PP1-binding Motif Research Articles

CAVPENET Peptide Inhibits Prostate Cancer Cells Proliferation and Migration through PP1γ-Dependent Inhibition of AKT Signaling.

Protein phosphatase 1 (PP1) complexes have emerged as promising targets for anticancer therapies. The ability of peptides to mimic PP1-docking motifs, and so modulate interactions with regulatory factors, has enabled the creation of highly selective modulators of PP1-dependent cellular processes that promote tumor growth. The major objective of this study was to develop a novel bioactive cell-penetrating peptide (bioportide), which, by mimicking the PP1-binding motif of caveolin-1 (CAV1), would regulate PP1 activity, to hinder prostate cancer (PCa) progression. The designed bioportide, herein designated CAVPENET, and a scrambled homologue, were synthesized using microwave-assisted solid-phase methodologies and evaluated using PCa cell lines. Our findings indicate that CAVPENET successfully entered PCa cells to influence both viability and migration. This tumor suppressor activity of CAVPENET was attributed to inhibition of AKT signaling, a consequence of increased PP1γ activity. This led to the suppression of glycolytic metabolism and alteration in lipid metabolism, collectively representing the primary mechanism responsible for the anticancer properties of CAVPENET. Our results underscore the potential of the designed peptide as a novel therapy for PCa patients, setting the stage for further testing in more advanced models to fully realize its therapeutic promise.

Protein phosphatase PP1 regulation of RNA polymerase II transcription termination and allelic exclusion of VSG genes in trypanosomes.

The genomes of Leishmania and trypanosomes are organized into polycistronic transcription units flanked by a modified DNA base J involved in promoting RNA polymerase II (Pol II) termination. We recently characterized a Leishmania complex containing a J-binding protein, PP1 protein phosphatase 1, and PP1 regulatory protein (PNUTS) that controls transcription termination potentially via dephosphorylation of Pol II by PP1. While T. brucei contains eight PP1 isoforms, none purified with the PNUTS complex, complicating the analysis of PP1 function in termination. We now demonstrate that the PP1-binding motif of TbPNUTS is required for function in termination in vivo and that TbPP1-1 modulates Pol II termination in T. brucei and dephosphorylation of the large subunit of Pol II. PP1-1 knock-down results in increased cellular levels of phosphorylated RPB1 accompanied by readthrough transcription and aberrant transcription of the chromosome by Pol II, including Pol I transcribed loci that are typically silent, such as telomeric VSG expression sites involved in antigenic variation. These results provide important insights into the mechanism underlying Pol II transcription termination in primitive eukaryotes that rely on polycistronic transcription and maintain allelic exclusion of VSG genes.

Substrate recognition determinants of human eIF2α phosphatases.

Phosphorylation of the translation initiation factor eIF2α is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2α phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Here, we generated a series of R15 truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A325-554 and R15B340-639. G-actin, which has been proposed to confer selectivity to R15 phosphatases, does not bind these regions, indicating that it is not required for substrate binding. Fragments containing the substrate-binding regions but lacking the PP1-binding motif trapped the phospho-substrate and caused accumulation of phosphorylated eIF2α in unstressed cells. Activity assays in cells showed that R15A325-674 and R15B340-713, encompassing the substrate-binding region and the PP1-binding region, exhibit wild-type activity. This work identifies the substrate-binding region in R15s, that functions as a phospho-substrate trapping mutant, thereby defining a key region of R15s for follow up studies.

PP1 regulatory subunit NIPP1 regulates transcription of E2F1 target genes following DNA damage.

