Sigurd Ørstavik

Cloning and characterization of a cDNA encoding an A‐kinase anchoring protein located in the centrosome, AKAP450

A combination of protein kinase A type II (RII) overlay screening, database searches and PCR was used to identify a centrosomal A‐kinase anchoring protein. A cDNA with an 11.7 kb open reading frame was characterized and found to correspond to 50 exons of genomic sequence on human chromosome 7q21‐22. This cDNA clone encoded a 3908 amino acid protein of 453 kDa, that was designated AKAP450 (DDBJ/EMBL/GenBank accession No. AJ131693). Sequence comparison demonstrated that the open reading frame contained a previously characterized cDNA encoding Yotiao, as well as the human homologue of AKAP120. Numerous coiled‐coil structures were predicted from AKAP450, and weak homology to pericentrin, giantin and other structural proteins was observed. A putative RII‐binding site was identified involving amino acid 2556 of AKAP450 by mutation analysis combined with RII overlay and an amphipatic helix was predicted in this region. Immunoprecipitation of RII from RIPA‐buffer extracts of HeLa cells demonstrated co‐precipitation of AKAP450. By immunofluorecent labeling with specific antibodies it was demonstrated that AKAP450 localized to centrosomes. Furthermore, AKAP450 was shown to co‐purify in centrosomal preparations. The observation of two mRNAs and several splice products suggests additional functions for the AKAP450 gene.

Identification of a human member of the Ly‐49 multigene family

Three classes of multigene family‐encoded receptors enable NK cells to discriminate between polymorphic MHC class I molecules: Ly‐49 homodimers, CD94/NKG2 heterodimers and the killer cell inhibitory receptors (KIR). Of these, CD94/NKG2 has been characterized in both rodents and humans. In contrast, Ly‐49 family members have hitherto been found only in rodents, and KIR molecules only in the human. In this report, we describe a human cDNA, termed Ly‐49L, that constitutes the first human member of the Ly‐49 multigene family. Compared with rodent Ly‐49 molecules, the Ly‐49L sequence contains a premature stop codon and predicts a truncated protein that lacks the distal part of a C‐terminal lectin domain. Evidence is presented that the premature stop codon results from incomplete excision of the intron between the first two lectin domain exons. Splice variants predicting a full‐size Ly‐49L protein were not detected. As demonstrated by Northern blot analysis, Ly‐49L was transcribed by IL‐2‐activated NK cells, but not by freshly isolated B or T cells. PCR screening of a 22‐clone yeast artificial chromosome contig localized the LY49L locus to the human NK gene complex on chromosome 12p12‐p13. Southern blot analysis of genomic DNA showed a simple pattern with a full‐length Ly‐49L probe at low stringency hybridization conditions, suggesting that Ly‐49L may be the only human member of the Ly‐49 multigene family.

EBNA-LP Associates with Cellular Proteins Including DNA-PK and HA95

ABSTRACT EBNA-LP-associated proteins were identified by sequencing proteins that immunoprecipitated with Flag epitope-tagged EBNA-LP (FLP) from lymphoblasts in which FLP was stably expressed. The association of EBNA-LP with Hsp70 (72/73) was confirmed, and sequences of DNA-PK catalytic subunit (DNA-PKcs), HA95, Hsp27, prolyl 4-hydroxylase α-1 subunit, α-tubulin, and β-tubulin were identified. The fraction of total cellular HA95 that associated with FLP was very high, while progressively lower fractions of the total DNA-PKcs, Hsp70, Hsp 27, α-tubulin, and β-tubulin specifically associated with EBNA-LP as determined by immunoblotting with antibodies to these proteins. EBNA-LP bound to two domains in the DNA-PKcs C terminus and DNA-PKcs associated with the EBNA-LP repeat domain. DNA-PKcs that was bound to EBNA-LP phosphorylated p53 or EBNA-LP in vitro, and the phosphorylation of EBNA-LP was inhibited by Wortmannin, a specific in vitro inhibitor of DNA-PKcs.

A Novel Isoform of Human Cyclic 3′,5′-Adenosine Monophosphate-Dependent Protein Kinase, Cα-s, Localizes to Sperm Midpiece

Abstract Using rapid amplification of cDNA ends, a cDNA encoding a novel splice variant of the human Cα catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal part of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine Cα gene. Sequence comparison revealed similarity to an ovine Cα variant characterized by protein purification and micropeptide sequencing, Cα-s, identifying the cloned human cDNA as the Cα-s isoform. The Cα-s mRNA was expressed exclusively in human testis and expression in isolated human pachytene spermatocytes was demonstrated. The Cα-s protein was present in ejaculated human sperm, and immunofluorescent labeling with a Cα-s-specific antibody indicated that Cα-s was localized in the midpiece region of the spermatozoon. The majority of Cα-s was particulate and could not be released from the sperm midpiece by cAMP treatment alone. Furthermore, detergent extraction solubilized approximately two-thirds of the Cα-s pool, indicating interaction both with detergent-resistant cytoskeletal and membrane structures. In addition, we recently identified the regulatory subunit isoforms RIα, RIIα, and an A-kinase anchoring protein, hAKAP220 in this region in sperm that could target Cα-s. This novel Cα-s splice variant appeared to have an independent anchor in the human sperm midpiece as it could not be completely solubilized even in the presence of both detergent and cAMP.

