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Published as doi: 10.1096/fj.07-092841.
 (The FASEB Journal. 2008;22:1660-1671.)
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Selective targeting of the {gamma}1 isoform of protein phosphatase 1 to F-actin in intact cells requires multiple domains in spinophilin and neurabin

Leigh C. Carmody*, Anthony J. Baucum, II*,{dagger},{ddagger}, Martha A. Bass* and Roger J. Colbran*,{dagger},{ddagger},1

* Department of Molecular Physiology and Biophysics,

{dagger} Center for Molecular Neuroscience, and

{ddagger} Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

1Correspondence: Robinson Research Bldg., Room 724,Vanderbilt University Medical Center, Nashville, TN 37232-0615, USA. E-mail: roger.colbran{at}vanderbilt.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein phosphatase 1 (PP1) catalytic subunits dephosphorylate specific substrates in discrete subcellular compartments to modulate many cellular processes. Canonical PP1-binding motifs (R/K-V/I-X-F) in a family of proteins mediate subcellular targeting, and the amino acids that form the binding pocket for the canonical motif are identical in all PP1 isoforms. However, PP1{gamma}1 but not PP1β is selectively localized to F-actin-rich dendritic spines in neurons. Although the F-actin-binding proteins neurabin I and spinophilin (neurabin II) also bind PP1, their role in PP1 isoform selective targeting in intact cells is poorly understood. We show here that spinophilin selectively targets PP1{gamma}1, but not PP1β, to F-actin-rich cortical regions of intact cells. Mutation of a PP1{gamma}1 selectivity determinant (N464EDYDRR 470 in spinophilin: conserved as residues 473–479 in neurabin) to VKDYDTW severely attenuated PP1{gamma}1 interactions with neurabins in vitro and in cells and disrupted PP1{gamma}1 targeting to F-actin. This domain is not involved in the weaker interactions of neurabins with PP1β. In contrast, mutation of the canonical PP1-binding motif attenuated interactions of neurabins with both isoforms. Thus, selective targeting of PP1{gamma}1 to F-actin by neurabins in intact cells requires both the canonical PP1-binding motif and an auxiliary PP1{gamma}1-selectivity determinant.—Carmody, L. C., Baucum II, A. J., Bass, M. A., Colbran, R. J. Selective targeting of the {gamma}1 isoform of protein phosphatase 1 to F-actin in intact cells requires multiple domains in spinophilin and neurabin.


Key Words: subcellular targeting • cytoskeleton • dendritic spine • synaptic plasticity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
FOUR PROTEIN PHOSPHATASE 1 (PP1) catalytic subunit isoforms ({alpha}, β, {gamma}1, and {gamma}2) (1) are ~80% identical in amino acid sequence and are major serine/threonine phosphatases in mammalian cells (1) . (A single PP1 isoform has been referred to as both PP1β and PP1{delta} in the literature, depending on the species and lab of origin. Herein, we identify this isoform as PP1β to be consistent with our previous work.) The PP1 catalytic subunits are targeted to discrete subcellular compartments and regulated by interactions with >50 proteins that typically contain a canonical PP1-binding motif with a consensus R/K-V/I-X-F sequence (2 , 3) . Residues that form the binding pocket for the canonical motif are identical in the 4 isoforms, yet the PP1 isoforms exhibit distinct subcellular localizations. For example, in neurons, PP1β is enriched in the soma and in the dendritic shaft, whereas PP1{alpha} and PP1{gamma}1 are enriched in dendritic spines (4 5 6) . Peptides that nonspecifically disrupt PP1 catalytic subunit interactions with canonical binding motifs profoundly regulate excitatory synaptic transmission, suggesting that targeting of PP1, presumably to dendritic spines, is critical for normal modulation of synaptic plasticity (7 8 9) . However, mechanisms underlying the isoform selectivity of PP1 targeting are unclear.

Dendritic spines are highly specialized F-actin-rich protrusions on dendrites that contain neurabin I and spinophilin (neurabin II), related F-actin- and PP1-binding proteins (10 11 12 13 14 15 16) . The neurabins homo- and heteromultimerize and also bind many other proteins involved in regulating spine morphology and other cellular functions (17 18 19 20 21 22) . Interestingly, neurabin, spinophilin, and many of their interacting partners are regulated by reversible protein phosphorylation, which can modulate assembly of the complex and/or activity of the associated proteins (23) . Therefore, neurabins modulate several signaling cascades by coordinating the assembly of multiprotein complexes that are regulated by phosphorylation.

