JBC INTERFERin siRNA transfection reagent

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Connor, J. H.
Right arrow Articles by Shenolikar, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Connor, J. H.
Right arrow Articles by Shenolikar, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 273, Issue 42, 27716-27724, October 16, 1998


Inhibitor-1 Interaction Domain That Mediates the Inhibition of Protein Phosphatase-1*

John H. ConnorDagger , Hai N. QuanDagger , Nadja T. Ramaswamy§, Lifang Zhang, Sailen Barikparallel , Jianhong Zheng§, John F. Cannon§, Ernest Y. C. Lee, and Shirish ShenolikarDagger **

From the Dagger  Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, the § Department of Molecular Microbiology and Immunology, University of Missouri, School of Medicine, Columbia, Missouri 65212, the  Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101, and the parallel  Department of Biochemistry and Molecular Biology, University of South Alabama School of Medicine, Mobile, Alabama 36688

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Inhibitor-1 (I-1), a cyclic AMP-regulated phosphoprotein, inhibits protein phosphatase-1 (PP1) activity in response to hormones. The molecular mechanism for PP1 inhibition by I-1 remains unknown. Mutation of nine acidic residues lining a proposed I-1-binding channel in rabbit PP1alpha yielded one mutant (E256A) slightly impaired in its inhibition by I-1, with the IC50 increased by 3-fold, and one mutant (E275R) located in the beta 12-beta 13 loop that showed 4-fold enhanced inhibition by I-1. Substituting Tyr-272, a proposed binding site for the toxins okadaic acid and microcystin-LR, in the beta 12-beta 13 loop with Trp, Phe, Asp, Arg, or Ala impaired PP1alpha inhibition by I-1 by 8-10-fold. Chemical mutagenesis of the Saccharomyces cerevisiae PP1 gene (GLC7) yielded 20 point mutations in the PP1 coding region. Two-hybrid analyses and biochemical assays of these yeast enzymes identified four additional residues in the beta 12-beta 13 loop that were required for PP1 binding and inhibition by I-1. Ten-fold higher concentrations of I-1 were required to inhibit these mutants. Finally, deletion of the beta 12-beta 13 loop from PP1alpha maintained full enzyme activity, but attenuated inhibition by I-1 by >100-fold. These data identified the beta 12-beta 13 loop in the PP1 catalytic subunit as a domain that mediates binding and enzyme inhibition by I-1.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Protein phosphatase-1 (PP1)1 is a major eukaryotic protein-serine/threonine phosphatase that regulates a multitude of physiological processes, including cell cycle, gene expression, protein synthesis, carbohydrate and lipid metabolism, and muscle contraction (1-3). Control of these functions, in response to hormones and growth factors, is likely to be mediated by interaction of the PP1 catalytic subunit with a growing number of regulatory subunits (4). To date, physiological evidence suggests that hormones control PP1 activity through changes in the endogenous protein inhibitors, such as inhibitor-1 (I-1) and its structural homologue, DARPP-32 (5). I-1 has been implicated in hormone signaling in many tissues, including skeletal muscle (6, 7), heart (8), adipose tissue (9), brain (10), and liver (11). Among the physiological processes controlled by I-1 are glycogen metabolism in skeletal muscle (12), neuronal plasticity in the hippocampal neurons (13), and proliferation of pituitary cells (14).

Despite the physiological evidence in favor of I-1 as a hormone-regulated PP1 inhibitor, the molecular basis for the selective and potent PP1 inhibition by I-1 remains unknown. Here, we describe a mutagenic approach that was taken to identify domain(s) in the PP1 catalytic subunit that associate with I-1 and mediate enzyme inhibition. Initially, we were guided by the x-ray crystallography of the PP1alpha catalytic subunit that had predicted an ionic interaction of the I-1 phosphorylation site sequence with several acidic residues that lined a putative substrate-binding channel (15). Mutagenesis of nine acidic amino acids was undertaken to eliminate their negative charge and to assess the impact on PP1 inhibition by I-1. The observation that I-1 competes with the toxins okadaic acid and microcystin-LR for PP1 inhibition (16) led to the suggestion of a common binding site for PP1 inhibitors. In this regard, a number of mutations in the beta 12-beta 13 loop drastically reduced the sensitivity of PP1 to toxins (17, 18). These mutants were also analyzed for their inhibition by I-1. Finally, we exploited the budding yeast Saccharomyces cerevisiae, which contains a single PP1 gene (GLC7) that is also essential for viability. This property allowed us to develop a novel mutagenic screen that eliminated inactive PP1 enzymes (19, 20) and focused on the structural determinants required for PP1 regulation. Random mutagenesis combined with biochemical assays of mutant PP1 enzymes identified a domain essential for PP1 inhibition and generated an I-1-resistant catalytic subunit that may be used to establish the physiological relevance of this signaling mechanism.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- DNA was routinely purified using Wizard Miniprep (Promega) or Qiaprep Spin (QIAGEN Inc.) kits. Restriction enzymes, T4 polynucleotide kinase, T4 DNA ligase, T4 DNA polymerase, and isopropyl-beta -D-thiogalactopyranoside were obtained from either New England Biolabs Inc. or Boehringer Mannheim. [gamma -32P]ATP, [alpha -32P]dCTP, and alpha -35S-ATP were purchased from Amersham Biotech Inc. Protein concentration was estimated using the Bio-Rad protein Assay with bovine serum albumin (Sigma) as standard.

Yeast Strains and Media-- Yeast strains and plasmids are listed in Table I. Plasmids that express mutant Glc7p (pKC886-X) or Gal4p-Glc7p (pAS1-GLC7-X) fusions were described by Ramaswamy et al. (20). As the chromosomal wild-type GLC7 locus was disrupted by insertion of the HIS3 nutritional marker, the JC821A/pKC886-X strains expressed Glc7p proteins exclusively from the pKC886 plasmid. YEPD medium contained 1% (w/v) yeast extract (Difco), 2% (w/v) glucose, and 2% (w/v) Bacto-peptone (Difco). Filter-sterilized 3-aminotriazole was added after autoclaving the media. Other selected media were used as described by Sherman et al. (24). Plasmid JZ303 was made by cloning a NcoI-SalI fragment from pGEM3ZF(-)-hI-1 into pACT2 digested with NcoI and XhoI.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Yeast strains and plasmids