DNA damage induces transcriptional repression of E2F1 target genes and a reduction in histone H3‐Thr11 phosphorylation (H3‐pThr11) at E2F1 target gene promoters. Dephosphorylation of H3‐pThr11 is partly mediated by Chk1 kinase and protein phosphatase 1γ (PP1γ) phosphatase. Here, we isolated NIPP1 as a regulator of PP1γ‐mediated H3‐pThr11 by surveying nearly 200 PP1 interactor proteins. We found that NIPP1 inhibits PP1γ‐mediated dephosphorylation of H3‐pThr11 both in vivo and in vitro. By generating NIPP1‐depleted cells, we showed that NIPP1 is required for cell proliferation and the expression of E2F1 target genes. Upon DNA damage, activated protein kinase A (PKA) phosphorylated the NIPP1‐Ser199 residue, adjacent to the PP1 binding motif (RVxF), and triggered the dissociation of NIPP1 from PP1γ, leading to the activation of PP1γ. Furthermore, the inhibition of PKA activity led to the activation of E2F target genes. Statistical analysis confirmed that the expression of NIPP1 was positively correlated with E2F target genes. Taken together, these findings demonstrate that the PP1 regulatory subunit NIPP1 modulates E2F1 target genes by linking PKA and PP1γ during DNA damage.

Exploring the Intracellular‐pH Sensitivity of the Electrogenic Sodium Bicarbonate Cotransporter NBCe1‐B when Co‐expressed with its Cytosolic Binding Partner IRBIT

The electrogenic Na/HCO3 cotransporter (NBCe1), by transferring HCO3− across the cell plasma membrane, plays a major role in regulating intracellular pH (pHi)—a parameter that must be maintained within a tight range to support normal physiological functions. In addition, NBCe1 mediates HCO3− movement across epithelial cells. Because of its role in pHi regulation, we ask whether the activity of this transporter depends on pHi. Recently, we addressed this question by investigating the pHi‐dependence of NBCe1‐A, the variant of NBCe1 that is most abundant in the kidney. Surprisingly, we found that, when expressed in oocytes, the activity of NBCe1‐A is not pHi sensitive in the pH range of 6.70–7.50 (Occhipinti and Boron, 2019 FASEB J, 33 (1): 544.2). Here, we extend the above study to NBCe1‐B, the variant of NBCe1 that is more widespread, but especially abundant in the pancreas. Because the activity of NBCe1‐B is naturally low when expressed by itself, we investigate the pHi‐dependence of NBCe1‐B when co‐expressed with its cytosolic binding partner IRBIT [inositol trisphosphate (IP3)‐receptor binding protein released with IP3], which is known to relieve autoinhibition by binding to the NH2‐terminus. As done previously, we employ ion‐sensitive microelectrodes and two‐electrode voltage clamping to record pHi and NBCe1‐conductance (GNBC) on Xenopus oocytes injected with either H2O (control) or cRNA encoding NBCe1‐B with IRBIT or super‐IRBIT (which lacks the PP1 binding motif). Again, we use out‐of‐equilibrium (OOE) CO2/HCO3− solutions to control pHi as we vary bulk extracellular [CO2] ([CO2]o) and keep [Na+]o, pHo, and [HCO3−]o constant. After exposing oocytes to ND96 (free of CO2 and HCO3−) and obtaining a first set of current‐voltage recordings (IV#1), we switch the superfusate to a “pure” HCO3− OOE solution (0% CO2/33 mM HCO3−/pH 7.50). In the case of NBCe1‐expressing oocytes, exposure to “pure” HCO3− leads to the entry of HCO3− into the cell, thereby raising pHi. Once pHi reaches ~7.50, we take a second set of current‐voltage recordings (IV#2). We then switch the bath solution to an OOE solution containing either 2.5% CO2 or 20% CO2 and, once pHi has reached its nadir (lower in the case of 20% CO2), take a third set of current‐voltage recordings (IV#3). For each experiment, we compute GNBC as the slope of the I‐V curve in the presence of HCO3− minus the slope of the I‐V curve in the absence of HCO3− (i.e., in ND96). We compute GNBC between −20 and +20 mV, where the net NBCe1‐mediated HCO3− flux is strongly inward. To account for variability in protein expression levels, we normalize GNBC from IV#3 to GNBC from IV#2. Analysis of normalized GNBC reveals that the activity of NBCe1‐B, whether co‐expressed with IRBIT or with super‐IRBIT, does not change between pHi ~6.60 (20% CO2, apparent [HCO3−]i = 16.6 mM) and ~7.60 (0% CO2, apparent [HCO3−]i = 0 mM). Assuming that NBCe1‐B is insensitive to CO2 and (while operating in the inward direction) insensitive to [HCO3−]i, we reach the surprising conclusion that NBCe1‐B—when activated by IRBIT or super‐IRBIT—is independent of pHi.Support or Funding InformationSupported by NIH K01‐DK107787 to RO and NIH R01‐DK113197, ONR N00014‐15‐1‐2060, ONR N00014‐16‐1‐2535 to WFB

