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Importance of the β12-β13 Loop in Protein Phosphatase-1 Catalytic Subunit for Inhibition by Toxins and Mammalian Protein Inhibitors

1999; Elsevier BV; Volume: 274; Issue: 32 Linguagem: Inglês

10.1074/jbc.274.32.22366

1083-351X

John H. Connor, Theresa A. Kleeman, Sailen Barik, Richard E. Honkanen, Shirish Shenolikar,

Enzyme Structure and Function

Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the β12-β13 loop attenuated the inhibition of the resulting PP1α catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with dose-response characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1α, the PP1α catalytic core, and CRHM2 with identical IC50. Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the β12-β13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the β12-β13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins. Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the β12-β13 loop attenuated the inhibition of the resulting PP1α catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with dose-response characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1α, the PP1α catalytic core, and CRHM2 with identical IC50. Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the β12-β13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the β12-β13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins. Type-1 protein serine/threonine phosphatases (PP1) 1The abbreviations used are: PP1, protein phosphatase-1; PP2A, protein phosphatase-2A; I-1, inhibitor-1; DARPP-32, dopamine- and cAMP-regulated phosphoprotein of apparentM r 32,000; I-2, inhibitor-2; NIPP-1, nuclear inhibitor of PP1; CRHM2, a chimera of PP11–273 and PP2A267–309; GM, skeletal muscle glycogen-targeting subunit; GST, glutathione S-transferase; WT, wild-type are expressed in all eukaryotic cells and have been implicated in the control of a variety of physiological processes, including carbohydrate and lipid metabolism, protein synthesis, and gene transcription (1Bollen M. Stalmans W. Biochem. J. 1994; 311: 17-29Google Scholar, 2Shenolikar S. Annu. Rev. Cell Biol. 1994; 10: 55-86Crossref PubMed Scopus (402) Google Scholar). PP2A, the major type-2 protein serine/threonine phosphatase, shares nearly 50% sequence identity with the PP1 catalytic subunit with most of this being in sequences that organize the three-dimensional structure of the catalytic site (3Barton G.J. Cohen P.T. Barford D. Eur. J. Biochem. 1994; 220: 225-237Crossref PubMed Scopus (154) Google Scholar). Microcystin-LR and other toxins that inhibit PP1 activity also inhibit PP2A, emphasizing the shared structural determinants at or near the catalytic site that mediate phosphatase inhibition by these natural compounds (4Holmes C.F.B. Boland M.P. Curr. Opin. Struct. Biol. 1993; 3: 934-943Crossref Scopus (64) Google Scholar). Despite these similarities between the two phosphatases, cellular mechanisms that regulate PP1 and PP2A show a high degree of specificity. For instance, the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and the nuclear inhibitor NIPP-1 uniquely inhibit PP1 activity. Moreover, these PP1 inhibitors are regulated by reversible phosphorylation so that they can modulate PP1 activity in response to hormonal stimuli. Physiological studies suggest that PP1 inhibitors function as molecular switches to control cellular signaling pathways (5Oliver C.J. Shenolikar S. Front. Biosci. 1998; 3: 961-972Crossref PubMed Google Scholar). For example, I-1 and its structural homologue, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of apparentM r 32,000), once phosphorylated by cAMP-dependent protein kinase, inhibit PP1 activity to elevate and maintain cellular proteins in their phosphorylated state. In this manner, I-1 and DARPP-32 amplify and prolong cAMP signals. The importance of PP1 inhibitors in cAMP signaling was highlighted by the disruption of the mouse DARPP-32 gene (6Fienberg A.A. Hiroi N. Mermelstein P.G. Song W. Snyder G.L. Nishi A. Cheramy A. O'Callaghan J.P. Miller D.B. Cole D.G. Corbett R. Haile C.N. Cooper D.C. Onn S.P. Grace A.A. Quimet C.C. White F.J. Hyman S.E. Surmeier D.J. Girault J. Nestler E.J. Greengard P. Science. 