Expression and Purification of Recombinant hI-1-- hI-1 cDNA was excised from pGEM3ZF(-)-hI-1 using a NcoI-SalI digest. The insert was excised from a 0.8% (w/v) agarose gel, purified using a QIAGEN column, and inserted into pT7-7 cut with NdeI-SalI. The ligated vector was transformed into Escherichia coli BL21(DE3). A seed culture of transformed BL21 bacteria grown overnight in LB medium (5 ml) containing ampicillin (50 µg/ml) at 37 °C was used to inoculate 250 ml of LB medium containing ampicillin. When the absorbance of the culture at 600 nm reached 0.6, hI-1 was induced by addition of 1 mM isopropyl-beta -D-thiogalactopyranoside, and growth was continued at 37 °C for 3 h. Bacteria were sedimented at 3000 × g for 15 min, resuspended in 20 ml of ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, containing 1% Nonidet P-40, 5 mM EDTA, 5 mM EGTA, 5 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride), and sonicated for 4 × 10 s using a Branson sonifier (30% duty cycle and 0.5 output power). The cell lysate was centrifuged at 3000 × g, and trichloroacetic acid was added to the supernatant to a final concentration of 1% (w/v). The mixture was centrifuged at 12,000 × g for 20 min at 4 °C, and the supernatant was adjusted to 15% (w/v) trichloroacetic acid. The 12,000 × g pellet was resuspended in 3 ml of 500 mM Tris-HCl, pH 7.5, and dialyzed overnight against 0.5 mM Tris-HCl, pH 7.5, containing 0.005% (w/v) Brij 35. Preparative SDS-polyacrylamide gel electrophoresis was used to obtain pure hI-1 (25). Purified hI-1 was lyophilized and stored at -80 °C.

Expression and Purification of Recombinant PP1alpha Catalytic Subunits-- Mutations in PP1alpha were made using double-stranded polymerase chain reaction (26) or Altered Site II mutagenesis (Promega). In the latter method, PP1 cDNA was inserted into pALTER-Ex1. The mutagenic oligonucleotide, the ampicillin repair oligonucleotide, and the tetracycline knockout oligonucleotide were phosphorylated and annealed to the template. Second strand DNA was initiated using T4 DNA polymerase and T4 DNA ligase in 10 mM Tris HCl, pH 7.5, containing 0.5 mM dNTPs, 1 mM ATP, and 2 mM dithiothreitol. The resulting heteroduplex double-stranded DNA was transformed into E. coli BMH71-18 or ES1301 mutS and grown on agar plates containing ampicillin (50 µg/ml). Plasmid DNA was isolated from individual colonies and transformed into JM109 to propagate the mutant plasmids. Mutations were confirmed by double-stranded sequencing (Sequenase II, U. S. Biochemical Corp.) and subcloned into pTACTAC for PP1 expression (27).

The cDNA for the rabbit skeletal muscle PP1alpha catalytic subunit was expressed in E. coli. Briefly, bacteria were grown in Terrific Broth containing 0.2 mM MnCl2 and 50 µg/ml ampicillin at 37 °C until the absorbance of the culture at 600 nm reached 0.3. Protein expression was initiated with 0.5 mM isopropyl-beta -D-thiogalactopyranoside, and bacterial growth was continued at 27 °C for 20 h. Cells were pelleted by centrifugation at 5000 × g for 20 min and lysed in 50 mM imidazole, pH 7.4, containing 1 mM EDTA, 2 mM dithiothreitol, and 2 mM MnCl2 using a French press (at 1000 p.s.i). The PP1 catalytic subunits were then purified to homogeneity according to Zhang et al. (27).

Isolation of Mutant PP1 (GLC7) Alleles-- pKC886 plasmid was mutagenized as described (20). Briefly, pKC886 was treated with hydroxylamine and transformed into JC821A. Transformants were replica-plated on media lacking tryptophan or uracil as well as on two YEPD plates. The YEPD plates were incubated at 30 and 37 °C and scored for glycogen accumulation and temperature sensitivity. DNA was prepared from uracil-minus colonies that also exhibited either altered glycogen accumulation or temperature sensitivity compared with the wild type. Mutant pKC886 was amplified in E. coli DH5alpha .

Purification of Yeast PP1-- Overnight cultures (100 ml) of JC821A/pKC886-X strains were inoculated into 1 liter of YEPD medium. Cells were grown to late log phase, harvested by centrifugation, and resuspended in 10 ml of 50 mM Tris-HCl, pH 7.5, containing 1 mM EDTA and 15 mM 2-mercaptoethanol. Cells were lysed using either Bead-Beater-6 or a French press at 1000 p.s.i. Extracts were centrifuged at 800 × g for 15 min, and the supernatant was brought to 70% saturation with ammonium sulfate. The pellet, obtained from centrifugation at 100,000 × g for 20 min, was resuspended in 10 ml of 50 mM Tris-HCl, pH 7.5, containing 1 mM EDTA and 15 mM 2-mercaptoethanol and dialyzed overnight against the same buffer. The dialyzed sample was applied to heparin-Sepharose (Amersham Pharmacia Biotech) equilibrated in the dialysis buffer. The column was washed with 10 volumes of the dialysis buffer containing 100 mM NaCl, and PP1 activity was eluted with buffer containing 250 mM NaCl and 10% (v/v) glycerol. The eluate was dialyzed against 50 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 15 mM 2-mercaptoethanol, and 40% (v/v) glycerol and stored at -20 °C. This procedure consistently yielded a preparation containing the 37-kDa PP1 catalytic subunit as ~30% of the total protein.

Analysis of I-1 as a PP1 Inhibitor-- hI-1 was phosphorylated with the protein kinase A catalytic subunit (28) using either ATP or ATPgamma S (25). Activated I-1 was analyzed for the inhibition of 0.02 unit of PP1 catalytic subunit using [32P]phosphorylase a as substrate. One unit of PP1 activity is defined as hydrolyzing 1 nmol of phosphorylase a in 1 min in this assay.

Two-hybrid Interactions-- Three methods were used to assay two-hybrid interactions in yeast. In the first method, Y190 was transformed with pAS1-GLC7 and pJZ203 (pACT2-hI-1) containing wild-type hI-1; derivatives containing the T35A or T35D mutant (Table I); or pCV6 (23), which expresses Gac1p fused to the Gal4p activation domain. Transformants bearing both plasmids were selected on media deficient in tryptophan and leucine. Y190 contained a GAL1 promoter-driven HIS3 gene that was activated following the association of two Gal4p fusion proteins. Positive interaction was noted as resistance to growth in the presence of the histidine analogue 3-aminotriazole (22).

