A protein phosphatase-1gamma1 isoform selectivity determinant in dendritic spine-associated neurabin

Protein phosphatase-1 (PP1) catalytic subunit isoforms interact with diverse proteins, typically containing a canonical (R/K)(V/I)XF motif. Despite sharing approximately 90% amino acid sequence identity, PP1beta and PP1gamma1 have distinct subcellular localizations that may be determined by selective interactions with PP1-binding proteins. Immunoprecipitation studies from brain and muscle extracts demonstrated that PP1gamma1 selectively interacts with spinophilin and neurabin, F-actin-targeting proteins, whereas PP1beta selectively interacted with G(M)/R(GL), the striated-muscle glycogen-targeting subunit. Glutathione S-transferase (GST) fusion proteins containing residues 146-493 of neurabin (GST-Nb-(146-493)) or residues 1-240 of G(M)/R(GL) (GST-G(M)-(1-240)) recapitulated these isoform selectivities in binding and phosphatase activity inhibition assays. Site-directed mutagenesis indicated that this isoform selectivity was not due to sequence differences between the canonical PP1-binding motifs (neurabin, (457)KIKF(460); G(M)/R(GL), (65)RVSF(68)). A chimeric GST fusion protein containing residues 1-64 of G(M)/R(GL) fused to residues 457-493 of neurabin (GST-G(M)/Nb) selectively bound to and inhibited PP1gamma1, whereas a GST-Nb/G(M) chimera containing Nb-(146-460) fused to G(M)-(69-240) selectively interacted with and weakly inhibited PP1beta, implicating domain(s) C-terminal to the (R/K)(V/I)XF motif as determinants of PP1 isoform selectivity. Deletion of Pro(464) and Ile(465) in neurabin (deltaPI) to equally space a conserved cluster of amino acids from the (R/K)(V/I)XF motif as in G(M)/R(GL) severely compromised the ability of neurabin to bind and inhibit both isoforms but did not affect PP1gamma1 selectivity. Further analysis of a series of C-terminal truncated GST-Nb-(146-493) proteins identified residues 473-479 of neurabin as containing a crucial PP1gamma1-selectivity determinant. In combination, these data identify a novel PP1gamma1-selective interaction domain in neurabin that may allow for selective regulation and/or subcellular targeting of PP1 isoforms.     

The neuronal actin-binding proteins,neurabin I and neurabin II,recruit specific isoforms of protein phosphatase-1 catalytic subunits

Neurabins are protein phosphatase-1 (PP1) targeting subunits that are highly concentrated in dendritic spines and post-synaptic densities. Immunoprecipitation of neurabin I and neurabin II/spinophilin from rat brain extracts sedimented PP1gamma1 and PP1alpha but not PP1beta. In vitro studies showed that recombinant peptides representing central regions of neurabins also preferentially bound PP1gamma1 and PP1alpha from brain extracts and associated poorly with PP1beta. Analysis of PP1 binding to chimeric neurabins suggested that sequences flanking a conserved PP1-binding motif altered their selectivity for PP1beta and their activity as regulators of PP1 in vitro. Assays using recombinant PP1 catalytic subunits and a chimera of PP1 and protein phosphatase-2A indicated that the C-terminal sequences unique to the PP1 isoforms contributed to their recognition by neurabins. Collectively, the results from several different in vitro assays established the rank order of PP1 isoform selection by neurabins to be PP1gamma1 > PP1alpha > PP1beta. This PP1 isoform selectivity was confirmed by immunoprecipitation of neurabin I and II from brain extracts from wild type and mutant PP1gamma null mice. In the absence of PP1gamma1, both neurabins showed enhanced association with PP1alpha but not PP1beta. These studies identified some of the structural determinants in PP1 and neurabins that together contribute to preferential targeting of PP1gamma1 and PP1alpha to the mammalian synapse.     

