Brain actin-associated protein phosphatase 1 holoenzymes containing spinophilin, neurabin, and selected catalytic subunit isoforms

We previously characterized PP1bp134 and PP1bp175, two neuronal proteins that bind the protein phosphatase 1 catalytic subunit (PP1). Here we purify from rat brain actin-cytoskeletal extracts PP1(A) holoenzymes selectively enriched in PP1gamma(1) over PP1beta isoforms and also containing PP1bp134 and PP1bp175. PP1bp134 and PP1bp175 were identified as the synapse-localized F-actin-binding proteins spinophilin (Allen, P. B., Ouimet, C. C., and Greengard, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9956-9561; Satoh, A., Nakanishi, H., Obaishi, H., Wada, M., Takahashi, K., Satoh, K., Hirao, K., Nishioka, H., Hata, Y., Mizoguchi, A., and Takai, Y. (1998) J. Biol. Chem. 273, 3470-3475) and neurabin (Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H. , Matsuura, Y., Mizoguchi, A., and Takai, Y. (1997) J. Cell Biol. 139, 951-961), respectively. Recombinant spinophilin and neurabin interacted with endogenous PP1 and also with each other when co-expressed in HEK293 cells. Spinophilin residues 427-470, or homologous neurabin residues 436-479, were sufficient to bind PP1 in gel overlay assays, and selectively bound PP1gamma(1) from a mixture of brain protein phosphatase catalytic subunits; additional N- and C-terminal sequences were required for potent inhibition of PP1. Immunoprecipitation of spinophilin or neurabin from crude brain extracts selectively coprecipitated PP1gamma(1) over PP1beta. Moreover, immunoprecipitation of PP1gamma(1) from brain extracts efficiently coprecipitated spinophilin and neurabin, whereas PP1beta immunoprecipitation did not. Thus, PP1(A) holoenzymes containing spinophilin and/or neurabin target specific neuronal PP1 isoforms, facilitating efficient regulation of synaptic phosphoproteins.     

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.     

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.     

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.     

Interactor-mediated nuclear translocation and retention of protein phosphatase-1

Protein Ser/Thr phosphatase-1 (PP1) is a ubiquitous eukaryotic enzyme that controls numerous cellular processes by the dephosphorylation of key regulatory proteins. PP1 is expressed in various cellular compartments but is most abundant in the nucleus. We have examined the determinants for the nuclear localization of enhanced green fluorescent protein-tagged PP1 in COS1 cells. Our studies show that PP1gamma(1) does not contain a functional nuclear localization signal and that its nuclear accumulation does not require Sds22, which has previously been implicated in the nuclear accumulation of PP1 in yeast (Peggie, M. W., MacKelvie, S. H., Bloecher, A., Knatko, E. V., Tatchell, K., and Stark, M. J. R. (2002) J. Cell Sci. 115, 195-206). However, the nuclear targeting of PP1 isoforms was alleviated by the mutation of their binding sites for proteins that interact via an RVXF motif. Moreover, one of the mutants with a cytoplasmic accumulation and decreased affinity for RVXF motifs (PP1gamma(1)-F257A) could be re-targeted to the nucleus by the overexpression of nuclear interactors (NIPP1 (nuclear inhibitor of PP1) and PNUTS (PP1 nuclear targeting subunit)) with a functional RVXF motif. Also, the addition of a synthetic RVXF-containing peptide to permeabilized cells resulted in the loss of nuclear enhanced green fluorescent protein-PP1gamma(1). Finally, NIPP1(-/-) mouse embryos showed a nuclear hyperphosphorylation on threonine, consistent with a role for NIPP1 in the nuclear targeting and/or retention of PP1. Our data suggest that both the nuclear translocation and the nuclear retention of PP1 depend on its binding to interactors with an RVXF motif.     

Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1.

The diverse forms of protein phosphatase 1 in vivo result from the association of its catalytic subunit (PP1c) with different regulatory subunits, one of which is the G-subunit (G(M)) that targets PP1c to glycogen particles in muscle. Here we report the structure, at 3.0 A resolution, of PP1c in complex with a 13 residue peptide (G(M[63-75])) of G(M). The residues in G(M[63-75]) that interact with PP1c are those in the Arg/Lys-Val/Ile-Xaa-Phe motif that is present in almost every other identified mammalian PP1-binding subunit. Disrupting this motif in the G(M[63-75]) peptide and the M(110[1-38]) peptide (which mimics the myofibrillar targeting M110 subunit in stimulating the dephosphorylation of myosin) prevents these peptides from interacting with PP1. A short peptide from the PP1-binding protein p53BP2 that contains the RVXF motif also interacts with PP1c. These findings identify a recognition site on PP1c, invariant from yeast to humans, for a critical structural motif on regulatory subunits. This explains why the binding of PP1 to its regulatory subunits is mutually exclusive, and suggests a novel approach for identifying the functions of PP1-binding proteins whose roles are unknown.     

