Dephosphorylation of cardiac myofibril C-protein by protein phosphatase 1 and protein phosphatase 2A

C-protein purified from chicken cardiac myofibrils was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase to nearly 3 mol [32P]phosphate/mol C protein. Digestion of 32P-labeled C-protein with trypsin revealed that the radioactivity was nearly equally distributed in three tryptic peptides which were separated by reversed-phase HPLC. Fragmentation of 32P-labeled C-protein with CNBr showed that the isotope was incorporated at different ratios in three CNBr fragments which were separated on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphorylation was present in both serine and threonine residues. Incubation of 32P-labeled C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of the [32P]phosphate. The major site(s) dephosphorylated by either one of the phosphatases was a phosphothreonine residue(s) apparently located on the same tryptic peptide and on the same CNBr fragment. CNBr fragmentation also revealed a minor phosphorylation site which was dephosphorylated by either of the phosphatases. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A at high concentrations could completely dephosphorylate C-protein. These results demonstrate that C-protein phosphorylated with cAMP-dependent protein kinase can be dephosphorylated by protein phosphatases 1 and 2A. It is suggested that the enzyme responsible for dephosphorylation of C-protein in vivo is phosphatase 2A.     

Activation by G protein beta gamma subunits of agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptors and rhodopsin.

We have partially purified a protein kinase that phosphorylates muscarinic receptors (mAChR) in the presence of agonists and have shown that the phosphorylation is stimulated by the beta gamma subunits of the GTP binding protein Go (Haga, K., and Haga, T. (1990) FEBS Lett. 268, 43-47). We report here that rhodopsin is also phosphorylated in a light-dependent manner by the same kinase preparation and that beta gamma subunits derived from Gs, Gi, and Go stimulate the phosphorylation of both rhodopsin and mAChRs. The rhodopsin- and mAChR-phosphorylating activities were eluted in the same fractions using a purification procedure that is essentially the same as that used for the purification of beta-adrenergic receptor kinase (Benovic, J.L., Strasser, R.H., Caron, M.G., and Lefkowitz, R.J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2797-2801) and were inhibited by low concentrations of heparin, an inhibitor of beta-adrenergic receptor kinase, (IC50 = 15 nM), suggesting that both mAChR and rhodopsin are phosphorylated by the same or very similar kinase(s) belonging to the beta-adrenergic receptor kinase family. G protein beta gamma subunits increased the Vmax of the phosphorylation of rhodopsin 12-fold. Kinetic data were consistent with the assumptions that the protein kinase (mAChR kinase) binds rhodopsin and beta gamma subunits in a random order and that the reaction rate is proportional to concentration of the ternary complex. By contrast, the light-dependent phosphorylation of rhodopsin by the rhodopsin kinase was not stimulated by the beta gamma subunits. These results indicate that beta gamma subunits may interact with and activate the mAChR kinase but not rhodopsin kinase and suggest that the beta gamma subunit of G proteins may take part in the desensitization of G protein-linked receptors.     

Regulation of transmembrane signaling by receptor phosphorylation

At least two major effects of receptor phosphorylation have been identified--regulation of receptor function, and regulation of receptor distribution. In many cases where phosphorylation directly alters the functions of receptors, this appears to be in a negative direction. Such decreases in receptor activity may reflect reduced ability to interact with biochemical effectors (e.g., the beta-adrenergic receptor, rhodopsin), reduced affinity for binding agonist ligands (EGF,IGF-I, insulin receptors) or reduced enzymatic activity (e.g., tyrosine kinase activity of the insulin or EGF receptor). In all instances, these negative modulations are associated with phosphorylation of serine and/or threonine residues of the receptor proteins. In contrast, the tyrosine kinase receptors also appear to be susceptible to positive modulation by phosphorylation. With these receptors, autophosphorylation of tyrosine residues may lead to enhanced protein-tyrosine kinase activity of the receptors and increased receptor function. In addition, the subcellular distribution of a receptor may be regulated by its phosphorylation status (e.g., the beta-adrenergic receptor, receptors for insulin, EGF, IGF-II, and transferrin). The emerging paradigm is that receptor phosphorylation may in some way promote receptor internalization into sequestered compartments where dephosphorylation occurs. The molecular and cellular mechanisms involved in translating changes in receptor phosphorylation into changes in receptor distribution remain to be elucidated. Moreover, the biological role of receptor internalization may be quite varied. Thus, in the case of the beta-adrenergic receptor, it may serve primarily as a mechanism for bringing the phosphorylated receptors into contact with intracellular phosphatases that dephosphorylate and resensitize it. By contrast, for the transferrin receptor and other receptors involved in receptor-mediated endocytosis, the internalization presumably functions to carry some specific ligand or metabolite into the cell. The role of phosphorylation in regulating receptor function dramatically extends the range of regulatory control of this important covalent modification.     