The association of PP1 presumably plays a key role in modulating the function of neurabin complexes. Indeed, PP1 is perhaps the best-studied interaction partner of the neurabins. Stable binding of PP1 requires canonical PP1-binding motifs in spinophilin (K448IHF451) and neurabin (K457IKF460) (10 , 24 , 25) . Interestingly, biochemical studies have shown that both of the neurabins preferentially bind PP1{gamma}1 over PP1β (13 , 25 , 26) . Moreover, spinophilin and neurabin are targeted to dendritic spines apparently by the N-terminal F-actin-binding domain (4 5 6 , 10 11 12 , 14 , 15) . These data are consistent with the idea that neurabins selectively target PP1{gamma}1 to the F-actin cytoskeleton in dendritic spines. However, the isoform selectivity of PP1 targeting by the neurabins in intact cells has not been established. Here we show that spinophilin selectively targets PP1{gamma}1 to the F-actin cytoskeleton in intact cells and that targeting requires both the nonisoform selective canonical PP1-binding motif and an additional domain C-terminal to the canonical PP1-binding motif.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
DNA constructs
Spinophilin and neurabin mutations were generated from pCMV4-myc vectors containing cDNAs encoding the full-length rat proteins (13) . Spinophilin F451A (Sp(F->A)) and neurabin F460A (Nb(F->A)) were generated using complementary forward (F) and reverse (R) primers containing the mutation: Sp(F->A) F: 5'-GCCCCGAGCCGGAAGATCCATGCTAGCACCGCACCG-3'; and Nb(F->A) F: 5'-GCAAATAGGAAAATTAAGGCTAGCTGTGCTCCGATTAAG-3'. All other mutations were made using partially overlapping primer pairs in a 2-step process, using methodology described by Zheng et al. (27) . The first set of primers were as follows: Nb(VK) (N473V, E475K), F: 5'-GTACTCCGTAAAAGACTATGACAGG-3' and R: 5'-GTCTTTTACGGAGTACGTGTTGAAAAC-3'; Sp(VK) (Sp N464V, E465K), F: 5'-CCTACTCCGTTAAGGACTATG-3' and R: 5'-CATAGTCCTTAACGGAGTAGGTGCTGAATAC-3'. The initial mutant constructs were sequenced and then used as templates with a second set of primers: Nb(VK/TW) (N473V, E475K, R478T, R479W), F: 5'-CTCCGTTAAAGACTATGACACGTGGAATGATGACGTTG-3' and R: 5'-CATTCCACGTGTCATAGTCTTTAACGGAGTACGTGTTG-3'; Sp(VK/TW) (N464V, E465K, R469T, R470W), F: 5'-CTCCGTTAAGGACTATGACACATGGAATGAGGATGTGG-3' and R: 5'-CATTCCATGTGTCATAGTCCTTAACGGAGTAGGTGCTG-3'. Similarly, GM(NE/RR) was generated in pGEX-4T-GM (1–240) using 2 steps: GM(ND), (V79N, K80D), F: 5'-CTTTCTCATCGTTGGACACAAGATTGAATC-3' and R: 5'- CTTTCTCATCGTTGGACACAAGATTGAATC-3'; GM(NE/RR), (V79N, K80E, T84R, W85R), F: 5'-GTGTCGAACGAAGAGTTTGATAGGAGGG-3' and R: 5'-CAAACTCTTCGTTCGACACAAGATTGAATCC-3'. To create neurabin GST-fusion proteins, the full-length wild-type myc-neurabin (myc-Nb(WT)) or PP1-binding domain mutations (Nb (F->A), Nb (VK/TW)) were used as templates to subclone residues 146–493 into pGEX-4T vector. The sequences of all constructs and mutations were confirmed by automated DNA-sequence analysis (GenHunter Sequencing; GenHunter Corporation, Nashville, TN, USA; http://www.genhunter.com/VUsequencing). Eukaryotic expression vectors for N-terminal GFP-tagged PP1{gamma}1 and PP1β were generous gifts from Dr. Mattieu Bollen (Katholieke Universiteit, Leuven, Belgium) (28) . The expression construct for the mCherry protein was a generous gift from the laboratory of Dr. David W. Piston (Vanderbilt University School of Medicine, Nashville, TN, USA).

Antibodies
Rabbit and sheep antibodies recognizing the C termini of PP1β or PP1{gamma}1, and rabbit antibodies recognizing spinophilin and neurabin were described previously (5 , 13) . Other antibodies were mouse monoclonal PP2A (BD Biosciences, San Jose, CA, USA), mouse monoclonal spinophilin (BD Biosciences), mouse monoclonal neurabin (BD Biosciences), mouse monoclonal GFP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse monoclonal myc (Zymed Laboratories, South San Francisco, CA, USA), donkey anti-mouse Cy5 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), donkey anti-rabbit Alexa594 (Invitrogen, Carlsbad, CA, USA), donkey anti-mouse Alexa647 (Invitrogen).

Phosphatase catalytic subunit preparation
A crude mixture of native protein phosphatase catalytic subunits was generated by ethanol precipitation of brain extracts, as described previously (13 , 29) . The final preparations contained a mixture of monomeric phosphatase catalytic subunits, including an approximately equimolar ratio of PP1β and PP1{gamma}1, as determined by immunoblotting with isoform-selective antibodies in comparison with bacterial PP1 isoform standards (a generous gift of Dr. Ernest Y. Lee, New York Medical College, Valhalla, NY, USA). Native PP1β and PP1{gamma}1 were purified from the catalytic subunit preparation by adsorption to isoform-specific antibodies conjugated to Affi-Gel-10 (Bio-Rad Laboratories, Hercules, CA, USA), eluted with sodium isothiocyanate, dialyzed, and then stored at –80°C in 50% glycerol (29) .

Glutathione-agarose cosedimentation assays
In the "standard" condition, the indicated GST fusion proteins (8 µg) were mixed for 1 h at 4°C with the crude phosphatase catalytic subunit mixture (15 µg total protein) in a final volume of 1 ml binding buffer (50 mM Tris-HCl, pH 7.5; 0.2 M NaCl; 0.1% Triton X-100; 0.25 mg/ml bovine serum albumin). The final concentration of GST fusion protein (~130 nM) was in excess of the approximate concentrations of PP1β and PP1{gamma}1 (~25 nM each). Cosedimentation assays also were performed using a "stringent" protocol in which the entire incubation was diluted 14-fold in the same buffer. GST protein complexes were immunoblotted for the presence of phosphatase catalytic subunits as described previously (26 , 29) .

PP1 inhibition assays
Activities of purified native PP1 isoforms were measured toward [32P]phosphorylase a substrate, essentially as described previously (13 , 26 , 29) .

Cell culture and transfections
293FT cells (Invitrogen) were transfected using FuGENE 6 (Roche Applied Science, Indianapolis, IN, USA) with 1 or more constructs. Transfected cells were grown in 100 mm dishes for 40–48 h, lysed with 600 µl lysis buffer (50 mM Tris-HCl, pH 7.5; 120 mM NaCl; 1 mM EDTA; 0.1% (v/v) Nonidet P-40; 0.1% (w/v) deoxycholate; 0.1 mM PMSF; 1 mM benzamidine; 20 mg/L soybean trypsin inhibitor; and 5 mg/L leupeptin), sonicated, and then centrifuged (5000 g, 10 min) to generate soluble cell extracts. Extracts were stored at –80°C until required.

Coimmunoprecipitations
293FT cell lysates were diluted to 1 mg/ml protein in IP buffer (50 mM Tris-HCl, pH 7.5; 0.15 M NaCl; 1 mM EDTA; 0.5% (v/v) Triton X-100; 1 mM PMSF; 5 mg/L leupeptin; 20 mg/L soybean trypsin inhibitor; and 0.5 mM benzamidine). Lysates were incubated with the indicated rabbit antibodies or equivalent amounts of nonimmune rabbit IgG for 2 h at 4°C. GammaBind Plus-Sepharose (Amersham Biosciences, Arlington Heights, IL, USA) beads were added and incubated for an additional 2 h. Resin was collected, and immune complexes and supernatants were analyzed by immunoblotting.