MEK1 and MEK2 regulate distinct functions by sorting ERK2 to different intracellular compartments

In this study, we provide novel insight into the mechanism of how ERK2 can be sorted to different intracellular compartments and thereby mediate different responses. MEK1‐activated ERK2 accumulated in the nucleus and induced proliferation. Conversely, MEK2‐activated ERK2 was retained in the cytoplasm and allowed survival. Localization was a determinant for ERK2 functions since MEK1 switched from providing proliferation to be a mediator of survival when ERK2 was routed to the cytoplasm by the attachment of a nuclear export site. MEK1‐mediated ERK2 nuclear translocation and proliferation were shown to depend on phosphorylation of S298 and T292 sites in the MEK1 proline‐rich domain. These sites are phosphorylated on cellular adhesion in MEK1 but not MEK2. Whereas p21‐activated kinase phosphorylates S298 and thus enhances the MEK1‐ERK2 association, ERK2 phosphorylates T292, leading to release of active ERK2 from MEK1. On the basis of these results, we propose that the requirement of adhesion for cells to proliferate in response to growth factors, in part, may be explained by the MEK1 S298/T292 control of ERK2 nuclear translocation. In addition, we suggest that ERK2 intracellular localization determines whether growth factors mediate proliferation or survival and that the sorting occurs in an adhesion‐dependent manner.—Skarpen, E., Flinder, L. I., Rosseland, C. M., Ørstavik, S., Wierød, L., Pedersen Oksvold, M., Skålhegg, B. S., Huitfeldt, H. S. MEK1 and MEK2 regulate distinct functions by sorting ERK2 to different intracellular compartments. FASEB J. 22, 466–476 (2008)

Protein Kinase A Associates with HA95 and Affects Transcriptional Coactivation by Epstein-Barr Virus Nuclear Proteins

ABSTRACT HA95, a nuclear protein homologous to AKAP95, has been identified in immune precipitates of the Epstein-Barr virus (EBV) coactivating nuclear protein EBNA-LP from EBV-transformed lymphoblastoid cells (LCLs). We now find that HA95 and EBNA-LP are highly associated in LCLs and in B-lymphoma cells where EBNA-LP is expressed by gene transfer. Binding was also evident in yeast two-hybrid assays. HA95 binds to the EBNA-LP repeat domain that is the principal coactivator of transcription. EBNA-LP localizes with HA95 and causes HA95 to partially relocalize with EBNA-LP in promyelocytic leukemia nuclear bodies. Protein kinase A catalytic subunit α (PKAcsα) is significantly associated with HA95 in the presence or absence of EBNA-LP. Although EBNA-LP is not a PKA substrate, HA95 or PKAcsα expression in B lymphoblasts specifically down-regulates the strong coactivating effects of EBNA-LP. The inhibitory effects of PKAcsα are reversed by coexpression of protein kinase inhibitor. PKAcsα also inhibits EBNA-LP coactivation with the EBNA-2 acidic domain fused to the Gal4 DNA binding domain. Furthermore, EBNA-LP- and EBNA-2-induced expression of the EBV oncogene, LMP1, is down-regulated by PKAcsα or HA95 expression in EBV-infected lymphoblasts. These experiments indicate that HA95 and EBNA-LP localize PKAcsα at nuclear sites where it can affect transcription from specific promoters. The role of HA95 as a scaffold for transcriptional regulation is discussed.

Identification, cloning and characterization of a novel nuclear protein, HA95, homologous to A-kinase anchoring protein 95⋆