Phosphoprotein phosphatase-catalyzed dephosphorylation of the 22,000 dalton phosphoprotein of cardiac sarcoplasmic reticulum.

The present study demonstrated the presence within the myocardium of phosphoprotein phosphatase activity which can account for dephosphorylation of a 22,000 dalton phosphoprotein of cardiac sarcoplasmic reticulum that has been associated with the stimulatory effects of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase on calcium transport (Tada, M., Kirchberger, M. A., and Katz, A. M. (1975) J. Biol. Chem. 250:2640-2647). Dog cardiac microsomes, consisting mainly of fragmented sarcomplasmic reticulum, were phosphorylated by incubation with cyclic AMP-dependent protein kinase and [gamma-32P]ATP, and subsequently washed with trichloroacetic acid or buffered KCl. Phosphorylated microsomes contained approximately 1 nmole of 32P bound per mg of microsomal protein, 32P labeling occurring almost exclusively at the 22,000 dalton component. Soluble phosphoprotein phosphatases, isolated from the cytosol, catalyzed dephosphorylation of 32P-labeled microsomes. The existence of a phosphoprotein phosphatase that is associated with the microsomes was demonstrated by the ability of the microsomes to dephosphorylate 32P-histone. This membrane-associated phosphatase activity can also account for a rapid decrease in the amount of 32P-labeling of the 22,000 dalton protein. The dephosphorylation of the phosphorylated 22,000 dalton protein by phosphoprotein phosphatase satisfies an important requirement for the phosphorylation of the 22,000 dalton protein to serve a physiological role, namely, its reversibility.     

Receptor-specific desensitization with purified proteins. Kinase dependence and receptor specificity of beta-arrestin and arrestin in the beta 2-adrenergic receptor and rhodopsin systems.

Homologous desensitization of beta-adrenergic receptors, as well as adaptation of rhodopsin, are thought to be triggered by specific phosphorylation of the receptor proteins. However, phosphorylation alone seems insufficient to inhibit receptor function, and it has been proposed that the inhibition is mediated, following receptor phosphorylation, by the additional proteins beta-arrestin in the case of beta-adrenergic receptors and arrestin in the case of rhodopsin. In order to test this hypothesis with isolated proteins, beta-arrestin and arrestin were produced by transient overexpression of their cDNAs in COS7 cells and purified to apparent homogeneity. Their functional effects were assessed in reconstituted receptor/G protein systems using either beta 2-adrenergic receptors with Gs or rhodopsin with Gt. Prior to the assays, beta 2-receptors and rhodopsin were phosphorylated by their specific kinases beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase, respectively. beta-Arrestin was a potent inhibitor of the function of beta ARK-phosphorylated beta 2-receptors. Half-maximal inhibition occurred at a beta-arrestin:beta 2-receptor stoichiometry of about 1:1. More than 100-fold higher concentrations of arrestin were required to inhibit beta 2-receptor function. Conversely, arrestin caused half-maximal inhibition of the function of rhodopsin kinase-phosphorylated rhodopsin when present in concentrations about equal to those of rhodopsin, whereas beta-arrestin at 100-fold higher concentrations had little inhibitory effect. The potency of beta-arrestin in inhibiting beta 2-receptor function was increased over 10-fold following phosphorylation of the receptors by beta ARK, but was not affected by receptor phosphorylation using protein kinase A. This suggests that beta-arrestin plays a role in beta ARK-mediated homologous, but not in protein kinase A-mediated heterologous desensitization of beta-adrenergic receptors. It is concluded that even though arrestin and beta-arrestin are similar proteins, they display marked specificity for their respective receptors and that phosphorylation of the receptors by the receptor-specific kinases serves to permit the inhibitory effects of the "arresting" proteins by allowing them to bind to the receptors and thereby inhibit their signaling properties. Furthermore, it is shown that this mechanism of receptor inhibition can be reproduced with isolated purified proteins.     