Fluorescent microscopy
HEK293 or 293FT cells (Invitrogen) were plated onto poly-D-lysine coated coverslips and transfected (as above). After 40–48 h, cells were fixed with 4% (v/v) paraformaldehyde and 4% (w/v) sucrose in PBS (10 min, 37°C) and permeabilized with PBS containing 0.1% (v/v) Triton X-100 for 5 min at room temperature, as described previously (5) . Coverslips were incubated with DRAQ5 (Biostatus Ltd., Shepshed, UK) in PBS, or mouse myc antibodies (1:100 dilution) in a PBS solution containing 2% (v/v) in normal donkey serum in PBS and 0.01% (v/v) Tween 20. Washed coverslips were incubated with anti-mouse Cy5 (1:800), and/or rhodamine-phalloidin (1:40; Molecular Probes, Inc., Eugene, OR, USA). Coverslips were mounted with Aqua Poly/Mount (Polysciences, Inc., Warrington, PA, USA) and imaged using a LSM 510 META inverted confocal microscope (Carl Zeiss Microimaging, Thornwood, NY, USA). Green fluorescent protein (GFP) and red fluorescent protein (not shown) autofluorescence was assessed. Controls were performed to verify that primary and secondary antibodies were specific and that fluorescent signals were restricted to the appropriate channel. Microscope settings were optimized to capture both bright and dim images, and the same settings were used to collect all images within each experimental condition. Images shown represent single optical sections (nominal 0.5 µm thickness) from the center of the cells. Each experiment contained 2 separate transfections for each condition, and 5–12 fields of view were selected for analysis of each condition. For GFP-PP1{gamma}1 samples, fields of view were randomly chosen. Because of lower transfection efficiency/expression, it was not possible to randomly choose fields of view for GFP-PP1β. Therefore, fields of view containing 3 or more GFP-expressing cells were chosen for quantification. Acquired images were uniformly adjusted by linearly reassigning the values of pixel intensities to use the full 8-bit range (0–255) and then thresholded based on control cells not expressing the transfected protein using Metamorph software (Molecular Devices Corp., Sunnyvale, CA, USA). Colocalization was scored as the percentage of thresholded GFP fluorescent pixels that overlap with thresholded rhodamine fluorescent pixels (percentage pixel overlap). An intensity ratio was then calculated to provide a measure of colocalization that included information about the relative amount of protein in the 2 pools. First, the percentage pixel overlap (green over red) was multiplied by the average GFP fluorescence signal intensity in those pixels. This value was then divided by the average GFP fluorescence signal intensity in the noncolocalized fraction multiplied by the percentage of nonoverlapping pixels. The quantified data shown were calculated from the entire field, and no correction was made for the presence of singly transfected cells, although a majority of cells were cotransfected (>80%). In preliminary experiments, limiting quantitative analyses to individually selected cotransfected cells did not improve our ability to distinguish targeting of GFP-PP1 isoforms by wild-type and mutated spinophilins, so we performed quantitative analyses on entire fields of view (see above) to avoid the potential for operator bias in selecting individual cells. For display purposes only, the contrast settings were increased to be able to see both dim and bright objects.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Spinophilin is a PP1{gamma}1-selective targeting subunit in intact cells
As an initial step toward investigating mechanisms controlling the subcellular localization of PP1 isoforms, we transiently expressed PP1{gamma}1 and PP1β as GFP fusion proteins in HEK293 cells. To monitor overall cell morphology, cells were cotransfected with a soluble monomeric red fluorescent protein (mCherry), fixed, and then nuclei were stained with DRAQ5. As reported previously (28) , confocal fluorescence microscopy revealed GFP-PP1{gamma}1 is strongly clustered within the cell. GFP-PP1{gamma}1 clusters partially overlap DRAQ5 in some cells but appear to be predominantly perinuclear in HEK293 cells. Parallel studies showed that GFP-PP1{gamma}1 is predominantly nuclear in HeLa cells (not shown). In contrast, GFP-PP1β is more diffusely, if unevenly, distributed throughout the nucleus and cytosol in HEK293 cells (Fig. 1 A) and HeLa cells (not shown). The overall morphology and size of HEK293 cells or HeLa cells was unaffected by expression of either GFP-PP1 isoform, as indicated by mCherry fluorescence (Fig. 1A ; data not shown).


Figure 1
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  		Figure 1. Preferential targeting of GFP-PP1{gamma}1 by spinophilin in intact cells. A) HEK293 cells were transfected to coexpress mCherry with enhanced GFP or the indicated GFP fusion proteins. Cells were fixed and counterstained with DRAQ5, a nuclear stain. Confocal microscope images of the intrinsic fluorescence of GFP, mCherry, and DRAQ5 are shown on a gray scale, with the last column representing an overlay of all 3 channels in the green, red, and blue channels, respectively. B) HEK293 cells were transfected to express myc-Sp and mCherry with either GFP-PP1β or GFP-PP1{gamma}1. Fixed cells were processed to detect myc immunofluorescence (blue) and intrinsic GFP (green) or mCherry (red) fluorescence by confocal microscopy as in A. Scale bars in top left images of panels A and B apply to all images in that panel.

Neurabins have been proposed to function as PP1-isoform-selective F-actin-targeting proteins. Expression of GFP-spinophilin in HEK293 cells resulted in predominant localization of GFP to submembrane cortical regions, quite distinct from the localization of either GFP-PP1β or GFP-PP1{gamma}1 (Fig. 1A ). The overall morphology of HEK293 and HeLa cells was unaffected by expression of GFP-spinophilin under these conditions.

For initial qualitative assessment of the effect of neurabins on PP1 localization, GFP-PP1 isoforms were coexpressed with myc-tagged spinophilin (myc-Sp). Coexpression of myc-Sp had little effect on the generally diffuse distribution of GFP-PP1β, and there was little overlap in their localization. In contrast, coexpression of myc-Sp resulted in the transition of GFP-PP1{gamma}1 fluorescence from predominant intracellular clusters to mostly peripheral submembranous localization, strongly overlapping with myc-Sp (Fig. 1B ). Quantitative data further supporting this interpretation and defining mechanisms for isoform-specific targeting are described below. Notably, the range of sizes and morphologies of HEK293 cells coexpressing myc-Sp and GFP-PP1 isoforms was not noticeably different from those expressing either protein alone. Coexpression of myc-neurabin (myc-Nb) resulted in noticeable, but only partial, redistribution of GFP-PP1{gamma}1 from intracellular puncta, presumably because myc-Nb was expressed at much lower levels than was myc-Sp (not shown). Taken together, these data directly demonstrate for the first time that neurabins preferentially target PP1{gamma}1 over PP1β in intact cells.