1998; 281: 838-842Crossref PubMed Scopus (396) Google Scholar), which severely attenuated and in some cases completely ablated dopamine signaling. I-2 (7Park I.-K. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 28919-28928Abstract Full Text PDF PubMed Google Scholar) and NIPP-1 (8Jagiello I. Beullens M. Stalmans W. Bollen M. J. Biol. Chem. 1995; 270: 17257-17263Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) are two potent PP1 inhibitors whose activity is attenuated by protein phosphorylation. Inactive complexes of PP1 with I-2 or NIPP-1 have been isolated from cell extracts. These can be fully reactivated following the phosphorylation of the inhibitors. Although the phosphorylated inhibitors remain bound to PP1, they no longer suppress enzyme activity (7Park I.-K. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 28919-28928Abstract Full Text PDF PubMed Google Scholar, 9Beullens M. Van Eynde A. Bollen M. Stalmans W. J. Biol. Chem. 1993; 268: 13172-13177Abstract Full Text PDF PubMed Google Scholar). Such different modes of PP1 regulation and the lack of structural homology between I-1, I-2, and NIPP-1 suggest that there may be a variety of different mechanisms for inhibiting PP1 activity. We recently undertook a mutagenesis screen to identify regions of the PP1 catalytic subunit required for its inhibition by I-1 (10Connor J.H. Quan H.Q. Ramaswamay N.T. Zhang L. Barik S. Zheng J. Cannon J.F. Lee E.Y.C. Shenolikar S. J. Biol. Chem. 1998; 273: 27716-27724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). These studies identified multiple residues in the β12-β13 loop, a structure close to the catalytic site, as essential for effective PP1 inhibition by I-1. Deletion of C-terminal sequences containing the β12-β13 loop severely attenuated PP1 inhibition by I-1. The β12-β13 loop is also a target of natural compounds that inhibit PP1 and other protein serine/threonine phosphatases. A point mutation, C269G, in the β12-β13 loop was identified in the PP2A catalytic subunit from okadaic acid-resistant cells (11Shima H. Tohda H. Aonuma S. Nakayasu M. DePaoli-Roach A.A. Sugimura T. Nagao M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9267-9271Crossref PubMed Scopus (47) Google Scholar). The mutant PP2A required higher concentrations of the toxin than wild-type PP2A to inhibit its activity. Subsequently, Zhang et al. (12Zhang L. Zhang Z. Long F. Lee E.Y.C. Biochemistry. 1996; 35: 1606-1611Crossref PubMed Scopus (68) Google Scholar) introduced point mutations throughout the β12-β13 loop in the PP1 catalytic subunit and identified Tyr-272 as the critical determinant of its sensitivity to several toxins. Yet other studies identified mutations in the yeast calcineurin or PP2B catalytic subunit that converted Thr-350 in the β12-β13 loop to either lysine or arginine and reduced its sensitivity to inhibition by the natural product and immunosuppressive drug cyclosporin (13Cardenas M.E. Muir R.S. Breuder T. Heitman J. EMBO J. 1995; 14: 2772-2783Crossref PubMed Scopus (108) Google Scholar). Together, these data point to the β12-β13 loop as a common site for inhibitory mechanisms that impinge on this family of protein serine/threonine phosphatases. The precise contribution of the β12-β13 loop in phosphatase inhibition by structurally diverse endogenous inhibitors and natural compounds remains unknown. Thus, we undertook a detailed biochemical analysis of a PP1α catalytic subunit from which the β12-β13 loop had been deleted and a chimeric PP1α catalytic subunit that incorporated the β12-β13 loop and C-terminal sequences from a different serine/threonine phosphatase, PP2A. These studies established the absolute requirement of the β12-β13 loop structure for PP1 inhibition by many different inhibitors and suggested that the toxins and endogenous protein inhibitors recognized distinct C-terminal sequences. The role of the β12-β13 loop in binding phosphatase inhibitors