Desensitization of turkey erythrocyte adenylate cyclase. Beta-adrenergic receptor phosphorylation is correlated with attenuation of adenylate cyclase activity

Preincubation of turkey erythrocytes with beta-adrenergic agonists leads to an attenuation of the responsiveness of adenylate cyclase to subsequent hormonal stimulation. Recently, our laboratory has shown (Stadel, J. M., Nambi, P., Shorr, R. G. L., Sawyer, D. D., Caron, M. G., and Lefkowitz, R. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3173-3177) using 32Pi incorporation that phosphorylation of the beta-adrenergic receptor accompanies this desensitization process. We now report that, as determined from intracellular [gamma-32P] ATP specific activity measurements, this phosphorylation reaction occurs in a stoichiometric fashion. Under basal conditions there exists 0.75 +/- 0.1 mol of phosphate per mol of receptor whereas under maximally desensitized conditions this ratio increases to 2.34 +/- 0.13 mol/mol. This phosphorylation of the receptor is dose-dependent with respect to isoproterenol and exhibits a dose-response curve coincidental with that for isoproterenol-induced desensitization of adenylate cyclase. The time courses for receptor phosphorylation and adenylate cyclase desensitization are identical. In addition, the rate of resensitization of adenylate cyclase activity is comparable to the rate of return of the phosphate/receptor stoichiometries to control levels. Both the phosphorylation and desensitization reactions are pharmacologically specific as indicated by the high degree of stereoselectivity, rank order of catecholamines, and blockade by the specific beta-adrenergic antagonist, propranolol. Incubation of turkey erythrocytes with cAMP and cAMP analogs maximally activates cAMP-dependent protein kinase but only partially mimics isoproterenol in promoting phosphorylation of the receptor in concordance with their partial effects in inducing desensitization. Conversely, activators or inhibitors of Ca2+/calmodulin kinase or protein kinase C do not affect the isoproterenol-induced desensitization. These results indicate that desensitization of turkey erythrocyte adenylate cyclase is highly correlated with phosphorylation of the beta-adrenergic receptor and that these events are mediated, at least partially, by cAMP.     

Asialoglycoprotein receptor phosphorylation and receptor-mediated endocytosis in hepatoma cells. Effect of phorbol esters

The asialoglycoprotein (ASGP) receptor on Hep G2 cells undergoes constitutive recycling and ligand endocytosis in the presence of phorbol dibutyrate, at a 50% reduced rate relative to control cells (Fallon, R. J., and Schwartz, A. L. (1986) J. Biol. Chem. 261, 15081-15089). The relevance of receptor phosphorylation to these events was investigated by selective immunoprecipitation of surface receptors with polyclonal anti-human ASGP antiserum and pulse-chase labeling with [32P]orthophosphate to identify subcellular locations of initial receptor phosphorylation events as well as the eventual fate of phosphorylated receptor during recycling. The surface immunoprecipitation method recovers greater than 95% of surface ASGP receptors and only 5% or less of intracellular (brief[35S]methionine pulse-labeled) receptors. With this assay we detected low levels of ASGP receptor phosphorylation at the cell surface in control cells (0.1 mol of P/mol of R) which were rapidly (less than 1 min) stimulated 20-fold by 400 nM phorbol dibutyrate addition (1.7 mol of P/mol of R). Staurosporine, a protein kinase C inhibitor, blocks this stimulation by phorbol. Receptor phosphorylation at early time points in the presence of phorbol esters was restricted to the plasma membrane. Subsequent chase in the presence of excess unlabeled phosphate and phorbol esters lowered [32P] ATPi specific activity by 68% at 1 h. Surface immunoprecipitation during this chase period showed the phosphorylated ASGP receptors were rapidly lost from the cell surface (t1/2 = 20 min). In contrast, examination of intracellular receptor during the pulse-chase experiment in phorbol dibutyrate-treated cells showed the presence of phosphorylated pool(s) of ASGP receptors which were detectable for 6 h of chase. Since no labeled receptor can be detected at the cell surface at this time, the described intracellular phosphorylated receptors are in a non-recycling pool.     