Neurabins contain multiple PP1-binding determinants
To investigate the mechanism for targeting PP1{gamma}1 in cells, we first characterized the biochemical basis for selective interactions of PP1 isoforms with neurabins. Mutation of the phenylalanine residue within canonical PP1-binding motifs in spinophilin (K448IHF451) and neurabin (K457IKF460) severely disrupts their interactions with PP1 (24 , 25) . Our previous truncation studies showed that residues 473–479 of neurabin are necessary for selective binding of the PP1{gamma}1 isoform in vitro (26) . This PP1{gamma}1 selectivity determinant is 100% conserved in spinophilin (residues 464–470), but critical residues within this domain were not identified. Therefore, we aligned amino acid sequences C-terminal to the canonical PP1-binding domain of spinophilin and neurabin with corresponding sequences from the muscle glycogen targeting subunit of PP1 (GM), which displays an inverse PP1 isoform selectivity (PP1β>PP1{gamma}1) (26) . (GM has also been designated RGL; ref. 30 ). Four amino acids within the PP1{gamma}1 selectivity determinant of the neurabins were not conserved in GM (Fig. 2 A). Consequently, we mutated the NEDYDRR sequence to VKDYDTW in both neurabins (Nb(VK/TW) and Sp(VK/TW)).


Figure 2
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  		Figure 2. Effects of mutagenesis on PP1 binding to neurabin fragments. A) Alignment of sequences surrounding the canonical PP1-binding motif (R/K-V/I-X-F/W, gray box) in neurabin/spinophilin and GM. Identical and conserved amino acids are indicated in black boxes (white and gray letters, respectively). The black rectangle outlines a cluster of residues C-terminal to the canonical motif that overlap a domain previously shown to be essential for PP1{gamma}1-selective interactions by truncation mutagenesis (26) . Residues marked with asterisks were targeted by site-directed mutagenesis in the present study. B–D) The indicated GST fusion proteins (8 µg) were incubated with a crude protein phosphatase catalytic subunit mixture (15 µg of total protein) under "standard" conditions (B, C; 1 ml) or "stringent" conditions (D, 14 ml; see Materials and Methods). The resulting complexes were collected using glutathione agarose and immunoblotted for PP1β, PP1{gamma}1, and PP2A. A sample of the protein phosphatase catalytic subunit mixture was analyzed in parallel (input). Prior to immunoblotting, nitrocellulose membranes were stained with Ponceau to reveal the amounts of GST fusion protein in each lane (protein stain). The arrowhead labeled BSA indicates bovine serum albumin carried over from the incubation buffer. B) Representative raw data obtained under standard conditions. C) Quantification of 3 separate experiments performed under standard conditions. D) Quantification of 5 separate experiments performed under stringent conditions. Bars = mean ± SE.

GST fusion proteins containing the PP1-binding domain (residues 146–493) of neurabin (wild-type: GST-Nb(WT), F460A mutant: GST-Nb(F->A), and NEDYDRR to VKDYDTW mutant: GST-Nb(VK/TW)) were used in glutathione agarose cosedimentation assays to compare the effects of mutating the PP1{gamma}1 selectivity determinant and the canonical PP1-binding motif. Parallel controls were performed with a GST fusion protein containing a fragment of GM with inverse PP1 isoform binding selectivity (GST-GM). Since recombinant PP1 isoforms do not typically display the full repertoire of biochemical properties of the native proteins (29 , 31) , we tested the interactions of these protein fragments with native rat brain catalytic subunit isoforms. GST-Nb(WT) interacts with both PP1β and PP1{gamma}1 (Fig. 2B ) but preferentially interacts with PP1{gamma}1 under our standard conditions (Fig. 2B, C ; see Materials and Methods). Under more stringent conditions (i.e., with 14-fold dilution), the selectivity of GST-Nb(WT) for PP1{gamma}1 becomes much more apparent (Fig. 2D ). This difference is not due to abnormal properties of the PP1β preparation because GST-GM selectively interacts with PP1β rather than PP1{gamma}1 (Fig. 2D ). Mutation of either the canonical motif or the selectivity determinant completely prevented binding of PP1{gamma}1 to GST-Nb under stringent incubation conditions (Fig. 2D ). Under "standard" conditions, mutation of the canonical binding motif greatly attenuated binding of both PP1 isoforms, but mutation of the selectivity determinant attenuated PP1{gamma}1 binding without affecting the weaker binding of PP1β (Fig. 2B, C ). These data identify residues Asn473, Glu474, Arg478, and Arg479 as crucial determinants for stable binding of PP1{gamma}1 to neurabin in vitro.

As an additional assessment of the effects of these mutations that does not require separation of complexes from excess unbound proteins, we assayed activities of purified native PP1 isoforms in the presence of various concentrations of the GST fusion proteins. GST-Nb(WT) is a much more potent inhibitor of PP1{gamma}1 (EC50%~0.6 nM) than PP1β (EC50%~40 nM) (Fig. 3 A), in agreement with previous studies (26) . In contrast, GST-GM preferentially inhibited PP1β over PP1{gamma}1, albeit only modestly (Fig. 3D ), consistent with previous studies. Mutation of the canonical binding motif in GST-Nb reduced the potency for inhibition of both isoforms by more than 100-fold, but it was readily apparent that the GST-Nb(F->A) retained similar selectivity for PP1{gamma}1 as the parent GST-Nb(WT) protein (Fig. 3B ). In contrast, GST-Nb(VK/TW) exhibited no significant PP1 isoform selectivity (Fig. 3C ). The potency of PP1{gamma}1 inhibition was decreased by ~100-fold, with only an ~2-fold effect on PP1β inhibition (compare Fig. 3A, C ). These data show that the canonical binding motif is important for interaction of neurabin fragments with both PP1 isoforms, whereas specific residues (Asn473, Glu474, Arg478, and Arg479) within a domain C-terminal to the canonical PP1-binding motif are critical only for high-affinity interactions of neurabin fragments with PP1{gamma}1.


