Expression and function of resolvin RvD1n-3 DPA receptors in oral epithelial cells.

Chronic inflammatory responses can inflict permanent damage to host tissues. Specialized pro-resolving mediators downregulate inflammation but also can have other functions. The aim of this study was to examine whether oral epithelial cells express the receptors FPR2/ALX and DRV1/GPR32, which bind RvD1n-3 DPA , a recently described pro-resolving mediator derived from omega-3 docosapentaenoic acid (DPA), and whether RvD1n-3 DPA exposure induced significant responses in these cells. Gingival biopsies were stained using antibodies to FPR2/ALX and DRV1/GPR32. Expression of FPR2/ALX and DRV1/GPR32 was examined in primary oral epithelial cells by qRT-PCR, flow cytometry, and immunofluorescence. The effect of RvD1n-3 DPA on intracellular calcium mobilization and transcription of beta-defensins 1 and 2, and cathelicidin was evaluated by qRT-PCR. FPR2/ALX and DRV1/GPR32 were expressed by gingival keratinocytes in situ. In cultured oral epithelial cells, FPR2/ALX was detected on the cell surface, whereas FPR2/ALX and DRV1/GPR32 were detected intracellularly. Exposure to RvD1n-3 DPA induced intracellular calcium mobilization, FPR2/ALX internalization, DRV1/GPR32 translocation to the nucleus, and significantly increased expression of genes coding for beta-defensin 1, beta-defensin 2, and cathelicidin. This shows that the signal constituted by RvD1n-3 DPA is recognized by oral keratinocytes and that this can strengthen the antimicrobial and regulatory potential of the oral epithelium.

RvD1n-3 DPA Downregulates the Transcription of Pro-Inflammatory Genes in Oral Epithelial Cells and Reverses Nuclear Translocation of Transcription Factor p65 after TNF-α Stimulation.

Specialized pro-resolving mediators (SPMs) are multifunctional lipid mediators that participate in the resolution of inflammation. We have recently described that oral epithelial cells (OECs) express receptors of the SPM resolvin RvD1n-3 DPA and that cultured OECs respond to RvD1n-3 DPA addition by intracellular calcium release, nuclear receptor translocation and transcription of genes coding for antimicrobial peptides. The aim of the present study was to assess the functional outcome of RvD1n-3 DPA-signaling in OECs under inflammatory conditions. To this end, we performed transcriptomic analyses of TNF-α-stimulated cells that were subsequently treated with RvD1n-3 DPA and found significant downregulation of pro-inflammatory nuclear factor kappa B (NF-κB) target genes. Further bioinformatics analyses showed that RvD1n-3 DPA inhibited the expression of several genes involved in the NF-κB activation pathway. Confocal microscopy revealed that addition of RvD1n-3 DPA to OECs reversed TNF-α-induced nuclear translocation of NF-κB p65. Co-treatment of the cells with the exportin 1 inhibitor leptomycin B indicated that RvD1n-3 DPA increases nuclear export of p65. Taken together, our observations suggest that SPMs also have the potential to be used as a therapeutic aid when inflammation is established.

PML protein organizes heterochromatin domains where it regulates histone H3.3 deposition by ATRX/DAXX.

Maintenance of chromatin homeostasis involves proper delivery of histone variants to the genome. The interplay between different chaperones regulating the supply of histone variants to distinct chromatin domains remains largely undeciphered. We report a role of promyelocytic leukemia (PML) protein in the routing of histone variant H3.3 to chromatin and in the organization of megabase-size heterochromatic PML-associated domains that we call PADs. Loss of PML alters the heterochromatic state of PADs by shifting the histone H3 methylation balance from K9me3 to K27me3. Loss of PML impairs deposition of H3.3 by ATRX and DAXX in PADs but preserves the H3.3 loading function of HIRA in these regions. Our results unveil an unappreciated role of PML in the large-scale organization of chromatin and demonstrate a PML-dependent role of ATRX/DAXX in the deposition of H3.3 in PADs. Our data suggest that H3.3 loading by HIRA and ATRX-dependent H3K27 trimethylation constitute mechanisms ensuring maintenance of heterochromatin when the integrity of these domains is compromised.

The PML-associated protein DEK regulates the balance of H3.3 loading on chromatin and is important for telomere integrity.