Neurabins recruit protein phosphatase-1 and inhibitor-2 to the actin cytoskeleton

Inhibitor-2 (I-2) bound protein phosphatase-1 (PP1) and several PP1-binding proteins from rat brain extracts, including the actin-binding proteins, neurabin I and neurabin II. Neurabins from rat brain lysates were sedimented by I-2 and its structural homologue, I-4. The central domain of both neurabins bound PP1 and I-2, and mutation of a conserved PP1-binding motif abolished neurabin binding to both proteins. Microcystin-LR, a PP1 inhibitor, also attenuated I-2 binding to neurabins. Immunoprecipitation of neurabin I established its association with PP1 and I-2 in HEK293T cells and suggested that PP1 mediated I-2 binding to neurabins. The C terminus of I-2, although not required for PP1 binding, facilitated PP1 recruitment by neurabins, which also targeted I-2 to polymerized F-actin. Mutations that attenuated PP1 binding to I-2 and neurabin I suggested distinct and overlapping sites for these two proteins on the PP1 catalytic subunit. Immunocytochemistry in epithelial cells and cultured hippocampal neurons showed that endogenous neurabin II and I-2 colocalized at actin-rich structures, consistent with the ability of neurabins to target the PP1.I-2 complex to actin cytoskeleton and regulate cell morphology.     

Interaction of inhibitor-2 with the catalytic subunit of type 1 protein phosphatase. Identification of a sequence analogous to the consensus type 1 protein phosphatase-binding motif

Inhibitor-2 (I-2) is the regulatory subunit of a cytosolic type 1 Ser/Thr protein phosphatase (PP1) and potently inhibits the activity of the free catalytic subunit (CS1). Previous work from the laboratory had proposed that the interaction of I-2 with CS1 involved multiple sites (Park, I. K., and DePaoli-Roach, A. A. (1994) J. Biol. Chem. 269, 28919-28928). The present study refines the earlier analysis and arrives at a more detailed model for the interaction between I-2 and CS1. Although the NH(2)-terminal I-2 regions containing residues 1-35 and 1-64 have no inhibitory activity on their own, they increase the IC(50) for I-2 by approximately 30-fold, indicating the presence of a CS1-interacting site. Based on several experimental approaches, we have also identified the sequence Lys(144)-Leu-His-Tyr(147) as a second site of interaction that corresponds to the RVXF motif present in many CS1-binding proteins. The peptide I-2(135-151) significantly increases the IC(50) for I-2 and attenuates CS1 inhibition. Replacement of Leu and Tyr with Ala abolishes the ability to counteract inhibition by I-2. The I-2(135-151) peptide, but not I-2(1-35), also antagonizes inhibition of CS1 by DARPP-32 in a pattern similar to that of I-2. Furthermore, a peptide derived from the glycogen-binding subunit, R(GL)/G(M)(61-80), which contains a consensus CS1-binding motif, completely counteracts CS1 inhibition by I-2 and DARPP-32. The NH(2)-terminal 35 residues of I-2 bind to CS1 at a site that is specific for I-2, whereas the KLHY sequence interacts with CS1 at a site shared with other interacting proteins. Other results suggest the presence of yet more sites of interaction. A model is presented in which multiple "anchoring interactions" serve to position a segment of I-2 such that it sterically occludes the catalytic pocket but need not make high affinity contacts itself.     

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 muscle-specific protein phosphatase PP1G/R(GL)(G(M))is essential for activation of glycogen synthase by exercise