and the potential role of conformation changes in this structure in PP1 inhibition are discussed. Tautomycin, microcystin-LR, okadaic acid, and calyculin A were purchased from Calbiochem. Phosphorylase kinase and phosphorylaseb were obtained from Life Technologies, Inc. [γ-32P]ATP was purchased from Amersham Pharmacia Biotech. The digoxygenin-labeling kit was obtained from Roche Molecular Biochemicals. I-2 and NIPP-1 (recombinant central domain) were kindly provided by Mathieu Bollen (Catholic University of Leuven, Belgium). I-2-Sepharose was provided by Ernest Y. C. Lee (New York Medical College). A bacterial expression vector for GST-GM, the N-terminal 215 amino acids of human skeletal muscle glycogen-targeting subunit, GM, fused to glutathione S-transferase, was provided by David L. Brautigan (University of Virginia, Charlottesville). The PP2A catalytic subunit purified from bovine brain was obtained from Brian Wadzinski of Vanderbilt University Medical School. Fostriecin was kindly provided by Parke-Davis. Recombinant human PP1α and CRHM2, a chimera consisting of residues 1–273 of human PP1α fused in-frame to residues 267–309 from the bovine PP2A catalytic subunit, were expressed as described by Walsh et al. (14Walsh A.H. Cheng A. Honkannen R.E. FEBS Lett. 1997; 416: 230-234Crossref PubMed Scopus (200) Google Scholar). Briefly, pKK223–2 containing the appropriate cDNA was transformed intoEscherichia coli JM105. The bacteria were grown in LB medium containing 1 mm MnCl2 and 50 μm isopropyl-β-d-thiogalactopyranoside for 48 h or until the absorbance at 600 nm was ∼0.6. The bacteria were sedimented by centrifugation, resuspended in 0.001 volume of 50 mm Tris-HCl, pH 7.5, containing 1 mm EDTA, 0.1% (v/v) Nonidet P-40, 0.1% (v/v) β-mercaptoethanol, and lysed by passing twice through the French press. The lysate was cleared by centrifugation (25 min at 15,000 × g). The supernatant was adjusted to 20% (v/v) glycerol and applied to heparin-Sepharose. The column was washed with 50 mm Tris-HCl, pH 7.5, containing 1 mm EDTA, 50 mm NaCl, 20% (v/v) glycerol, and 0.1% (v/v) β-mercaptoethanol. The PP1 catalytic subunit was eluted with the same buffer containing 500 mmNaCl. Fractions containing phosphorylase phosphatase activity were pooled and dialyzed against 50 mm Tris-HCl, pH 7.5, containing 1 mm EDTA, 50 mm NaCl, 20% (v/v) glycerol, and 0.1% (v/v) β-mercaptoethanol before loading on to a second heparin-Sepharose column. This time, the phosphatase was eluted using a linear gradient of the same buffer containing 50–500 mm NaCl. Both WT PP1 and CRHM2 were eluted from this column between 300 and 400 mm NaCl. Fractions containing PP1 activity were pooled, concentrated using Centricon-10, and further purified by gel filtration on Sephadex 200. This yielded a highly purified PP1 catalytic subunit represented by a 37-kDa polypeptide that accounted for 50–90% of the total protein. The PP1α catalytic core (residues 41–269) was expressed in bacteria and purified as described previously (15Ansai T. Dupuy L.C. Barik S. J. Biol. Chem. 1996; 271: 24401-24407Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Recombinant human inhibitor-1 was expressed, purified, and phosphorylated as described by Connor et al. (16Connor J.H. Oliver C.O. Quan H.N. Shenolikar S. Methods Mol. Biol. 1998; 93: 41-58PubMed Google Scholar). GST-GM was produced according to Wu et al. (17Wu J. Kleiner U. Brautigan D. Biochemistry. 1996; 35: 13858-13864Crossref PubMed Scopus (30) Google Scholar). Protein phosphatase activity was assayed by the release of [32P]phosphate from phosphorylase a as described by Shenolikar and Ingebritsen (18Shenolikar S. Ingebritsen T.S. Methods Enzymol. 