The second method assayed for beta -galactosidase activity in diploids produced by mating JC981 with JC993. The GAL1 promoter-driven lacZ gene was activated in these strains on the reconstitution of Gal4p activity. JC981 × JC993 diploids containing two-hybrid plasmids were grown on solid sucrose minimal medium lacking tryptophan and leucine for 48 h. Equivalent amounts of cells were transferred to a nitrocellulose filter using a dot-blot manifold. Filters were floated in liquid nitrogen for 5 s and placed on Whatman No. 3MM filters soaked with Z buffer (29) containing 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside (1 mg/ml). The filters were incubated at 37 °C for 2-24 h before drying and photography.

In the third method, the JC981 × JC993 diploids were grown in liquid glucose minimal medium lacking leucine and tryptophan until the absorbance of the culture reached ~1.0. The cells were harvested, permeabilized with chloroform and SDS, and assayed using o-nitrophenyl beta -D-galactoside as substrate (30). Units of beta -galactosidase activity were calculated in triplicate from independent cultures and varied by <10%.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Mutation of Surface Acidic Residues in PP1alpha -- Surface channels emanating from the PP1 catalytic center were widely speculated to function as sites for binding phosphoprotein substrates and inhibitors (15). Modeling a phosphopeptide conserved in I-1 and DARPP-32 suggested that the four basic residues preceding the phosphothreonine in these PP1 inhibitors made ionic interactions with some of the nine acidic amino acids lining this putative substrate/inhibitor-binding channel. Thus, we mutated Asp-208, Asp-210, Asp-212, Glu-218, Asp-220, Glu-252, Asp-253, Glu-256, and Glu-275 to alanines (Fig. 1), expressed the mutant PP1alpha proteins in E. coli, and analyzed the inhibition of the purified enzymes by hI-1. As noted by Zhang and Lee (26), the specific activities of the six mutants D210A, D212A, E252A, D253N, E256A, and E275R were identical to that of wild-type PP1alpha using phosphorylase a and other substrates. Dose-dependent inhibition of mutants D210A and D212A (Fig. 2A) and mutants E252A and D253N (Fig. 2B) by hI-1 was also identical to that of wild-type PP1alpha . Only E256A showed a 3-fold right shift in its dose response for hI-1 (Fig. 2B). The double mutants D210N/D212N and E252N/D253N also showed no change in their IC50 for hI-1 (Fig. 2C). The one surprising finding was that mutant E275R increased the sensitivity of PP1 to I-1 by ~4-fold (data not shown).


View larger version (123K):
[in this window]
[in a new window]
  		Fig. 1.   Location of the acidic groove in the PP1alpha catalytic subunit. The crystal structure of the recombinant PP1alpha catalytic subunit (15) is shown. Nine acidic residues (numbered) line the putative substrate-binding groove that is postulated to interact with four consecutive arginines near the phosphorylation site in I-1. Two partially hidden metals (filled spheres) locate the catalytic center of PP1alpha .


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 2.   Analysis of PP1alpha with mutations in the acidic groove. Oligonucleotide-directed mutagenesis of rabbit PP1alpha cDNA was carried out as described under "Experimental Procedures." Mutant cDNAs were inserted in the pTACTAC vector and expressed in E. coli (27). Dose-dependent inhibition of PP1 enzymes with charged-to-alanine substitutions, D210A and D212A (A) and E252A, D253A, and D256A (B), is shown using thiophosphorylated hI-1. The double mutations D210N/D212N and E252N/D253N were also analyzed (C). The symbols, shown in the insets, identify the individual mutants. Each point represents the average of three values and varied by <10%. wt, wild type.

The other mutants, D208A, E218A, and D220A, possessed 7-10-fold lower specific activity than wild-type PP1alpha . D208A, in particular, was highly unstable. Overnight storage under conditions (0.1 mg/ml protein in buffers containing 10% glycerol at 4 °C) that maintained 100% wild-type activity resulted in complete loss of D208A activity. E218A and D220A showed no difference in their IC50 values for hI-1 compared with the wild type. Freshly prepared D208A, on the other hand, showed a 10-fold increased sensitivity to hI-1 (data not shown).

Toxin-resistant PP1 Catalytic Subunits-- Numerous toxins inhibit PP1 and PP2A (31), with PP2A being 10-100-fold more sensitive to okadaic acid than PP1. PP2A is also characterized by its complete insensitivity to I-1. A mutation (C269G) in the PP2A catalytic subunit was identified in an okadaic acid-resistant cell line (32). To test the possibility that Cys-269 and the surrounding sequence may represent a toxin-binding site, Zhang et al. (17) substituted the corresponding sequence, GEFD, in PP1alpha with YRC269G, the PP2A sequence. This enhanced the sensitivity of PP1 to okadaic acid, making it more PP2A-like (17). Okadaic acid competes with other PP1 inhibitors (33), suggesting a common inhibitor-binding site. Thus, we postulated that the chimeric PP1 enzyme may also be more PP2A-like in showing reduced sensitivity or no inhibition by I-1. We analyzed YRCG-containing chimeric PP1alpha , but found no difference in its dose-response curve for hI-1 compared with wild-type PP1alpha (data not shown). Mutations of individual residues within this sequence (F276A and D277G) also had no effect on the sensitivity of PP1alpha to hI-1 (data not shown).

In later studies, Zhang et al. (18) undertook a systematic mutagenesis of the beta 12-beta 13 loop in PP1alpha and identified Tyr-272 as the primary determinant of PP1 inhibition by toxins. Substituting Tyr-272 with other amino acids reduced the sensitivity of PP1 to toxins by several hundred- to thousand-fold. These mutants also showed small reductions in their sensitivity to I-2, suggesting that the protein inhibitors of PP1 may also recognize Tyr-272. Thus, we also analyzed several different substitutions in place of Tyr-272 for their effects on PP1alpha inhibition by hI-1. Conservative substitutions, Y272W (Fig. 3A) and Y272F, as well as nonconservative mutations, Y272A, Y272E, and Y272D, all impaired PP1alpha inhibition by hI-1 (Fig. 3B), increasing the IC50 by 8-10-fold (data not shown).


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3.   Mutations of tyrosine 272 in PP1alpha . Site-directed mutagenesis substituted several amino acids in place of Tyr-272 in PP1alpha . The mutant enzymes were expressed in E. coli and purified to homogeneity. A shows dose-response curves for the inhibition of the Y272W mutant (solid line) and wild-type PP1A (shaded line) by hI-1. B shows the comparison of several substitutions in place of Tyr-272 at a fixed concentration of hI-1 (3.0 µM). The average of three independent experiments is shown with S.E.