Structural basis for spinophilin-neurabin receptor interaction

Neurabin and spinophilin are neuronal scaffolding proteins that play important roles in the regulation of synaptic transmission through their ability to target protein phosphatase 1 (PP1) to dendritic spines where PP1 dephosphorylates and inactivates glutamate receptors. However, thus far, it is still unknown how neurabin and spinophilin themselves are targeted to these membrane receptors. Spinophilin and neurabin contain a single PDZ domain, a common protein-protein interaction recognition motif, which are 86% identical in sequence. We report the structures of both the neurabin and spinophilin PDZ domains determined using biomolecular NMR spectroscopy. These proteins form the canonical PDZ domain fold. However, despite their high degree of sequence identity, there are distinct and significant structural differences between them, especially between the peptide binding pockets. Using two-dimensional 1H-15N HSQC NMR analysis, we demonstrate that C-terminal peptide ligands derived from glutamatergic AMPA and NMDA receptors and cytosolic proteins directly and differentially bind spinophilin and neurabin PDZ domains. This peptide binding data also allowed us to classify the neurabin and spinophilin PDZ domains as the first identified neuronal hybrid class V PDZ domains, which are capable of binding both class I and II peptides. Finally, the ability to bind to glutamate receptor subunits suggests that the PDZ domains of neurabin and spinophilin are important for targeting PP1 to C-terminal phosphorylation sites in AMPA and NMDA receptor subunits.     

Distinct Roles of Protein Phosphatase 1 Bound on Neurabin and Spinophilin and Its Regulation in AMPA Receptor Trafficking and LTD Induction

Protein phosphatase-1 (PP1) constrains learning and memory formation in part through its effects on the induction threshold of long-term potentiation (LTP) and depression (LTD). LTD induction requires both the enzymatic activity of PP1 and its proper anchoring to synaptic spines. We have shown previously that neurabin, a major synaptic scaffolding protein, targets PP1 to synapses for LTD induction. Here, we show that PP1 bound on spinophilin, a close homolog of neurabin and another major synaptic PP1 anchoring protein, does not play a role in LTD induction, which suggests that neurabin plays a privileged role in nanodomain targeting of PP1 in LTD induction. We found that protein kinase A can significantly weaken the neurabin-PP1 interaction in neurons via phosphorylation of neurabin at serine 461, a phosphorylation site adjacent to the PP1-binding motif that is not conserved in spinophilin. Finally, we found that a neurabin mutation (S461E), which mimics phosphorylation, blocked AMPA receptor endocytosis and LTD induction. The results indicate the critical importance of nanodomain targeting of PP1 within synaptic spines and its regulation in LTD induction.     

Association of protein phosphatase 1 gamma 1 with spinophilin suppresses phosphatase activity in a Parkinson disease model

Sustained nigrostriatal dopamine depletion increases the serine/threonine phosphorylation of multiple striatal proteins that play a role in corticostriatal synaptic plasticity, including Thr(286) phosphorylation of calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha). Mechanisms underlying these changes are unclear, but protein phosphatases play a critical role in the acute modulation of striatal protein phosphorylation. Here we show that dopamine depletion for periods ranging from 3 weeks to 10 months significantly reduces the total activity of protein phosphatase (PP) 1, but not of PP2A, in whole lysates of rat striatum, as measured using multiple substrates, including Thr(286)-autophosphorylated CaMKIIalpha. Striatal PP1 activity is partially inhibited by a fragment of the PP1-binding protein neurabin-I, Nb-(146-493), because of the selective inhibition of the PP1gamma(1) isoform. The fraction of PP1 activity that is insensitive to Nb-(146-493) was unaffected by dopamine depletion, demonstrating that dopamine depletion specifically reduces the activity of PP1 isoforms that are sensitive to Nb-(146-493) (i.e. PP1gamma(1)). However, total striatal levels of PP1gamma(1) or any other PP1 isoform were unaffected by dopamine depletion, and our previous studies showed that total levels of the PP1 regulatory/targeting proteins DARPP-32, spinophilin, and neurabin were also unchanged. Rather, co-immunoprecipitation experiments demonstrated that dopamine depletion increases the association of PP1gamma(1) with spinophilin in striatal extracts. In combination, these data demonstrate that striatal dopamine depletion inhibits a specific synaptic phosphatase by increasing PP1gamma(1) interaction with spinophilin, perhaps contributing to hyperphosphorylation of synaptic proteins and disruptions of synaptic plasticity and/or dendritic morphology.     