Phosphorylation of highly purified growth hormone receptors by a growth hormone receptor-associated tyrosine kinase

We have shown previously that growth hormone (GH) promotes the phosphorylation of its receptor on tyrosyl residues (Foster, C. M., Shafer, J. A., Rozsa, F. W., Wang, X., Lewis, S. D., Renken, D. A., Natale, J. E., Schwartz, J., and Carter-Su, C. (1988) Biochemistry 27, 326-334). In the present study, we investigated the possibility that a tyrosine kinase is specifically associated with the GH receptor. GH-receptor complexes were first partially purified from GH-treated 3T3-F442A fibroblasts, a GH-responsive cell, by immunoprecipitation using anti-GH antiserum. 35S-Labeled proteins of Mr = 105,000-125,000 were observed in the immunoprecipitate from GH-treated cells labeled metabolically with 35S-amino-acids. These proteins were not observed in immunoprecipitates from cells not exposed to GH or when non-immune serum replaced the anti-GH antiserum, consistent with the proteins being GH receptors. GH receptors appeared to be phosphorylated, as evidenced by the presence of 32P-labeled bands, comigrating with the 105-125 kDa 35S-labeled proteins, in the immunoprecipitate of GH-treated cells labeled metabolically with [32P]Pi. When partially purified GH receptor preparation was incubated with [gamma-32P]ATP (7-15 microM) for 10 min at 30 degrees C in the presence of MnCl2, a protein of Mr = 121,000 was phosphorylated exclusively on tyrosyl residues. As expected for the GH receptor, this protein was not observed in immunoprecipitates when cells had not been treated with GH nor when non-immune serum replaced the anti-GH antiserum. GH-receptor complexes were also purified to near homogeneity by sequential immunoprecipitation with phosphotyrosyl-binding antibody followed by anti-GH antiserum. When cells were labeled metabolically with 35S-amino acids, the 35S label migrated almost exclusively as an Mr = 105,000-125,000 protein. This protein also incorporated 32P into tyrosyl residues when incubated in solution with [gamma-32P]ATP. These results show that highly purified GH receptor preparations undergo tyrosyl phosphorylation, suggesting that either the GH receptor itself is a tyrosine kinase or is tightly associated with a tyrosine kinase.     

A novel endogenous PP2C-like phosphatase dephosphorylates casein kinase II-phosphorylated Physarum fragmin

Plasmodial fragmin, a Physarum polycephalum F-actin severing and capping protein, is phosphorylated by casein kinase II at Ser(266) (De Corte, V., Gettemans, J., De Ville, Y., Waelkens, E., and Vandekerckchove, J. (1996), Biochemistry 35, 5472-5480). In this study, we report the purification and characterization of the corresponding fragmin phosphatases. One of the enzymes was purified to near homogeneity from a cytosolic extract; it dephosphorylates CKII-phosphorylated fragmin, a peptide encompassing the CKII phosphorylation site of fragmin as well as histone 2A, CKII-phosphorylated casein and the CKII model-peptide substrate: R(3)E(3)S(P)E(3). Its activity was highly stimulated by Mn(2+) and Mg(2+), and based on its lack of sensitivity toward phosphatase effectors we could exclude similarities with PP1, PP2A and PP2B phosphatases. All biochemical properties of the phosphatase point to a PP2C-like enzyme. A second phosphatase dephosphorylating fragmin was identified as a Physarum alkaline phosphatase.     

Purification and characterization of a novel protein phosphatase highly specific for ribosomal protein S6