1984; 107: 102-129Crossref PubMed Scopus (75) Google Scholar). The recombinant phosphatases were incubated with 10 μm phosphorylase a in 50 mmTris-HCl, pH 7.0, 1 mg/ml bovine serum albumin, 1 mmMnCl2, 0.3% (v/v) β-mercaptoethanol (total volume 60 μl) at 37 °C for 10 min. The reaction was terminated by the addition of 0.2 ml of 20% (w/v) trichloroacetic acid and 50 μl of bovine serum albumin (6–10 mg/ml). Following centrifugation at 15,000 × g for 5 min, the supernatant (200 μl) was analyzed for 32P release by liquid scintillation counting.32P release was restricted to 15–20% of the total counts present in the assay. One unit of phosphorylase phosphatase activity is defined as releasing 0.2 nmol of phosphate in 1 min in the standard assay. In assays with toxins, I-1, and NIPP1, which inhibit PP1 activity almost instantaneously, the reaction was initiated by the addition of enzyme to the substrate/inhibitor mixture preincubated at 37 °C for 10 min. However, for I-2, which shows a time-dependent inhibition of enzyme activity, the enzyme and inhibitor were preincubated for 20 min at 37 °C to ensure maximal inhibition and the assay initiated by the addition of the radiolabeled substrate. PP1 binding to microcystin-LR-Sepharose (19Campos M. Fadden P. Alms G. Qian Z. Haystead T.A.J. J. Biol. Chem. 1996; 271: 28478-28484Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), I-2-Sepharose (20Zhang Z. Zhao S. Zirattu S.D. Bai G. Lee E.Y.C. Arch. Biochem. Biophys. 1994; 308: 37-41Crossref PubMed Scopus (15) Google Scholar), and NIPP-1-Sepharose (21Beullens M. Van Eynde A. Vulsteke V. Connor J. Shenolikar S. Stalmans W. Bollen M. J. Biol. Chem. 1999; 274: 14053-14061Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) was carried out as described previously. Briefly, a 40-μl packed volume of affinity matrix was washed three times with 10 volumes of 50 mm Tris-HCl, pH 7.0, 1 mg/ml bovine serum albumin, 1 mm MnCl2, and 0.3% (v/v) β-mercaptoethanol. The beads were then incubated in 200 μl of PP1 core, WT PP1α, or CRHM2 (10 units/ml) at 4 °C for 30 min. The beads were then pelleted by centrifugation, and the supernatant (20 μl) was assayed for residual PP1 activity using phosphorylasea as substrate. PP1α and CRHM2 were covalently modified using the digoxygenin labeling kit (Roche Molecular Biochemicals). The PP1-binding proteins were separated by 10% (w/v) polyacrylamide gel electrophoresis in the presence of 0.1% (w/v) SDS and then electrophoretically transferred to the polyvinylidene difluoride membrane. The membrane was incubated with blocking solution containing 3% (w/v) dry milk in 50 mmTris-HCl, pH 7.5, containing 150 mm NaCl, and the PP1-binding proteins were detected by incubation with digoxygenin-conjugated PP1α or CRHM2 followed by an anti-digoxygenin antibody as described by Jagiello et al. (8Jagiello I. Beullens M. Stalmans W. Bollen M. J. Biol. Chem. 1995; 270: 17257-17263Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). PP1 enzymes from many species, including rabbit (22Endo S. Zhou X. Connor J. Wang B. Shenolikar S. Biochemistry. 1996; 35: 5220-5228Crossref PubMed Scopus (150) Google Scholar), fly (23Dombradi V. Gergely P. Bot G. Friederich P. Biochem. Biophys. Res. Commun. 1987; 144: 1175-1181Crossref PubMed Scopus (12) Google Scholar), and yeast (24Cohen P. Schelling D.L. Stark M.J. FEBS Lett. 1989; 250: 601-606Crossref PubMed Scopus (90) Google Scholar), are all inhibited by nanomolar concentrations of rabbit muscle I-1 and I-2. This is consistent with the high degree of structural conservation seen in human PP1α (25Barker H.M. Jones T.A. da Cruz e Silva E.F. Spurr N.K. Sheer D. Cohen P.T. Genomics. 1990; 7: 159-166Crossref PubMed Scopus (69) Google Scholar),Drosophila 87B (26Dombradi V. Axton J.M. Glover D.M. Cohen P.T. Eur. J. Biochem. 1989; 183: 603-610Crossref PubMed Scopus (56) Google Scholar), and Saccharomyces cerevisiaeGLC7 (27Feng Z.H. Wilson S.E. Peng Z.Y. Schlender K.K. Reimann E.M. Trumbly R.J. J. Biol. Chem. 1991; 266: 23796-23801Abstract Full Text PDF PubMed Google Scholar). The most significant differences in their primary sequences are restricted to their extreme N termini (3Barton G.J. Cohen P.T. Barford D. Eur. J. Biochem. 