Mutagenesis of Snu114 domain IV identifies a developmental role in meiotic splicing

ABSTRACTSnu114, a component of the U5 snRNP, plays a key role in activation of the spliceosome. It controls the action of Brr2, an RNA-stimulated ATPase/RNA helicase that disrupts U4/U6 snRNA base-pairing prior to formation of the spliceosome’s catalytic centre. Snu114 has a highly conserved domain structure that resembles that of the GTPase EF-2/EF-G in the ribosome. It has been suggested that the regulatory function of Snu114 in activation of the spliceosome is mediated by its C-terminal region, however, there has been only limited characterisation of the interactions of the C-terminal domains. We show a direct interaction between protein phosphatase PP1 and Snu114 domain ‘IVa’ and identify sequence ‘YGVQYK’ as a PP1 binding motif. Interestingly, this motif is also required for Cwc21 binding. We provide evidence for mutually exclusive interaction of Cwc21 and PP1 with Snu114 and show that the affinity of Cwc21 and PP1 for Snu114 is influenced by the different nucleotide-bound states of Snu114. Moreover, we identify a novel mutation in domain IVa that, while not affecting vegetative growth of yeast cells, causes a defect in splicing transcripts of the meiotic genes, SPO22, AMA1 and MER2, thereby inhibiting an early stage of meiosis.

Aurora B opposes PP1 function in mitosis by phosphorylating the conserved PP1-binding RVxF motif in PP1 regulatory proteins.

Protein phosphatase 1 (PP1) is a highly conserved protein phosphatase that performs most of the serine- and threonine-dephosphorylation reactions in eukaryotes and opposes the actions of a diverse set of serine and threonine (Ser-Thr) protein kinases. PP1 gains substrate specificity through binding to a large number (>200) of regulatory proteins that control PP1 localization, activity, and interactions with substrates. PP1 recognizes the well-characterized RVxF binding motif that is present in many of these regulatory proteins, thus generating a multitude of distinct PP1 holoenzymes. We showed that a subset of the RVxF binding motifs, in which x is a phosphorylatable amino acid (RV[S/T]F), was phosphorylated specifically during mitosis and that this phosphorylation event abrogated the interaction of PP1 with the regulatory protein. We determined that this phosphorylation was primarily governed by the mitotic protein kinase Aurora B and that high phosphorylation site stoichiometry of these sites maintained the phosphorylation of PP1 substrates during mitosis by disrupting the assembly of PP1 holoenzymes. We generated an antibody that recognizes the phosphorylated form of the RV[S/T]F motif (RVp[S/T]F) and used it to identify known PP1 regulatory proteins (KNL1, CDCA2, and RIF1) and multiple proteins that could potentially act as PP1 binding partners (UBR5, ASPM, SEH1, and ELYS) governed by this mechanism. Together, these data suggest a general regulatory mechanism by which the coordinated activities of Aurora B and PP1 control mitotic progression.

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.

Characterization of the interactions between inhibitor-1 and recombinant PP1 by NMR spectroscopy

Inhibitor-1 is converted into a potent inhibitor of native protein phosphatase-1 (PP1) when Thr35 is phosphorylated by cAMP-dependent protein kinase (PKA). However, PKA-phosphorylated form of inhibitor-1 displayed a weak activity in inhibition of recombinant PP1. The mechanism for the impaired activity of PKA-phosphorylated inhibitor-1 toward inhibition of recombinant PP1 remained elusive. By using NMR spectroscopy in combination with site-directed mutagenesis and inhibitory assay, we found that the interaction between recombinant PP1 and the consensus PP1-binding motif of PKA-thiophosphorylated form of inhibitor-1 was unexpectedly weak. Unlike binding to native PP1, the subdomains 1 (residues around and including the phosphorylated Thr35) and 2 (the consensus PP1-binding motif) of PKA-thiophosphorylated form of inhibitor-1 do not exhibit a synergistic effect in inhibition of recombinant PP1. This finding implied that a slight structural discrepancy exists between native and recombinant PP1, resulting in PKA-thiophosphorylated form of inhibitor-1 displaying a different affinity to native and recombinant enzyme.