Ribosomal protein S6 is the principal phosphoprotein of the eucaryotic ribosome that becomes multiply phosphorylated on serine residues in response to a wide variety of mitogenic stimuli. In this paper the principal protein phosphatases able to dephosphorylate S6 were characterized in Xenopus laevis ovary and eggs. Two enzymes termed peak I and peak II were found to account for most S6 phosphatase activity in both oocytes and eggs. The peak I enzyme had an apparent Mr of 200,000 on gel filtration, dephosphorylated the beta subunit of phosphorylase kinase and phosphorylase a, and was inhibited by inhibitor 1 and inhibitor 2, suggesting it was similar to protein phosphatase 1. The peak II enzyme was purified over 12,000-fold and had an apparent Mr = 55,000 on glycerol gradient centrifugation. This phosphatase could dephosphorylate all sites in S6 but was unable to dephosphorylate phosphorylase a or phosphorylase kinase. However, it was inhibited by nanomolar concentrations of inhibitor 1 and inhibitor 2. These results indicate the peak II enzyme represents a new class of highly specific protein phosphatase and suggest that inhibition of dephosphorylation in cellular extracts by inhibitor 1 and inhibitor 2 is not a sufficient criterion for implicating protein phosphatase 1 in a cellular process.     

Isolation and characterization of a tyrosyl phosphatase activator from rabbit skeletal muscle and Xenopus laevis oocytes

PTPA, a specific phosphotyrosyl phosphatase activator of the PCSH2 and PCSL protein phosphatases, was purified up to apparent homogeneity from Xenopus laevis ovaries and rabbit skeletal muscle and highly purified from dog liver. PTPA appears as a 40-kDa protein in gel filtration, as well as in sucrose gradient centrifugation, and as a 37-39-kDa protein doublet in SDS-PAGE. Its estimated cellular concentration of 0.75 microM in oocytes or 0.25 microM in rabbit skeletal muscle is suggestive of an important role in the regulation of the cellular PTPase activity. The PTPase activation reaction of the PCSL phosphatase is time-dependent, ATP and Mg2+ being essential cofactors [A50(ATP) = 0.12 mM in the presence of 5 mM MgCl2]. With RCM lysozyme as substrate, the specific activity of the PTPA-activated PCSL phosphatase is 700 nmol of Pi/(min.mg). The pH optimum of the PTPase shifts from 8.5-9 in basal conditions to a neutral pH (7-7.5), and the A50 for the essential metal ion Mg2+ is decreased (3 mM). The activation is rapidly reversed in the presence of the substrate, and more slowly after removal of ATP.Mg. The PTPA-activated PCSL phosphatase represents a major PTPase activity in the cytosol of X. laevis oocytes (at least 50% of the measurable PTPase with RCM lysozyme phosphorylated on tyrosyl residues). The PTPA activation is specific for the PTPase activity of the PCSL and PCSH2 phosphatases, without affecting their phosphoseryl/threonyl phosphatase activity. However, effectors of the phosphorylase phosphatase activity, such as polycations and okadaic acid, also influence the PTPase activity. Phosphorylase alpha inhibits the activated PTPase activity (I50 = 5 microM). The PTPase activity of the other oligomeric PCS phosphatases (PCSH1 and PCSM) is not influenced, suggesting an inhibitory role for some of their subunits. This activation is compared with the recently described PTPase stimulation of the PCS phosphatases by ATP/PPi [Goris, J., Pallen, C. J., Parker, P. J., Hermann, J., Waterfield, M. D., & Merlevede, W. (1988) Biochem. J. 256, 1029-1034] and by tubulin [Jessus, C., Goris, J., Cayla, X., Hermann, J., Hendrix, P., Ozon, R., & Merlevede, W. (1989) Eur. J. Biochem. 180, 15-22].     

Cyclic phosphorylation-dephosphorylation of rhodopsin in retina by protein kinase FA (the activator of ATP.Mg-dependent protein phosphatase).

The ATP.Mg-dependent protein phosphatase activating factor (protein kinase FA) was identified to exist in bovine retina. Furthermore, rhodopsin, the visual light pigment associated with rod outer segments in retina, could be well phosphorylated by kinase FA to about 0.9 mol of phosphates per mol of protein. Moreover, more than 90% of the phosphates in [32P]-rhodopsin could be completely removed by ATP.Mg-dependent protein phosphatase and the rhodopsin phosphatase activity was strictly kinase FA-dependent. Taken together, the results provide initial evidence that a cyclic phosphorylation-dephosphorylation of rhodopsin can be controlled by the retina-associated protein kinase FA, representing an efficient cyclic cascade mechanism possibly involved in the rapid regulation of rhodopsin function in retina.     