1994; 220: 225-237Crossref PubMed Scopus (154) Google Scholar) and C-terminal sequences extending beyond the β12-β13 loop (Fig.1 A). To establish the importance of the β12-β13 loop in PP1 regulation, we expressed the PP1α "catalytic core," residues 41–269, which excluded the β12-β13 loop as well as the divergent N- and C-terminal sequences. The C-terminal sequence in PP1α differs significantly from that of PP2A (3Barton G.J. Cohen P.T. Barford D. Eur. J. Biochem. 1994; 220: 225-237Crossref PubMed Scopus (154) Google Scholar), which is characterized by its insensitivity to mammalian PP1 inhibitors. Yet, PP1 and PP2A share the sequence FSAPNYC, which constitutes the N-terminal half of the β12-β13 loop (Fig.1 B). To investigate the role of PP1-specific sequences in the β12-β13 loop in enzyme inhibition by endogenous inhibitors, we produced a chimeric PP1 catalytic subunit termed CRHM2 that incorporated the N terminus of PP1α (residues 1–273) and C-terminal PP2A sequences (residues 267–309) extending beyond the conserved FSAPNYC sequence. WT PP1α, the PP1α catalytic core, and CRHM2 were expressed in E. coli, purified to near homogeneity, and analyzed for their inhibition by phosphatase inhibitors along with the PP2A catalytic subunit purified from bovine brain (Fig. 1 C). Our recent studies (10Connor J.H. Quan H.Q. Ramaswamay N.T. Zhang L. Barik S. Zheng J. Cannon J.F. Lee E.Y.C. Shenolikar S. J. Biol. Chem. 1998; 273: 27716-27724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) described the PP1α catalytic core, which is indistinguishable from WT PP1α as a phosphorylasea phosphatase. However, unlike WT PP1α (IC50200–300 nm), the PP1α core was not inhibited by thiophosphorylated I-1 at concentrations up to 20 μm. To determine if the lack of inhibition of the PP1α core also applied to other PP1 inhibitors, we analyzed its inhibition by I-2 and NIPP-1, which unlike I-1, do not require phosphorylation to inhibit phosphatase activity. Wild-type human PP1α was potently inhibited by NIPP-1 and I-2 with the half-maximal concentrations at or below 1 nm. By comparison, the PP1α core was not inhibited by either protein at greater than 100-fold higher concentrations (data not shown). Earlier studies (15Ansai T. Dupuy L.C. Barik S. J. Biol. Chem. 1996; 271: 24401-24407Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) reported that the PP1α core was resistant to selected concentrations of the toxins, okadaic acid and microcystin-LR. More detailed dose-response curves established that WT PP1α was inhibited by microcystin-LR with an IC50 of approximately 1 nm. In contrast, the PP1α core was not inhibited by microcystin-LR at concentrations exceeding 2 μm (data not shown). The PP1α core also showed a greater than 1000-fold reduction in its sensitivity to other xenobiotic compounds, including tautomycin and calyculin A (data not shown). The antitumor compound, fostriecin, inhibits PP2A (14Walsh A.H. Cheng A. Honkannen R.E. FEBS Lett. 1997; 416: 230-234Crossref PubMed Scopus (200) Google Scholar) and PP4 (28Hastie C.J. Cohen P.T. FEBS Lett. 1998; 431: 357-361Crossref PubMed Scopus (99) Google Scholar) at nanomolar concentrations but is a weaker PP1 inhibitor. Fostriecin inhibited wild-type PP1α and the catalytic core with identical IC50 slightly above 0.2 mm (Fig.2 A). In this regard, fostriecin differed from microcystin-LR and all other toxins tested. This was further emphasized by the fact that fostriecin did not effectively compete with microcystin-LR for PP1 inhibition (Fig.2 B). The IC50 for WT PP1α inhibition by microcystin-LR was identical (approximately 1 nm) in the presence or absence of 0.2 mm fostriecin. To define the specific elements within the β12-β13 loop required for PP1 inhibition, we analyzed CRHM2, which consisted of the N terminus of human PP1α (residues 1–273) fused to the C terminus (residues 267–309) of bovine PP2A Cα. Microcystin-LR inhibited WT PP1α and CRHM2 with essentially identical IC50 values of approximately 1 nm(Fig. 3 A). Tautomycin, which inhibits PP1 with a 50-fold lower IC50 than PP2A, inhibited CRHM2 with an IC50 similar to WT PP1α (Fig.3 B). Calyculin, which also shows a 10-fold preference as a PP1 inhibitor, also inhibited CRHM2 like WT PP1α with an IC50 of approximately 0.1 nm (data not shown). Finally, we