The alpha-helical propensity of the cytoplasmic domain of phospholamban: a molecular dynamics simulation of the effect of phosphorylation and mutation

We have used molecular dynamics simulations to investigate the effect of phosphorylation and mutation on the cytoplasmic domain of phospholamban (PLB), a 52-residue protein that regulates the calcium pump in cardiac muscle. Simulations were carried out in explicit water systems at 300 K for three peptides spanning the first 25 residues of PLB: wild-type (PLB(1-25)), PLB(1-25) phosphorylated at Ser16 and PLB(1-25) with the R9C mutation, which is known to cause human heart disease. The unphosphorylated peptide maintains a helical conformation from 3 to 15 throughout a 26-ns simulation, in agreement with spectroscopic data. Comparison with simulations of a fourth peptide truncated at Pro21 showed the importance of the region from 17 to 21 in preventing local unfolding of the helix. The results suggest that residues 11-16 are more likely to unfold when specific capping motifs are not present. It is proposed that protein kinase A exploits the intrinsic flexibility of the 11-21 region when binding PLB. In agreement with available CD and NMR data, the simulations show a decrease in the helical content upon phosphorylation. The phosphorylated peptide is characterized by helix spanning residues 3-11, followed by a turn that optimizes the salt-bridge interaction between the side chains of the phosphorylated Ser-16 and Arg-13. Replacing Arg-9 with Cys results in unfolding of the helix from C9 and an overall decrease of the helical conformation. The simulations show that initiation of unfolding is due to increased solvent accessibility of the backbone atoms near the smaller Cys. It is proposed that the loss of inhibitory potency upon Ser-16 phosphorylation or R9C mutation of PLB is due to a similar mechanism, in which the partial unfolding of the cytoplasmic helix of PLB results in a conformation that interacts with the cytoplasmic domain of the calcium pump to relieve its inhibition.     

Spatial requirements for location of basic residues in peptide substrates for smooth muscle myosin light chain kinase

The requirement of basic residues as substrate specificity determinants for the chicken gizzard myosin light chain kinase has been studied using synthetic peptide analogs of the local phosphorylation site sequence in the myosin light chains, Lys-Lys-Arg13-Pro-Gln-Arg16-Ala-Thr-Ser19-Asn-Val-Phe- Ala. The basic residue, Arg-16, was found to have a strong influence on the kinetics of phosphorylation similar to that reported previously for the three adjacent residues, Lys-11, Lys-12, and Lys-13 (Kemp, B. E., Pearson, R. B., and House, C. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7471-7475). The location of Arg-16 in relation to Ser-19 as well as the distance between Arg-13 and Arg-16 had a profound effect on both the kinetics and the site specificity of phosphorylation. Placement of Arg-16 at position 15 resulted in a complete switch in phosphorylation site specificity from Ser-19 to Thr-18. Increasing the number of alanine residues between Arg-13 and Arg-16 in the model peptide, Lys-Lys-Arg-(Ala)n-Arg-Ala-Thr-Ser-Asn-Val-Phe-Ala, also influenced the kinetics and site specificity of peptide phosphorylation. With two or three alanines (n = 2 or 3), the apparent Km was 7.5 and 10 microM, respectively, and 97% of the phosphate was esterified to Ser-19. Increasing or decreasing the number of alanines (n = O to n = 4) was accompanied by an increase in the apparent Km and phosphorylation of both Thr-18 and Ser-19. These results support the concept that both the presence and location of basic residues play an essential role in the substrate specificity of the smooth muscle myosin light chain kinase.     

Implications of threonine hydrogen bonding in the glycophorin A transmembrane helix dimer

The transmembrane helix of glycophorin A contains a seven-residue motif, LIxxGVxxGVxxT, that mediates protein dimerization. Threonine is the only polar amino acid in this motif with the potential to stabilize the dimer through hydrogen-bonding interactions. Polarized Fourier transform infrared spectroscopy is used to establish a robust protocol for incorporating glycophorin A transmembrane peptides into membrane bilayers. Analysis of the dichroic ratio of the 1655-cm(-1) amide I vibration indicates that peptides reconstituted by detergent dialysis have a transmembrane orientation with a helix crossing angle of <35 degrees. Solid-state nuclear magnetic resonance spectroscopy is used to establish high resolution structural restraints on the conformation and packing of Thr-87 in the dimer interface. Rotational resonance measurement of a 2.9-A distance between the gamma-methyl and backbone carbonyl carbons of Thr-87 is consistent with a gauche- conformation for the chi1 torsion angle. Rotational-echo double-resonance measurements demonstrate close packing (4.0 +/- 0.2 A) of the Thr-87 gamma-methyl group with the backbone nitrogen of Ile-88 across the dimer interface. The short interhelical distance places the beta-hydroxyl of Thr-87 within hydrogen-bonding range of the backbone carbonyl of Val-84 on the opposing helix. These results refine the structure of the glycophorin A dimer in membrane bilayers and highlight the complementary role of small and polar residues in the tight association of transmembrane helices in membrane proteins.     