Dephosphorylation of Microtubule Proteins by Brain Protein Phosphatases 1 and 2A, and Its Effect on Microtubule Assembly

Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), tau factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, tau factor, and tubulin were compared. The Km values were in the ranges of 1.6-2.7 microM for MAP2 and tau factor. The Km value for tubulin decreased from 25 to 10-12.5 microM in the presence of 1.0 mM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, tau factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and tau factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.     

Dephosphorylation of endogenous GABAB receptor R2 subunit and AMPK α subunits which were measured by in vitro method using transfer membrane

Protein phosphorylation can be regulated by changes in kinase activity, phosphatase activity, or both. GABA_B receptor R2 subunit (GABA_BR2) is phosphorylated at S783 by 5′-AMP-activated-protein kinase (AMPK), and this phosphorylation modulates GABA_B receptor desensitization. Since the GABA_B receptor is an integral membrane protein, solubilizing GABA_BR2 is difficult. To circumvent this problem and to identify specific phosphatases that dephosphorylate S783, we employed an in vitro assay based on dephosphorylation of proteins on PVDF membranes by purified phosphatases. Our method allowed us to demonstrate that S783 in GABA_BR2 is directly dephosphorylated by PP2A (but not by PP1, PP2B nor PP2C) in a dose-dependent and okadaic acid-sensitive manner. We also show that the level of phosphorylation of the catalytic subunit of AMPK at T172 is reduced by PP1, PP2A and PP2C. Our data indicate that PP2A dephosphorylates GABA_BR2(S783) less efficiently than AMPK(T172), and that additional phosphatases might be involved in S783 dephosphorylation.     

Regulation of bovine kidney branched-chain 2-oxoacid dehydrogenase complex by reversible phosphorylation

Bovine kidney mitochondrial branched-chain 2-oxoacid dehydrogenase complex is inactivated by covalent phosphorylation catalysed by a specific protein kinase intrinsic to the complex. It has been shown previously [Cook, K.G., Lawson, R. and Yeaman, S.J. (1983) FEBS Lett. 157, 59-62] that tryptic digestion of phosphorylated complex releases three phosphopeptides, indicative of multisite phosphorylation. In this communication we report several findings. (a) These three tryptic peptides contain only two sites of phosphorylation which are closely grouped on the alpha subunit of the E1 component of the complex. (b) The amino acid sequence of the phosphorylated region has been determined. (c) Conditions have been developed which allow investigation of the phosphorylation and dephosphorylation of the two sites. (d) Both sites can be dephosphorylated at significant rates in vitro by two cytosolic protein phosphatases, namely phosphatases 2A and 2C. Dephosphorylation of one site correlates closely with re-activation of the complex.     

Photoaffinity labeling of the beta-adrenergic receptor

A new photoactive beta-adrenergic antagonist, p-azidobenzylcarazolol (pABC) has been synthesized by combining a carbazole moiety with a p-azido-benzyl substituent. The compound has been labeled with tritium to a specific activity of 26 Ci/mmol. In frog erythrocyte membranes, [3H]p-azido-benzylcarazolol binds to the beta-adrenergic receptor with the expected beta 2 specificity and with high affinity (KD congruent to 100 +/- 10 pM). Unlabeled p-azido-benzylcarazolol can irreversibly inactivate the [3H]dihydroalprenolol-binding activity of frog erythrocyte membranes in a photodependent manner which can be prevented by beta-adrenergic agents. Incubation of frog erythrocyte membranes or digitonin-solubilized preparations of these membranes or digitonin-solubilized preparations of these membranes which had been enriched in beta-adrenergic receptors by a Sepharose-alprenolol chromatography step led to covalent incorporation of radioactivity into a Mr = 58,000 peptide. Specific incorporation of [3H]pABC into the Mr = 58,000 peptide could be prevented by both beta-adrenergic agonists and antagonists. This peptide has previously been purified and shown to contain the beta-adrenergic receptor-binding site (Shorr, R. G. L., Lefkowitz, R. J., and Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826). Thus, photoaffinity labeling of the beta-adrenergic receptor protein directly identifies the same hormone-binding subunit as has been isolated by conventional purification techniques.     

Specificity of protein phosphatases in the dephosphorylation of protein kinase C.