Histone variant H3.3 is deposited in chromatin at active sites, telomeres, and pericentric heterochromatin by distinct chaperones, but the mechanisms of regulation and coordination of chaperone-mediated H3.3 loading remain largely unknown. We show here that the chromatin-associated oncoprotein DEK regulates differential HIRA- and DAAX/ATRX-dependent distribution of H3.3 on chromosomes in somatic cells and embryonic stem cells. Live cell imaging studies show that nonnucleosomal H3.3 normally destined to PML nuclear bodies is re-routed to chromatin after depletion of DEK. This results in HIRA-dependent widespread chromatin deposition of H3.3 and H3.3 incorporation in the foci of heterochromatin in a process requiring the DAXX/ATRX complex. In embryonic stem cells, loss of DEK leads to displacement of PML bodies and ATRX from telomeres, redistribution of H3.3 from telomeres to chromosome arms and pericentric heterochromatin, induction of a fragile telomere phenotype, and telomere dysfunction. Our results indicate that DEK is required for proper loading of ATRX and H3.3 on telomeres and for telomeric chromatin architecture. We propose that DEK acts as a "gatekeeper" of chromatin, controlling chromatin integrity by restricting broad access to H3.3 by dedicated chaperones. Our results also suggest that telomere stability relies on mechanisms ensuring proper histone supply and routing.

Localized Movement and Levels of 53BP1 Protein Are Changed by γ-irradiation in PML Deficient Cells.

We studied epigenetics, distribution pattern, kinetics, and diffusion of proteins recruited to spontaneous and γ-radiation-induced DNA lesions. We showed that PML deficiency leads to an increased number of DNA lesions, which was accompanied by changes in histone signature. In PML wt cells, we observed two mobile fractions of 53BP1 protein with distinct diffusion in spontaneous lesions. These protein fractions were not detected in PML-deficient cells, characterized by slow-diffusion of 53BP1. Single particle tracking analysis revealed limited local motion of 53BP1 foci in PML double null cells and local motion 53BP1 foci was even more reduced after γ-irradiation. However, radiation did not change co-localization between 53BP1 nuclear bodies and interchromatin granule-associated zones (IGAZs), nuclear speckles, or chromocenters. This newly observed interaction pattern imply that 53BP1 protein could be a part of not only DNA repair, but also process mediated via components accumulated in IGAZs, nuclear speckles, or paraspeckles. Together, PML deficiency affected local motion of 53BP1 nuclear bodies and changed composition and a number of irradiation-induced foci. J. Cell. Biochem. 117: 2583-2596, 2016. © 2016 Wiley Periodicals, Inc.

DAXX-dependent supply of soluble (H3.3-H4) dimers to PML bodies pending deposition into chromatin.

Replication-independent chromatin deposition of histone variant H3.3 is mediated by several chaperones. We report a multistep targeting of newly synthesized epitope-tagged H3.3 to chromatin via PML bodies. H3.3 is recruited to PML bodies in a DAXX-dependent manner, a process facilitated by ASF1A. DAXX is required for enrichment of ATRX, but not ASF1A or HIRA, with PML. Nonetheless, the chaperones colocalize with H3.3 at PML bodies and are found in one or more complexes with PML. Both DAXX and PML are necessary to prevent accumulation of a soluble, nonincorporated pool of H3.3. H3.3 targeting to PML is enhanced with an (H3.3-H4)2 tetramerization mutant of H3.3, suggesting H3.3 recruitment to PML as an (H3.3-H4) dimer rather than as a tetramer. Our data support a model of DAXX-mediated recruitment of (H3.3-H4) dimers to PML bodies, which may function as triage centers for H3.3 deposition into chromatin by distinct chaperones.

Distinct kinetics of DNA repair protein accumulation at DNA lesions and cell cycle-dependent formation of γH2AX- and NBS1-positive repair foci.