In vivo phosphorylation of regulatory light chain of myosin II during mitosis of cultured cells

Phosphorylation of the regulatory light chain of myosin II (MLC) controls the contractility of actomyosin in nonmuscle and muscle cells. It has been reported that cdc2 phosphorylates MLC in vitro at Ser-1 or Ser-2 and Thr-9 which protein kinase C phosphorylates (Satterwhite, L. L., M. J. Lohka, K. L. Wilson, T. Y. Scherson, L. K. Cisek, J. L. Corden, and T. D. Pollard. 1992 J. Cell Biol. 118:595-605). We have examined in vivo phosphorylation of MLC during mitosis and after the release of mitotic arrest. Phosphate incorporation of MLC in mitotic cells is found to be 6-12 times greater than that in nonmitotic cells. Phosphopeptide maps have revealed that the MLC from mitotic cells is phosphorylated at Ser-1 and/or Ser-2 (Ser-1/2), but not at Thr-9. MLC is also phosphorylated to a much lesser extent at Ser-19 which myosin light chain kinase phosphorylates. On the other hand, MLC of nonmitotic cells is phosphorylated at Ser-19 but not at Ser-1/2. The extent of phosphate incorporation is doubled at 30 min after the release of mitotic arrest when some cells start cytokinesis. Phosphopeptide analyses have revealed that the phosphorylation at Ser-19 is increased 20 times, while the phosphorylation at Ser-1/2 is decreased by half. This high extent of MLC phosphorylation at Ser-19 is maintained for another 30 min and gradually decreased to near the level of interphase cells as cells complete spreading at 180 min. On the other hand, phosphorylation at Ser-1/2 is decreased to 18% at 60 min, and is practically undetectable at 180 min after the release of mitotic arrest. The stoichiometry of MLC phosphorylation has been determined by quantitation of phosphorylated and unphosphorylated forms of MLC separated on 2D gels. The molar ratio of phosphorylated MLC to total MLC is found to be 0.16 +/- 0.06 and 0.31 +/- 0.05 in interphase and mitotic cells, respectively. The ratio is increased to 0.49 +/- 0.05 at 30 min after the release of mitotic arrest. These results suggest that the change in the phosphorylation site from Ser-1/2 to Ser-19 plays an important role in signaling cytokinesis.     

Identification of in vivo phosphorylation sites and their functional significance in the sodium iodide symporter

The Na+/I- symporter (NIS)-mediated iodide uptake activity is the basis for targeted radioiodide ablation of thyroid cancers. Although it has been shown that NIS protein is phosphorylated, neither the in vivo phosphorylation sites nor their functional significance has been reported. In this study, Ser-43, Thr-49, Ser-227, Thr-577, and Ser-581 were identified as in vivo NIS phosphorylation sites by mass spectrometry. Kinetic analysis of NIS mutants of the corresponding phosphorylated amino acid residue indicated that the velocity of iodide transport of NIS is modulated by the phosphorylation status of Ser-43 and Ser-581. We also found that the phosphorylation status of Thr-577 may be important for NIS protein stability and that the phosphorylation status of Ser-227 is functionally silent. Thr-49 appears to be critical for proper local structure/conformation of NIS because mutation of Thr-49 to alanine, aspartic acid, or serine results in reduced NIS activity without alterations in total or cell surface NIS protein levels. Taken together, we showed that NIS protein levels and functional activity could be modulated by phosphorylation through distinct mechanisms.     

