TOF and Western blot analysis. The enzyme showed a high affinity for Mg-ATP (Km = 39 microM) and Kemptide (Km = 31 microM) and was strongly inhibited by the PKA specific inhibitors PKI 5-24 and H89. Manduca sexta PKA only recognized serine residues as phosphate acceptor; theronine or tyrosine containing peptides were not phosphorylated. Purified fat body TG-lipase proved to be a good substrate of the purified kinase. However, phosphorylation of the lipase did not enhance the lipolytic activity of the enzyme in vitro. These results suggest that, besides lipase phosphorylation, the mechanism of AKH-induced activation of the lipolysis requires the involvement of other proteins and/or signals.     

The localization of the cAMP-dependent protein kinase phosphorylation site in the platelet rat protein, rap 1B

Rap 1B is a low molecular weight G protein which is phosphorylated by cAMP-dependent protein kinase. In order to identify the site of phosphorylation by cAMP-dependent protein kinase, purified rap 1B from human platelets was phosphorylated and subjected to limited proteolysis with trypsin. Single digestion fragment containing the phosphorylation site was obtained and purified by reversed-phase HPLC. Sequence analysis of the phosphorylated digestion fragment demonstrated that the sequence of the phosphorylation site was -Lys-Lys-Ser-Ser-. This sequence is near the carboxy terminus and is adjacent to the site of membrane attachment of the protein.     

rap1B, a cAMP-dependent protein kinase substrate, associates with the platelet cytoskeleton

rap1B is a member of the ras superfamily of low molecular weight GTP binding proteins which constitutes a focal point of GTP and cAMP signal transduction systems. Like other members of this superfamily, rap1B is membrane-associated in resting platelets, presumably through polyisoprenylation. The studies presented here were undertaken to determine the subcellular changes in rap1B localization during cell activation. Activated and unactivated platelets were fractionated by Triton X-100 lysis followed by differential centrifugation to obtain a 10,000 x g cytoskeleton fraction, a 100,000 x g membrane skeleton fraction, and a 100,000 x g supernatant fraction containing solubilized proteins. In unactivated platelets, rap1B was present in the 100,000 x g supernatant fraction. In contrast, in platelets activated with 1 unit/ml alpha-thrombin or with the calcium ionophore, A23187, rap1B was quantitatively recovered in the 10,000 x g cytoskeleton fraction. rap1B was absent from the 100,000 x g fraction containing the membrane skeleton and could not be detected in the 100,000 x g supernatant containing cytosolic proteins and solubilized membrane components. These results indicate that rap1B associates with the cytoskeleton during cell activation.     

The cytosolic protein kinase CK2 phosphorylates cardiac calsequestrin in intact cells

The luminal SR protein CSQ2 contains phosphate on roughly half of the serines found in its C-terminus. The sequence around phosphorylation sites in CSQ2 suggest that the in vivo kinase is protein kinase CK2, even though this enzyme is thought to be present only in the cytoplasm and nucleus. To test whether CSQ2 kinase is CK2, we combined approaches that reduced CK2 activity and CSQ2 phosphorylation in intact cells. Tetrabromocinnamic acid, a specific inhibitor of CK2, inhibited both the CSQ2 kinase and CK2 in parallel across a range of concentrations. In intact primary adult rat cardiomyocytes and COS cells, 24 h of drug treatment reduced phosphorylation of overexpressed CSQ2 by 75%. Down-regulation of CK2α subunits in COS cells using siRNA, produced a 90% decrease in CK2α protein levels, and CK2-silenced COS cells exhibited a twofold reduction in CSQ2 kinase activity. Phosphorylation of CSQ2 overexpressed in CK2-silenced cells was also reduced by a factor of two. These data suggested that CSQ2 in intact cells is phosphorylated by CK2, a cytosolic kinase. When phosphorylation site mutants were analyzed in COS cells, the characteristic rough endoplasmic reticulum form of the CSQ2 glycan (GlcNAc2Man9,8) underwent phosphorylation site dependent processing such that CSQ2-nonPP (Ser to Ala mutant) and CSQ2-mimPP (Ser to Glu mutant) produced apparent lower and greater levels of ER retention, respectively. Taken together, these data suggest CK2 can phosphorylate CSQ2 co-translationally at biosynthetic sites in rough ER, a process that may result in changes in its subsequent trafficking through the secretory pathway.     