Targeting protein phosphatase 1 (PP1) to the actin cytoskeleton: the neurabin I/PP1 complex regulates cell morphology

Neurabin I, a neuronal actin-binding protein, binds protein phosphatase 1 (PP1) and p70 ribosomal S6 protein kinase (p70S6K), both proteins implicated in cytoskeletal dynamics. We expressed wild-type and mutant neurabins fused to green fluorescent protein in Cos7, HEK293, and hippocampal neurons. Biochemical and cellular studies showed that an N-terminal F-actin-binding domain dictated neurabin I localization at actin cytoskeleton and promoted disassembly of stress fibers. Deletion of the C-terminal coiled-coil and sterile alpha motif domains abolished neurabin I dimerization and induced filopodium extension. Immune complex assays showed that neurabin I recruited an active PP1 via a PP1-docking sequence,(457)KIKF(460). Mutation of the PP1-binding motif or PP1 inhibition by okadaic acid and calyculin A abolished filopodia and restored stress fibers in cells expressing neurabin I. In vitro and in vivo studies suggested that the actin-binding domain attenuated protein kinase A (PKA) phosphorylation of neurabin I. Modification of a major PKA site, serine-461, impaired PP1 binding. Finally, p70S6K was excluded from neurabin I/PP1 complexes and required the displacement of PP1 for recruitment to neurabin I. These studies provided new insights into the assembly and regulation of a neurabin I/PP1 complex that controls actin rearrangement to promote spine development in mammalian neurons.     

The Rho-specific GEF Lfc interacts with neurabin and spinophilin to regulate dendritic spine morphology

Neurabin and spinophilin are homologous protein phosphatase 1 and actin binding proteins that regulate dendritic spine function. A yeast two-hybrid analysis using the coiled-coil domain of neurabin revealed an interaction with Lfc, a Rho GEF. Lfc was highly expressed in brain, where it interacted with either neurabin or spinophilin. In neurons, Lfc was largely found in the shaft of dendrites in association with microtubules but translocated to spines upon neuronal stimulation. Moreover, expression of Lfc resulted in reduction in spine length and size. Both the translocation and the effect on spine morphology depended on the coiled-coil domain of Lfc. Coexpression of neurabin or spinophilin with Lfc resulted in their clustering together with F-actin, a process that depended on Rho activity. Thus, interaction between Lfc and neurabin/spinophilin selectively regulates Rho-dependent organization of F-actin in spines and is a link between the microtubule and F-actin cytoskeletons in dendrites.     

Flexibility in the PP1:spinophilin holoenzyme

Protein phosphatase 1 (PP1) interacts with ∼200 regulatory proteins to form holoenzymes, which target PP1 to specific locations and regulate its specificity. While it is known that many PP1 regulatory proteins are dynamic in the unbound state, much less is known about the residual flexibility after PP1 holoenzyme formation. Here, we have used small angle X-ray scattering to investigate the flexibility of the PP1:spinophilin holoenzyme in solution. Collectively, our data shows that the PP1:spinophilin holoenzyme is dynamic in solution, which allows for an increased capture radius of spinophilin and is likely important for its biological role.

Structured summary

MINT-8057915: PP1-alpha (uniprotkb:P62136) and Spinophilin (uniprotkb:O35274) bind (MI:0407) by x ray scattering (MI:0826)     

Association of Brain Protein Phosphatase 1 with Cytoskeletal Targeting/Regulatory Subunits

