Phosphorylation of RII subunit and attenuation of cAMP-dependent protein kinase activity by proline-directed protein kinase.

Previous studies identified proline-directed protein kinase (PDPK) as a growth factor-sensitive serine/threonine protein kinase that is active in the cytosol of proliferative cells and tissues during interphase. In this communication, we report that the regulatory subunit (RII) of bovine cardiac muscle cAMP-dependent protein kinase (PKA) is a putative substrate for the multifunctional PDPK. Purified RII is readily phosphorylated by PDPK in vitro in a time-dependent, enzyme-dependent manner to a stoichiometry approaching 0.7 mol phosphate/mol RII subunit protein. The major RII phosphorylation site is identified as a threonine residue located within a large hydrophobic tryptic peptide that is predicted to contain the cAMP binding domains. In contrast to the reported effects of RII autophosphorylation, kinetic analysis of RII function following phosphorylation by PDPK indicates that the inhibitory potency of RII toward the catalytic subunit of PKA in a reassociation assay is increased in proportion to the degree of phosphorylation. Further studies indicate that the cAMP-dependent activation of the RII2C2 holoenzyme is inhibited by PDPK phosphorylation. Taken together, the results of these studies indicate that phosphorylation of RII by PDPK attenuates the activity of PKA. This antagonistic interaction suggests a biochemical mechanism by which a growth factor-activated signaling system may function to modulate cAMP-dependent cellular responses.     

The phosphoform of the regulatory subunit RII of cyclic AMP-dependent protein kinase possesses intrinsic topoisomerase activity

The phosphoform of the type II regulatory subunit (phospho-RII-cAMP) of cAMP-dependent protein kinase from rat liver was found to possess intrinsic topoisomerase activity towards several DNA substrates such as phi X174, pBR322, SV40, and M13. Like the type I topoisomerases from several eukaryotic cells, phospho-RII X cAMP can relax both positive and negative superhelical turns of phi X174 DNA. Topological isomers with a decreasing number of superhelical turns can be identified as transient products. Conditions under which phospho-RII X cAMP relaxes superhelical phi X174 DNA lead to transient formation of a DNA-phospho-RII X cAMP complex via DNA strand breakage and covalent attachment of the DNA to a tyrosine residue of phospho-RII X cAMP via a phospho-RII X cAMP depends on the presence of cAMP and is altered by changes in the degree of phosphorylation of RII. Both dephosphorylation and removal of cAMP from phospho-RII X cAMP abolish its topoisomerase activity.     

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.     

Regulation of protein phosphatase-1G from rabbit skeletal muscle. 2. Catalytic subunit translocation is a mechanism for reversible inhibition of activity toward glycogen-bound substrates

The glycogen-associated form of protein phosphatase-1 (PP-1G) comprises a 37-kDa catalytic (C) subunit and a 161-kDa glycogen-binding (G) subunit. In the preceding paper in this issue of the journal we showed that the C subunit is released from PP-1G in response to phosphorylation of the G subunit by cAMP-dependent protein kinase. We now show that at 0.15-0.2 M KCl the phosphorylase phosphatase activity of glycogen-bound PP-1G is 5-8 times higher than that of released C subunit or unbound PP-1G, which are strongly inhibited at these ionic strengths. The activity of glycogen-bound PP-1G towards glycogen synthase was about 5-fold higher than that of released C subunit at 0.15M KCl. Studies with glycogen-bound substrates and myosin P-light chain (which does not interact with glycogen) indicated that PP-1G activity is only enhanced compared to free C subunit at near physiological ionic strength and when both PP-1G and substrate are glycogen-associated. The inhibition by increasing ionic strength and enhanced activity upon binding to glycogen reflected changes in K'm, but not Vmax. From the determined specificity constant, k'cat/K'm approximately 4 x 10(6) s-1 M-1, it was calculated that at physiological levels of glycogen-bound PP-1G (200 nM) and phosphorylase (70 microM), dephosphorylation of the latter could occur with a half time of 15 s, sufficient to account for inactivation rates in vivo. The much higher catalytic efficiency of glycogen-bound PP-1G toward the glycogen-metabolising enzymes at physiological ionic strength compared to free C subunit substantiates the role of PP-1G in the regulation of these substrates, and establishes a novel mechanism for selectively regulating their phosphorylation states in response to adrenalin and other factors affecting phosphorylation of the G subunit.     