Phosphorylation of rap1GAP in vivo and by cAMP-dependent kinase and the cell cycle p34cdc2 kinase in vitro.

rap1GAP is a GTPase activating protein that specifically stimulates the GTP hydrolytic rate of the ras-related protein p21rap1.rap1GAP undergoes post-translational modification that causes a substantial change in its mobility on sodium dodecyl sulfate-polyacrylamide gels. At least part of this modification is due to the phosphorylation. Expression of a rap1GAP cDNA in insect cells labeled with 32Pi resulted in high level incorporation of radioactivity into serine residues of the expressed protein. Purified rap1GAP was phosphorylated in vitro by cAMP-dependent kinase and the cell cycle p34cdc2 kinase. The molar ratio of incorporated phosphate/rap1GAP was approximately 3 by cAMP-dependent kinase and 2 by p34cdc2. The sites of phosphorylation by both kinases were localized to a 100-residue segment contained in the carboxyl-terminal region of the predicted primary structure of rap1GAP. Highly favorable recognition sequences for the two kinases are contained within this fragment and are proposed as the sites of phosphorylation. Treatment of SK-MEL-3 cells with dibutyryl cAMP promoted phosphorylation of rap1GAP in vivo. Based on the results of comparative phosphopeptide mapping the sites of phosphorylation in vivo and in vitro are identical.     

Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro

The Snf1/AMP-activated protein kinase (AMPK) family is important for metabolic regulation and is highly conserved from yeast to mammals. The upstream kinases are also functionally conserved, and the AMPK kinases LKB1 and Ca2+/calmodulin-dependent protein kinase kinase activate Snf1 in mutant yeast cells lacking the native Snf1-activating kinases, Sak1, Tos3, and Elm1. Here, we exploited the yeast genetic system to identify members of the mammalian AMPK kinase family by their function as Snf1-activating kinases. A mouse embryo cDNA library in a yeast expression vector was used to transform sak1Delta tos3Delta elm1Delta yeast cells. Selection for a Snf+ growth phenotype yielded cDNA plasmids expressing LKB1, Ca2+/calmodulin-dependent protein kinase kinase, and transforming growth factor-beta-activated kinase (TAK1), a member of the mitogen-activated protein kinase kinase kinase family. We present genetic and biochemical evidence that TAK1 activates Snf1 protein kinase in vivo and in vitro. We further show that recombinant TAK1, fused to the activation domain of its binding partner TAB1, phosphorylates Thr-172 in the activation loop of the AMPK catalytic domain. Finally, expression of TAK1 and TAB1 in HeLa cells or treatment of cells with cytokines stimulated phosphorylation of Thr-172 of AMPK. These findings indicate that TAK1 is a functional member of the Snf1/AMPK kinase family and support TAK1 as a candidate for an authentic AMPK kinase in mammalian cells.     

Association of type II cAMP-dependent protein kinase with p34cdc2 protein kinase in human fibroblasts

Previous independent studies suggested that type II cAMP-dependent protein kinase and the p34cdc2 protein kinase cell cycle regulator co-localize at centrosomes. In order to investigate whether there is an association of type II cAMP-dependent protein kinase with p34cdc2 in human fibroblasts, we used three different approaches. First, the regulatory subunits RI and RII were photoaffinity-labeled with 8-N3-[32P]cAMP, and anti-p34cdc2 immunoprecipitates were screened for the presence of either RI or RII regulatory subunits by one- or two-dimensional gel electrophoresis. Second, anti-RII alpha immunoprecipitates were screened for the presence of p34cdc2 by Western blot using three different affinity-purified antibodies recognizing different domains of human p34cdc2. Conversely, anti-p34cdc2 immunoprecipitates (three different antibodies), as well as the material retained on p13suc1-Sepharose Bio-Beads, which binds specifically p34cdc2, were screened for the presence of RII alpha. Finally, we have looked for cAMP-dependent protein kinase activity specifically inhibited by PKI in immunoprecipitates obtained from extracts treated with different anti-p34cdc2 antibodies. All these experiments gave concordant results and demonstrate that at least at G0/G1, human fibroblasts contain a complex of active type II cAMP-dependent protein kinase associated through its RII alpha subunit with p34cdc2.     

