Identification and characterization of AtI-2, an Arabidopsis homologue of an ancient protein phosphatase 1 (PP1) regulatory subunit

PP1 (protein phosphatase 1) is among the most conserved enzymes known, with one or more isoforms present in all sequenced eukaryotic genomes. PP1 dephosphorylates specific serine/threonine phosphoproteins as defined by associated regulatory or targeting subunits. In the present study we performed a PP1-binding screen to find putative PP1 interactors in Arabidopsis thaliana and uncovered a homologue of the ancient PP1 interactor, I-2 (inhibitor-2). Bioinformatic analysis revealed remarkable conservation of three regions of plant I-2 that play key roles in binding to PP1 and regulating its function. The sequence-related properties of plant I-2 were compared across eukaryotes, indicating a lack of I-2 in some species and the emergence points from key motifs during the evolution of this ancient regulator. Biochemical characterization of AtI-2 (Arabidopsis I-2) revealed its ability to inhibit all plant PP1 isoforms and inhibitory dependence requiring the primary interaction motif known as RVXF. Arabidopsis I-2 was shown to be a phosphoprotein in vivo that was enriched in the nucleus. TAP (tandem affinity purification)-tag experiments with plant I-2 showed in vivo association with several Arabidopsis PP1 isoforms and identified other potential I-2 binding proteins.     

Identification of a novel cell-adhesive protein spatiotemporally expressed in the basement membrane of mouse developing hair follicle

We used PCR-based cDNA subtraction to screen for genes up-regulated during mouse hair morphogenesis. One gene selected was predominantly expressed at the tip of developing hair follicles and encoded a protein characterized by the presence of twelve tandem repeats of approximately 120 amino acids and a novel N-terminal domain containing an Arg-Gly-Asp cell-adhesive motif. Immunohistochemistry demonstrated that the protein encoded by this gene, named QBRICK, was localized at the basement membrane zone of embryonic epidermis and hair follicles, in which it was more enriched at the tip rather than the stalk region. Cell adhesion assays showed that QBRICK was active in mediating cell-substratum adhesion through integrins containing alphav or alpha8 chain, but not integrin alpha5beta1. Immunohistochemistry showed that QBRICK colocalized with alphav-containing integrins in the interfollicular region, but with the alpha8-containing integrin at the tip region of developing hair follicles. These results, together, indicate that QBRICK is an adhesive ligand of basement membrane distinctively recognized by cells in the embryonic skin and hair follicles through different types of integrins directed to the Arg-Gly-Asp motif.     

MARCH-XI, a novel transmembrane ubiquitin ligase implicated in ubiquitin-dependent protein sorting in developing spermatids

A mechanism by which ubiquitinated cargo proteins are sorted into multivesicular bodies (MVBs) from plasma and trans-Golgi network (TGN) membranes is well established in yeast and mammalian somatic cells. However, the ubiquitin-dependent sorting pathway has not been clearly defined in germ cells. In this study we identified a novel member of the transmembrane RING-finger family of proteins, termed membrane-associated RING-CH (MARCH)-XI, that is expressed predominantly in developing spermatids and weakly in brain and pituitary. MARCH-XI possesses an E3 ubiquitin ligase activity that targets CD4 for ubiquitination. Immunoelectron microscopy of rat round spermatids showed that MARCH-XI is localized to TGN-derived vesicles and MVBs. Fluorescence staining of rat round spermatids and immunoprecipitation of rat testis demonstrated that MARCH-XI forms complexes with the adaptor protein complex-1 and with fucose-containing glycoproteins including ubiquitinated forms. Furthermore, the C-terminal region of MARCH-XI mediates its interaction with mu1-adaptin and Veli through a tyrosine-based motif and a PDZ binding motif, respectively. Our data suggest that MARCH-XI acts as a ubiquitin ligase with a role in ubiquitin-mediated protein sorting in the TGN-MVB transport pathway, which may be involved in mammalian spermiogenesis.     

Identification of phostensin, a PP1 F-actin cytoskeleton targeting subunit

We have identified a novel protein, protein phosphatase 1 F-actin cytoskeleton targeting subunit (phostensin). This protein is encoded by KIAA1949 and was found to associate with protein phosphatase 1 (PP1) in the yeast two-hybrid assay, co-immunoprecipitation, and GST pull-down assay. Northern blot analysis revealed that phostensin mRNA was predominantly distributed in leukocytes and spleen, and phostensin protein was present in crude extracts of human peripheral leukocytes. Immunofluorescence microscopic analysis revealed that the phostensin/PP1 complex was conspicuously localized with the actin cytoskeleton at the cell periphery in Madin-Darby canine kidney (MDCK) epithelial cells. Taken together, our data shows that phostensin targets PP1 to F-actin cytoskeleton. The phostensin/PP1 complex may play a vital role in modulation of actin rearrangements.     