Coelution of the type II holoenzyme form of cAMP-dependent protein kinase with regulatory subunits of the type I form of cAMP-dependent protein kinase

The types and subunit composition of cAMP-dependent protein kinases in soluble rat ovarian extracts were investigated. Results demonstrated that three peaks of cAMP-dependent kinase activity could be resolved using DEAE-cellulose chromatography. Based on the sedimentation of cAMP-dependent protein kinase and regulatory subunits using sucrose density gradient centrifugation, identification of 8-N3[32P]cAMP labeled RI and RII in DEAE-cellulose column and sucrose gradient fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Scatchard analysis of the cAMP-stimulated activation of the eluted peaks of kinase activity, the following conclusions were drawn regarding the composition of the three peaks of cAMP-dependent protein kinase activity: peak 1, eluting with less than or equal to 0.05 M potassium phosphate, consisted of the type I form of cAMP-dependent protein kinase; peak 2, eluting with 0.065-0.11 M potassium phosphate, consisted of free RI and a type II tetrameric holoenzyme; peak 3, eluting with 0.125 M potassium phosphate, consisted of an apparent RIIC trimer, followed by the elution with 0.15 M potassium phosphate of free RII. The regulatory subunits were confirmed as authentic RI and RII based upon their molecular weights and autophosphorylation characteristics. The more basic elution of the type II holoenzyme with free RI was not attributable to the ionic properties of the regulatory subunits, based upon the isoelectric points of photolabeled RI and RII and upon the elution location from DEAE-cellulose of RI and RII on dissociation from their respective holoenzymes by cAMP. This is the first report of a type II holoenzyme eluting in low salt fractions with free RI, and of the presence of an apparent RIIC trimer in a soluble tissue extract.     

cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors

Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor.     

Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase

The phosphorylation of the calmodulin-dependent enzyme myosin light chain kinase, purified from bovine tracheal smooth muscle and human blood platelets, by the catalytic subunit of cAMP-dependent protein kinase and by cGMP-dependent protein kinase was investigated. When myosin light chain kinase which has calmodulin bound is phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of tracheal myosin light chain kinase or platelet myosin light chain kinase, with no effect on the catalytic activity. Phosphorylation when calmodulin is not bound results in the incorporation of 2 mol of phosphate and significantly decreases the activity. The decrease in myosin light chain kinase activity is due to a 5 to 7-fold increase in the amount of calmodulin required for half-maximal activation of both tracheal and platelet myosin light chain kinase. In contrast to the results with the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase cannot phosphorylate tracheal myosin light chain kinase in the presence of bound calmodulin. When calmodulin is not bound to tracheal myosin light chain kinase, cGMP-dependent protein kinase phosphorylates only one site, and this phosphorylation has no effect on myosin light chain kinase activity. On the other hand, cGMP-dependent protein kinase incorporates phosphate into two sites in platelet myosin light chain kinase when calmodulin is not bound. The sites phosphorylated by the two cyclic nucleotide-dependent protein kinases were compared by two-dimensional peptide mapping following extensive tryptic digestion of the phosphorylated myosin light chain kinases. With respect to the tracheal myosin light chain kinase, the single site phosphorylated by cGMP-dependent protein kinase when calmodulin is not bound appears to be the same site phosphorylated in the tracheal enzyme by the catalytic subunit of cAMP-dependent protein kinase when calmodulin is bound. With respect to the platelet myosin light chain kinase, the additional site that was phosphorylated by cGMP-dependent protein kinase when calmodulin was not bound was different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.     