In skeletal muscle both insulin and contractile activity are physiological stimuli for glycogen synthesis, which is thought to result in part from the dephosphorylation and activation of glycogen synthase (GS). PP1G/R(GL)(G(M)) is a glycogen/sarcoplasmic reticulum-associated type 1 phosphatase that was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P. G., Zhang, H., Jie Yang, J., Cooper, L. D., Steele, M., Kennedy, A., Bock, C., Scrimgeour, A., Lawrence, J. C. Jr., L., and DePaoli-Roach, A. A. (2001) Mol. Cell. Biol. 21, 2683-2694) that insulin activates GS in muscle of R(GL)(G(M)) knockout (KO) mice similarly to the wild type (WT). To determine whether PP1G is involved in glycogen metabolism during muscle contractions, R(GL) KO and overexpressors (OE) were subjected to two models of contraction, in vivo treadmill running and in situ electrical stimulation. Both procedures resulted in a 2-fold increase in the GS -/+ glucose-6-P activity ratio in WT mice, but this response was completely absent in the KO mice. The KO mice, which also have a reduced GS activity associated with significantly reduced basal glycogen levels, exhibited impaired maximal exercise capacity, but contraction-induced activation of glucose transport was unaffected. The R(GL) OE mice are characterized by enhanced GS activity ratio and an approximately 3-4-fold increase in glycogen content in skeletal muscle. These animals were able to tolerate exercise normally. Stimulation of GS and glucose uptake following muscle contraction was not significantly different as compared with WT littermates. These results indicate that although PP1G/R(GL) is not necessary for activation of GS by insulin, it is essential for regulation of glycogen metabolism under basal conditions and in response to contractile activity, and may explain the reduced muscle glycogen content in the R(GL) KO mice, despite the normal insulin activation of GS.     

Analysis of Ppp1cc-null mice suggests a role for PP1gamma2 in sperm morphogenesis

Serine/threonine protein phosphatase 1 (PP1) consists of four ubiquitously expressed major isoforms, two of which, PP1gamma1 and PP1gamma2, are derived by alternative splicing of a single gene, Ppp1cc. PP1gamma2 is the most abundant isoform in the testis, and is a key regulator of sperm motility. Targeted disruption of the Ppp1cc gene causes male infertility in mice due to impaired spermiogenesis. This study was undertaken to determine the expression patterns of specific PP1 isoforms in testes of wild-type mice and to establish how the defects produced in Ppp1cc-null developing sperm are related to the loss of PP1gamma isoform expression. We observed that PP1gamma2 was prominently expressed in the cytoplasm of secondary spermatocytes and round spermatids as well as in elongating spermatids and testicular and epididymal spermatozoa, whereas its expression was weak or absent in spermatogonia, pachytene spermatocytes, and interstitial cells. In contrast, a high level of PP1gamma1 expression was observed in interstitial cells, whereas much weaker expression was observed in all stages of spermatogenesis. Another PP1 isoform, PP1alpha, was predominant in spermatogonia, pachytene spermatocytes, and interstitial cells. Examining the temporal expression of PP1 enzymes in testes revealed a striking postnatal increase in PP1gamma2 levels compared with other isoforms. Testicular sperm tails from Ppp1cc-null mice showed malformed mitochondrial sheaths and extra outer dense fibers in both the middle and principal pieces. These data suggest that in addition to its previously documented role in motility, PP1gamma2 is involved in sperm tail morphogenesis.     

Reversal of diet-induced glucose intolerance by hepatic expression of a variant glycogen-targeting subunit of protein phosphatase-1.

Glycogen-targeting subunits of protein phosphatase-1 facilitate interaction of the phosphatase with enzymes of glycogen metabolism. Expression of one family member, PTG, in the liver of normal rats improves glucose tolerance without affecting other plasma variables but leaves animals unable to reduce hepatic glycogen stores in response to fasting. In the current study, we have tested whether expression of other targeting subunit isoforms, such as the liver isoform G(L), the muscle isoform G(M)/R(Gl), or a truncated version of G(M)/R(Gl) termed G(M)DeltaC in liver ameliorates glucose intolerance in rats fed on a high fat diet (HF). HF animals overexpressing G(M)DeltaC, but not G(L) or G(M)/R(Gl), exhibited a decline in blood glucose of 35-44 mg/dl relative to control HF animals during an oral glucose tolerance test (OGTT) such that levels were indistinguishable from those of normal rats fed on standard chow at all but one time point. Hepatic glycogen levels were 2.1-2.4-fold greater in G(L)- and G(M)DeltaC-overexpressing HF rats compared with control HF animals following OGTT. In a second set of studies on fed and 20-h fasted HF animals, G(M)DeltaC-overexpressing rats lowered their liver glycogen levels by 57% (from 402 +/- 54 to 173 +/- 27 microg of glycogen/mg of protein) in the fasted versus fed states compared with only 44% in G(L)-overexpressing animals (from 740 +/- 35 to 413 +/- 141 microg of glycogen/mg of protein). Since the OGTT studies were performed on 20-h fasted rats, this meant that G(M)DeltaC-overexpressing rats synthesized much more glycogen than G(L)-overexpressing HF rats during the OGTT (419 versus 117 microg of glycogen/mg of protein, respectively), helping to explain why G(M)DeltaC preferentially enhanced glucose clearance. We conclude that G(M)DeltaC has a unique combination of glycogenic potency and responsiveness to glycogenolytic signals that allows it to be used to lower blood glucose levels in diabetes.     