Mutations of Yeast PP1 with Altered Interactions with hI-1-- S. cerevisiae is unique among eukaryotes in having a single PP1 gene. The GLC7 gene encodes a PP1 catalytic subunit with >80% identity to mammalian PP1alpha (34) and is essential for yeast viability (35). A GLC7-containing plasmid, randomly mutagenized with hydroxylamine, was screened in a strain that contained a disruption in the chromosomal copy of the GLC7 gene. We analyzed 20 different alleles that maintained yeast viability but resulted in a wide range of phenotypes, suggesting altered Glc7p function or regulation (20). Two-hybrid analysis of mutant GLC7 cDNAs inserted in the pAS1 vector with the hI-1 cDNA in pACT2 (22) showed effective PP1/I-1 binding with most alleles. Insertion of these cDNAs in the opposing vectors showed no significant binding, suggesting that the fusion of the Gal4p DNA-binding domain to the N terminus of hI-1 and/or of the transactivation domain to the C terminus of PP1 impaired PP1/I-1 binding. The Glc7p/hI-1 association was first monitored by the ability of Y190 diploid strains to grow in the presence of 30 mM 3-aminotriazole, indicating the activation of the GAL1 promoter-driven HIS3 gene (Fig. 4). In this growth assay, wild-type Glc7p interacted with hI-1 with equal avidity compared with Gac1p (23, 37), a yeast homologue of the PP1 glycogen-targeting subunit. Substitution of threonine 35, whose phosphorylation by protein kinase A is essential for PP1 inhibition in vitro (25), with either alanine or aspartic acid abolished Glc7p/hI-1 interaction, suggesting that threonine 35 phosphorylation was also necessary for the binding of hI-1 to Glc7p in intact yeast. We have overexpressed hI-1 in yeast and, using a phospho-specific antibody (38), have shown that threonine 35 is phosphorylated (data not shown).


View larger version (41K):
[in this window]
[in a new window]
  		Fig. 4.   Association of S. cerevisiae PP1 (Glc7p) with hI-1 in the two-hybrid assay. Interaction of Glc7p with hI-1 was analyzed using the two-hybrid assay as described under "Experimental Procedures." Association of wild-type (wt) hI-1 with Glc7p was defined by growth of Y190 transformants on control YEPD plates (A) and on plates containing 30 mM 3-aminotriazole (B). The role of threonine 35 phosphorylation in Glc7p/I-1 binding was assessed by substituting the non-phosphorylated residues alanine (T35A) and aspartic acid (T35D). Yeast growth resulting from Glc7p binding to Gac1p, a known yeast PP1 regulator, was used as a control.

We also monitored the expression of a beta -galactosidase reporter construct (GAL1 promoter-driven lacZ) in the two-hybrid assay using a convenient solid-phase or dot-blot assay (Fig. 5A). In this assay, wild-type hI-1 bound Glc7p with equal or better avidity than Snf1p and Snf4p, two well characterized interacting proteins. We identified eight Glc7p mutants that showed impaired binding to hI-1. These proteins were also analyzed for binding to two other PP1 regulators: Glc8p, a yeast I-2 homologue (39), and Gac1p (Fig. 5B). Three mutants (P82S, P195F, and R245Q) failed to bind all three baits (hI-1, Glc8p, and Gac1p), but, in other studies (20), were shown to bind Sds22p, a nuclear PP1 regulator. Two Glc7p mutants (R42K and Q48K) showed enhanced hI-1 binding. The particular focus of these studies was the five negative or weak I-1-interacting Glc7p proteins that represented point mutations near the C terminus (domain III in Fig. 5B). Quantitative analysis of hI-1 binding to all Glc7p mutants using a more sensitive permeabilized cell assay emphasized the complete inability of some C-terminal Glc7p mutants to interact with hI-1 in the two-hybrid assay (Fig. 6, asterisks). Four of these mutations (A268T, P269L, A278V, and G279S) were localized to the beta 12-beta 13 loop and flanked Tyr-272.


View larger version (37K):
[in this window]
[in a new window]
  		Fig. 5.   Comparison of Glc7p binding to hI-1 and yeast PP1 regulators. Association of the 19 mutant Glc7p proteins (the modified amino acids shown in the left panel), generated by hydroxylamine mutagenesis, with hI-1 was analyzed by the dot-blot assay (A) as described under "Experimental Procedures." ts120 is a temperature-sensitive GLC7 allele that results from conversion of the consensus TACTAAC intron splice sequence to TAATAAC. Association of Snf1p and Snf4p was used as a positive control, and Glc7p and Snf4p were used as a negative control. The dot-blot assay was also used to compare Glc7p binding to hI-1 as well as Glc8p and Gac1p, two known yeast PP1 regulators (B). + represents wild-type signal; +/- represents inconsistent or weak signal; and - indicates no signal in four independent assays. The primary structure of yeast PP1 is schematically shown, with the locations of the individual mutations marked by asterisks. The central or core domain of PP1 (shown as a broad bar) is highly conserved in all eukaryotes, with greater diversity present at their N and C termini (shown by thin lines). Three domains (indicated by roman numerals) were assigned by the preponderance of PP1 mutations that increased hI-1 binding (domain I), resulted in little or no change (domain II), or significantly reduced Glc7p binding to hI-1 (domain III). WT, wild type.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 6.   Quantitation of beta -galactosidase expression and Glc7p/hI-1 binding. Quantitative analysis of reporter gene expression following Glc7p/hI-1 binding was undertaken using a liquid assay as described under "Experimental Procedures." Assays were carried out in triplicate. Asterisks indicate diploid strains that failed to induce beta -galactosidase. The ts120 allele, described in the legend for Fig. 5, is indicated here as intron. WT, wild type.

To determine whether loss of PP1/I-1 binding in the two-hybrid assay was synonymous with defective PP1 inhibition, we purified wild-type and mutant yeast PP1 enzymes for in vitro biochemical assays. In the absence of divalent cations, wild-type Glc7p was inhibited by hI-1 with IC50 = 2 nM, a value similar to that for PP1 from mammalian tissues (Fig. 7A). Mutants P269L and A278V showed a significant loss of inhibition by hI-1, with 10-fold right shifts in their dose-response curves (Fig. 7A). Similar results were obtained with A268T and G279S (data not shown).


View larger version (55K):
[in this window]
[in a new window]
  		Fig. 7.   Inhibition of mutant Glc7p phosphatases by hI-1. Glc7p proteins that failed to bind or weakly bound hI-1 in the yeast two-hybrid assay were purified as described under "Experimental Procedures." The purified yeast enzymes were assayed for inhibition by thiophosphorylated hI-1 using phosphorylase a as substrate. Results for two mutants (A278V (closed diamonds) and P269L (open diamonds)) and wild-type Glc7p (wt; open squares) are shown in A. Each data point represents the average of three values. These studies identified the beta 12-beta 13 loop (highlighted in black in B) as a determinant of hI-1-mediated PP1 inhibition.