KT5823 inhibits cGMP-dependent protein kinase activity in vitro but not in intact human platelets and rat mesangial cells

Many signal transduction pathways are mediated by the second messengers cGMP and cAMP, cGMP- and cAMP-dependent protein kinases (cGK and PKA), phosphodiesterases, and ion channels. To distinguish among the different cGMP effectors, inhibitors of cGK and PKA have been developed including the K-252 compound KT5823 and the isoquinolinesulfonamide H89. KT5823, an in vitro inhibitor of cGK, has also been used in numerous studies with intact cells to implicate or rule out the involvement of this protein kinase in a given cellular response. However, the efficacy and specificity of KT5823 as cGK inhibitor in intact cells or tissues have never been demonstrated. Here, we analyzed the effects of both KT5823 and H89 on cyclic-nucleotide-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in intact human platelets and rat mesangial cells. These two cell types both express high levels of cGK. KT5823 inhibited purified cGK. However, with both intact human platelets and rat mesangial cells, KT5823 failed to inhibit cGK-mediated serine 157 and serine 239 phosphorylation of VASP induced by nitric oxide, atrial natriuretic peptide, or the membrane-permeant cGMP analog, 8-pCPT-cGMP. KT5823 enhanced 8-pCPT-cGMP-stimulated VASP phosphorylation in platelets and did not inhibit forskolin-stimulated VASP phosphorylation in either platelets or mesangial cells. In contrast H89, an inhibitor of both PKA and cGK, clearly inhibited 8-pCPT-cGMP and forskolin-stimulated VASP phosphorylation in the two cell types. The data indicate that KT5823 inhibits purified cGK but does not affect a cGK-mediated response in the two different cell types expressing cGK I. These observations indicate that data that interpret the effects of KT5823 in intact cells as the major or only criteria supporting the involvement of cGK clearly need to be reconsidered.     

Phospholamban is a good substrate for cyclic GMP-dependent protein kinase in vitro, but not in intact cardiac or smooth muscle.

Protein residualizing labels facilitate localization of tissue sites of protein catabolism and the quantification of protein accumulation because of their prolonged intracellular retention of protein accumulation because of their prolonged intracellular retention times. Radioiodinated residualizing labels have been used to define the metabolism of a wide variety of proteins, but this has necessitated destructive analysis. Here we describe the implementation and validation of a novel 19F-containing residualizing label for protein, NN-dilactitol-3,5-bis(trifluoromethyl)benzylamine (DLBA), that permits the non-invasive assessment of protein accumulation and catabolism by n.m.r. spectroscopy in vivo. DLBA comprises a reporter molecule containing six equivalent 19F atoms. 19F is strongly n.m.r.-active, has 100% natural abundance, and is present in minimal background concentrations in soft tissues. We validated the use of DLBA as a protein-labelling compound by coupling to asialofetuin (ASF), a protein that is recognized exclusively by hepatic tissue via a saturable receptor-mediated process. Coupling of DLBA to ASF by reductive amination had no effect on the physiological receptor-mediated uptake of the protein in rat liver in vivo. The 19F-n.m.r. spectrum of DLBA exhibited a single peak that was subject to a small chemical-shift change and broadening after coupling to ASF. Pronase digestion of DLBA-ASF was performed to simulate intracellular degradation products, and resulted in a narrower set of resonances, with chemical shifts intermediate between those of uncoupled DLBA and DLBA-ASF. Intravenous administration of DLBA-ASF to rats followed by quantification of 19F in homogenates of liver tissue indicated that the half-life of residence time of degradation products from DLBA-ASF in liver was approx. 2 days. This intracellular half-life was comparable with that described for similar residualizing labels that contain radioiodide as a reporter. Similar results for the half-life of retention were obtained non-destructively and non-invasively in situ with the use of a whole-body radio-frequency antenna to acquire sequential spectra over 80 h after intravenous administration of DLBA-ASF. Quantification of these spectra demonstrated an initial accumulation of DLBA-ASF in liver followed by an expected gradual loss of 19F-labelled degradation products. The approach developed offers promise for the sequential and longitudinal characterization of metabolism of specific proteins in individual experimental animals and ultimately in human subjects.     