confirmed the findings of Walsh et al. (14Walsh A.H. Cheng A. Honkannen R.E. FEBS Lett. 1997; 416: 230-234Crossref PubMed Scopus (200) Google Scholar) that fostriecin inhibited both WT PP1α and CRHM2 in an identical manner requiring high micromolar concentrations of the drug. By comparison, nanomolar concentrations of fostriecin inhibited PP2A activity. Thus, the sensitivity of CRHM2 to several different toxins was identical to WT PP1α and was therefore most likely defined by the N-terminal 271 residues. The PP1/PP2A chimera, CRHM2, was also analyzed for inhibition by three different mammalian PP1-specific inhibitors, I-1, I-2, and NIPP1. As I-1 is only a PP1 inhibitor when phosphorylated by cAMP-dependent protein kinase, we utilized constitutively active thiophosphorylated I-1. This avoided the possibility that CRHM2 may acquire the ability of PP2A to dephosphorylate and inactivate I-1 in the assay. As previously noted (29Endo S. Connor J.H. Forney B. Zhang L. Ingebritsen T.S. Lee E.Y.C. Shenolikar S. Biochemistry. 1997; 36: 6986-6992Crossref PubMed Scopus (40) Google Scholar), the recombinant human PP1α was less sensitive to inhibition by I-1, with an IC50 of approximately 300 nm, than PP1 catalytic subunit purified rabbit skeletal muscle (IC50 1 nm). CRHM2 behaved more like PP2A than PP1 and was not inhibited by thiophosphorylated I-1 at several hundred-fold higher concentrations (data not shown). On the other hand, I-2 inhibited WT PP1α with an IC50 of approximately 1 nm (Fig. 4), a value that is essentially identical to that obtained with the native PP1 catalytic subunit isolated from mammalian tissues. NIPP-1 was also an equally potent inhibitor of recombinant PP1α and native PP1 catalytic subunits (data not shown) with an IC50 below 1 nm. However, neither I-2 (Fig. 4) nor NIPP-1 (data not shown) inhibited CRHM2 activity at several hundred-fold higher concentrations. Thus, in contrast to toxins, where CRHM2 largely demonstrated the properties of PP1α, the presence of PP2A C-terminal sequences severely impaired CRHM2 inhibition by the mammalian PP1 inhibitors, making it more like PP2A. Early studies showed that PP1 was proteolyzed in muscle extracts to yield a 35-kDa rather than 37-kDa catalytic subunit, which lacked C-terminal sequences and was destabilized in its association with I-2 (30Tung H.Y. Cohen P. Eur. J. Biochem. 1984; 145: 57-64Crossref PubMed Scopus (68) Google Scholar). To evaluate the contribution of PP1 C-terminal sequences eliminated in the PP1α catalytic core or substituted with PP2A C-terminal sequences in CRHM2, in its association with phosphatase inhibitors, we analyzed the direct binding of these enzymes to microcystin-LR, NIPP-1, I-2, and thiophosphorylated I-1 immobilized to Sepharose. Consistent with the inhibition of WT PP1α and CRHM2 by microcystin-LR, both activities were readily adsorbed on microcystin-LR-Sepharose, which removed more than 98% of the proteins from solution (Fig. 5 A). The enzyme binding to the affinity matrix was confirmed by immunoblot analysis using an anti-PP1 monoclonal antibody (data not shown) following their release in SDS-sample buffer. The PP1α core, which was insensitive to microcystin-LR, failed to bind the immobilized toxin. Greater than 95% of enzyme activity remained in solution even after prolonged incubation with micro- cystin-LR-Sepharose. The immobilized NIPP-1, I-2, and thiophosphorylated I-1 all effectively adsorbed WT PP1α, removing between 70 and 90% of enzyme activity, as expected by the ability of these proteins to inhibit enzyme activity (Fig. 5 A). Of the three affinity matrices, thiophosphorylated I-1-Sepharose was the least effective in depleting PP1 activity, perhaps reflecting the loss in affinity of recombinant PP1 catalytic subunits for I-1 discussed above (29Endo S. Connor J.H. Forney B. Zhang L. Ingebritsen T.S. Lee E.Y.C. Shenolikar S. Biochemistry. 1997; 36: 6986-6992Crossref PubMed Scopus (40) Google Scholar, 31Alessi D.R. Street A.J. Cohen P. Cohen P.T. Eur. J. Biochem. 