Protein kinase C can autophosphorylate in vitro and has also been shown to be phosphorylated in vivo. In order to investigate the factors that may determine the phosphorylation state of protein kinase C in vivo, we determined the ability of the ATP + Mg2+-dependent phosphatase and the polycation-stimulated (PCS) phosphatases to dephosphorylate protein kinase C in vitro. These studies show that all the oligomeric forms of the PCS phosphatases (PCSH1, PCSH2, PCSM and PCSL phosphatases) are effective in the dephosphorylation of protein kinase C, showing 34-82% of the activity displayed with phosphorylase a as substrate. In contrast both the catalytic subunit of the PCS phosphatase and that of the ATP+Mg2+-dependent phosphatase showed only weak activity with protein kinase C as substrate. All these phosphatases, however, were activated by protamine (Ka 14-16 micrograms/ml) through what appears to be a substrate-directed effect. The relative role of these phosphatases in the control of protein kinase C is discussed.     

Neuronal Cyclin-Dependent Kinase-5 Phosphorylation Sites in Neurofilament Protein (NF-H) Are Dephosphorylated by Protein Phosphatase 2A

Abstract: Neurofilament (NF) protein [high molecular mass (NF-H)] is extensively phosphorylated in vivo. The phosphorylation occurs mainly in its characteristic KSP (Lys-Ser-Pro) repeat motifs. There are two major types of KSP motifs in the NF-H tail domain: KSPXKX and KSPXXX. Recent studies by two different laboratories have demonstrated the presence of a cdc2-like kinase [cyclin-dependent kinase-5 (cdk5)] in nervous tissue that selectively phosphorylates KSPXKX and XS/TXK motifs in NF-H and lysine-rich histone (H1). This article describes the identification of phosphatases dephosphorylating three different substrates: histone (H1), NF-H in a NF preparation, and a bacterially expressed C-terminal tail domain of NF-H, each containing KSPXKX repeats phosphorylated in vitro by cdk5. Among various phosphatases identified, protein phosphatase (PP) 2A from rabbit skeletal muscle appeared to be the most effective phosphatase in in vitro assays. Three phosphatase activity peaks—P1, P2, and P3—were partially purified from frozen rat spinal cord by ion exchange and size exclusion column chromatography and then characterized on the basis of biochemical, pharmacological, and immunochemical studies. One of the three peaks was identified as PP2A, whereas the others were mixtures of both PP2A and PP1. These three peaks could dephosphorylate cdk5-phosphorylated ³²P-histone (H1), ³²P-NF-H in the NF preparation, and ³²P-NF-H tail fusion protein. These studies suggest the involvement of PP2A or a PP2A-like activity in the regulation of the phosphorylation state of KSPXKX motifs in NF-H.     

Selective activation of the two catalytic sites in the ATP.Mg-dependent phosphoprotein phosphatase by kinase Fa and Mn2+ ion

1. Although Mn2+ could mimic kinase FA/ATP.Mg to activate ATP.Mg-dependent protein phosphatase, strong indications have been obtained that the Mn2(+)-activated and FA/ATP.Mg-activated phosphatase forms are not identical in terms of their substrate specificities and catalytic properties. 2. Both Mn2(+)-activated and FA/ATP.Mg-activated phosphatase forms readily dephosphorylate 32P-labeled phosphorylase a and myelin basic protein (MBP), however the Mn2(+)-activated phosphatase displays activity preferentially against [32P]MBP and FA/ATP.Mg-activated phosphatase preferentially dephosphorylates [32P]phosphorylase a, representing a unique control mechanism to regulate the substrate specificity of multisubstrate protein phosphatase in mammalian tissues.     

p42 MAP kinase phosphorylation sites in microtubule-associated protein tau are dephosphorylated by protein phosphatase 2A1. Implications for Alzheimer's disease [corrected]

The paired helical filament (PHF), which comprises the major fibrous element of the neurofibrillary tangle of Alzheimer's disease, is composed of abnormally phosphorylated microtubule-associated protein tau. Here we show that p42 MAP kinase phosphorylates recombinant tau and converts it to a form which is similar to PHF tau. Of the major serine/threonine protein phosphatases found in mammalian tissues only protein phosphatase 2A (PP2A) could dephosphorylate tau phosphorylated in this manner, with PP2A1 being the most effective form of the enzyme.