Function of serine-52 and serine-80 in the catalytic mechanism of Escherichia coli aspartate transcarbamoylase

Carbamoyl phosphate is held in the active site of Escherichia coli aspartate transcarbamoylase by a variety of interactions with specific side chains of the enzyme. In particular, oxygens of the phosphate of carbamoyl phosphate interact with Ser-52, Thr-53 (backbone), Arg-54, Thr-55, and Arg-105 from one catalytic chain, as well as Ser-80 and Lys-84 from an adjacent chain in the same catalytic subunit. In order to define the role of Ser-52 and Ser-80 in the catalytic mechanism, two mutant versions of the enzyme were created with Ser-52 or Ser-80 replaced by alanine. The Ser-52----Ala holoenzyme exhibits a 670-fold reduction in maximal observed specific activity, and a loss of both aspartate and carbamoyl phosphate cooperativity. This mutation also causes 23-fold and 5.6-fold increases in the carbamoyl phosphate and aspartate concentrations required for half the maximal observed specific activity, respectively. Circular dichroism spectroscopy indicates that saturating carbamoyl phosphate does not induce the same conformational change in the Ser-52----Ala holoenzyme as it does for the wild-type holoenzyme. The kinetic properties of the Ser-52----Ala catalytic subunit are altered to a lesser extent than the mutant holoenzyme. The maximal observed specific activity is reduced by 89-fold, and the carbamoyl phosphate concentration at half the maximal observed velocity increases by 53-fold while the aspartate concentration at half the maximal observed velocity increases 6-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of multiple in vivo phosphorylation sites in rabbit myelin basic protein

Myelin basic protein of rabbit brain (Mr = 18,200) was initially freed of the bulk of the nonphosphorylated species (mainly component 1) by Cm-cellulose chromatography at high pH. The remainder of the protein was subjected to peptic digestion at pH 6.00, which resulted in specific, essentially complete cleavage at several bonds (Phe-44--Phe-45, Phe-87--Phe-88, Leu-109--Ser-110, and Leu-151--Phe-152) and partial cleavage at the Tyr-14--Leu-15 bond. Gel filtration of the digest through Sephadex G-25 (fine) yielded three fractions, the first containing primarily peptides 1-44 and 45-87, the second peptides 15-44, 88-109, and 110-151, and the third peptides 1-14 and 152-168. Each fraction was chromatographed on Cm-cellulose at pH 8.2, and the resulting subfractions and partially purified peptides were analyzed for phosphoserine and phosphothreonine. Materials containing significant amounts of the phosphoamino acids were subsequently chromatographed on Cm-cellulose at pH 4.65, and the analyses for phosphoserine and phosphothreonine were repeated. The resulting purified peptic phosphopeptides were identified by amino acid analysis and tryptic peptide mapping. Comparison of the maps with those of the unphosphorylated counterparts located the tryptic phosphopeptides. These were recovered and their identities were established by amino acid analysis. In those cases where the phosphopeptide contained 2 Ser residues, the position of the phosphoserine was established by aminopeptidase M digestion. Five phosphorylation sites were found: Ser-7, Ser-56, Thr-96, Ser-113, and Ser-163. Only a small fraction of these sites was phosphorylated in the total basic protein, with values ranging from about 2 (ser-113) to 6% (Thr-96). With the possible exception of Ser-56, these sites are not the ones that have been reported to be phosphorylated in vitro by cyclic AMP-dependent protein kinase.     

Casein kinase II-catalysed phosphorylation of calmodulin is altered by amino acid deletions in the central helix of calmodulin.

Calmodulin is phosphorylated by casein kinase II on Thr-79, Ser-81, Ser-101 and Thr-117. To determine the consensus sequences for casein kinase II in intact calmodulin, we examined casein kinase II-mediated phosphorylation of engineered calmodulins with 1-4 deletions in the central helical region (positions 81-84). Total casein kinase II-catalyzed phosphate incorporation into all deleted calmodulins was similar to control calmodulin. Neither CaM delta 84 (Glu-84 deleted) nor CaM delta 81-84 (Ser-81 to Glu-84 deleted) has phosphate incorporated into Thr-79 or Ser-81, but both exhibit increased phosphorylation of residues Ser-101 and Thr-117. These data suggest that phosphoserine in the +2 position may be a specificity determinant for casein kinase II in intact proteins and/or secondary structures are important in substrate recognition by casein kinase II.     