I-1-resistant PP1alpha Catalytic Subunit-- The central core of the PP1 catalytic subunit has been highly conserved through evolution (34). Attempts to truncate the N or C termini in mammalian (40) or yeast (19) PP1 failed to yield active enzymes. Ansai et al. (41) combined N- or C-terminal truncations to successfully express an active PP1alpha core in E. coli. This enzyme lacked the beta 12-beta 13 loop, a potential mediator of PP1 inhibition by hI-1. We expressed the recombinant PP1alpha core in E. coli and analyzed the purified enzyme for its regulation by I-1. The specific activity of the core enzyme was indistinguishable from that of wild-type PP1alpha with phosphorylase a as substrate (Fig. 8A). Wild-type PP1 and the core also dephosphorylated I-1 at similar rates (Fig. 8B). In contrast to wild-type PP1alpha , which was inhibited by thiophosphorylated hI-1 with an IC50 of ~300 nM, the PP1 core was not significantly inhibited by I-1 concentrations up to 20 µM (Fig. 8C). Use of thiophosphorylated hI-1 precluded the possibility that dephosphorylation and inactivation of hI-1 accounted for the apparent insensitivity of the PP1alpha core to I-1. The PP1alpha core was not inhibited by several other phosphatase inhibitors, including 10 µM okadaic acid, 1.0 µM microcystin-LR, and 2 µM I-2 (data not shown).


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 8.   Inhibition of the PP1alpha core by hI-1. The core structure of PP1alpha (residues 41-269) is conserved in all eukaryotic PP1 catalytic subunits and is equivalent to the smallest active protein phosphatase identified, that from bacteriophage lambda  (41). The specific activities of the PP1 core and wild-type (wt) PP1alpha were compared using phosphorylase a as substrate (A). The time-dependent dephosphorylation of hI-1 (B) was also compared using the core (closed diamonds) and wild-type PP1alpha (open squares). The dose-dependent inhibition of phosphorylase phosphatase activity (C) of wild-type PP1alpha (open squares) and the PP1 core (closed diamonds) was undertaken using thiophosphorylated hI-1. All experiments were carried out in triplicate.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Several endogenous inhibitors control PP1 activity in mammalian cells. These include I-1 (42), DARPP-32 (43), I-2 (33), the ribosomal protein RIPP-1 (44), and the nuclear protein NIPP-1 (45). To define the molecular mechanism for PP1 inhibition, early attention focused on I-1 and its neuronal homologue, DARPP-32, two proteins that control PP1 activity in response to both hormones and neurotransmitters (5). Structure-function studies of I-1 (46, 47) and DARPP-32 (43, 48, 49) established that protein kinase A phosphorylation of a conserved threonine was required but not sufficient for PP1 inhibition. The phosphorylated threonine acts in concert with an N-terminal tetrapeptide sequence (KIQF) to inhibit PP1 activity. The KIQF sequence represents a PP1-binding motif present in other PP1 regulators (47, 49, 50). Synthetic peptides modeled on this motif in skeletal muscle glycogen-binding subunit GM or smooth muscle myosin-binding subunit M110 partially mimic the ability of the parent proteins to modulate PP1 activity (51). Co-crystallization of the GM decapeptide with PP1 (50) showed that it binds some distance from the catalytic site and suggested that the peptide induces a conformational change that modifies the substrate specificity of PP1. However, the three-dimensional structure of PP1 in the PP1-peptide complex is virtually identical to that of the isolated catalytic subunit (52). So, the molecular basis for functional effects induced by PP1-binding peptides remains unknown. Whereas both DARPP-32 (49, 53) and I-1 (25), in their dephosphorylated state, bind the PP1 catalytic subunit, most likely through the KIQF sequence, only dephospho-DARPP-32 inhibits the phosphatase (49). Dephosphorylated hI-1, even at much higher concentrations, has no effect on PP1 activity (25). This places greater emphasis on the region of I-1 near the phosphothreonine as critical for PP1 inhibition.

X-ray crystallography of human PP1gamma 1 localized the catalytic site by the presence of a metal-bound tungstate (52). Co-crystallization of rabbit PP1alpha with microcystin-LR identified the same region as the catalytic site (15). To define the interactions of phospho-I-1 at or near the catalytic site, we first examined the model of the DARPP-32 phosphopeptide bound within the PP1 catalytic site (15). Four consecutive arginines that precede the phosphothreonine in I-1 and DARPP-32 were proposed to interact with multiple acidic residues lining a groove that emanates from the catalytic center. By site-directed mutagenesis, we eliminated the negative charge at six of these residues (at positions 210, 212, 218, 220, 252 and 253), but found no measurable change in the sensitivity of PP1 to hI-1. Indeed, the double mutants D210N/D212N and E252N/D253N, which lacked adjacent charged residues, also showed no detriment in their regulation by I-1. This result was in contrast to the findings of Huang et al. (54), who showed that the double mutation E252A/D253A right-shifted the dose-response curve for DARPP-32 by 10-fold, suggesting that the loss of negative charge impaired the interaction of PP1 with the I-1 homologue.

The more remarkable finding was that PP1 mutants D208A and E275R showed enhanced sensitivity to hI-1 (10- and 3-fold, respectively). The same mutations also enhanced PP1 inhibition by DARPP-32 by 30- and 50-fold, respectively (54). This unexpected increase in affinity for hI-1 and DARPP-32 may, at least for Asp-208, be due to its contribution to packing or organization of the PP1 metal-binding centers. Asp-208 is partially buried and participates in the architecture of metal-binding site 2 (15, 52). This may explain why D208A lowers enzyme activity and was unstable. The other mutation, E275R, is located in the beta 12-beta 13 loop and is more difficult to explain. Glu-275 is not predicted to interact with the four basic residues in I-1 and DARPP-32. Moreover, the chimeric enzyme that exchanged the sequence GEFD in PP1 for YRCG in PP2A also incorporated E275R, but had no effect on the sensitivity of PP1 to I-1. Point mutations in the flanking residues (F276C and D277G) also had no effect on PP1 inhibition by I-1. So, at this time, it is unclear why E275R enhanced PP1 inhibition by I-1 and DARPP-32.

Only one mutation (E256A) lining the proposed I-1/DARPP-32-binding groove reduced the sensitivity of PP1 to I-1, and then only by a modest 3-fold. This result also contrasted with the findings of Huang et al. (54), who introduced a more drastic change in this position (E256R) and found the opposite result, namely a 10-fold increase in the potency of DARPP-32 as a PP1alpha inhibitor. Examination of PP1 structure provides no clues to the differing effects of the Glu-256 mutations on the sensitivity of PP1 to DARPP-32 and I-1, and it may take the co-crystallization of these proteins to resolve this issue.