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman

The sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na(+)/K(+)-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca(2+) binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5-8Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.

An eIF2α-binding motif in protein phosphatase 1 subunit GADD34 and its viral orthologs is required to promote dephosphorylation of eIF2α

Transient protein synthesis inhibition, mediated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), is an important protective mechanism cells use during stress conditions. Following relief of the stress, the growth arrest and DNA damage-inducible protein GADD34 associates with the broadly acting serine/threonine protein phosphatase 1 (PP1) to dephosphorylate eIF2α. Whereas the PP1-binding motif on GADD34 has been defined, it remains to be determined how GADD34 directs PP1 to specifically dephosphorylate eIF2α. In this report, we map a novel eIF2α-binding motif to the C terminus of GADD34 in a region distinct from where PP1 binds to GADD34. This motif is characterized by the consensus sequence Rx[Gnl]x(1-2)Wxxx[Arlv]x[Dn][Rg]xRFxx[Rlvk][Ivc], where capital letters are preferred and x is any residue. Point mutations altering the eIF2α-binding motif impair the ability of GADD34 to interact with eIF2α, promote eIF2α dephosphorylation, and suppress PKR toxicity in yeast. Interestingly, this eIF2α-docking motif is conserved among viral orthologs of GADD34, and is necessary for the proteins produced by African swine fever virus, Canarypox virus, and Herpes simplex virus to promote eIF2α dephosphorylation. Taken together, these data indicate that GADD34 and its viral orthologs direct specific dephosphorylation of eIF2α by interacting with both PP1 and eIF2α through independent binding motifs.

Ki67 Antigen Contributes to the Timely Accumulation of Protein Phosphatase 1γ on Anaphase Chromosomes

Ki67 is a protein widely used as cell-proliferation marker, with its cellular functions being hardly unveiled. In this paper, we present the direct interaction between Ki67 and PP1γ, a protein phosphatase showing characteristic accumulation on anaphase chromosomes via the canonical PP1-binding motif within Ki67. In cells depleted of Ki67, PP1γ is targeted to anaphase chromosomes less efficiently. Additionally, overexpression of Ki67, but not a mutant form without the ability to bind PP1γ, induced ectopic localization of PP1γ οn metaphase chromosomes. These observations demonstrate that Ki67 is one factor that defines the cellular behavior of PP1γ in anaphase. To explore the specific roles of the subset of PP1γ recruited on chromosome via its interaction with Ki67 (PP1γ-Ki67), endogenous Ki67 was replaced with a Ki67 mutant deficient in its ability to interact with PP1γ. Although no obvious defects in the progression of mitosis were observed, the timing of dephosphorylation of the mutant Ki67 in anaphase was delayed, indicating that Ki67 itself is one of the substrates of PP1γ-Ki67.

The K898E germline variant in the PP1-binding motif of BRCA1 causes defects in DNA Repair.

BRCA1 is a phosphoprotein involved in many biological processes, including transcription, ubiquitination, checkpoint control, homologous recombination, and DNA repair. We have demonstrated that protein phosphatase 1α (PP1α) interacts with BRCA1 via a PP1-binding motif 898KVTF901, and can dephosphorylate multiple serine residues phosphorylated by checkpoint kinases. A K898E germline missense variant in the PP1-binding motif of BRCA1 has been found in an Ashkenazi patient and a non-Ashkenazi Argentinean patient with breast and ovarian cancer, but its clinical significance is still unknown. Here we report that the lysine residue in the PP1-binding motif of BRCA1 is highly conserved across many mammalian species. The K898E mutation interferes with the interaction between BRCA1 and PP1α. Moreover, while the expression of wild-type BRCA1 in Brca1-deficient cells improved cell survival after DNA damage induced by ionizing radiation (IR), expression of BRCA1 K898E proved unable to enhance cell survival. DNA damage repair mechanisms remained defective in these BRCA1 K898E-reconstituted cells, as revealed by the comet assay and IR-induced Rad51 foci formation assay. These results reflect the significance of the interaction between BRCA1 and PP1, and indicate that the K898E variant may render carriers susceptible to DNA damage and malignant transformation.