Figure 3
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  		Figure 3. Effects of mutagenesis on inhibition of native PP1 isoforms by neurabin fragments. Activities of native PP1β (gray lines/triangles) and PP1{gamma}1 (black lines/squares) were assayed in the presence of the various concentrations of the indicated GST fusion proteins. Each data point represents mean ± SE of 2–5 observations. GST alone (3 µM) had no significant effect on PP1β or PP1{gamma}1 (data not shown).

Association of PP1 isoforms with the neurabins in cells
To investigate the relative importance of the canonical PP1-binding motif and the isoform selectivity determinant for binding of PP1 to full-length neurabins in intact cells, we compared the interaction of GFP-PP1{gamma}1 with coexpressed myc-tagged wild-type or mutated full-length versions of both proteins (WT, F->A, VK/TW). Initially, neurabin or spinophilin were immunoprecipitated from cell extracts and immune complexes were probed for PP1{gamma}1. Both endogenous PP1{gamma}1 and GFP-PP1{gamma}1 coprecipitated with myc-Nb(WT) (Fig. 4 A, lane 4) and myc-Sp(WT) (Fig. 4B , lane 2). Neither form of PP1{gamma}1 could be detected in immunoprecipitates of myc-Sp(F->A) mutant (canonical PP1-binding motif mutant) (Fig. 4B , lane 4). However, small amounts of both endogenous PP1{gamma}1 and GFP-PP1{gamma}1 were coprecipitated with the myc-Nb(F->A) mutant (Fig. 4A , lane 6). This result may result from coprecipitation of low levels of endogenous spinophilin in the 293FT cells because a similar amount of endogenous PP1{gamma}1 was immunoprecipitated with the neurabin antibody in the absence of overexpressed myc-Nb(WT) (Fig. 4A , compare lanes 2, 6). Most significantly, mutating the PP1 isoform selectivity domain (VK/TW) in neurabin or spinophilin resulted in a similar reduction in the coprecipitation of endogenous PP1{gamma}1 and GFP-PP1{gamma}1 as the canonical PP1-binding motif mutation (Fig. 4A , lane 8; B, lane 6). These data suggest that the canonical motif and the PP1{gamma}1 selectivity determinant are equally important for PP1{gamma}1 interaction with full-length spinophilin or neurabin in intact cells.


Figure 4
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  		Figure 4. Effects of mutagenesis on PP1-binding to full-length neurabin (Nb) and spinophilin (Sp) in intact cells. Soluble extracts of 293FT cells expressing GFP-PP1{gamma}1, GFP-PP1β, and/or myc-tagged, full-length neurabins (wild-type or containing the indicated mutations) were immunoprecipitated (IP) using rabbit antibodies to Nb or Sp, or using equivalent amounts of control rabbit IgG (IgG). Immune complexes (P) and supernatants (S) were immunoblotted with mouse antibodies to Nb, Sp, or GFP or sheep antibodies to PP1{gamma}1 (PP1{gamma}1 blots in panels A and B indicate an approximate molecular weight range from 31 kDa at the bottom to 66kDa at the top). A) Cells expressing GFP-PP1{gamma}1 with or without wild-type or mutated neurabin. B) Cells expressing GFP-PP1{gamma}1 with wild-type or mutated Sp. C) Cells expressing GFP-PP1β or GFP-PP1{gamma}1 with wild-type or mutated Sp.

To determine how these mutations affect interactions of PP1β with spinophilin in cells, we coexpressed wild-type or mutated myc-Sp with GFP-PP1β and immunoprecipitated myc-Sp from the cell extracts. Immunoblotting using GFP antibodies detected GFP-PP1β in myc-Sp(WT) immune complexes, but the levels of GFP-PP1β were apparently lower than those of GFP-PP1{gamma}1 in parallel immunoprecipitations (Fig. 4C , compare lanes 2, 8). Moreover, although myc-Nb(WT) and myc-Sp(WT) immunoprecipitates contained endogenous PP1{gamma}1 in addition to GFP-PP1{gamma}1 (Fig. 4A, B ), we failed to detect endogenous HEK293 cell PP1β using our PP1β antibodies (data not shown), consistent with prior studies showing that PP1β is not significantly associated with spinophilin or neurabin in the brain (13) . These data show that full-length spinophilin displays a similar preference for coexpressed GFP-PP1 and endogenous HEK293 cell PP1 isoforms as do neurabin fragments for purified brain PP1 isoforms in vitro (Figs. 2C and 3) . Mutation of the canonical PP1-binding motif (myc-Sp(F->A)) appeared to abrogate the coprecipitation of GFP-PP1β. However, GFP-PP1β was readily detected in myc-Sp(VK/TW) immune complexes using antibodies to GFP (Fig. 4C , lanes 4, 6). In fact, we detected a similar ratio of GFP-PP1β to myc-Sp in myc immune complexes isolated from cells expressing the wild-type or VK/TW mutated proteins. Taken together, these data demonstrate that in the context of full-length protein in intact cells, the canonical PP1-binding motifs in neurabin and spinophilin are important for binding both isoforms, whereas the selectivity determinant only affects association of PP1{gamma}1.