X-ray crystallography of two protein-serine/threonine phosphatases, PP1 (15, 52) and PP2B/calcineurin (55), drew attention to grooves emanating from the catalytic centers as potential sites for phosphoprotein binding (56). Our studies focused on the acidic groove in PP1alpha that was postulated to be particularly important for accommodating the multiple basic residues near the phosphorylation site of I-1. However, mutations in all nine acidic residues failed to provide convincing evidence for their role in either substrate (phosphorylase) or inhibitor (I-1) binding. These experiments did highlight differences in the structurally related PP1 inhibitors (I-1 and DARPP-32) and could indicate that the two proteins, which are regulated differently by protein kinases and phosphatases (48), may also interact differently with the PP1 catalytic subunit.

Co-crystallization of PP1alpha with microcystin-LR (15) positioned this cyclic heptapeptide toxin in close proximity to the beta 12-beta 13 loop, where it forms a covalent link with cysteine 273. Docking the three-dimensional structure of okadaic acid, a structurally distinct inhibitor, into the PP1alpha catalytic site also suggests key interactions with the beta 12-beta 13 loop (57). PP1/PP2A chimeras that interchanged segments of the beta 12-beta 13 loop (18) and systematic mutagenesis of the beta 12-beta 13 loop (40) confirmed that selected residues in this domain play crucial roles in defining the sensitivity of PP1 to okadaic acid, microcystin, and other xenobiotic inhibitors. Competition between microcystin-LR and okadaic acid for PP1 binding (58) or with I-1 for PP1 inhibition (16) fostered the idea of a common binding site for PP1 inhibitors. We analyzed numerous mutations in the beta 12-beta 13 loop. The chimeric PP1alpha that exchanged a tetrapeptide sequence in the beta 12-beta 13 loop with that present in the I-1-insensitive phosphatase (PP2A) showed no change in its I-1 sensitivity. We also examined several substitutions of tyrosine 272, a key residue for toxin binding. All of these substitutions, conservative or nonconservative, impaired PP1 regulation by I-1 and pointed to a unique role for tyrosine 272 in binding I-1 and other PP1 inhibitors.

PP1 regulates numerous processes in S. cerevisiae, including glycogen metabolism, protein synthesis, glucose repression, sporulation, and cell cycle, and is encoded by a single essential GLC7 gene (36). This offered a unique opportunity to develop mutagenesis screens (19, 20) and to identify structural determinants for PP1 regulators. In this regard, yeast PP1, like the mammalian enzyme, is potently inhibited by I-1 (59). Chemical mutagenesis of the GLC7 gene yielded numerous low glycogen mutants (20), some of which were impaired in their interaction with hI-1 in two-hybrid analyses. Biochemical assays of these mutant PP1 enzymes identified four residues (Ala-268, Pro-269, Ala-278, and Gly-279) in the beta 12-beta 13 loop required for both PP1 binding and inhibition by I-1. Surprisingly, Glc7p proteins incapable of binding to hI-1 in the two-hybrid assay were still inhibited by this protein in the in vitro biochemical assays. Several factors may contribute to this. First, N-terminal fusion of GAL4 to hI-1, like glutathione S-transferase fusion (25), may reduce the potency of I-1 as a PP1 inhibitor. Second, the failure of hI-1 mutants T35A and T35D to bind PP1 points to the fact that as in the enzyme assay, hI-1 must be phosphorylated on this threonine to interact with PP1 in yeast. Here, we took no specific steps to enhance hI-1 phosphorylation, and so the protein may be only partially phosphorylated in yeast. The combined effect would be to increase the sensitivity of the two-hybrid assay to mutations that impair PP1/hI-1 interactions. In any case, we analyzed 9 of the 12 amino acids that compose the beta 12-beta 13 loop (Table II) and identified six residues that modified the sensitivity of PP1 to I-1.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Functional analysis of the beta 12-beta 13 loop in the PP1 catalytic subunit
Residues 268-279 form a loop connecting the beta 12 and beta 13 strands in the PP1 catalytic subunit and are conserved in all PP1 enzymes. The N-terminal half of this sequence is also conserved (indicated by colons) in other protein phosphatases, PP2A and PP2B (Part A). Mutagenesis of rabbit PP1alpha (Part B) and S. cerevisiae GLC7 (Part C) analyzed many of these residues for their role in PP1 regulation by hI-1. Substitution of five residues (shown in boldface) attenuated PP1 inhibition by hI-1, whereas one mutation, E275R (boldface and underlined), enhanced PP1 inhibition.

An interesting finding of these studies is that yeast PP1 is active in vitro in the absence of divalent cations. Thus, it resembles PP1 isolated from mammalian tissues rather than the enzymes expressed in bacteria, which are inactive in the absence of added cations. Moreover, Glc7p is inhibited by hI-1 with an IC50 of 1-2 nM, a value similar to that for mammalian PP1 (25). In contrast, bacterially expressed PP1 appears to be partially compromised, displaying IC50 values for I-1 of 50-600 nM (47, 60). This suggests that yeast is an excellent system for expressing mutant PP1 enzymes. However, budding yeast is highly sensitive to changes in Glc7p, such that both reduction and elevation of PP1 activity inhibit growth. This makes yeast less suitable for large-scale expression of PP1 enzymes and ideal for mutagenic screens for novel GLC7 alleles. Combining the speed and convenience of such screens with subsequent expression of specific mutants in bacteria or insect cells for more detailed biochemical studies may provide the most effective approach to analyze PP1 regulators like I-1.

To establish the functional importance of the beta 12-beta 13 loop in PP1 inhibition, we expressed a truncated PP1alpha (41) that lacked all but one residue in this loop. The resulting "core" enzyme was essentially insensitive to I-1 and several other PP1 inhibitors. As the PP1 core still recognized I-1 as a substrate, it clearly retained a subset of interactions with I-1 at or near the catalytic site, but lacking the beta 12-beta 13 loop, was poorly inhibited. This confirmed a pivotal role for the beta 12-beta 13 loop in PP1 inhibition and demonstrated that I-1 and the toxin inhibitors recognized elements of this domain to mediate their inhibitory effects. As the PP1 core was insensitive to high micromolar concentrations of hI-1, and the highest levels of I-1 in mammalian tissue is ~1 µM (42), the core may be classified as an "I-1-resistant" PP1 and could be useful in elucidating the physiological importance of this regulator in hormone signaling.