The effects of changing the site of activating phosphorylation in CDK2 from threonine to serine

Cyclin-dependent kinases (CDKs) that control cell cycle progression are regulated in many ways, including activating phosphorylation of a conserved threonine residue. This essential phosphorylation is carried out by the CDK-activating kinase (CAK). Here we examine the effects of replacing this threonine residue in human CDK2 by serine. We found that cyclin A bound equally well to wild-type CDK2 (CDK2(Thr-160)) or to the mutant CDK2 (CDK2(Ser-160)). In the absence of activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were more active than wild-type CDK2(Thr-160)-cyclin A complexes. In contrast, following activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were less active than phosphorylated CDK2(Thr-160)-cyclin A complexes, reflecting a much smaller effect of activating phosphorylation on CDK2(Ser-160). The kinetic parameters for phosphorylating histone H1 were similar for mutant and wild-type CDK2, ruling out a general defect in catalytic activity. Interestingly, the CDK2(Ser-160) mutant was selectively defective in phosphorylating a peptide derived from the C-terminal domain of RNA polymerase II. CDK2(Ser-160) was efficiently phosphorylated by CAKs, both human p40(MO15)(CDK7)-cyclin H and budding yeast Cak1p. In fact, the k(cat) values for phosphorylation of CDK2(Ser-160) were significantly higher than for phosphorylation of CDK2(Thr-160), indicating that CDK2(Ser-160) is actually phosphorylated more efficiently than wild-type CDK2. In contrast, dephosphorylation proceeded more slowly with CDK2(Ser-160) than with wild-type CDK2, either in HeLa cell extract or by purified PP2Cbeta. Combined with the more efficient phosphorylation of CDK2(Ser-160) by CAK, we suggest that one reason for the conservation of threonine as the site of activating phosphorylation may be to favor unphosphorylated CDKs following the degradation of cyclins.     

Mammalian target of rapamycin-dependent phosphorylation of PHAS-I in four (S/T)P sites detected by phospho-specific antibodies

The role and control of the four rapamycin-sensitive phosphorylation sites that govern the association of PHAS-I with the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), were investigated by using newly developed phospho-specific antibodies. Thr(P)-36/45 antibodies reacted with all three forms of PHAS-I that were resolved when cell extracts were subjected to SDS-polyacrylamide gel electrophoresis. Thr(P)-69 antibodies bound the forms of intermediate and lowest mobility, and Ser(P)-64 antibodies reacted only with the lowest mobility form. A portion of PHAS-I that copurified with eIF4E reacted with Thr(P)-36/45 and Thr(P)-69 antibodies but not with Ser(P)-64 antibodies. Insulin and/or amino acids increased, and rapamycin decreased, the reactivity of all three antibodies with PHAS-I in both HEK293 cells and 3T3-L1 adipocytes. Immunoprecipitated epitope-tagged mammalian target of rapamycin (mTOR) phosphorylated Thr-36/45. mTOR also phosphorylated Thr-69 and Ser-64 but only when purified immune complexes were incubated with the activating antibody, mTAb1. Interestingly, the phosphorylation of Thr-69 and Ser-64 was much more sensitive to inhibition by rapamycin-FKBP12 than the phosphorylation of Thr-36/45, and the phosphorylation of Ser-64 by mTOR was facilitated by phosphorylation of Thr-36, Thr-45, and Thr-69. In these respects the phosphorylation of PHAS-I by mTOR in vitro resembles the ordered phosphorylation of PHAS-I in cells.     

Protein kinase C phosphorylation of desmin at four serine residues within the non-alpha-helical head domain

We reported that phosphorylation by either cAMP-dependent protein kinase or protein kinase C (Ca2+/phospholipid-dependent enzyme) in vitro induces disassembly of the desmin filaments (Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., and Sato, C. (1988) J. Biol. Chem. 263, 5970-5978). For this subunit protein, Ser-29, Ser-35, and Ser-50 within the non-alpha-helical head domain were shown to be the sites of phosphorylation for cAMP-dependent protein kinase (Geisler, N., and Weber, K. (1988) EMBO J. 7, 15-20). In the present work, we identified the sites of desmin phosphorylated in vitro by other protein kinase which affects the filament structure. The protein kinase C-phosphorylated desmin was hydrolyzed with trypsin, and the phosphorylated peptides were isolated by reverse-phase chromatography. Sequential analysis of the purified phosphopeptides, together with the known primary sequence, revealed that Ser-12, Ser-29, Ser-38, and Ser-56 were phosphorylated by protein kinase C. All four sites are located within the non-alpha-helical head domain of desmin. Ser-12, Ser-38, and Ser-56, specifically phosphorylated by protein kinase C, have arginine residues at the carboxyl-terminal side (Arg-14, Arg-42, and Arg-59, respectively). Ser-29 phosphorylated by both protein kinase C and cAMP-dependent protein kinase has arginine residues at the amino and carboxyl termini (Arg-27 and Arg-33). These findings support the view that the head domain-specific phosphorylation strongly influences desmin filament structure; however, each protein kinase differed with regard to site recognition on this domain.     