Importance of a surface hydrophobic pocket on protein phosphatase-1 catalytic subunit in recognizing cellular regulators

Cellular functions of protein phosphatase-1 (PP1), a major eukaryotic serine/threonine phosphatase, are defined by the association of PP1 catalytic subunits with endogenous protein inhibitors and regulatory subunits. Many PP1 regulators share a consensus RVXF motif, which docks within a hydrophobic pocket on the surface of the PP1 catalytic subunit. Although these regulatory proteins also possess additional PP1-binding sites, mutations of the RVXF sequence established a key role of this PP1-binding sequence in the function of PP1 regulators. WT PP1alpha, the C-terminal truncated PP1alpha-(1-306), a chimeric PP1alpha containing C-terminal sequences from PP2A, another phosphatase, PP1alpha-(1-306) with the RVXF-binding pocket substitutions L289R, M290K, and C291R, and PP2A were analyzed for their regulation by several mammalian proteins. These studies established that modifications of the RVXF-binding pocket had modest effects on the catalytic activity of PP1, as judged by recognition of substrates and sensitivity to toxins. However, the selected modifications impaired the sensitivity of PP1 to the inhibitor proteins, inhibitor-1 and inhibitor-2. In addition, they impaired the ability of PP1 to bind neurabin-I, the neuronal regulatory subunit, and G(M), the skeletal muscle glycogen-targeting subunit. These data suggested that differences in RVXF interactions with the hydrophobic pocket dictate the affinity of PP1 for cellular regulators. Substitution of a distinct RVXF sequence in inhibitor-1 that enhanced its binding and potency as a PP1 inhibitor emphasized the importance of the RVXF sequence in defining the function of this and other PP1 regulators. Our studies suggest that the diversity of RVXF sequences provides for dynamic physiological regulation of PP1 functions in eukaryotic cells.     

Repo-Man recruits PP1 gamma to chromatin and is essential for cell viability

Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase regulating many cellular processes. PP1alpha and -gamma are closely related isoforms with distinct localization patterns, shown here by time-lapse microscopy of stably expressed fluorescent protein fusions. A pool of PP1gamma is selectively loaded onto chromatin at anaphase. Using stable isotope labeling and proteomics, we identified a novel PP1 binding protein, Repo-Man, which selectively recruits PP1gamma onto mitotic chromatin at anaphase and into the following interphase. This approach revealed both novel and known PP1 binding proteins, quantitating their relative distribution between PP1alpha and -gamma in vivo. When overexpressed, Repo-Man can also recruit PP1alpha to chromatin. Mutating Repo-Man's PP1 binding domain does not disrupt chromatin binding but abolishes recruitment of PP1 onto chromatin. RNA interference-induced knockdown of Repo-Man caused large-scale cell death by apoptosis, as did overexpression of this dominant-negative mutant. The data indicate that Repo-Man forms an essential complex with PP1gamma and is required for the recruitment of PP1 to chromatin.     

Eliminating phosphorylation sites of the parathyroid hormone receptor type 1 differentially affects stimulation of phospholipase C and receptor internalization

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTH1R) belongs to family B of seven-transmembrane-spanning receptors and is activated by PTH and PTHrP. Upon PTH stimulation, the rat PTH1R becomes phosphorylated at seven serine residues. Elimination of all PTH1R phosphorylation sites results in prolonged cAMP accumulation and impaired internalization in stably transfected LLC-PK1 cells. The present study explores the role of individual PTH1R phosphorylation sites in PTH1R signaling through phospholipase C, agonist-dependent receptor internalization, and regulation by G protein-coupled receptor kinases. By means of transiently transfected COS-7 cells, we demonstrate that the phosphorylation-deficient (pd) PTH1R confers dramatically enhanced coupling to G(q/11) proteins upon PTH stimulation predominantly caused by elimination of Ser(491/492/493), Ser(501), or Ser(504). Reportedly, impaired internalization of the pd PTH1R, however, is not dependent on a specific phosphorylation site. In addition, we show that G protein-coupled receptor kinase 2 interferes with pd PTH1R signaling to G(q/11) proteins at least partially by direct binding to G(q/11) proteins.     