Antagonism of the phosphatase PP1 by the measles virus V protein is required for innate immune escape of MDA5.

The cytosolic sensor MDA5 is crucial for antiviral innate immune defense against various RNA viruses including measles virus; as such, many viruses have evolved strategies to antagonize the antiviral activity of MDA5. Here, we show that measles virus escapes MDA5 detection by targeting the phosphatases PP1α and PP1γ, which regulate MDA5 activity by removing an inhibitory phosphorylation mark. The V proteins of measles virus and the related paramyxovirus Nipah virus interact with PP1α/γ, preventing PP1-mediated dephosphorylation of MDA5 and thereby its activation. The PP1 interaction with the measles V protein is mediated by a conserved PP1-binding motif in the C-terminal region of the V protein. A recombinant measles virus expressing a mutant V protein deficient in PP1 binding is unable to antagonize MDA5 and is growth impaired due to its inability to suppress interferon induction. This identifies PP1 antagonism as a mechanism employed by paramyxoviruses for evading innate immune recognition.

Abstract 14: Caskin2 is a Novel Regulator of Endothelial Cell Quiescence

Maintenance of vascular homeostasis is an active process that is dependent on continuous signaling by the quiescent endothelial cells (ECs) that line mature vessels. The signaling pathways that are active in quiescent ECs are distinct from those that regulate angiogenesis but are comparatively poorly understood. Here we demonstrate that the previously uncharacterized scaffolding protein Caskin2 is a novel regulator of EC quiescence. Caskin2 is highly expressed in ECs from various vascular beds both in vitro and in vivo. Morpholino knockdown of Caskin2 in zebrafish results in abnormal vascular development characterized by overly branched intersegmental vessels and failure to form the dorsal longitudinal vessels. Interestingly, Caskin2 knockout mice are viable and fertile. However, compared to wild-type mice, adult Caskin2 knockout mice have significantly more abdominal adipose and higher fasting blood glucose levels even when fed a standard chow diet. In vitro, Caskin2 overexpression inhibits serum-induced EC proliferation and DNA synthesis but promotes EC survival during serum starvation. Caskin2 localizes to the nucleus as well as the cytoplasm and promotes the downregulation of genes associated with EC activation (e.g., IL-8, VEGFR2, ANG2) and the upregulation of genes associated with EC quiescence (e.g., Notch1, KLF2/4, ANG1). More broadly, pathway analysis of microarray data demonstrates that Caskin2 primarily regulates cell cycle and metabolic genes. Additional data suggest that these effects result from the interaction of Caskin2 with the Ser/Thr phosphatase PP1alpha, which is mediated by a consensus PP1 binding motif at the Caskin2 N-terminus. Taken together, our data demonstrate that Caskin2 is sufficient to suppress EC proliferation in vitro and necessary to prevent dysregulated angiogenesis in at least one in vivo model. These findings indicate that Caskin2 is a novel regulator of EC quiescence and suggest a role for Caskin2 in the prevention of EC dysfunction in vivo. Understanding Caskin2’s function on the molecular level may lead to the development of novel pharmacological approaches to prevent inappropriate angiogenesis, normalize dysfunctional vessels, and improve vascular function in a variety of cardiovascular diseases.

A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice

DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.