Altered phosphorylation of free and bound forms of monkey p53 and simian virus 40 large T antigen during lytic infection.

We have identified the phosphorylation sites in monkey p53 as well as specific changes in the phosphorylation state of free and complexed forms of simian virus 40 (SV40) large T antigen (T) and monkey p53 isolate from SV40 lytically infected CV1 cells. Phosphopeptide analyses of free T and p53 (To and p53o) and complexed T and p53 (T+ and p53+) fractions indicated several quantitative increases in the specific phosphorylation of complexed forms of both proteins. The N terminus of monkey p53+ is phosphorylated at Ser-9, Ser-15, Ser-20, either Ser-33 or Ser-37, and at least one of Ser-90 to Ser-99. The C-terminal sites are Ser-315 and Ser-392. On comparing p53+ with p53o, we found that labeling of the two N-terminal phosphotryptic peptides encompassing residues 1 to 20 and 33 to 101 was increased fivefold and that Ser-315 was sevenfold more labeled than was Ser-392. When T+ was compared with To, the N-terminal peptide containing phosphorylation sites Ser-106 through Thr-124 was twofold more labeled, the peptide containing Ser-657 through Ser-679 was sixfold more labeled and contained up to four phosphorylated serine residues, and Ser-639 and Thr-701 appeared unchanged. Overall, T+ molecules appeared to contain 3.5 mol more of labeled phosphate than did To, with the N-terminal peptide appearing fully phosphorylated. The phosphopeptide patterns obtained for lytic T+ and To fractions were nearly identical to those found for wild-type SV40 T (stably complexed with mouse p53) and mutant 5080 T (defective for p53 binding) expressed in transformed C3H10T1/2 cells (L. Tack, C. Cartwright, J. Wright, A. Srinivasan, W. Eckhart, K. Peden, and J. Pipas, J. Virol. 63:3362-3367, 1989). These results indicate that increases in specific phosphorylation sites in both T+ and p53+ correlate with the association of T with p53. The enhanced phosphorylation state may be a consequence of complex formation between T and p53 or reflect an increased affinity of p53 for highly phosphorylated forms of T.     

Identification of novel phosphorylation sites on Xenopus laevis Aurora A and analysis of phosphopeptide enrichment by immobilized metal-affinity chromatography

Mass spectrometric analysis of proteolytically derived phosphopeptides has developed into a widespread technique for the identification of phosphorylated amino acids. Using liquid chromatography-electrospray ionization tandem mass spectrometry, 14 phosphorylation sites were identified on Xenopus laevis His6-Aurora A, a highly conserved regulator of centrosome maturation and cell division. These included seven novel phosphorylation sites, Ser-12, Thr-21, Thr-103, Ser-116, Thr-122, Tyr-155, and Thr-294, as well as the previously identified regulatory sites, Ser-53, Thr-295, and Ser-349. The identification of these novel phosphorylation sites will be important for future studies aimed at elucidating the mechanisms of Aurora A regulation by phosphorylation. Furthermore, we demonstrate that a "kinase-inactive" mutant of Aurora A, K169R, still retains 10% of activity of the wild-type enzyme in vitro along with occupancy of Thr-295 and Ser-12. However, mutation of Asp-281 to Ala completely abolishes activity of the enzyme and should therefore be used preferentially as a genuine kinase-dead construct. Because of the abundance of phosphorylated residues on His6-Aurora A, we found this protein to be an ideal tool for the characterization of immobilized metal-affinity chromatography (IMAC) as a method for phosphopeptide enrichment from complex mixtures. We present a detailed analysis of the binding and elution properties of both the phosphopeptides and unphosphorylated peptides of His6-Aurora A to Fe3+-IMAC before and after methyl esterification. Moreover, we demonstrate a significant difference in enrichment of phosphopeptides when different resins are used for Fe3+-IMAC and characterize the strengths and limitations of this methodology for the study of phosphoproteomics.     