Finally, a host of targeting proteins dictate the subcellular localization and substrate specificity of PP1 (61). These include several glycogen-binding proteins: GM in skeletal muscle (62), GL in liver (63), PTG (protein targeting to glycogen) in adipose tissue (64), and the myosin-binding complex in smooth muscle (65). PP1 also binds to ribosomal protein L5 (66); an RNA splicing factor (67); and three nuclear proteins, p53BP2 (68), Sds22p (69), and the retinoblastoma gene product pRB (22). Affinity purification of PP1 complexes (70) and interaction cloning with PP1 as bait (71) suggest that many PP1-binding proteins remain undiscovered. Libraries of PP1 mutants might be used to analyze the unique elements that mediate binding of potential PP1 regulators and provide new tools to establish their physiological role.

    ACKNOWLEDGEMENTS

We thank our many colleagues, in particular John York (Duke University), for thoughtful comments on the manuscript and John Kuriyan (Rockefeller University) for providing the coordinates for the PP1alpha crystal structure. We also thank Miriam Khalil for help with some of the yeast two-hybrid experiments.

    FOOTNOTES

* This work was supported by the Duke Comprehensive Cancer Center and United States Public Health Service Grants DK52054 (to S. S.), GM40326 (to J. F. C.), DK18512 (to E. Y. C. L.), and AI37938 (to S. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Duke University Medical Center, P. O. Box 3813, Durham, NC 27710. Tel.: 919-681-6178; Fax: 919-684-8922; E-mail: sheno001{at}mc.duke.edu.