Reduced levels of cardiac cAMP-dependent protein kinase in spontaneously hypertensive rat

Cardiac cAMP-dependent protein kinases were compared between the spontaneously hypertensive rat and the age-matched normotensive Wistar-Kyoto rat by DEAE-cellulose chromatography, photoaffinity labeling with 8-N3[32P]cAMP, and Western blots using the antiregulatory and 125I-anticatalytic subunit antibodies. DEAE-cellulose chromatography revealed that the ratio of type I to type II cAMP-dependent protein kinase was 3:1 in the cytoplasmic soluble proteins from the heart of normotensive rat. In contrast, the ratio of type I to type II was 1:1 in the heart of hypertensive rat. Type I protein kinase was reduced by 3-fold in hypertensive rat compared to normotensive rat. The levels of type II protein kinase were similar in both normotensive and hypertensive rats. The ratio of regulatory subunits of type I (RI) to type II (RII) cAMP-dependent protein kinase was 2.5 in the soluble proteins from the heart of normotensive rat compared to a ratio of 0.62 for hypertensive rat. RI was reduced by 4-fold in hypertensive rat compared to normotensive rat. The decrease in RI from hypertensive rat was also demonstrated by photoaffinity labeling with 8-N3[32P] cAMP. Western blot analysis of the catalytic subunit revealed a 2-fold decrease in catalytic subunit (C) in the soluble proteins from the hypertensive rat compared to normotensive rat. These results show that the reduced level of activity of cardiac type I protein kinase in hypertensive rat was the result of a decrease in both the RI and C subunits, thus reducing the number of type I cAMP-dependent protein kinase holoenzyme molecules. Comparison of type I protein kinase from "prehypertensive" and "hypertensive" stages of hypertensive rat indicated that the type I protein kinase was reduced by 3-fold before an increase in the blood pressure was detectable. Cardiac type I protein kinase is predominantly associated with the cytoplasmic proteins in both the normotensive and hypertensive rats. The levels of RI, RII, and C associated with the membrane-solubilized proteins were not affected in the hypertensive rat. The levels of RII were similar in the brain tissue of normotensive and hypertensive rats, suggesting that the decrease in type I protein kinase is specific in hypertensive rat. In conclusion, a decrease in cardiac type I cAMP-dependent protein kinase may affect the degree of phosphorylation of cardiac regulatory proteins, thus impairing normal cardiac physiology in hypertensive rat.     

Autophosphorylation of rat liver type II cAMP-dependent protein kinase

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.     

The glycogen-binding subunit of protein phosphatase-1G from rabbit skeletal muscle. Further characterisation of its structure and glycogen-binding properties

The glycogen-bound form of protein phosphatase-1 (PP-1G) was previously purified as a heterodimer composed of a 37-kDa catalytic (C) subunit and a proteolytically sensitive 103-kDa glycogen-binding (G) subunit [Str?hlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295-303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161-kDa protein, and that the 103-kDa species (now termed G') is itself a product of proteolysis. A second phosphorylation site for cAMP-dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP-1 at a slow rate, whereas site 1 was resistant to autodephosphorylation. PP-1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen-protein particles during washing with a variety of solvents. The PP-1G holoenzyme was released from glycogen-protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP-1G. Binding experiments with purified PP-1G and glycogen indicated a bimolecular process with Kapp values corresponding to about 8 nM glycogen and 4 nM PP-1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high-affinity glycogen-binding domain on the G subunit, and indicate that a physiological concentrations of phosphatase and glycogen, PP-1G should be almost entirely bound to glycogen.     

Phosphorylation of smooth muscle myosin light chain kinase by protein kinase C. Comparative study of the phosphorylated sites