Abstract: Protein phosphatase 1 catalytic subunit (PP1_C) is highly enriched in isolated rat postsynaptic densities. Gel overlay analyses using digoxigenin (DIG)-labeled PP1_C revealed four major rat brain PP1_C-binding proteins (PP1bps) with molecular masses of ≈216, 175, 134, and 75 kDa, which were (1) more abundant in brain than other rat tissues; (2) differentially expressed in microdissected brain regions; and (3) enriched in isolated cortex postsynaptic densities. PP1bp175, PP1bp134, PP1bp75, and PP1_C were partially released from forebrain particulate extracts by incubation at low ionic strength, which destabilizes the actin cytoskeleton. Size-exclusion chromatography of solubilized extracts separated two main PP1 activities (≈600 and ≈100 kDa). PP1bps and PP1_Cγ₁ were enriched in the ≈600-kDa peak, but PP1_Cβ was enriched in the ≈100-kDa peak. Furthermore, PP1bp175 and PP1bp134 exhibited lower binding of recombinant DIG-PP1_Cβ than recombinant DIG-PP1_Cγ₁ or DIG-PP1_Cα. Solubilized PP1bp175 and PP1bp134 interact with PP1_C under native conditions, because they both (1) coeluted from size-exclusion and ion-exchange columns; (2) bound to microcystin-LR-Sepharose; and (3) coprecipitated using PP1_C antibodies. Trypsinolysis of the ≈600-kDa form of PP1 increased phosphorylase a phosphatase activity approximately fourfold, suggesting that interaction of PP1_C with these PP1bps modulates its activity. Thus, brain PP1 activity is likely targeted to the cytoskeleton, including postsynaptic densities, by isoform-selective binding of PP1_C to these targeting/regulatory subunits, contributing to the specificity of its physiological roles.     

Spinophilin: from partners to functions

Spinophilin/neurabin 2 has been isolated independently by two laboratories as a protein interacting with protein phosphatase 1 (PP1) and F-actin. Gene analysis and biochemical approaches have contributed to define a number of distinct modular domains in spinophilin that govern protein-protein interactions such as two F-actin-, three potential Src homology 3 (SH3)-, a receptor- and a PP1-binding domains, a PSD95/DLG/zo-1 (PDZ) and three coiled-coil domains, and a potential leucine/isoleucine zipper (LIZ) motif. More than 30 partner proteins of spinophilin have been discovered, including cytoskeletal and cell adhesion molecules, enzymes, guanine nucleotide exchange factors (GEF) and regulator of G-protein signalling protein, membrane receptors, ion channels and others proteins like the tumour suppressor ARF. The physiological relevance of some of these interactions remains to be demonstrated. However, spinophilin structure suggests that the protein is a multifunctional protein scaffold that regulates both membrane and cytoskeletal functions. Spinophilin plays important functions in the nervous system where it is implicated in spine morphology and density regulation, synaptic plasticity and neuronal migration. Spinophilin regulates also seven-transmembrane receptor signalling and may provide a link between some of these receptors and intracellular mitogenic signalling events dependent on p70(S6) kinase and Rac G protein-GEF. Strikingly a role for spinophilin in cell growth was demonstrated and this effect was enhanced by its interaction with ARF. Here we review the current knowledge of the protein partners of spinophilin and present the available data that are contributing to the appreciation of spinophilin functions.     

Characterization and site-directed mutagenesis of a low M(r) GTP-binding protein, ram p25, expressed in Escherichia coli.

The ram gene encodes a GTP-binding protein with a M(r) of 25,068 (Nagata, K., Satoh, T., Itoh, H., Kozasa, T., Okano, Y., Doi, T., Kaziro, Y., and Nozawa, Y. (1990) FEBS Lett. 275, 29-32). It has a putative effector domain very similar to that of yeast SEC4 protein, and shares 40% identity and 60% homology with it, respectively. In order to analyze the biochemical properties, ram cDNA was engineered and inserted into a bacterial expression vector; this allowed the production at a high level of soluble recombinant ram p25 in Escherichia coli. The purified ram p25 contained an equimolar amount of GDP. The purified protein bound approximately 1 mol of [35S]guanosine 5'-O-(thiotriphosphate) GTP gamma S)/mol of protein, with a Kd value of 120 nM. [35S]GTP gamma S binding to this protein was inhibited by GTP and GDP, but not by ATP and ADP. In the presence of 10 mM Mg2+, the dissociation of [8,5'-3H]GDP and [35S]GTP gamma S from ram p25 occurred with rates of 0.015 min-1 and 0.004 min-1, respectively, showing that the ram p25 has a higher affinity for GTP than GDP. The rate of release of Pi from [gamma-32P]GTP-bound ram p25 was calculated to be 0.011 min-1. The contribution of guanine nucleotide-binding and GTP-hydrolysis domains of the protein to its biochemical activities was investigated by site-directed mutagenesis. Substitution of Val for Gly at position 19 resulted in disappearance of [35S]GTP gamma S- and [3H]GDP-binding activity in spite of good expression of the protein. Mutations of Thr41 to Ser, Ala76 to Thr, and Asn133 to His slightly increased the rates of [35S] GTP gamma S binding and [3H]GDP dissociation, but had almost no effects on the manner of [gamma-32P]GTP hydrolysis. Replacement of Gln78 with Leu significantly increased the [3H]GDP dissociation rate (7-fold) and decreased GTP hydrolytic activity considerably.     