The abbreviations used are: PP1, protein phosphatase-1; PP2A, protein phosphatase-2A; I-1, inhibitor-1; hI-1, human I-1; I-2, inhibitor-2; ATPgamma S, adenosine 5'-O-(thiotriphosphate).
    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Shenolikar, S. (1994) Annu. Rev. Cell Biol. 10, 55-86[CrossRef]
  2. DePaoli-Roach, A. A., Park, I. K., Cerovsky, V., Csortos, C., Durbin, S. D., Kuntz, M. J., Sitikov, A., Tang, P. M., Verin, A., and Zolnierowicz, S. (1994) Adv. Enzyme Regul. 34, 199-224[CrossRef][Medline] [Order article via Infotrieve]
  3. Wera, S., and Hemmings, B. A. (1995) Biochem. J. 311, 17-29
  4. Bollen, M., and Stalmans, W. (1992) Mol. Biol. (Mosc.) 27, 227-281
  5. Shenolikar, S. (1995) Semin. Cancer Biol. 6, 219-227[CrossRef][Medline] [Order article via Infotrieve]
  6. Khatra, B. S., Chiasson, J. L., Shikama, H., Exton, J. H., and Soderling, T. R. (1980) FEBS Lett. 114, 253-256[CrossRef][Medline] [Order article via Infotrieve]
  7. Foulkes, J. G., Jefferson, L. S., and Cohen, P. (1980) FEBS Lett. 112, 21-24[CrossRef][Medline] [Order article via Infotrieve]
  8. Gupta, R. C., Neumann, J., Watanabe, A. M., Lesch, M., and Sabbah, H. N. (1996) Am. J. Physiol. 270, H1159-H1164[Abstract/Free Full Text]
  9. Nemenoff, R. A., Blackshear, P. J., and Avruch, J. (1983) J. Biol. Chem. 258, 9437-9443[Abstract/Free Full Text]
  10. Snyder, G. L., Girault, J. A., Chen, J. Y., Czernik, A. J., Kebabian, J. W., Nathanson, J. A., and Greengard, P. (1992) J. Neurosci. 12, 3071-3083[Abstract]
  11. MacDougall, L. K., Campbell, D. G., Hubbard, M. J., and Cohen, P. (1989) Biochim. Biophys. Acta 1010, 218-226[Medline] [Order article via Infotrieve]
  12. Nakielny, S., Campbell, D. G., and Cohen, P. (1991) Eur. J. Biochem. 199, 713-722[Medline] [Order article via Infotrieve]
  13. Mulkey, R. M., Endo, S., Shenolikar, S., and Malenka, R. C. (1994) Nature 369, 486-488[CrossRef][Medline] [Order article via Infotrieve]
  14. Florio, T., Perrino, B. A., and Stork, P. J. (1996) Endocrinology 137, 4409-4418[Abstract]
  15. Goldberg, J., Huang, H. B., Kwon, Y. G., Greengard, P., Nairn, A. C., and Kuriyan, J. (1995) Nature 376, 745-753[CrossRef][Medline] [Order article via Infotrieve]
  16. MacKintosh, C., Beattie, K. A., Klumpp, S., Cohen, P., and Codd, G. A. (1990) FEBS Lett. 264, 187-192[CrossRef][Medline] [Order article via Infotrieve]
  17. Zhang, Z., Zhao, S., Bai, G., and Lee, E. Y. (1994) J. Biol. Chem. 269, 13766-13770[Abstract/Free Full Text]
  18. Zhang, J., Zhang, Z., Brew, K., and Lee, E. Y. (1996) Biochemistry 35, 6276-6282[CrossRef][Medline] [Order article via Infotrieve]
  19. Baker, S. H., Frederick, D. L., Bloecher, A., and Tatchell, K. (1997) Genetics 145, 615-626[Abstract]
  20. Ramaswamy, N. T., Li, L., Khalil, M., and Cannon, J. F. (1998) Genetics 149, 57-72[Abstract/Free Full Text]
  21. Dalley, B. K., and Cannon, J. F. (1996) Oncogene 13, 1209-1220[Medline] [Order article via Infotrieve]
  22. Durfee, T., Becherer, K., Chen, P. L., Yeh, S. H., Yang, Y., Kilburn, A. E., Lee, W. H., and Elledge, S. J. (1993) Genes Dev. 7, 555-569[Abstract/Free Full Text]
  23. Stuart, J. S., Frederick, D. L., Varner, C. M., and Tatchell, K. (1994) Mol. Cell. Biol. 14, 896-905[Abstract/Free Full Text]
  24. Sherman, F., Fink, G. R., and Hicks, J. B. (1986) Laboratory Course Manual for Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  25. Endo, S., Zhou, X., Connor, J., Wang, B., and Shenolikar, S. (1996) Biochemistry 35, 5220-5228[CrossRef][Medline] [Order article via Infotrieve]
  26. Zhang, L., and Lee, E. Y. C. (1997) Biochemistry 36, 8209-8214[CrossRef][Medline] [Order article via Infotrieve]
  27. Zhang, Z., Zhao, S., Deans-Zirattu, S., Bai, G., and Lee, E. Y. (1993) Mol. Cell. Biochem. 128, 113-119[CrossRef]
  28. Beavo, J. A., Bechtel, P. J., and Krebs, E. G. (1974) Methods Enzymol. 38, 299-308[Medline] [Order article via Infotrieve]
  29. Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  30. Yocum, R. R., Hanley, S., West, R., and Ptashne, M. (1984) Mol. Cell. Biochem. 4, 1985-1988
  31. Holmes, C. F. B., and Boland, M. P. (1993) Curr. Opin. Struct. Biol. 3, 934-943[CrossRef]
  32. Shima, H., Tohda, H., Aonuma, S., Nakayasu, M., DePaoli-Roach, A. A., Sugimura, T., and Nagao, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9267-9271[Abstract/Free Full Text]
  33. Park, I. K., and DePaoli-Roach, A. A. (1994) J. Biol. Chem. 269, 28919-28928[Abstract/Free Full Text]
  34. Barton, G. J., Cohen, P. T., and Barford, D. (1994) Eur. J. Biochem. 220, 225-237[Medline] [Order article via Infotrieve]
  35. Cohen, P. T., and Cohen, P. (1989) Biochem. J. 260, 931-934[Medline] [Order article via Infotrieve]
  36. Stark, M. J. (1996) Yeast 12, 1647-1675[CrossRef][Medline] [Order article via Infotrieve]
  37. Francois, J. M., Thompson-Jaeger, S., Skroch, J., Zellenka, U., Spevak, W., and Tatchell, K. (1992) EMBO J. 11, 87-96[Medline] [Order article via Infotrieve]
  38. Blitzer, R. D., Connor, J. H., Brown, G. P., Wong, T., Shenolikar, S., Iyengar, R., and Landau, E. M. (1998) Science 28, 1940-1943
  39. Cannon, J. F., Pringle, J. R., Fiechter, A., and Khalil, M. (1994) Genetics 136, 485-503[Abstract]
  40. Zhang, Z., Zhao, S., Long, F., Zhang, L., Bai, G., Shima, H., Nagao, M., and Lee, E. Y. (1994) J. Biol. Chem. 269, 16997-17000[Abstract/Free Full Text]
  41. Ansai, T., Dupuy, L. C., and Barik, S. (1996) J. Biol. Chem. 271, 24401-24407[Abstract/Free Full Text]
  42. Nimmo, G. A., and Cohen, P. (1978) Eur. J. Biochem. 87, 341-351[Medline] [Order article via Infotrieve]
  43. Hemmings, H. C., Nairn, A. C., Elliott, J. I., and Greengard, P. (1990) J. Biol. Chem. 265, 20369-20376[Abstract/Free Full Text]
  44. Beullens, M., Stalmans, W., and Bollen, M. (1996) Eur. J. Biochem. 239, 183-189[Medline] [Order article via Infotrieve]
  45. Van Eynde, A., Wera, S., Beullens, M., Torrekens, S., Van Leuven, F., Stalmans, W., and Bollen, M. (1995) J. Biol. Chem. 270, 28068-28074[Abstract/Free Full Text]
  46. Aitken, A., and Cohen, P. (1982) FEBS Lett. 147, 54-58[CrossRef][Medline] [Order article via Infotrieve]
  47. Endo, S., Connor, J. H., Forney, B., Zhang, L., Ingebritsen, T. S., Lee, E. Y. C., and Shenolikar, S. (1997) Biochemistry 36, 6986-6992[CrossRef][Medline] [Order article via Infotrieve]
  48. Desdouits, F., Cohen, D., Nairn, A. C., Greengard, P., and Girault, J. A. (1995) J. Biol. Chem. 270, 8772-8778[Abstract/Free Full Text]
  49. Kwon, Y. G., Huang, H. B., Desdouits, F., Girault, J. A., Greengard, P., and Nairn, A. C. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3536-3541[Abstract/Free Full Text]
  50. Egloff, M. P., Johnson, D. F., Moorhead, G., Cohen, P. T., Cohen, P., and Barford, D. (1997) EMBO J. 16, 1876-1887[CrossRef][Medline] [Order article via Infotrieve]
  51. Johnson, D. F., Moorhead, G., Caudwell, F. B., Cohen, P., Chen, Y. H., Chen, M. X., and Cohen, P. T. (1996) Eur. J. Biochem. 239, 317-325[Medline] [Order article via Infotrieve]
  52. Egloff, M. P., Cohen, P. T., Reinemer, P., and Barford, D. (1995) J. Mol. Biol. 254, 942-959[CrossRef][Medline] [Order article via Infotrieve]
  53. Desdouits, F., Cheetham, J. J., Huang, H. B., Kwon, Y. G., da Cruz e Silva, E. F., Denefle, P., Ehrlich, M. E., Nairn, A. C., Greengard, P., and Girault, J. A. (1995) Biochem. Biophys. Res. Commun. 206, 652-658[CrossRef][Medline] [Order article via Infotrieve]
  54. Huang, H. B., Horiuchi, A., Goldberg, J., Greengard, P., and Nairn, A. C. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3530-3535[Abstract/Free Full Text]
  55. Kissinger, C. R., Parge, H. E., Knighton, D. R., Lewis, C. T., Pelletier, L. A., Tempczyk, A., Kalish, V. J., Tucker, K. D., Showalter, R. E., Moomaw, E. W., Gastinel, L. N., Habuka, N., Chen, X., Maldonado, F., Barker, J. E., Bacquet, R., and Villafranca, J. E. (1995) Nature 378, 641-644[CrossRef][Medline] [Order article via Infotrieve]
  56. Lohse, D. L., Denu, J. M., and Dixon, J. E. (1996) Structure 3, 987-990[CrossRef]
  57. Bagu, J. R., Sykes, B. D., Craig, M. M., and Holmes, C. F. (1997) J. Biol. Chem. 272, 5087-5097[Abstract/Free Full Text]
  58. Matsushima, R., Yoshizawa, S., Watanabe, M. F., Harada, K., Furusawa, M., Carmichael, W. W., and Fujiki, H. (1990) Biochem. Biophys. Res. Commun. 171, 867-874[CrossRef][Medline] [Order article via Infotrieve]
  59. Cohen, P., Schelling, D. L., and Stark, M. J. (1989) FEBS Lett. 250, 601-606[CrossRef][Medline] [Order article via Infotrieve]
  60. Alessi, D. R., Street, A. J., Cohen, P., and Cohen, P. T. (1993) Eur. J. Biochem. 213, 1055-1066[Medline] [Order article via Infotrieve]
  61. Hubbard, M. J., and Cohen, P. (1993) Trends Biochem. Sci. 18, 172-177