BACKGROUND INFORMATION: The DNA damage response is a fundamental, well-regulated process that occurs in the genome to recognise DNA lesions. Here, we studied kinetics of proteins involved in DNA repair pathways and their recruitment to DNA lesions during the cell cycle. In non-irradiated and irradiated cells, we analysed the distribution pattern and spatiotemporal dynamics of γH2AX, 53BP1, BMI1, MDC1, NBS1, PCNA, coilin and BRCA1 proteins. RESULTS: We observed that spontaneous and irradiation-induced foci (IRIF) demonstrated a high abundance of phosphorylated H2AX, which was consistent with 53BP1 and BMI1 protein accumulation. However, NBS1 and MDC1 proteins were recruited to nuclear bodies (NBs) to a lesser extent. Irradiation by γ-rays significantly increased the number of 53BP1- and γH2AX-positive IRIF, but cell cycle-dependent differences were only observed for γH2AX-positive foci in both non-irradiated and γ-irradiated cells. In non-irradiated cells, the G2 phase was characterised by an increased number of spontaneous γH2AX-foci; this increase was more pronounced after γ-irradiation. Cells in G2 phase had the highest number of γH2AX-positive foci. Similarly, γ-irradiation increased the number of NBS1-positive NBs only in G2 phase. Moreover, NBS1 accumulated in nucleoli after γ-irradiation showed the slowest recovery after photobleaching. Analysis of protein accumulation kinetics at locally induced DNA lesions showed that in HeLa cells, BMI1, PCNA and coilin were rapidly recruited to the lesions, 10-15 s after UVA-irradiation, whereas among the other proteins studied, BRCA1 demonstrated the slowest recruitment: BRCA1 appeared at the lesion 20 min after local micro-irradiation by UVA laser. CONCLUSION: We show that the kinetics of the accumulation of selected DNA repair-related proteins is protein specific at locally induced DNA lesions, and that the formation of γH2AX- and NBS1-positive foci, but not 53BP1-positive NBs, is cell cycle dependent in HeLa cells. Moreover, γH2AX is the most striking protein present not only at DNA lesions, but also spreading out in their vicinity. SIGNIFICANCE: Our conclusions highlight the significant role of the spatiotemporal dynamics of DNA repair-related proteins and their specific assembly/disassembly at DNA lesions, which can be cell type- and cell cycle dependent.

Optic atrophy 1 is an A-kinase anchoring protein on lipid droplets that mediates adrenergic control of lipolysis.

Adrenergic stimulation of adipocytes yields a cAMP signal that activates protein kinase A (PKA). PKA phosphorylates perilipin, a protein localized on the surface of lipid droplets that serves as a gatekeeper to regulate access of lipases converting stored triglycerides to free fatty acids and glycerol in a phosphorylation-dependent manner. Here, we report a new function for optic atrophy 1 (OPA1), a protein known to regulate mitochondrial dynamics, as a dual-specificity A-kinase anchoring protein associated with lipid droplets. By a variety of protein interaction assays, immunoprecipitation and immunolocalization experiments, we show that OPA1 organizes a supramolecular complex containing both PKA and perilipin. Furthermore, by a combination of siRNA-mediated knockdown, reconstitution experiments using full-length OPA1 with or without the ability to bind PKA or truncated OPA1 fused to a lipid droplet targeting domain and cellular delivery of PKA anchoring disruptor peptides, we demonstrate that OPA1 targeting of PKA to lipid droplets is necessary for hormonal control of perilipin phosphorylation and lipolysis.

Investigation of Roles of SLC38A1 in Proliferation and Differentiation of Mouse Tongue Epithelium and Expression in Human Oral Tongue Squamous Cell Carcinoma.

The aerobic glycolytic pathway, boosting lactate formation, and glutamine addiction are two hallmarks of cancer pathophysiology. Consistent with this, several cell membrane glutamine transporters, belonging to different solute carrier (SLC) families, have been shown to be upregulated in a cell-specific manner to furnish the cells with glutamine and glutamine-derived metabolic intermediates. Among them, the system A transporter Slc38a1 has a higher affinity for glutamine compared to other SLC transporters, and it undergoes highly multifaceted regulation at gene and protein levels. The current study aimed to investigate the functional role of Slc38a1 in the proliferation and maturation of the mouse tongue epithelium. Secondly, we aimed to examine the expression of SLC38A1 and its regulation in human tongue oral squamous cell carcinoma (OTSCC). Employing Slc38a1 wild-type and knockout mice, we showed that Slc38a1 was not directly linked to the regulation of the proliferation and differentiation of the mouse tongue epithelium. External transcriptomic datasets and Western blot analyses showed upregulation of SLC38A1 mRNA/protein in human OTSCC and oral cancer cell lines as compared to the corresponding controls. Further, an investigation of external datasets indicated that mechanisms other than the amplification of the SLC38A1 chromosomal locus or hypomethylation of the SLC38A1 promoter region might be important for the upregulation of SLC38A1 in OTSCC.

Infectious pancreatic necrosis virus (IPNV) from salmonid fish enters, but does not replicate in, mammalian cells.