Smooth muscle myosin light chain kinase is phosphorylated in vitro by protein kinase C purified from human platelets. When myosin light chain kinase which has calmodulin bound is phosphorylated by protein kinase C, 0.8-1.1 mol of phosphate is incorporated per mol of myosin light chain kinase with no effect on its enzyme activity. Phosphorylation of myosin light chain kinase with no calmodulin bound results in the incorporation of 2-2.4 mol of phosphate and significantly decreases the rate of myosin light chain kinase activity. The decrease in myosin light chain kinase activity is due to a 3.3-fold increase in the concentration of calmodulin necessary for the half-maximal activation of myosin light chain kinase. The sites phosphorylated by protein kinase C and the catalytic subunit of cAMP-dependent protein kinase were compared by two-dimensional peptide mapping following extensive tryptic digestion of phosphorylated myosin light chain kinase. The single site phosphorylated by protein kinase C when calmodulin is bound to myosin light chain kinase (site 3) is different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 1). The additional site that is phosphorylated by protein kinase C when calmodulin is not bound appears to be the same site phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 2). These studies confirm the important role of site 2 in binding calmodulin to myosin light chain kinase. Sequential studies using both protein kinase C and the catalytic subunit of cAMP-dependent protein kinase suggest that the phosphorylation of site 1 also plays a part in decreasing the affinity of myosin light chain kinase for calmodulin.     

Regulatory subunit of the type I cAMP-dependent protein kinase as an inhibitor and substrate of the cGMP-dependent protein kinase

The regulatory subunit of the type I cAMP-dependent protein kinase (Rt) serves as a substrate for the phosphotransferase reaction catalyzed by cGMP-dependent protein kinase (Km = 2.2 microM). The reaction is stimulated by cGMP when RI . cAMP is the substrate, but not when nucleotide-free RI is used. The cGMP-dependent protein kinase catalyzes the incorporation of 2 mol of phosphate/mol of RI dimer in the presence of cAMP and a self-phosphorylation reaction to the extent of 4 mol of phosphate/mol of enzyme dimer. In the absence of cAMP, RI is a competitive inhibitor of the phosphorylation of histone H2B (Ki = 0.25 microM) and of the synthetic peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly (Ki = 0.15 microM) by the cGMP-dependent enzyme. Nucleotide-free RI also inhibits the intramolecular self-phosphorylation of cGMP-dependent protein kinase. The inhibition of the phosphorylation reactions are reversed by cAMP. The catalytic subunit of cAMP-dependent protein kinase does not catalyze the phosphorylation of RIand does not significantly alter the ability of RI to serve as a substrate or an inhibitor of cGMP-dependent protein kinase. These observations are consistent with the concept that the cGMP- and cAMP-dependent protein kinases are closely related proteins whose functional domains may interact.     

In vitro phosphorylation of type I collagen by cyclic AMP-dependent protein kinase

Both the triple-helical and denatured forms of nonfibrillar bovine dermal type I collagen were tested as substrates for the catalytic subunit of cAMP-dependent protein kinase in an in vitro reaction. Native, triple-helical collagen was not phosphorylated, but collagen that had been thermally denatured into individual alpha chains was a substrate for the protein kinase. Catalytic subunit of cAMP-dependent protein kinase phosphorylated denatured collagen to between 3 to 4 mol of phosphate/mol of (alpha 1(I)2 alpha 2(I). Pepsin-solubilized and intact collagens were phosphorylated similarly, as long as each was in a nonhelical conformation. The first 2 mol of phosphate incorporated into type I collagen by the protein kinase were present in the alpha 2(I) chain. The alpha 1(I) chain was only phosphorylated during long incubations in which the stoichiometry exceeded 2 mol of phosphate/mol of (alpha 1(I)2 alpha 2(I). Phosphoserine was the only phosphoamino acid identified in collagen that had been phosphorylated to any degree by the protein kinase. The 2 mol of phosphate incorporated into the alpha 2(I) chain were localized to the alpha 2(I)CB4 cyanogen bromide fragment. The catalytic subunit of cAMP-dependent protein kinase phosphorylated denatured pepsin-solubilized collagen with a Km of 8 microM and a Vmax of approximately 0.1 mumol/min/mg of enzyme. Denatured, but not triple-helical, type I collagen was also phosphorylated by cGMP-dependent protein kinase, although it was a poorer substrate for this enzyme than for the cAMP-dependent protein kinase. Collagen was not a substrate for phospholipid-sensitive Ca2+-dependent protein kinase. These results suggest the potential for nascent alpha chains of type I collagen to be susceptible to phosphorylation by cAMP-dependent protein kinase in vivo prior to triple-helix formation. Such a phosphorylation of collagen could be relevant to the action of cAMP to increase the intracellular degradation of newly synthesized collagen.     