Identification and characterization of a novel serine-arginine-rich splicing regulatory protein

We have identified an 86-kDa protein containing a single amino-terminal RNA recognition motif and two carboxy-terminal domains rich in serine-arginine (SR) dipeptides. Despite structural similarity to members of the SR protein family, p86 is clearly unique. It is not found in standard SR protein preparations, does not precipitate in the presence of high magnesium concentrations, is not recognized by antibodies specific for SR proteins, and cannot complement splicing-defective S100 extracts. However, we have found that p86 can inhibit the ability of purified SR proteins to activate splicing in S100 extracts and can even inhibit the in vitro and in vivo activation of specific splice sites by a subset of SR proteins, including ASF/SF2, SC35, and SRp55. In contrast, p86 activates splicing in the presence of SRp20. Thus, it appears that pairwise combination of p86 with specific SR proteins leads to altered splicing efficiency and differential splice site selection. In all cases, such regulation requires the presence of the two RS domains and a unique intervening EK-rich region, which appear to mediate direct protein-protein contact between these family members. Full-length p86, but not a mutant lacking the RS-EK-RS domains, was found to preferentially interact with itself, SRp20, ASF/SF2, SRp55, and, to a slightly lesser extent, SC35. Because of the primary sequence and unique properties of p86, we have named this protein SRrp86 for SR-related protein of 86 kDa.     

Identification of a novel WD repeat-containing gene predominantly expressed in developing and regenerating neurons

In the present study, we have identified a novel gene, NDRP (for neuronal differentiation-related protein), which is predominantly expressed in developing and regenerating neurons. The predicted NDRP comprises 1,019 amino acid residues and has 6 WD repeats in the N-terminal half and multiple potential nuclear localization signals (NLSs) at the C-terminal part. This molecule shows no significant structural similarity with any other molecules in available databases. In situ hybridization and immunohistochemistry revealed the highest expression of NRDP in sensory neurons, for instance, olfactory epithelia and neural layer of retina during embryonic development, as well as in perinatal dorsal root ganglions. The expression of this gene in intact motor neurons such as in the hypoglossal nerve was undetectable but became obvious after axotomy. These results suggest that the product of this gene might be involved in the development of sensory neurons as well as the regeneration of motor neurons.     

PP6 regulatory subunit R1 is bidentate anchor for targeting protein phosphatase-6 to DNA-dependent protein kinase

DNA-dependent protein kinase (DNA-PK) becomes activated in response to DNA double strand breaks, initiating repair by the non-homologous end joining pathway. DNA·PK complexes with the regulatory subunit SAPSR1 (R1) of protein phosphatase-6 (PP6). Knockdown of either R1 or PP6c prevents DNA-PK activation in response to ionizing radiation-induced DNA damage and radiosensitizes glioblastoma cells. Here, we demonstrate that R1 is necessary for and bridges the interaction between DNA-PK and PP6c. Using R1 deletion mutants, DNA-PK binding was mapped to two distinct regions of R1 spanning residues 1-326 and 522-700. Either region expressed alone was sufficient to bind DNA-PK, but only deletion of residues 1-326, not 522-700, eliminated interaction of R1 with DNA-PK. We assign 1-326 as the dominant domain and 522-700 as the supporting region. These results demonstrate that R1 acts as a bidentate anchor to DNA-PK and recruits PP6c. Targeting the dominant interface with small molecule or peptidomimetic inhibitors could specifically prevent activation of DNA-PK and thereby sensitize cells to ionizing radiation and other genotoxic agents.     

Identification of the regulatory subunit of a cAMP-dependent protein kinase in Dictyostelium discoideum

The occurrence of a cytosolic cAMP-binding protein of an approximate molecular weight of 41,000 daltons was monitored in vegetative and developing amoebae of $\text{Dictyostelium}\text{discoideum}$ by the use of the photoaffinity probe (³²P) 8N₃-cAMP. There was a large apparent increase in the amount of this binding protein during development; its molecular weight remained constant, if appropriate methods were employed for the disruption of the amoebae. Comigration during electrophoresis on two-dimensional gels identifies this cAMP-binding protein, photoaffinity-labeled in crude extracts, as the regulatory subunit of the cAMP-dependent protein kinase of $\text{D.}\text{discoideum}$.     

Identification and characterization of a novel regulatory factor: IgA-inducing protein

IgA is the predominant Ig isotype in mucosal secretions and thus plays a pivotal role in host defense. The mechanisms by which IgA expression is regulated may differ among species and involve multiple pathways. Various cytokines and costimulators have been identified which regulate expression of this isotype, including IL-10, IL-2, vasoactive intestinal peptide, and TGF-beta. We have tested a wide array of known factors, but only under very limited conditions do these factors mediate substantial IgA production in vitro from bovine B cells. In response to these findings, we generated a cDNA library in a mammalian expression vector from activated cells derived from bovine gut-associated lymphoid tissues (Peyer's patch and mesenteric lymph node cells) as a source of soluble factor(s) that may regulate IgA production. We have identified a novel factor, IgA-inducing protein, which stimulates relatively high levels of IgA production in vitro following CD40 stimulation in coculture with IL-2. Our data suggest that IgA-inducing protein regulates IgA by acting as a switch or differentiation factor and is expressed in a variety of lymphoid and nonlymphoid tissues.     