ADP-ribosylation of the bovine brain rho protein by botulinum toxin type C1

We have separated at least six GTP-binding proteins (G proteins) with Mr values between 20,000 and 25,000 from bovine brain crude membranes (Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ideda, K., Tanimoto, T., and Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904). Three of these G proteins were copurified with the proteins ADP-ribosylated by botulinum toxin type C1. One G protein ADP-ribosylated by this toxin was identified to be the bovine brain rho protein (rho p20) which was purified to near homogeneity (Yamamoto, K., Kondo, J., Hishida, T., Teranishi, Y., and Takai, Y. (1988) J. Biol. Chem. 263, 9926-9932). rho p20 was ADP-ribosylated by botulinum toxin type C1 in time- and dose-dependent manners. About 0.4 mol of ADP-ribose was maximally incorporated into 1 mol of rho p20. The ADP-ribosylation of rho p20 was dependent on the presence of Mg2+. GTP enhanced the ADP-ribosylation in the presence of a low concentration (50 nM) of Mg2+ but not in the presence of a high concentration (0.5 mM) of Mg2+. The high concentration of Mg2+ fully stimulated the ADP-ribosylation even in the absence of GTP. The ADP-ribosylation of rho p20 did not affect its GTP gamma S-binding and GTPase activities. These results indicate that there are at least three G proteins ADP-ribosylated by botulinum toxin type C1 in bovine brain crude membranes and that one of them is rho p20. Two other G proteins have not yet been identified, but neither the c-ras protein, ADP-ribosylation factor for Gs, nor a G protein with a Mr of 24,000 was ADP-ribosylated by this toxin.     

Purification, identification, and characterization of two GTP-binding proteins with molecular weights of 25,000 and 21,000 in human platelet cytosol. One is the rap1/smg21/Krev-1 protein and the other is a novel GTP-binding protein

We have purified, characterized, and identified two GTP-binding proteins with Mr of 25,000 (c25KG) and 21,000 (c21KG) from the cytosol fraction of human platelets. These two proteins were not copurified with the beta gamma subunits of heterotrimeric GTP-binding proteins. Amino acid sequences of tryptic fragments of c21KG completely matched with those of rap1 protein (Pizon, V., Chardin, P., Lerosey, I., Olofsson, B., and Tavitian, A. (1988) Oncogene 3, 201-204), smg p21 (Kawata, M., Matsui, Y., Kondo, J., Hishida, T., Teranishi, Y., and Takai, Y. (1988) J. Biol. Chem. 263, 18965-18971), and Krev-1 protein (Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M. (1989) Cell 56, 77-84). The partial amino acid sequence analysis of c25KG revealed that this protein was different from any low Mr GTP-binding proteins already reported. c25KG bound about 1 mol of [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein, with a Kd value of about 45 nM. [35S]GTP gamma S-binding to c25KG was specifically inhibited by guanine nucleotides, GTP and GDP, but not by adenine nucleotides such as ATP and adenyl-5'-yl beta, gamma-imidodiphosphate. The binding activity was not inhibited by pretreatment with N-ethylmaleimide. c25KG hydrolyzed GTP to librate Pi with the specific activity of 1.8 mmol of Pi/mol of protein/min, which are different from the activities of the already purified low Mr GTP-binding proteins. We conclude that c25KG is a novel GTP-binding protein and c21KG is a rap1/smg p21/Krev-1 product.     