Protein kinase C isoforms differentially phosphorylate human choline acetyltransferase regulating its catalytic activity

Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons; regulation of its activity or response to physiological stimuli is poorly understood. We show that ChAT is differentially phosphorylated by protein kinase C (PKC) isoforms on four serines (Ser-440, Ser-346, Ser-347, and Ser-476) and one threonine (Thr-255). This phosphorylation is hierarchical, with phosphorylation at Ser-476 required for phosphorylation at other serines. Phosphorylation at some, but not all, sites regulates basal catalysis and activation. Ser-476 with Ser-440 and Ser-346/347 maintains basal ChAT activity. Ser-440 is targeted by Arg-442 for phosphorylation by PKC. Arg-442 is mutated spontaneously (R442H) in congenital myasthenic syndrome, rendering ChAT inactive and causing neuromuscular failure. This mutation eliminates phosphorylation of Ser-440, and Arg-442, not phosphorylation of Ser-440, appears primarily responsible for ChAT activity, with Ser-440 phosphorylation modulating catalysis. Finally, basal ChAT phosphorylation in neurons is mediated predominantly by PKC at Ser-476, with PKC activation increasing phosphorylation at Ser-440 and enhancing ChAT activity.     

Tandem phosphorylation of Ser-911 and Thr-912 at the C terminus of yeast plasma membrane H+-ATPase leads to glucose-dependent activation

In recent years there has been growing interest in the post-translational regulation of P-type ATPases by protein kinase-mediated phosphorylation. Pma1 H(+)-ATPase, which is responsible for H(+)-dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5-10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896-918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal (14)N/(15)N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H(+)-ATPase.     

Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban.

The protein phosphatases which dephosphorylate native, sarcoplasmic reticulum (SR)-associated phospholamban were studied in cardiac muscle extracts and in a Triton fraction prepared by detergent extraction of myofibrils, the latter fraction containing 70-80% of the SR-associated proteins present in the tissue. At physiological concentrations of free Mg2+ (1 mM), protein phosphatase 1 (PP1) accounted for approximately 70% of the total phospholamban phosphatase activity in these fractions towards either Ser-16 (the residue labelled by cAMP-dependent protein kinase, PK-A) or Thr-17 (the residue phosphorylated by an SR-associated Ca2+/calmodulin-dependent protein kinase). Protein phosphatase 2A (PP2A) and protein phosphatase 2C (PP2C) accounted for the remainder of the activity. A major form of cardiac PP1, present in comparable amounts in both the extract and Triton fraction, was similar, if not identical, to skeletal muscle protein phosphatase 1G (PP1G), which is composed of the PP1 catalytic (C) subunit complexed to a G subunit of approximately 160 kDa, responsible for targeting PP1 to both the SR and glycogen particles of skeletal muscle. This conclusion was based on immunoblotting experiments using antibody to the G subunit, ability to bind to glycogen and the release of PP1 activity from glycogen upon incubation with PK-A and MgATP. PP1 accounted for approximately 90% of the phospholamban (Ser-16 or Thr-17) phosphatase activity in the material sedimented by centrifugation at 45,000 x g, a fraction prepared from cardiac extracts which is enriched in SR membranes. The G subunit in this fraction could be solubilised by Triton X-100, but not with 0.5 M NaCl or digestion with alpha-amylase, indicating that it is bound to membranes and not to glycogen. By analogy with the situation in skeletal muscle, the PK-A catalysed phosphorylation of the G subunit, with ensuing release of the C subunit from the SR, may prevent PP1 from dephosphorylating SR-bound substrates and represent one of the mechanisms by which adrenalin increases the phosphorylation of cardiac phospholamban (Ser-16 and Thr-17) in vivo. Hearts left in situ post mortem lose 85-95% of their PP1 activity within 20-30 min. This remarkable disappearance of PP1 may partly explain why the importance of this enzyme in cardiac muscle metabolism has not been recognized previously.     