BACKGROUND: The aquatic birnavirus infectious pancreatic necrosis virus (IPNV) causes infectious pancreatic necrosis (IPN), a severe disease in farmed salmonid fish. IPNV has a very broad host range and infects many different species of fish as well as molluscs and crustaceans. Investigation of the host reservoir of a virus may reveal important molecular mechanisms governing the infection processes such as receptors and entry mechanisms. In the present work we have studied whether IPNV is able to infect cells with different mammalian origin. RESULTS: IPNV bound in a specific manner to a membrane protein of the rabbit kidney cell line RK-13 as shown by the use of a virus overlay protein binding assay (VOPBA). Six different mammalian cell lines were inoculated with IPNV and incubated in parallels at different temperatures. At 7 days post inoculation (dpi), IPNV was detected by indirect immunofluorescent antibody test (IFAT) in all the cell lines. Confocal microscopy confirmed intracellular presence of the virus. No apparent cytopathic effect (cpe) was observed in any of the cultures, and no viral replication was demonstrated with real-time RT-PCR. CONCLUSION: Our results show that IPNV is able to enter into a wide range of mammalian cells, and virus entry is most likely receptor mediated. We found no indication of IPNV replication in any of the mammalian cell lines tested.

The protein phosphatase 1 regulator PNUTS is a new component of the DNA damage response.

The function of protein phosphatase 1 nuclear-targeting subunit (PNUTS)--one of the most abundant nuclear-targeting subunits of protein phosphatase 1 (PP1c)--remains largely uncharacterized. We show that PNUTS depletion by small interfering RNA activates a G2 checkpoint in unperturbed cells and prolongs G2 checkpoint and Chk1 activation after ionizing-radiation-induced DNA damage. Overexpression of PNUTS-enhanced green fluorescent protein (EGFP)--which is rapidly and transiently recruited at DNA damage sites--inhibits G2 arrest. Finally, γH2AX, p53-binding protein 1, replication protein A and Rad51 foci are present for a prolonged period and clonogenic survival is decreased in PNUTS-depleted cells after ionizing radiation treatment. We identify the PP1c regulatory subunit PNUTS as a new and integral component of the DNA damage response involved in DNA repair.

Mutually exclusive binding of PP1 and RNA to AKAP149 affects the mitochondrial network.

A-kinase-anchoring protein 149 (AKAP149) is a membrane protein of the mitochondrial and endoplasmic reticulum/nuclear envelope network. AKAP149 controls the subcellular localization and temporal order of protein phosphorylation by tethering protein kinases and phosphatases to these compartments. AKAP149 also includes an RNA-binding K homology (KH) domain, the loss of function of which has been associated in other proteins with neurodegenerative syndromes. We show here that protein phosphatase 1 (PP1) binding occurs through a conserved RVXF motif found in the KH domain of AKAP149 and that PP1 and RNA binding to this same site is mutually exclusive and controlled through a novel, phosphorylation-dependent mechanism. A collapse of the mitochondrial network is observed upon introduction of RNA-binding deficient mutants of AKAP149, pointing to the importance of RNA tethering to the mitochondrial membrane by AKAP149 for mitochondrial distribution.

Cyclic Di-adenosine Monophosphate Regulates Metabolism and Growth in the Oral Commensal Streptococcus mitis.

Cyclic di-adenosine monophosphate (c-di-AMP) has emerged as an important bacterial signaling molecule that functions both as an intracellular second messenger in bacterial cells and an extracellular ligand involved in bacteria-host cross-talk. In this study, we identify and characterize proteins involved in controlling the c-di-AMP concentration in the oral commensal and opportunistic pathogen Streptococcusmitis (S. mitis). We identified three known types of c-di-AMP turnover proteins in the genome of S. mitis CCUG31611: a CdaA-type diadenylate cyclase as well as GdpP-, and DhhP-type phosphodiesterases. Biochemical analyses of purified proteins demonstrated that CdaA synthesizes c-di-AMP from ATP whereas both phosphodiesterases can utilize c-di-AMP as well as the intermediary metabolite of c-di-AMP hydrolysis 5'-phosphadenylyl-adenosine (pApA) as substrate to generate AMP, albeit at different catalytic efficiency. Using deletion mutants of each of the genes encoding c-di-AMP turnover proteins, we show by high resolution MS/MS that the intracellular concentration of c-di-AMP is increased in deletion mutants of the phosphodiesterases and non-detectable in the cdaA-mutant. We also detected pApA in mutants of the DhhP-type phosphodiesterase. Low and high levels of c-di-AMP were associated with longer and shorter chains of S. mitis, respectively indicating a role in regulation of cell division. The deletion mutant of the DhhP-type phosphodiesterase displayed slow growth and reduced rate of glucose metabolism.