Activation of a Ca2+-inhibitable protein kinase that phosphorylates microtubule-associated protein 2 in vitro by growth factors, phorbol esters, and serum in quiescent cultured human fibroblasts

Treatment of quiescent human embryonic lung fibroblastic cells (TIG-3) with 10 nM epidermal growth factor (EGF) resulted in 4-6-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein 2 (MAP2) on serine and threonine residues in vitro. The half-maximal activation of the kinase activity occurred within 5 min after EGF treatment, and the maximal level was attained at 15 min. Casein and histone were very poor substrates for this EGF-stimulated MAP2 kinase activity. The activation of the kinase activity persisted after brief dialysis. Interestingly, the EGF-stimulated MAP2 kinase activity was sensitive to micromolar concentrations of free Ca2+; it was inhibited 50% by 0.5 microM Ca2+ and almost totally inhibited by 2 microM Ca2+. The activated MAP2 kinase activity was recovered in flow-through fractions on phosphocellulose column chromatography, while kinase activities that phosphorylate 40 S ribosomal protein S6 (S6 kinase activities) were mostly retained on the column and eluted at 0.5 M NaCl. Platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-I, insulin, phorbol esters (12-O-tetradecanoylphorbol 13-acetate and phorbol 12,13-dibutyrate), and fresh fetal calf serum also induced activation of the MAP2 kinase in the quiescent TIG-3 cells. The activated MAP2 kinase activity in cells stimulated by platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-I, insulin, 12-O-tetradecanoylphorbol 13-acetate, phorbol 12,13-dibutyrate, or fetal calf serum was almost completely inhibited by 2 microM Ca2+, like the EGF-stimulated kinase. In addition, MAP2 phosphorylated by the kinase activated by different stimuli gave very similar phosphopeptide mapping patterns. These results suggest that several growth factors, phorbol esters, and serum activate a common, Ca2+-inhibitable protein kinase which is distinct from S6 kinase in quiescent human fibroblasts.     

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.     

Phorbol ester prevents the thyroid-stimulating-hormone-induced but not the forskolin-induced decrease of cAMP-dependent protein kinase activity in thyroid cell cultures

The potent tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA) affects several thyroid cell functions and interacts with thyroid-stimulating hormone (TSH) either by inhibiting or potentiating its action on different cellular parameters. Since phorbol ester acts mainly through the activation of protein kinase C, which is its receptor, we studied this activation and its interaction with TSH and forskolin in suspension cultures of porcine thyroid cells. In thyroid cell cultures, TPA has a dual effect on protein kinase C activity: immediately (2-5 min) after exposure of cells to TPA, it began to be translocated from the cytosol to the particulate fraction. The transfer of the cytosolic enzyme was total and could occur with or without a loss of activity. The translocated enzyme still needed Ca2+ and phospholipids for its activation. The basal activity increased transiently (2-4 h) in both the cytosol and particulate fractions during translocation. The peak activity in the particulate fraction was reached 10-30 min after exposure of cells to TPA, and was followed by down-regulation of protein kinase C and almost complete disappearance of its activity. The residual activity was about 13% of control after a 2-day exposure to TPA. It was unequally distributed between cytosol (4%) and particulate fraction (9%). Prolonged exposure of cells to TPA did not affect either the activity or the subcellular distribution of the cAMP-dependent protein kinase activity. TPA interacted with TSH and prevented the decrease of this activity induced by prolonged exposure of cells to the hormone not only when it was introduced simultaneously with TSH, but also when it was added 24 h after TSH. However, the forskolin-induced decrease in cAMP-dependent protein kinase activity was not prevented by the presence of TPA. TPA also affected the increases in cAMP accumulation mediated by TSH and forskolin. The TSH-induced increase was significantly stimulated by TPA after short contacts (5-15 min), while longer preincubations of cells with TPA provoked a very strong inhibition of the TSH action. However, the forskolin-induced stimulation of the cAMP accumulation was maintained and even further increased in the presence of TPA. Consequently, the actions of TSH and TPA are apparently interdependent, while those of forskolin and TPA seem to be parallel and independent. Neither TSH nor forskolin prevented the TPA-induced down regulation of protein kinase C. The biologically inactive phorbol ester analogue 4 alpha-phorbol 12,13-didecanoate had no effect on protein kinase C activity, and did not interact with either TSH or forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)     

The host protein required for in vitro replication of poliovirus is a protein kinase that phosphorylates eukaryotic initiation factor-2

The HeLa cell protein (host factor) required for in vitro replication of poliovirus has been identified as a 67,000 dalton phosphoprotein. The purified protein displays three activities in vitro: stimulation of poliovirus RNA synthesis in the presence of poliovirus replicase, apparent self-phosphorylation, and phosphorylation of the alpha-subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2). All three activities can be removed or inhibited by an antibody to host factor. Partially purified preparations of reticulocyte eIF-2 contain a similar phosphoprotein and display host factor activity in the viral RNA synthesis assay in vitro. In vitro phosphorylation of the 67 kd protein can be stimulated by low concentrations of double-stranded RNA. Addition of phosphorylated host factor in an in vitro RNA synthesis assay significantly changes the kinetics of viral RNA synthesis, indicating that protein phosphorylation may play an important role in viral RNA replication.     