Characterization and quantitation of phospholamban and its phosphorylation state using antibodies

Quantitative immunoassays to discriminate and quantitate phospholamban and its phosphorylation states in heart homogenates were developed using known amounts of protein determined by amino acid analysis. Synthetic 1-52 phospholamban, the hydrophilic 1-25 peptide, and 1-25 phosphopeptides containing P-Ser(16), P-Thr(17), and dually phosphorylated (P-Ser(16), P-Thr(17)) were used to calibrate immunoblot systems. In addition, synthetic 1-52 peptide was phosphorylated using cAMP-dependent protein kinase (P-Ser(16)) or Ca(2+)-calmodulin protein kinase (P-Thr(17)) and then separated from unphosphorylated 1-52 by HPLC prior to quantitation. Further, canine cardiac sarcoplasmic reticulum was phosphorylated in vitro using [gamma-(32)P]-ATP with cAMP-dependent protein kinase and/or Ca(2+)-calmodulin-dependent protein kinase as well as sequential phosphorylation in both orders to assess the veracity of antibody recognition of phosphorylated forms. Western blots proved useful in characterizing the reactivity of the different antibodies to phospholamban and phosphorylated phospholamban, but were inefficient for accurate quantitation and problems with antibody recognition of dually phosphorylated phospholamban were found. mAb 1D11 recognized all forms of phospholamban, polyclonal antibodies 285 and PS-16 were highly selective for P-Ser(16) phospholamban but had diminished reactivity to diphosphorylated (P-Ser(16), P-Thr(17)) phospholamban, and polyclonal antibody PT-17, although selective for P-Thr(17) phospholamban, generated very weak signals on Western blots and reacted poorly with diphosphorylated phospholamban. Results in quantitative immunodot blot experiments were even more compelling. None of the phosphorylation specific antibodies reacted with the diphospho 1-25 phospholamban peptide. Transgenic mouse hearts expressing varying levels of PLB and ferret heart biopsy samples taken before and after isoproterenol perfusion were analyzed. In all samples containing phospholamban, a basal level of Ser(16) phosphorylation (about 4% of the total PLB population) and a lesser amount of Thr(17) phosphorylation was observed. Upon isoproterenol perfusion, Ser(16) phosphorylation increased only to 17% of the total phospholamban population with a similar change in Thr(17) phosphorylation. This suggests that phospholamban phosphorylation may serve as an electrostatic switch that dissociates inactive calcium pump complexes into catalytically active units. Thus, direct correlations between phospholamban phosphorylation state and contractile parameters may not be valid.     

Sequence specificity and role of proximal amino acids of the histone H3 tail on catalysis of murine G9A lysine 9 histone H3 methyltransferase

The activity of recombinant murine G9a toward lysine 9 of histone H3 was investigated. GST fusion proteins containing various lengths of the histone H3 amino-terminal tail were used as substrates in the presence of recombinant G9a enzyme and AdoMet cosubstrate. The minimal substrate methylated by G9a contained seven amino acids (TARKSTG) of the histone H3 tail. Furthermore, mutational analysis of the minimal substrate was performed to identify the amino acids essential for G9a-mediated methylation. All amino acids except Thr-11 were indispensable for the methylation reaction. Steady-state kinetic analysis of the wild-type and histone H3 point mutants, lysine 4 changed to alanine (K4A) or lysine 27 changed to alanine (K27A), with purified G9a revealed similar catalytic efficiency but a reduction in turnover number (k(cat)) from 78 to 58 h(-)(1). G9a methylated synthetic peptide substrates containing the first 13 amino acids of histone H3 efficiently, although methylation, acetylation, or mutation of proximal Lys-4 amino acids reduced Lys-9 methylation. The k(cat) for wild-type peptide substrate vs Lys-4 acetyl- or trimethyl-modified peptide were 88 and 32 h(-)(1), respectively, and the K(m) for the peptides varied from 0.6 to 2.2 muM, resulting in a large difference (15-91) in catalytic efficiency. Ser-10 or Thr-11 phosphorylation resulted in poor methylation by G9a. Immunoprecipitation of unmodified and Ser-10 and Thr-11 phosphorylated histone H3 displayed mostly Lys-4 dimethylation. Dimethylated Lys-9 was reduced in Ser-10 and Thr-11 immunoprecipitated phosphorylated histones as compared to nonphosphorylated H3. In an immunocytochemical assay, GFP fusion SUV39H1 or G9a did not colocalize with phosphorylated histone H3. Thus, Ser-10/Thr-11 phosphorylation impairs Lys-9 methylation. These data suggest that the sequence context of the modified residue affects G9a activity and the modification in the proximal amino acids influences methylation.