Effect of PP1-binding site mutations on PP1 isoform targeting
To better understand the mechanisms underlying neurabin modulation of PP1 isoform localization, we used 2 different approaches to quantify GFP-PP1 isoform targeting to F-actin from fluorescent images. GFP-PP1 isoforms were coexpressed with myc-Sp (wild-type or mutated), and rhodamine-phalloidin was used to stain F-actin. Importantly, we chose HEK293 cells for these studies because these transfections had little effect on cell size and morphology under our culture conditions (Fig. 1) , thus avoiding potentially misleading effects due to morphological changes. Images of random fields from each transfection were collected using identical microscope settings in order to avoid potential operator bias in selecting individual cells. Consistent with data in Fig. 1B , immunofluorescence signals for myc-Sp(WT) strongly overlapped rhodamine-phalloidin fluorescence in the cortical region of transfected cells, confirming previous studies showing that neurabins associate with F-actin-rich structures in numerous cell types. In the absence of myc-Sp(WT), GFP-PP1{gamma}1 fluorescence clustered in the center of cells and clearly did not strongly overlap with F-actin cytoskeletal elements (rhodamine-phalloidin) (Fig. 5 A); in fact, only ~15% of GFP-PP1{gamma}1-positive pixels overlapped with rhodamine phalloidin-positive pixels (Fig. 5B ). Coexpression of myc-Sp(WT) resulted in a dramatic redistribution of GFP-PP1{gamma}1 to the cell periphery such that GFP-PP1{gamma}1 colocalized with both myc-Sp(WT) and the F-actin cytoskeleton in the transfected cells: ~70% of GFP-PP1{gamma}1-positive pixels overlapped with rhodamine phaloidin in the presence of myc-Sp(WT), representing an ~5-fold increase in PP1{gamma}1 targeting to F-actin by myc-Sp(WT). However, these pixel overlap scores do not take into account the relative strength of GFP signals in the 2 subcellular compartments (phalloidin vs. nonphalloidin). Therefore, we also quantified the ratio of GFP fluorescence intensity in F-actin-localized vs. non-F-actin-localized pools, as defined by the presence or absence of rhodamine-phalloidin fluorescence. By this measure, myc-Sp(WT) enhanced the localization of GFP-PP1{gamma}1 to F-actin by ~18-fold. In combination, these 2 semiquantitative measures show that spinophilin targets PP1{gamma}1 to F-actin in intact cells.


Figure 5
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  		Figure 5. The canonical PP1-binding motif and the selectivity determinant are required for selective targeting of GFP-PP1{gamma}1 to F-actin. A) Coexpression of GFP-PP1{gamma}1 and wild-type or mutated myc-Sp in 293FT cells. Cells expressing GFP-PP1{gamma}1 without or with the indicated myc-Sp were fixed and stained with rhodamine-phalloidin (red). Antibodies to the myc epitope were used to detect spinophilin (blue), and GFP was detected by autofluorescence (green). White scale bars in merged images indicate 10 µm. B) Quantification of colocalization of GFP-PP1{gamma}1 with F-actin. Top panel: percentage colocalization scores are the fraction of GFP containing pixels that overlap with rhodamine-phalloidin pixels, providing a colocalization measurement that is independent of relative signal intensity. Bottom panel: the intensity ratio is the ratio of GFP fluorescence intensity in pixels that overlap with rhodamine to GFP fluorescence intensity in pixels that do not overlap with rhodamine. C) Coexpression of GFP-PP1β and wild-type Sp in 293FT cells. Cells expressing GFP-PP1β without or with myc-Sp(WT) were fixed, stained, and imaged as for panel A. D) Quantification of colocalization of GFP-PP1β with F-actin. (See panel B for details.) Data are plotted as mean ± SE and were analyzed by 1-way ANOVA using Bonferroni post hoc tests. *P < 0.05 vs. GFP-PP1 isoform alone; #P < 0.05 vs. cotransfection of GFP-PP1{gamma}1 and Sp(WT).

Similar studies were performed with GFP-PP1β in order to examine the isoform selectivity of PP1 targeting by spinophilin in intact cells. In the absence of myc-Sp, GFP-PP1β was relatively diffusely localized throughout the cell (Fig. 5C ), consistent with Fig. 1A , such that it was not distinctly excluded from the F-actin cytoskeleton. This result was reflected in somewhat higher colocalization scores by both pixel overlap and intensity ratio measures than observed when GFP-PP1{gamma}1 was expressed alone (~35% and ~0.5 vs. ~15% and ~0.1 for GFP-PP1β and GFP-PP1{gamma}1, respectively). Coexpression of myc-Sp(WT) resulted in a significant, but much more subtle, enrichment of GFP-PP1β at the F-actin cytoskeleton, compared with the dramatic redistribution of GFP-PP1{gamma}1 induced by myc-Sp(WT) (compare Fig. 5A, C ). Although myc-Sp(WT)-enhanced F-actin localization of GFP-PP1β was statistically significant by both pixel overlap and intensity ratio measures, there was only an ~2-fold increased enrichment of GFP-PP1β at F-actin (Fig. 5D ) compared with the ~18-fold enrichment of GFP-PP1{gamma}1, representing a 9-fold selectivity for GFP-PP1{gamma}1 by this measure. Thus, the combined data show that spinophilin selectively targets PP1{gamma}1 to the F-actin cytoskeleton in intact cells.

The importance of different domains in spinophilin for PP1 targeting was then determined by coexpressing GFP-tagged PP1 isoforms with spinophilins containing mutations in the canonical PP1-binding motif (myc-Sp(F->A)) or in the PP1{gamma}1 selectivity determinant (myc-Sp(VK/TW)). Neither mutation had a noticeable effect on F-actin-targeting of spinophilin itself (Fig. 5A , blue channels), but both disrupted the localization of GFP-PP1{gamma}1 to the F-actin cytoskeleton in a similar manner. In both cases, a fraction of GFP-PP1{gamma}1 colocalized with the mutated proteins and rhodamine-phalloidin at the cell periphery, but GFP-PP1{gamma}1 fluorescence was prominent in central intracellular clusters (Fig. 5A ), similar to those observed in the absence of myc-Sp (Fig. 1) . Quantitative measures of the pixel overlap and fluorescence intensity ratios revealed that mutation of the canonical motif or the PP1{gamma}1 selectivity determinant partially disrupted F-actin targeting of GFP-PP1{gamma}1 to a similar extent (Fig. 5B ). In contrast, the modest enhancement of F-actin targeting of GFP-PP1β by myc-Sp(WT) was sensitive to mutation of the canonical motif but not to mutation in the C-terminal selectivity determinant (Fig. 5C, D ). In combination, these data show that the canonical PP1-binding motif in spinophilin is critical for interactions with both PP1 isoforms in intact cells. However, a domain C-terminal to this motif (residues 473–479) is also critical for the selective and strong targeting of GFP-PP1{gamma}1 to F-actin.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Dendritic spines are highly enriched with F-actin and F-actin regulatory proteins such as neurabin, spinophilin, and PP1{gamma}1 (4 5 6 , 11 , 12 , 14 , 15) . N-terminal F-actin-binding domains target spinophilin and neurabin to dendritic spines (11 , 12 , 32 , 33) . Previous studies have suggested that interactions of several other proteins with neurabins modulate F-actin dynamics and cell morphology (21 , 34) . In addition, cell morphology and dendritic spine dynamics are affected by interactions of undefined PP1 isoforms with overexpressed truncated neurabin fragments (32 , 35) , although the physiological relevance of these observations is unclear. PP1 targeting by neurabins also is important for normal regulation of synaptic and extrasynaptic AMPA and N-methyl-D-aspartate-type glutamate receptors (7 , 9 , 36) . While these studies infer an important role for PP1 binding to neurabins in situ, subcellular targeting of PP1 by the neurabins has not been directly demonstrated, and the isoform selectivity of targeting is not clearly defined. Here we examine the targeting of PP1 isoforms to F-actin in HEK293 cells, which are relatively resistant to morphological changes induced by our experimental manipulations, thus avoiding potential complications in experimental interpretation. Our data show that spinophilin selectively targets PP1{gamma}1 over PP1β to F-actin in intact cells and identify specific amino acids in an auxiliary interaction domain that are required for selective targeting.