Pescadillo, a novel cell cycle regulatory protein abnormally expressed in malignant cells

Using a culture model of glial tumorigenesis, we identified a novel gene that was up-regulated in malignant mouse astrocytes following the loss of p53. The gene represents the murine homologue of pescadillo, an uncharacterized gene that is essential for embryonic development in zebrafish. Pescadillo is a strongly conserved gene containing unique structural motifs such as a BRCA1 C-terminal domain, clusters of acidic amino acids and consensus motifs for post-translational modification by SUMO-1. Pescadillo displayed a distinct spatial and temporal pattern of gene expression during brain development, being detected in neural progenitor cells and postmitotic neurons. Although it is not expressed in differentiated astrocytes in vivo, the pescadillo protein is dramatically elevated in malignant human astrocytomas. Yeast strains harboring temperature-sensitive mutations in the pescadillo gene were arrested in either G(1) or G(2) when grown in nonpermissive conditions, demonstrating that pescadillo is an essential gene in yeast and is required for cell cycle progression. Consistent with the latter finding, DNA synthesis was only observed in mammalian cells expressing the pescadillo protein. These results suggest that pescadillo plays a crucial role in cell proliferation and may be necessary for oncogenic transformation and tumor progression.     

The alpha4 regulatory subunit exerts opposing allosteric effects on protein phosphatases PP6 and PP2A

The protein Ser/Thr phosphatase family contains three enzymes called PP2A, PP4, and PP6 with separate biological functions inferred from genetics of the yeast homologues Pph21/22, Pph3, and Sit4. These catalytic subunits associate with a common subunit called alpha4 (related to yeast Tap42). Here, we characterized recombinant PP6 and PP2A catalytic monomers and alpha4.phosphatase heterodimers. Monomeric PP6 and PP2A showed identical kinetics using either p-nitrophenyl phosphate (pNPP) or 32P-myelin basic protein (MBP) as substrates, with matching Km and Vmax values. Using pNPP as substrate, PP6 and PP2A gave the same IC50 with active site inhibitors okadaic acid, microcystin-LR, calyculin A, and cantharidin. However, with MBP as substrate, PP6 was inhibited at 5-fold lower concentrations of toxins relative to PP2A, suggesting PP6 might be a preferred in vivo target of toxins. Heterodimeric alpha4.PP6 and alpha4.PP2A were starkly different. With MBP as substrate the alpha4.PP2A heterodimer had a 100-fold higher Vmax than alpha4.PP6, and neither heterodimer was active with pNPP. Thus, these phosphatases are distinguished by their different responses to allosteric binding of the common regulatory subunit alpha4. Transient expression of alpha4 differentially increased or decreased phosphorylation of endogenous phosphoproteins, consistent with opposing effects on PP2A and PP6.     

Identification of a novel protein phosphatase catalytic subunit by cDNA cloning

A cDNA encoding a novel protein phosphatase catalytic subunit (protein phosphatase X) has been isolated from a rabbit liver library. It codes for a protein having 45% and 65% amino acid sequence identity, respectively, to the catalytic subunits of protein phosphatase 1 and protein phosphatase 2A from skeletal muscle. The enzyme is neither the hepatic form of protein phosphatase 1 or 2A, nor is it protein phosphatase 2B or 2C. The possible identity of protein phosphatase X is discussed.     

PAL31, a novel nuclear protein, expressed in the developing brain

We cloned a cDNA encoding a novel protein (PAL31) predominantly expressed in the fetal rat brain by differential display. PAL31 contains leucine-rich repeat domains, a highly acidic region and a putative nuclear localization signal. PAL31 has 50-70% similarity to SSP29, APRIL, LANP, PHAP I, and PP32. Expression of PAL31 mRNA in the brain was high during the fetal period and decreased after birth. Immunohistochemical studies showed that PAL31 is expressed in the entire embryonic brain, whereas in the adult brain its expression is restricted to the subventricular zone where there are neural progenitor cells. It was also revealed that PAL31 is colocalized with PCNA in the nucleus, indicating that the PAL31 expression is developmentally regulated. Considering the primary structure of PAL31 and its spatiotemporal expression pattern, PAL31 is a novel nuclear protein related to the development of the brain through the proliferation of neuronal cells.     

Identification and characterization of Veph, a novel gene encoding a PH domain-containing protein expressed in the developing central nervous system of vertebrates

We isolated Veph, a novel gene encoding a pleckstrin homology (PH) domain-containing protein from a mouse. Veph was strongly expressed in the embryonic brain, and its expression level gradually decreased in later stages. In situ hybridization analysis of sectioned embryo brains revealed that Veph was expressed exclusively in the ventricular zone. We then isolated a zebrafish orthologue of Veph (zVeph). As observed in the mouse gene, zVeph was expressed in the ventricular zone of developing brain and spinal cord. Blockage of zVeph expression by injection of zVeph-specific morpholino antisense oligo into zebrafish fertilized eggs resulted in a defect in the midbrain-hindbrain boundary and otic vesicle formation, suggesting the important function of zVeph in central nervous system (CNS) development. On the other hand, homozygous knockout mice of Veph showed no significant defect in the CNS, pointing to possible different functions of Veph between the zebrafish and mouse.