Summary— Previously, we have identified and characterized nuclear AKAP95 from man which targets cyclic AMP (cAMP)‐dependent protein kinase (PKA)‐type II to the condensed chromatin/spindle region at mitosis. Here we report the cloning of a novel nuclear protein with an apparent molecular mass of 95 kDa that is similar to AKAP95 and is designated HA95 (homologous to AKAP95). HA95 cDNA sequence encodes a protein of 646 amino acids that shows 61% homology to the deduced amino acid sequence of AKAP95. The HA95 gene is located on chromosome 19p13.1 immediately upstream of the AKAP95 gene. Both HA95 and AKAP95 genes contain 14 exons encoding similar regions of the respective proteins, indicating a previous gene duplication event as the origin of the two tandem genes. Despite their apparent similarity, HA95 does not bind RII in vitro. HA95 contains a putative nuclear localization signal in its N‐terminal domain. It is localized exclusively into the nucleus as demonstrated in cells transfected with HA95 fused to either green fluorescence protein or the c‐myc epitope. In the nucleus, the HA95 protein is found as complexes directly associated with each other or indirectly associated via other nuclear proteins. In interphase, HA95 is co‐localized with AKAP95, but the two proteins are not biochemically associated. At metaphase, both proteins co‐localize with condensed chromosomes. The similarity in sequence and localization of HA95 and AKAP95 suggests that the two molecules constitute a novel family of nuclear proteins that may exhibit related functions.

Identification and characterization of novel PKA holoenzymes in human T lymphocytes

Cyclic AMP‐dependent protein kinase (PKA) is a holoenzyme that consists of a regulatory (R) subunit dimer and two catalytic (C) subunits that are released upon stimulation by cAMP. Immunoblotting and immunoprecipitation of T‐cell protein extracts, immunofluorescence of permeabilized T cells and RT/PCR of T‐cell RNA using C subunit‐specific primers revealed expression of two catalytically active PKA C subunits Cα1 (40 kDa) and Cβ2 (47 kDa) in these cells. Anti‐RIα and Anti‐RIIα immunoprecipitations demonstrated that both Cα1 and Cβ2 associate with RIα and RIIα to form PKAI and PKAII holoenzymes. Moreover, Anti‐Cβ2 immunoprecipitation revealed that Cα1 coimmunoprecipitates with Cβ2. Addition of 8‐CPT‐cAMP which disrupts the PKA holoenzyme, released Cα1 but not Cβ2 from the Anti‐Cβ2 precipitate, indicating that Cβ2 and Cα1 form part of the same holoenzyme. Our results demonstrate for the first time that various C subunits may colocate on the same PKA holoenzyme to form novel cAMP‐responsive enzymes that may mediate specific effects of cAMP.

Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine Cβ2

BackgroundTwo main genes encoding the catalytic subunits Cα and Cβ of cyclic AMP dependent protein kinase (PKA) have been identified in all vertebrates examined. The murine, bovine and human Cβ genes encode several splice variants, including the splice variant Cβ2. In mouse Cβ2 has a relative molecular mass of 38 kDa and is only expressed in the brain. In human and bovine Cβ2 has a relative molecular mass of 47 kDa and is mainly expressed in lymphoid tissues.ResultsWe identified a novel 47 kDa splice variant encoded by the mouse Cβ gene that is highly expressed in lymphoid cells. Cloning, expression, and production of a sequence-specific antiserum and characterization of PKA catalytic subunit activities demonstrated the 47 kDa protein to be a catalytically active murine homologue of human and bovine Cβ2. Based on the present results and the existence of a human brain-specifically expressed Cβ splice variant designated Cβ4 that is identical to the former mouse Cβ2 splice variant, the mouse splice variant has now been renamed mouse Cβ4.ConclusionMurine lymphoid tissues express a protein that is a homologue of human and bovine Cβ2. The murine Cβ gene encodes the splice variants Cβ1, Cβ2, Cβ3 and Cβ4, as is the case with the human Cβ gene.

Structural and functional organization of the gene encoding the human thyrotropin-releasing hormone receptor.

Abstract : The thyrotropin‐releasing hormone (TRH) receptor (TRHR) is widely distributed throughout the central and peripheral nervous systems. In addition to its role in controlling the synthesis and secretion of thyroid‐stimulating hormone and prolactin from the anterior pituitary, TRH is believed to act as a neurotransmitter as well as a neuromodulator. We have isolated genomic λ and P1‐derived artificial chromosome clones encoding the human TRHR. The gene was found to be 35 kb with three exons and two introns. A 541‐bp intron 1 (‐629 to ‐89 relative to the translation start site) is conserved between human and mouse. A large intron 2 of 31 kb disrupts the open reading frame (starting in position +790) in the sequence encoding the supposed junction between the third intracellular loop and the putative sixth transmembrane domain. A similar intron was found in chimpanzee and sheep but not in rat and mouse. Promoter analysis of upstream regions demonstrated cell type‐specific reporter activation, and sequencing of 2.5 kb of the promoter revealed putative cis‐acting regulatory elements for several transcription factors that may contribute to the regulation of the TRHR gene expression. Functional analysis of potential response elements for the anterior pituitary‐specific transcription factor Pit‐1 revealed cell type‐specific binding that was competed out with a Pit‐1 response element from the GH gene promoter.