AMY-1 interacts with S-AKAP84 and AKAP95 in the cytoplasm and the nucleus,respectively, and inhibits cAMP-dependent protein kinase activity by preventing binding of its catalytic subunit to A-kinase-anchoring protein (AKAP) complex

We have reported that a novel c-Myc-binding protein, AMY-1, binds to cAMP-dependent protein kinase-anchoring protein 149 (AKAP149) and its splicing variant, AKAP84 and is localized in the mitochondria in a complex with RII, a regulatory subunit of cAMP-dependent protein kinase (PKA) (Furusawa, M., Ohnishi, T., Taira, T., Iguchi-Ariga, S. M. M., and Ariga, H. (2001) J. Biol. Chem. 276, 36647-36651). In this study, we further found that AMY-1 competitively bound to either AKAP95 or AKAP84 in the nucleus and the cytoplasm, respectively, in a concentration-dependent manner of either AKAP. Like AKAP84, AMY-1 was found to bind to the RII-binding region of AKAP95 in vivo and in vitro and to make a ternary complex with RII. It was also found that the formation of the complex of AMY-1 with AKAP84/95 and RII prevented a catalytic subunit from binding to this AKAP complex, leading to suppression of PKA activity. These findings suggest that AMY-1 is an important modulator of PKA.     

Nuclear proteins--substrates for cAMP-dependent protein kinase

The phosphorylation of nuclear proteins of porcine brain cAMP-dependent protein kinase was studied. Some nuclear proteins after extraction from the nuclei served as substrates for protein kinase. Lysine-rich histones H1, H2a and H2b were found to accept phosphate during chromatin phosphorylation by cAMP-dependent protein kinase. Phosphorylation of intact nuclei revealed that in such a system only histone H1 is a substrate for cAMP-dependent protein kinase. In the presence of DNA the histones are phosphorylated by cAMP-dependent protein kinase in a different manner. It was concluded that DNA can determine the accessibility of protein substrates for the catalytic subunit of cAMP-dependent protein kinase.     

Inhibitory effect of the regulatory subunit of type I cAMP-dependent protein kinase on phosphoprotein phosphatase

The regulatory subunit of type I cAMP-dependent protein kinase (RI) from rabbit skeletal muscle inhibited the activity of a low molecular weight phosphoprotein phosphatase. The inhibition was concentration and time dependent. A maximum inhibition, about 70%, was observed at 2 microM of RI with an apparent Ki of 0.8 microM. Inhibition was associated with a decrease in Vmax with no change in Km for substrate, phosphorylase a. On the other hand, cAMP-dependent protein kinase holoenzyme or its catalytic subunit was without any effect. The inhibition of phosphoprotein phosphatase by RI may be of physiological significance since the dissociation of cAMP-dependent protein kinase by cAMP would result in a simultaneous increase in the phosphorylation and decrease in the dephosphorylation rates of target proteins.     

Regulation of protein phosphatase-1G from rabbit skeletal muscle. 1. Phosphorylation by cAMP-dependent protein kinase at site 2 releases catalytic subunit from the glycogen-bound holoenzyme