OPA1-anchored PKA phosphorylates perilipin 1 on S522 and S497 in adipocytes differentiated from human adipose stem cells.

Optic atrophy 1 (OPA1) is the A-kinase anchoring protein targeting the pool of protein kinase A (PKA) responsible for perilipin 1 phosphorylation, a gatekeeper for lipolysis. However, the involvement of OPA1-bound PKA in the downstream regulation of lipolysis is unknown. Here we show up-regulation and relocation of OPA1 from mitochondria to lipid droplets during adipocytic differentiation of human adipose stem cells. We employed various biochemical and immunological approaches to demonstrate that OPA1-bound PKA phosphorylates perilipin 1 at S522 and S497 on lipolytic stimulation. We show that the first 30 amino acids of OPA1 are essential for its lipid droplet localization as is OMA1-dependent processing. Finally, our results indicate that presence of OPA1 is necessary for lipolytic phosphorylation of downstream targets. Our results show for the first time, to our knowledge, how OPA1 mediates adrenergic control of lipolysis in human adipocytes by regulating phosphorylation of perilipin 1.

AKAP95 interacts with nucleoporin TPR in mitosis and is important for the spindle assembly checkpoint.

Faithful chromosome segregation during mitosis relies on a proofreading mechanism that monitors proper kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) is based on the concerted action of numerous components that maintain a repressive signal inhibiting transition into anaphase until all chromosomes are attached. Here we show that A-Kinase Anchoring Protein 95 (AKAP95) is necessary for proper SAC function. AKAP95-depleted HeLa cells show micronuclei formed from lagging chromosomes at mitosis. Using a BioID proximity-based proteomic screen, we identify the nuclear pore complex protein TPR as a novel AKAP95 binding partner. We show interaction between AKAP95 and TPR in mitosis, and an AKAP95-dependent enrichment of TPR in the spindle microtubule area in metaphase, then later in the spindle midzone area. AKAP95-depleted cells display faster prometaphase to anaphase transition, escape from nocodazole-induced mitotic arrest and show a partial delocalization from kinetochores of the SAC component MAD1. Our results demonstrate an involvement of AKAP95 in proper SAC function likely through its interaction with TPR.

In vitro reprogramming of nuclei and cells.

Directly turning a somatic cell type into another would be beneficial for producing replacement cells for therapeutic purposes. To this end, novel cell reprogramming strategies are being developed. We describe here methods for functionally reprogramming a somatic cell using an extract derived from another somatic cell type. The procedure involves reversible permeabilization of 293T fibroblasts, incubation of the permeabilized cells in a nuclear and cytoplasmic extract of T-cells, resealing of the "reprogrammed" cells, and culture for assessment of reprogramming. Reprogramming has been evidenced by nuclear uptake and assembly of transcription factors, induction of activity of a chromatin remodeling complex, changes in chromatin composition, activation of lymphoid cell-specific genes, and expression of T-cell-specific surface molecules. The system is likely to constitute a powerful tool to examine the processes of nuclear reprogramming, at least as they occur in vitro.

PNUTS enhances in vitro chromosome decondensation in a PP1-dependent manner.

PP1 (protein phosphatase-1) is a serine/threonine phosphatase involved in mitosis exit and chromosome decondensation. In the present study, we characterize the subcellular and subnuclear localization of PNUTS (PP1 nuclear targeting subunit), a nuclear regulatory subunit of PP1, and report a stimulatory role of PNUTS in the decondensation of prometaphase chromosomes in two in vitro systems. In interphase, PNUTS co-fractionates, together with a fraction of nuclear PP1, primarily with micrococcal nuclease-soluble chromatin. Immunofluorescence analysis shows that PNUTS is targeted to the reforming nuclei in telophase following the assembly of nuclear membranes and concomitantly with chromatin decondensation. In interphase cytosolic extract, ATP-dependent decondensation of prometaphase chromosomes is blocked by PP1-specific inhibitors. In contrast, a recombinant PNUTS(309-691) fragment accelerates chromosome decondensation. This decondensation-promoting activity requires the consensus RVXF PP1-binding motif of PNUTS, whereas a secondary, inhibitory PP1-binding site is dispensable. In a defined buffer system, PNUTS(309-691) also elicits decondensation in an exogenous PP1-dependent manner and, as in the cytosolic extract, a W401A (Thr401-->Ala) mutation that destroys PP1 binding abolishes this activity. The results illustrate an involvement of the PNUTS:PP1 holoenzyme in chromosome decondensation in vitro and argue that PNUTS functions as a PP1-targeting subunit in this process. We hypothesize that targeting of PNUTS to reforming nuclei in telophase may be a part of a signalling event promoting chromatin decondensation as cells re-enter interphase.