Protein Phosphatase 1γ Isoforms Linked Interactions in the Brain

Posttranslational protein modifications, in particular reversible protein phosphorylation, are important regulatory mechanisms involved in cellular signaling transduction pathways. Thousands of human proteins are phosphorylatable and the tight regulation of phosphorylation states is crucial for cell maintenance and development. Protein phosphorylation occurs primarily on serine, threonine, and tyrosine residues, through the antagonistic actions of protein kinases and phosphatases. The catalytic subunit of protein phosphatase 1 (PP1), a major Ser/Thr-phosphatase, associates with a large variety of regulatory subunits that define substrate specificity and determine specific cellular pathway responses. PP1 has been shown to bind to different proteins in the brain in order to execute key and differential functions. This work reports the identification of proteins expressed in the human brain that interact with PP1γ1 and PP1γ2 isoforms by the yeast two-hybrid method. An extensive search of PP1-binding motifs was performed for the proteins identified, revealing already known PP1 regulators but also novel interactors. Moreover, our results were integrated with the data of PP1γ interacting proteins from several public web databases, permitting the development of physical maps of the novel interactions. The PP1γ interactome thus obtained allowed for the identification of novel PP1 interacting proteins, supporting novel functions of PP1γ isoforms in the human brain.

Protein Phosphatase-1α Interacts with and Dephosphorylates Polycystin-1

Polycystin signaling is likely to be regulated by phosphorylation. While a number of potential protein kinases and their target phosphorylation sites on polycystin-1 have been identified, the corresponding phosphatases have not been extensively studied. We have now determined that polycystin-1 is a regulatory subunit for protein phosphatase-1α (PP1α). Sequence analysis has revealed the presence of a highly conserved PP1-interaction motif in the cytosolic, C-terminal tail of polycystin-1; and we have shown that transfected PP1α specifically co-immunoprecipitates with a polycystin-1 C-tail construct. To determine whether PP1α dephosphorylates polycystin-1, a PKA-phosphorylated GST-polycystin-1 fusion protein was shown to be dephosphorylated by PP1α but not by PP2B (calcineurin). Mutations within the PP1-binding motif of polycystin-1, including an autosomal dominant polycystic kidney disease (ADPKD)-associated mutation, significantly reduced PP1α-mediated dephosphorylation of polycystin-1. The results suggest that polycystin-1 forms a holoenzyme complex with PP1α via a conserved PP1-binding motif within the polycystin-1 C-tail, and that PKA-phosphorylated polycystin-1 serves as a substrate for the holoenzyme.

C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1

Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

Ikaros, CK2 kinase, and the road to leukemia

Ikaros encodes a zinc finger protein that is essential for hematopoiesis and that acts as a tumor suppressor in leukemia. Ikaros function depends on its ability to localize to pericentromeric-heterochromatin (PC-HC). Ikaros protein binds to the upstream regulatory elements of target genes, aids in their recruitment to PC-HC, and regulates their transcription via chromatin remodeling. We identified four novel Ikaros phosphorylation sites that are phosphorylated by CK2 kinase. Using Ikaros phosphomimetic and phosphoresistant mutants of the CK2 phosphorylation sites, we demonstrate that (1) CK2-mediated phosphorylation inhibits Ikaros' localization to PC-HC; (2) dephosphorylation of Ikaros at CK2 sites increases its binding to the promoter of the terminal deoxynucleotidetransferase (TdT) gene, leading to TdT repression during thymocyte differentiation; and (3) hyperphosphorylation of Ikaros promotes its degradation by the ubiquitin/proteasome pathway. We show that Ikaros is dephosphorylated by Protein Phosphatase 1 (PP1) via interaction at a consensus PP1-binding motif, RVXF. Point mutations that abolish Ikaros-PP1 interaction result in functional changes in DNA-binding affinity and subcellular localization, similar to those observed in hyperphosphorylated Ikaros and/or Ikaros phosphomimetic mutants. Phosphoresistant Ikaros mutations at CK2 sites restored Ikaros' DNA-binding activity and localization to PC-HC and prevented accelerated Ikaros degradation. These results demonstrate the role of CK2 kinase in lymphocyte differentiation and in regulation of Ikaros' function, and suggest that CK2 promotes leukemogenesis by inhibiting the tumor suppressor activity of Ikaros. We propose a model whereby a balance between CK2 kinase and PP1 phosphatase is essential for normal lymphocyte differentiation and for the prevention of malignant transformation.