The human tumor suppressor arf interacts with spinophilin/neurabin II, a type 1 protein-phosphatase-binding protein

The INK4a gene, one of the most often disrupted loci in human cancer, encodes two unrelated proteins, p16(INK4a) and p14(ARF) (ARF) both capable of inducing cell cycle arrest. Although it has been clearly demonstrated that ARF inhibits cell cycle via p53 stabilization, very little is known about the involvement of ARF in other cell cycle regulatory pathways, as well as on the mechanisms responsible for activating ARF following oncoproliferative stimuli. In search of factors that might associate with ARF to control its activity or its specificity, we performed a yeast two-hybrid screen. We report here that the human homologue of spinophilin/neurabin II, a regulatory subunit of protein phosphatase 1 catalytic subunit specifically interacts with ARF, both in yeast and in mammalian cells. We also show that ectopic expression of spinophilin/neurabin II inhibits the formation of G418-resistant colonies when transfected into human and mouse cell lines, regardless of p53 and ARF status. Moreover, spinophilin/ARF coexpression in Saos-2 cells, where ARF ectopic expression is ineffective, somehow results in a synergic effect. These data demonstrate a role for spinophilin in cell growth and suggest that ARF and spinophilin could act in partially overlapping pathways.     

Regulation of neurabin I interaction with protein phosphatase 1 by phosphorylation.

Neurabin I is a brain-specific actin-binding protein. Here we show that neurabin I binds protein phosphatase 1 (PP1) and inhibits PP1 activity. Neurabin I interacted with PP1alpha in an overlay assay, in yeast two-hybrid interaction analysis, and in coprecipitation and co-immunoprecipitation experiments. Neurabin I also copurified with both the alpha and gamma isoforms of PP1. A glutathione S-transferase (GST)-neurabin I fusion protein (residues 318-661) containing the putative PP1 binding domain (residues 456-460) inhibited PP1 activity (K(i) = 2.7 +/- 1.2 nM). This fusion protein was also rapidly phosphorylated in vitro by PKA (K(m) = 6 microM) to a stoichiomtry of 1 mol/mol. The phosphorylated residue was identified as serine 461 by HPLC-MS analysis of a tryptic digest. Phosphorylation of GST-neurabin I (residues 318-661) by PKA significantly reduced its binding to PP1 by overlay and by glutathione-Sepharose coprecipitation assays. A 35-fold decrease in inhibitory potency was also observed using a S461E mutant, which mimics phosphorylation of S461. These findings identify a signaling mechanism involving the regulation of PP1 activity and localization mediated by the cAMP pathway.     

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin.

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.     

Origin of apparent negative cooperativity of F(1)-ATPase

In order to get insight into the origin of apparent negative cooperativity observed for F(1)-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F(1)-ATPase, alpha((W463F)3)beta((Y341W)3)gamma and alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma. For alpha((W463F)3)beta((Y341W)3)gamma, apparent K(m)'s of ATPase kinetics (4.0 and 233 microM) did not agree with apparent K(m)'s deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 microM). On the other hand, in case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, which lacks noncatalytic nucleotide binding sites, the apparent K(m) of ATPase activity (10 microM) roughly agreed with the highest K(m) of fluorescence measurements (27 microM). The results indicate that in case of alpha((W463F)3)beta((Y341W)3)gamma, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K(m) of ATPase activity does not represent the ATP binding to a catalytic site. In case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, the K(m) of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma was rather linear compared with that of alpha((W463F)3)beta((Y341W)3)gamma, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F(1)-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K(m)'s of 100-300 microM and 1-30 microM range for wild-type F(1)-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.     