Protein phosphatase 1 regulators in DNA damage signaling.

In response to DNA damaging agents and endogenous DNA lesions, human cells activate signaling cascades and repair mechanisms to help maintain genomic integrity. Phosphorylation plays a major role in DNA damage signaling, and the role of Ser/Thr kinases, including ATM, ATR, CHK1, CHK2 and DNA-PK, is particularly well documented. While these kinases have taken the center stage in DNA damage signaling until now, a role for Ser/Thr phosphatases is emerging, including Protein Phosphatase 1 (PP1). PP1 substrate specificity is regulated by its binding to a large number of different targeting subunits, and several of these have recently been identified as regulators of DNA damage responses. Here we review recent progress regarding the involvement of PP1 and its binding partners in DNA damage signaling.

Chromatin environment of histone variant H3.3 revealed by quantitative imaging and genome-scale chromatin and DNA immunoprecipitation.

In contrast to canonical histones, histone variant H3.3 is incorporated into chromatin in a replication-independent manner. Posttranslational modifications of H3.3 have been identified; however, the epigenetic environment of incorporated H3.3 is unclear. We have investigated the genomic distribution of epitope-tagged H3.3 in relation to histone modifications, DNA methylation, and transcription in mesenchymal stem cells. Quantitative imaging at the nucleus level shows that H3.3, relative to replicative H3.2 or canonical H2B, is enriched in chromatin domains marked by histone modifications of active or potentially active genes. Chromatin immunoprecipitation of epitope-tagged H3.3 and array hybridization identified 1649 H3.3-enriched promoters, a fraction of which is coenriched in H3K4me3 alone or together with H3K27me3, whereas H3K9me3 is excluded, corroborating nucleus-level imaging data. H3.3-enriched promoters are predominantly CpG-rich and preferentially DNA methylated, relative to the proportion of methylated RefSeq promoters in the genome. Most but not all H3.3-enriched promoters are transcriptionally active, and coenrichment of H3.3 with repressive H3K27me3 correlates with an enhanced proportion of expressed genes carrying this mark. H3.3-target genes are enriched in mesodermal differentiation and signaling functions. Our data suggest that in mesenchymal stem cells, H3.3 targets lineage-priming genes with a potential for activation facilitated by H3K4me3 in facultative association with H3K27me3.

Association of PP1 with its regulatory subunit AKAP149 is regulated by serine phosphorylation flanking the RVXF motif of AKAP149.

Reformation of the nuclear envelope at the end of mitosis involves the recruitment of the B-type lamin phosphatase PP1 to nuclear membranes by A-kinase anchoring protein AKAP149. PP1 remains associated to AKAP149 throughout G1 but dissociates from AKAP149 when AKAP149 is phosphorylated at the G1/S transition. We examine here the role of phosphorylation of serines flanking the RVXF PP1-binding motif of AKAP149, on PP1 anchoring. The use of AKAP149 peptides encompassing the RVXF motif and five flanking serines, either wild type (wt) or bearing S-->A or S-->D mutations, specifically shows that phosphorylation of S151 or S159 abolishes PP1 binding to immobilized AKAP149. Peptides with S151 or S159 as the only wt serine residue trigger dissociation of PP1 from immunoprecipitated AKAP149, whereas S151/159D mutants are ineffective. Furthermore, immunoprecipitated AKAP149 from purified G1-phase nuclear envelopes binds PKA and PKC in overlay assays. PKA binding to AKAP149 in vitro is unaffected by the presence of PKC or PP1, and similarly, PKC binding is independent of PKA or PP1. The immunoprecipitated AKAP149 complex contains PKA and PKC activities. Both AKAP149-associated PKA and PKC serine-phosphorylate immunoprecipitated AKAP149 in vitro; however, only PKC-mediated phosphorylation promotes dissociation of PP1 from the AKAP. The results suggest a putative temporally and spatially controlled mechanism promoting release of PP1 from AKAP149. AKAP149 emerges as a scaffolding protein for multiple protein kinases and phosphatases that may be involved in the integration of intracellular signals that converge at the nuclear envelope.