The role of autophosphorylation of cAMP-dependent protein kinase II in the inhibition of protein phosphatase-1

1. The inhibition of the catalytic subunit of protein phosphatase-1 (PP-1c) by the regulatory subunit of cAMP-dependent protein kinase II (RII) was studied. 2. Phosphorylation or thiophosphorylation of RII increased its inhibitory potency up to 4- and 6-fold and rendered it competitive with respect to the substrate of PP-1c, phosphorylase a. The Ki values for thiophospho-RII and phospho-RII were 200 and 500 nM, respectively. 3. Though PP-1c was able to release phosphate from phospho-RII, its activity once incubated with phospho-RII, remained inhibited even 80% of the phosphate was released from phospho-RII. 4. The catalytic subunit of cAMP-dependent protein kinase was effective in suspending the inhibition employed either before or after the addition of phospho-RII to PP-1c. 5. No exclusive bindings of thiophospho-RII and heat-stable protein inhibitors to the PP-1c could be proved by double inhibition studies, however some synergism was observed in their effect.     

Serum, like phorbol esters, rapidly activates protein kinase C in intact quiescent fibroblasts.

Addition of serum to quiescent cultures of Swiss 3T3 cells and mouse embryo fibroblasts causes a rapid increase in the phosphorylation of an 80 000 mol. wt. cellular protein (termed 80 K). The effect is dose- and time-dependent; enhancement in 80 K phosphorylation can be detected as early as 10-15 s after adding serum. In contrast, platelet-derived growth factor elicits the response after a lag of 1.5 min suggesting that this growth factor does not mediate the response to serum. Recently a rapid increase in the phosphorylation of an 80 K cellular protein following treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact fibroblasts. The 80 K phosphoproteins generated in response to serum and to phorbol dibutyrate (PBt2) co-migrated in one- and two-dimensional PAGE and produced identical phosphopeptide fragments when subjected to partial digestion with Staphyloccocus aureus V8 protease. These observations suggest that the same 80 K protein is generated in response to serum and PBt2. We conclude that activation of protein kinase C in intact cells is one of the earliest effects elicited by serum in quiescent fibroblasts.     

Regulation of rat alveolar type 2 cell proliferation in vitro involves type II cAMP-dependent protein kinase

To elucidate the role of cAMP and different cAMP-dependent protein kinases (PKA; A-kinase) in lung cell proliferation, we investigated rat alveolar type 2 cell proliferation in relation to activation or inhibition of PKA and PKA regulatory subunits (RIIalpha and RIalpha). Both the number of proliferating type 2 cells and the level of different regulatory subunits varied during 7 days of culture. The cells exhibited a distinct peak of proliferation after 5 days of culture. This proliferation peak was preceded by a rise in RIIalpha protein level. In contrast, an inverse relationship between RIalpha and type 2 cell proliferation was noted. Activation of PKA increased type 2 cell proliferation if given at peak RIIalpha expression. Furthermore, PKA inhibitors lowered the rate of proliferation only when a high RII level was observed. An antibody against the anchoring region of RIIalpha showed cell cycle-dependent binding in contrast to antibodies against other regions, possibly related to altered binding to A-kinase anchoring protein. Following activation of PKA, relocalization of RIIalpha was confirmed by immunocytochemistry. In conclusion, it appears that activation of PKA II is important in regulation of alveolar type 2 cell proliferation.     

Regulatory subunit of type II cAMP-dependent protein kinase as substrate and inhibitor of protein phosphatase-1 and -2A

The dissociated regulatory subunit (RII) of autophosphorylated cAMP-dependent protein kinase II was dephosphorylated by the catalytic subunits of protein phosphatase-1 and -2A (phosphatase-1c and -2Ac) and by a high-Mr polycation-dependent form of phosphatase-2A (2Ao) with Km values of 5, 0.3 and 1 microM, respectively. Dissociation of protein kinase by cAMP preferentially increased the dephosphorylation of RII by phosphatase-1c, whereas polycations (histone Hl or polybrene) markedly stimulated phosphatase-2Ac and -2Ao even in the absence of cAMP. Thiophosphorylated RII inhibited the dephosphorylation of phosphorylase a by these phosphatases with half-maximum inhibitory concentrations of 0.1-0.36 microM.