The neurabins contain a canonical R/K-V/I-X-F/W PP1-binding motif, and structures of PP1 catalytic subunits bound to proteins/peptides containing this motif have been elucidated (37 38 39) . Variants of the canonical motif exhibit different affinities for PP1. Mutagenesis studies with mGluR7b and inhibitor-1 suggest that isoleucine may confer a lower affinity interaction than valine at the V/I position (38 , 40) . In contrast, exchanging neurabin’s motif (KIKF) for that of GM (RVSF) attenuated PP1 binding (26) . This discrepancy may reflect different sequence contexts of the canonical motifs and/or the presence of different auxiliary interactions (see below). Alternatively, it may reflect the use of different sources of PP1 isoforms. We studied interactions with native PP1 isoforms because recombinant proteins expressed in bacteria typically do not exhibit the full repertoire of native enzymatic functions (31) . Most significantly in this context, native brain PP1{gamma}1 is inhibited by GST-Nb (residues 146–493) ~100-fold more potently than is recombinant PP1{gamma}1 from bacteria (29) . One interesting aspect of the present study is that the magnitude of the effect of the F->A mutation in the canonical PP1-binding motif depends on the assay used to monitor the interactions. The F->A mutation appears to almost completely block the interaction in pull-down and coimmunoprecipitation assays that require extensive washing to remove excess unbound PP1 from the complexes (Figs. 2 and 4) . In contrast, it is clear from activity and colocalization assays that monitor steady state interaction that the F->A mutant neurabins retain significant interactions with PP1 (Figs. 3 and 5) . It is likely that higher protein concentrations, combined with the lack of washing to remove unbound PP1, allows for detection of the residual weak interactions in enzyme activity assays and in intact cells. These data suggest caution in interpreting data obtained by comparing effects of wild-type PP1-binding proteins with F->A mutated proteins as revealing a functional role (or lack thereof) for the PP1 interaction. More extensive mutations of the canonical motif might be required to completely block PP1 binding in intact cells. Despite these differences, our data are consistent with the consensus view that the Phe residue within the canonical motif is critical for high-affinity PP1 binding and that the canonical motif does not influence PP1 isoform selectivity.

Canonical PP1-binding motifs may provide an anchor to facilitate auxiliary secondary interactions between PP1 and its targeting/regulatory subunits (2) . For example, the auxiliary domains in DARPP-32 and inhibitor-1 contain protein kinase A phosphorylation sites, allowing the phosphorylated proteins to potently inhibit PP1 (41 42 43) . Similarly, the myosin-targeting subunit contains a structural domain that has extensive secondary interactions with the C-terminal domain of PP1β (39) . Previous studies showed that residues N-terminal to the canonical motif stabilize weak interactions of neurabin with PP1β (25) . In contrast, analysis of chimeric proteins containing domains from neurabin and GM, the PP1β-selective glycogen targeting subunit, showed that residues C-terminal to the canonical motif are critical for overall PP1{gamma}1 selectivity in vitro, and this idea was supported by truncation mutagenesis studies that specifically implicated residues 473–479 as a PP1{gamma}1 selectivity determinant (26) . Here, we identified specific amino acids in neurabin and spinophilin that are important for high affinity, selective interactions with PP1{gamma}1 in vitro and show that these same residues are important in the selective targeting of PP1{gamma}1 in cells. Comparison of amino acid sequences surrounding the canonical PP1-binding motifs in neurabins and GM revealed weak overall similarity, with the exception of a cluster of residues overlapping the putative PP1{gamma}1 selectivity determinant that are 70% identical. Notably, 4 amino acids in neurabin within the putative PP1{gamma}1 selectivity determinant (Asn473, Glu474, Arg478, and Arg479) are identical in spinophilin but are not conserved in GM (Fig. 2A ). Mutating neurabin residues 473, 474, 478, and 479 to the corresponding residues from GM (VK/TW mutant) disrupted PP1{gamma}1 binding to fragments of neurabin in vitro but had little to no effect on PP1β binding (Figs. 2 and 3) . In the context of both full-length neurabin and full-length spinophilin, the corresponding VK/TW mutation strongly affected association of PP1{gamma}1 in coimmunoprecipitation assays but had little to no effect on the much weaker association of GFP-PP1β. Moreover, the VK/TW mutation substantially reduced the selective targeting of PP1{gamma}1 to F-actin with no detectable effect on the much weaker targeting of GFP-PP1β. Thus, residues 473–479 of neurabin and residues 464–470 of spinophilin are critical for PP1{gamma}1-selective binding to neurabins in vitro and in intact cells.

In combination, our results suggest a model in which at least 2 domains in the neurabins are needed for selective targeting of PP1{gamma}1 to F-actin in intact cells. The canonical PP1-binding motif can interact with both PP1 isoforms, but an auxiliary selectivity determinant strongly stabilizes binding of PP1{gamma}1 but not PP1β, accounting for selective targeting (Fig. 6 , top row). Mutation of the canonical motif weakens interactions with both isoforms such that the selectivity ratio is unchanged, presumably because the PP1{gamma}1 selectivity determinant is intact (Fig. 6 , middle row). Lastly, mutation of the selectivity determinant in either neurabin or spinophilin disrupts binding of PP1{gamma}1 but does not affect binding of PP1β, thereby abrogating selectivity (Fig. 6 , bottom row).