The glycogen-associated form of protein phosphatase-1 (PP-1G) is a heterodimer comprising a 37-kDa catalytic (C) subunit and a 161-kDa glycogen-binding (G) subunit, the latter being phosphorylated by cAMP-dependent protein kinase at two serine residues (site 1 and site 2). Here the amino acid sequence surrounding site 2 has been determined and this phosphoserine shown to lie 19 residues C-terminal to site 1 in the primary structure. The sequence in this region is: (sequence; see text) At physiological ionic strength, phosphorylation of glycogen-bound PP-1G was found to release all the phosphatase activity from glycogen. The released activity was free C subunit, and not PP-1G, while the phospho-G subunit remained bound to glycogen. Dissociation reflected a greater than or equal to 4000-fold decrease in affinity of C subunit for G subunit and was readily reversed by dephosphorylation. Phosphorylation and dephosphorylation of site 2 was rate-limiting for dissociation and reassociation of C subunit. Release of C subunit was also induced by the binding of anti-site-1 Fab fragments to glycogen-bound PP-1G. At near physiological ionic strength, PP-1G and glycogen concentration, site 2 was autodephosphorylated by PP-1G with a t0.5 of 2.6 min at 30 degrees C, approximately 100-fold slower than the t0.5 for dephosphorylation of glycogen phosphorylase under the same conditions. Site 2 was a good substrate for all three type-2 phosphatases (2A, 2B and 2C) with t0.5 values less than those toward the alpha subunit of phosphorylase kinase. At the levels present in skeletal muscle, the type-2A and type-2B phosphatases are potentially capable of dephosphorylating site 2 in vivo within seconds. Site 1 was at least 10-fold less effective than site 2 as a substrate for all four phosphatases. In conjunction with information presented in the following paper in this issue of this journal, the results substantiate the hypothesis that PP-1 activity towards the glycogen-metabolising enzymes is regulated in vivo by reversible phosphorylation of a targetting subunit (G) that directs the C subunit to glycogen--protein particles. The efficient dephosphorylation of site 2 by the Ca2+/calmodulin-stimulated protein phosphatase (2B) provides a potential mechanism for regulating PP-1 activity in response to Ca2+, and represents an example of a protein phosphatase cascade.     

The structure, role, and regulation of type 1 protein phosphatases.

Type 1 protein phosphatases (PP-1) comprise a group of widely distributed enzymes that specifically dephosphorylate serine and threonine residues of certain phosphoproteins. They all contain an isoform of the same catalytic subunit, which has an extremely conserved primary structure. One of the properties of PP-1 that allows one to distinguish them from other serine/threonine protein phosphatases is their sensitivity to inhibition by two proteins, termed inhibitor 1 and inhibitor 2, or modulator. The latter protein can also form a 1:1 complex with the catalytic subunit that slowly inactivates upon incubation. This complex is reactivated in vitro by incubation with MgATP and protein kinase FA/GSK-3. In the cell the type 1 catalytic subunit is associated with noncatalytic subunits that determine the activity, the substrate specificity, and the subcellular location of the phosphatase. PP-1 plays an essential role in glycogen metabolism, calcium transport, muscle contraction, intracellular transport, protein synthesis, and cell division. The activity of PP-1 is regulated by hormones like insulin, glucagon, alpha- and beta-adrenergic agonists, glucocorticoids, and thyroid hormones.     

Molecular determinants of nuclear protein phosphatase-1 regulation by NIPP-1.

NIPP-1 is a subunit of the major nuclear protein phosphatase-1 (PP-1) in mammalian cells and potently inhibits PP-1 activity in vitro. Using yeast two-hybrid and co-sedimentation assays, we mapped a PP-1-binding site and the inhibition function to the central one-third domain of NIPP-1. Full-length NIPP-1 (351 residues) and the central domain, NIPP-1(143-217), were equally potent PP-1 inhibitors (IC50 = 0.3 nM). Synthetic peptides spanning the central domain of NIPP-1 further narrowed the PP-1 inhibitory function to residues 191-200. A second, noninhibitory PP-1-binding site was identified by far-Western assays with digoxygenin-conjugated catalytic subunit (PP-1C) and included a consensus RVXF motif (residues 200-203) found in many other PP-1-binding proteins. The substitutions, V201A and/or F203A, in the RVXF motif, or phosphorylation of Ser199 or Ser204, which are established phosphorylation sites for protein kinase A and protein kinase CK2, respectively, prevented PP-1C-binding by NIPP-1(191-210) in the far-Western assay. NIPP-1(191-210) competed for PP-1 inhibition by full-length NIPP-1(1-351), inhibitor-1 and inhibitor-2, and dissociated PP-1C from inhibitor-1- and NIPP-1(143-217)-Sepharose but not from full-length NIPP-1(1-351)-Sepharose. Together, these data identified some of the key elements in the central domain of NIPP-1 that regulate PP-1 activity and suggested that the flanking sequences stabilize the association of NIPP-1 with PP-1C.