Molecular investigations of the structure and function of the protein phosphatase 1-spinophilin-inhibitor 2 heterotrimeric complex

Regulation of the major Ser/Thr phosphatase protein phosphatase 1 (PP1) is controlled by a diverse array of targeting and inhibitor proteins. Though many PP1 regulatory proteins share at least one PP1 binding motif, usually the RVxF motif, it was recently discovered that certain pairs of targeting and inhibitor proteins bind PP1 simultaneously to form PP1 heterotrimeric complexes. To date, structural information for these heterotrimeric complexes and, in turn, how they direct PP1 activity is entirely lacking. Using a combination of NMR spectroscopy, biochemistry, and small-angle X-ray scattering (SAXS), we show that major structural rearrangements in both spinophilin (targeting) and inhibitor 2 (I-2, inhibitor) are essential for the formation of the heterotrimeric PP1-spinophilin-I-2 (PSI) complex. The RVxF motif of I-2 is released from PP1 during the formation of PSI, making the less prevalent SILK motif of I-2 essential for complex stability. The release of the I-2 RVxF motif allows for enhanced flexibility of both I-2 and spinophilin in the heterotrimeric complex. In addition, we used inductively coupled plasma atomic emission spectroscopy to show that PP1 contains two metals in both heterodimeric complexes (PP1-spinophilin and PP1-I-2) and PSI, demonstrating that PSI retains the biochemical characteristics of the PP1-I-2 holoenzyme. Finally, we combined the NMR and biochemical data with SAXS and molecular dynamics simulations to generate a structural model of the full heterotrimeric PSI complex. Collectively, these data reveal the molecular events that enable PP1 heterotrimeric complexes to exploit both the targeting and inhibitory features of the PP1-regulatory proteins to form multifunctional PP1 holoenzymes.     

Partial characterization of a glycoprotein comprising the NH2-terminal region of mouse tumor cell pro-adrenocorticotropic hormone/endorphin.

The glycoprotein accounting for most of the nonadrenocorticotropic hormone (ACTH), non-beta-lipotropin (beta LPH) region of mouse tumor cell pro-ACTH/endorphin was purified from tumor cell culture medium and shown to contain 1/2 cystine residues. Preparations of the 16,000-dalton fragment-related material (referred to as 16K fragment) were heterogeneous with respect to size and charge. Despite this heterogeneity, a partial amino acid sequence for the NH2-terminal region of the molecule was determined by automated Edman degradationof the 16K fragment labeled by reduction and alkylation with [3H]iodoacetic acid or labeled biosynthetically with [3H]tryptophan. The sequence of 1/2 cystine and tryptophan residues in the mouse tumor 16K fragment can be aligned with one region of the amino acid sequence predicted from the cDNA for a bovine precursor to ACTH/beta LPH (Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A.C.Y., Cohen, S.N., and Numa, S. (1979) Nature 278, 423--427).     

Characterisation of two protein phosphatase 2A holoenzymes from maize seedlings

Two holoenzymes of protein phosphatase 2A (PP2A), designated PP2AI and PP2AII, were purified from maize seedlings. The subunit composition of maize holoenzymes generally resembled those of animal PP2A. Using SDS/PAGE and Western blots with antibodies generated against peptides derived from animal PP2A, we established the subunit composition of plant protein phosphatase 2A. In both maize holoenzymes, a 38000 catalytic (PP2Ac) and a 66000 constant regulatory subunit (A) constituting the core dimer of PP2A were present. In addition, PP2AI (180000-200000) contained a protein of 57000 which reacted with antibodies generated against the peptide (EFDYLKSLEIEE) conserved in all eukaryotic Balpha regulatory subunits. In contrast, none of the proteins visualised in PP2AII (140000-160000) by double staining reacted with these antibodies. The activity of PP2AI measured with (32)P-labelled phosphorylase a in the presence of protamine and ammonium sulfate is about two times higher than that of PP2AII. PP2AI and PP2AII displayed different patterns of activation by protamine, polylysine and histone H1 and exhibit high sensitivity toward inhibition by okadaic acid. The data obtained provide direct biochemical evidence for the existence in plants of PP2A holoenzymes composed of a catalytic subunit complexed with one or two regulatory subunits.