Figure 6
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  		Figure 6. Working model of PP1 interactions with neurabin/spinophilin. Neurabin/spinophilin associates with PP1{gamma}1 via a canonical binding motif (KI(K/H)F) and a PP1{gamma}1-selectivity determinant (NE/RR). XXX indicates mutations of each motif. The weight of the arrows in each row indicates the relative binding of PP1{gamma}1 and PP1β to each form of spinophilin.

Although the NE/RR motif is completely conserved in all mammalian neurabins, it is unclear whether other PP1-binding proteins employ a similar mechanism to selectively interact with PP1{gamma}1 over other PP1 isoforms. In an effort to determine whether the NE/RR motif is sufficient to enhance binding of PP1{gamma}1 in another context, the VK/TW residues C-terminal to the canonical PP1-binding motif in GM were mutated to NE/RR in GST-GM. However, this mutation failed to enhance binding or inhibitory potency toward PP1{gamma}1 (data not shown). Thus, the NE/RR motif may require the specific context of the neurabins in order to confer PP1{gamma}1 binding selectivity. Notably, the NE/RR motif in GST-GM(1–240: NE/RR) is 2 amino acids closer to the canonical PP1-binding motif than the natural NE/RR motif in neurabins, and our previous studies have suggested that precise spacing between these motifs is critical for binding of PP1{gamma}1 because deletion of 2 amino acids between the canonical PP1-binding and NE/RR selectivity motif in neurabin severely compromised PP1{gamma}1 binding (26) . Interestingly, inhibitor 3 displays some selectivity for PP1{gamma}1 in cells (44) and also contains an NEHMGRR motif C-terminal to the canonical PP1-binding motif, but the spacing of the 2 domains is 6 residues closer than in neurabin. Thus, the functional role of this motif in the context of inhibitor 3 is unclear. No other known PP1-binding proteins appear to contain similarly located NE/RR-like motifs. These considerations lead us to suggest that employment of an NE/RR motif to confer PP1{gamma}1 isoform selectivity may be contextual and relatively unique to the neurabins.

PP1 catalytic subunit isoforms exhibit similar activities in vitro (31) but are differentially localized in neurons and other cells (4 5 6 , 28 , 45 , 46) , suggesting that they play distinct biological roles. Although interactions of PP1{alpha} with neurabins were not investigated here because of technical limitations, previous coimmunoprecipitation data indicated that neurabins bind PP1{gamma}1>PP1{alpha}>> PP1β (25) . Thus, it appears that spinophilin binds mostly to PP1{gamma}1 in the brain, perhaps in part because PP1{gamma}1 is expressed at somewhat higher levels than PP1{alpha} (47) . Interestingly, the amount of PP1{alpha} associated with neurabins increased in PP1{gamma}1 knockout mice (25) . Thus, even though PP1{alpha} and PP1{gamma}1 appear to be differentially localized within spines (6) , our data cannot exclude the possibility that neurabins can target both isoforms (4 , 6) . In contrast, PP1β is enriched in the soma and dendritic shafts, where it appears to associate with microtubules (5 , 6) .

Multiple lines of evidence suggest that neurabins mediate PP1-dependent effects on the morphology of some cells, including actin stress fiber formation, filopodia, and the development of neuronal dendrites and dendritic spines (11 , 32 , 33 , 35 , 36) . The present data suggest that PP1{gamma}1 (or perhaps PP1{alpha}) but not PP1β regulates signaling pathways responsible for these morphological effects by regulating protein phosphorylation/dephosphorylation in neurabin complexes. Phosphorylation of neurabins modulates binding to both PP1 and F-actin (35 , 48 49 50) . Thus neurabin and additional neurabin binding partners may be dephosphorylated by bound PP1{gamma}1. For example, PP1 inactivates Tiam1 (51 , 52) , a rac-specific guanine nucleotide exchange factor that interacts with spinophilin and regulates dendritic spine development (20 , 53) . Interestingly, Tiam1 facilitates the activation of p70 S6 kinase, another spinophilin/neurabin-associated protein that competes with PP1 for binding to neurabin (20 , 35) . However, despite the overall similarity between neurabin and spinophilin, recent analyses using knockout mice showed that spinophilin is critical for long-term depression whereas neurabin is critical for long-term potentiation, presumably because synaptic targeting of PP1 isoforms is critical for regulation of multiple glutamate receptors (36 , 54) . Thus, differential targeting of PP1 isoforms by spinophilin and neurabin likely mediates distinct biological responses.

In summary, our data show that neurabins strongly target PP1{gamma}1 to the F-actin cytoskeleton in intact cells but have little effect on the localization of PP1β. While canonical PP1-binding motifs in spinophilin and neurabin affect binding of all isoforms, a domain C-terminal to the canonical domain is essential for selective interactions with PP1{gamma}1 in vitro and for selective targeting in intact cells. Thus, coordinated interactions of PP1{gamma}1 with 2 domains in the neurabins are essential for specific targeting of PP1{gamma}1 to F-actin, and presumably to dendritic spines in neurons, to facilitate dynamic modulation of the F-actin cytoskeleton, dendritic spine morphology, and synaptic plasticity.


   ACKNOWLEDGMENTS
 
This work was supported by the U.S. National Institutes of Health (NIH), grant NIH-PO1-NS44282. A.J.B. was supported by the Neurogenomics Training Program (T32-MH65215). Fluorescence microscopy data were collected using the VUMC Cell Imaging Shared Resource (supported by NIH grants CA68485, DK20593, DK58404, HD15052, DK59637, and EY08126) with excellent advice from Drs. S. Wells and D. Kilkenny-Rocheleau concerning quantification of images using Metamorph software. We appreciate the advice and assistance of K. Betke in some of the cell imaging studies. In addition, we thank Dr. B. Wadzinski, N. Garbarini, and N. Byun for their critical comments on drafts of this manuscript, as well as Dr. A. Brady and members of the Colbran Lab for fruitful discussions.

Received for publication June 21, 2007. Accepted for publication December 6, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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J. L. McConnell and B. E. Wadzinski
Targeting Protein Serine/Threonine Phosphatases for Drug Development
Mol. Pharmacol., June 1, 2009; 75(6): 1249 - 1261.
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