1993; 213: 1055-1066Crossref PubMed Scopus (167) Google Scholar). To our surprise, CRHM2, which was insensitive to the three protein inhibitors, bound all affinity matrices, albeit with slightly reduced efficacy when compared with WT PP1α. The decreased binding of CRHM2 was most notable with thiophosphorylated I-1-Sepharose. The PP1α core failed to bind any of the immobilized protein inhibitors, as expected given the inability of the mammalian PP1 inhibitors to inhibit the mutant enzyme even at very high concentrations. I-1 (22Endo S. Zhou X. Connor J. Wang B. Shenolikar S. Biochemistry. 1996; 35: 5220-5228Crossref PubMed Scopus (150) Google Scholar), I-2 (7Park I.-K. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 28919-28928Abstract Full Text PDF PubMed Google Scholar), and NIPP-1 (21Beullens M. Van Eynde A. Vulsteke V. Connor J. Shenolikar S. Stalmans W. Bollen M. J. Biol. Chem. 1999; 274: 14053-14061Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) possess multiple sites of interaction with the PP1 catalytic subunit. One key interaction is mediated through a conserved tetrapeptide (RVXF) motif present in many other PP1 regulators. Cocrystallization of the PP1 catalytic subunit with a synthetic peptide from the skeletal muscle glycogen-targeting subunit, GM, identified the RVXF-binding pocket (32Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (538) Google Scholar). Comparison of PP1α and CRHM2 predicted a substitution, cysteine 291 to tyrosine, within the RVXF-binding pocket in the chimeric enzyme, which could account for the insensitivity of CRHM2 to I-1, I-2, and NIPP-1 and its slightly reduced binding to the immobilized inhibitors. Thus, we analyzed PP1 binding to GM, whose sequence first defined the RVXF-binding pocket using a far western assay with digoxygenin-derivatized WT PP1α and CRHM2. This assay identified numerous PP1-binding proteins containing the conserved motif (19Campos M. Fadden P. Alms G. Qian Z. Haystead T.A.J. J. Biol. Chem. 1996; 271: 28478-28484Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and appears to principally monitor the association of PP1 with the RVXF motif (21Beullens M. Van Eynde A. Vulsteke V. Connor J. Shenolikar S. Stalmans W. Bollen M. J. Biol. Chem. 1999; 274: 14053-14061Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). WT PP1α and CRHM2 bound a range of concentrations of GST-GM in an identical manner (Fig.5 B). Substitution of two key residues, Val and Phe, within the RVXF motif with Ala abolished PP1α binding to GST-GM (data not shown), confirming that the association was mediated through the RVXF sequence. Thus, the several hundred-fold reduced sensitivity of CRHM2 to I-1, I-2, and NIPP-1 was not attributable to their diminished binding in the RVXF-binding pocket. Structure-function studies of four mammalian PP1 inhibitors, I-1 (22Endo S. Zhou X. Connor J. Wang B. Shenolikar S. Biochemistry. 1996; 35: 5220-5228Crossref PubMed Scopus (150) Google Scholar), DARPP-32 (33Kwon Y.-G. Huang H.-B. Desdouits F. Girault J.-A. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3536-3541Crossref PubMed Scopus (109) Google Scholar), I-2 (7Park I.-K. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 28919-28928Abstract Full Text PDF PubMed Google Scholar), and NIPP-1 (21Beullens M. Van Eynde A. Vulsteke V. Connor J. Shenolikar S. Stalmans W. Bollen M. J. Biol. Chem. 1999; 274: 14053-14061Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), established that multiple domains in these proteins are required to inhibit PP1 activity. However, the cognate regions of PP1 catalytic subunit recognized by the inhibitors remained unknown. I-1 and DARPP-32 are structural homologues, which share little sequence homology with I-2 or NIPP-1. The sole common feature in the four PP1 inhibitors is a tetrapeptide sequence known as the RVXF motif. Deletion of this sequence inactivates I-1 (22Endo S. Zhou X. Connor J. Wang B. Shenolikar S. Biochemistry. 1996; 35: 5220-5228Crossref PubMed Scopus (150) Google Scholar) and DARPP-32 (33Kwon Y.-G. Huang H.-B. Desdouits F. Girault J.-A. Greengard P. Nairn A.C