A testis specific isoform of endophilin B1, endophilin B1t, interacts specifically with protein phosphatase-1c gamma2 in mouse testis and is abnormally expressed in PP1c gamma null mice

Male mice homozygous for a null mutation in the protein phosphatase-1c gamma (PP1c gamma) gene are infertile, displaying a severe impairment in spermatogenesis that is not compensated by the presence of PP1c alpha and PP1c beta in mutant testes. A lack of the PP1c gamma2 splice variant seems the most likely cause of the mutant phenotype, as it is the most heavily expressed PP1c gamma isoform in wild type testes. Yeast two-hybrid screening using PP1c gamma2 has identified several new binding partners, including endophilin B1t, a testis enriched isoform of endophilin B1a which differs from the somatic form by virtue of a carboxy terminal deletion spanning the last 10 amino acids. The testis isoform did not show an interaction with PP1c alpha, or with a truncated PP1c gamma2 mutant lacking the unique carboxy terminus. In contrast, somatic endophilin B1a did not interact with any of the PP1c isoforms. Sedimentation and co-immunoprecipitation experiments using native testis proteins verified binding of endophilin B1t to PP1c gamma2. Immunohistochemistry on wild type testis sections revealed a stage specific expression pattern for endophilin that appeared concentrated at discrete puncta throughout the seminiferous epithelium. Punctate endophilin expression in cells adjacent to the lumen was absent in PP1c gamma null mice. Phosphatase assays indicate that chimeric endophilin B1t is able to inhibit recombinant PP1c gamma2 activity toward phosphorylase a while having little effect on the activity of PP1c alpha. A potential role for endophilin B1t in mammalian spermatogenesis is discussed within the context of the PP1c gamma knockout testis phenotype.     

Differential expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest

In the present study, we examined the expression and cytolocalization of protein phosphatase type 1 (PP1) isoforms and nucleolin in human osteoblastic cell line MG63 cells at two boundaries in the cell cycle. We treated MG63 cells with hydroxyurea and nocodazole to arrest the cells at the G(1)/S and G(2)/M boundaries, respectively. As judged from the results of Western blot analysis, PP1 isoforms were expressed differently at each boundary of the cell cycle. Nucleolin was also shown to have a different expression pattern at each boundary. In the hydroxyurea-treated cells, nucleolus-like bodies were bigger in size and decreased in number compared with those in asynchronized cells. However, the subcellular localization of PP1s and nucleolin was not changed. Anti-nucleolin antibody interacted with 110-kDa and 95-kDa proteins present in asynchronized cells and in the cells treated with hydroxyurea. Treatment of the cells with nocodazole decreased the level of the 95-kDa form of nucleolin. In the nocodazole-treated cells, it was impossible to distinguish the distribution of each protein. The phosphorylation status of nucleolin in the cell cycle arrested samples was examined by 2D-IEF-PAGE followed by Western blot analysis. In the case of asynchronized cells or hydroxyurea-treated ones, nucleolin was located at a basic isoelectric point (dephosphorylated status); whereas in the G(2)/M arrest cells, the isoelectric point of nucleolin shifted to an acidic status, indicating that nucleolin was phosphorylated. The present results indicate that PP1 and nucleolin were differently expressed at G(1)/S and G(2)/M boundaries of the cell cycle and acted in a different fashion during cell-cycle progression.     

Regulation of MYPT1 stability by the E3 ubiquitin ligase SIAH2

Myosin phosphatase target subunit 1 (MYPT1), together with catalytic subunit of type1 δ isoform (PP1cδ) and a small 20-kDa regulatory unit (M20), form a heterotrimeric holoenzyme, myosin phosphatase (MP), which is responsible for regulating the extent of myosin light chain phosphorylation. Here we report the identification and characterization of a molecular interaction between Seven in absentia homolog 2 (SIAH2) and MYPT1 that resulted in the proteasomal degradation of the latter in mammalian cells, including neurons and glia. The interaction involved the substrate binding domain of SIAH2 (aa 116-324) and a central region of MYPT1 (aa 445-632) containing a degenerate consensus Siah-binding motif RLAYVAP (aa 493-499) evolutionally conserved from fish to humans. These findings suggest a novel mechanism whereby the ability of MP to modulate myosin light chain might be regulated by the degradation of its targeting subunit MYPT1 through the SIAH2-ubiquitin-proteasomal pathway. In this manner, the turnover of MYPT1 would serve to limit the duration and/or magnitude of MP activity required to achieve a desired physiological effect.     

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.