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PubMed2006

AKAP signaling complexes: getting to the heart of the matter.

Subcellular compartmentalization of protein kinases and phosphatases through their interaction with A-kinase anchoring proteins (AKAPs) provides a mechanism to control signal transduction events at specific sites within the cell. Recent findings suggest that these anchoring proteins dynamically assemble different cAMP effectors to control the cellular actions of cAMP spatially and temporally. In the heart, signaling events such as the onset of cardiac hypertrophy are influenced by muscle-specific mAKAP signaling complexes that target protein kinase A (PKA), the cAMP-responsive guanine-nucleotide exchange factor EPAC and cAMP-selective phosphodiesterase 4 (PDE4). Mediation of signaling events by AKAPs might also have a role in the control of lipolysis in adipocytes, where insulin treatment reduces the association of AKAPs with G-protein-coupled receptors. These are only two examples of how AKAPs contribute to specificity in cAMP signaling. This review will explore recent development that illustrates the role of multiprotein complexes in the regulation of cAMP signaling.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2001

The histone acetyltransferase activity of PCAF cooperates with the brahma/SWI2-related protein BRG-1 in the activation of the enhancer A of the MHC class I promoter.

Major histocompatibility complex (MHC) class I proteins are an essential component of the immune system allowing the organism to protect from viral infections and neoplastic transformation. Expression of the MHC class I genes is regulated by a variety of cis-regulatory promoter elements among which the enhancer A is of particular importance. This enhancer is synergistically activated through AP-1/ATF and NF-kappa B transcription factors. NF-kappa B recruits the histone acetyltransferase (HAT) p300/CREB-binding protein (CBP) to the multiprotein complex bound to the enhancer A. Here we present evidence that acetylation and deacetylation processes are involved in the activation of the enhancer A. The p300/CBP associated factor PCAF, but not p300/CBP, counteracts the repression of the enhancer A mediated by the histone deacetylase HDAC1. Furthermore, overexpression of PCAF results in an increase in the acetylation of histone H4 bound to the enhancer A and HDAC1 counteracts the PCAF-mediated H4 acetylation. The activation function of PCAF requires the p300/CBP binding motif indicating that PCAF might be recruited to the enhancer A through an association with p300/CBP. Moreover, PCAF and the Brahma/SWI2-related protein BRG-1, which is a key factor of the human ATP-dependent chromatin remodelling complex SWI/SNF, synergistically up-regulate the enhancer A. Synergistic activation requires the HAT domain of PCAF. Taken together our data suggest that members of two different groups of chromatin modifying complexes are involved in the activation of the enhancer A of the MHC class I promoter.

Acetylation↗

PubMed2000

Phosphorylation of c-Crk II on the negative regulatory Tyr222 mediates nerve growth factor-induced cell spreading and morphogenesis.

The Crk family of adaptor proteins participate in diverse signaling pathways that regulate growth factor-induced proliferation, anchorage-dependent DNA synthesis, and cytoskeletal reorganization, important for cell adhesion and motility. Using kidney epithelial 293T cells for transient co-transfection studies and the nerve growth factor (NGF)-responsive PC12 cell line as a model system for neuronal morphogenesis, we demonstrate that the non-receptor tyrosine kinase c-Abl is an intermediary for NGF-inducible c-Crk II phosphorylation on the negative regulatory Tyr(222). Transient expression of a c-Crk II Tyr(222) point mutant (c-Crk Y222F) in 293T cells induces hyperphosphorylation of paxillin on Tyr(31) and enhances complex formation between c-Crk Y222F and paxillin as well as c-Crk Y222F and c-Abl, suggesting that c-Crk II Tyr(222) phosphorylation induces both the dissociation of the Crk SH2 domain from paxillin and the Crk SH3 domain from c-Abl. Interestingly, examination of the early kinetics of NGF stimulation in PC12 cells showed that c-Crk II Tyr(222) phosphorylation preceded paxillin Tyr(31) phosphorylation, followed by a transient initial dissociation of the c-Crk II paxillin complex. PC12 cells overexpressing c-Crk Y222F manifested a defect in cellular adhesion and neuritogenesis that led to detachment of cells from the extracellular matrix, thus demonstrating the biological significance of c-Crk II tyrosine phosphorylation in NGF-dependent morphogenesis. Whereas previous studies have shown that Crk SH2 binding to paxillin is critical for cell adhesion and migration, our data show that the phosphorylation cycle of c-Crk II determines its dynamic interaction with paxillin, thereby regulating turnover of multiprotein complexes, a critical aspect of cytoskeletal plasticity and actin dynamics.

Animals↗

PubMed2005

Trichostatin A induces transforming growth factor beta type II receptor promoter activity and acetylation of Sp1 by recruitment of PCAF/p300 to a Sp1.NF-Y complex.

Transforming growth factor beta type II receptor (TbetaRII) is a tumor suppressor gene that can be transcriptionally silenced by histone deacetylases (HDACs) in cancer cells. In this report, we demonstrated the mechanism by which trichostatin A (TSA), an inhibitor of HDAC, induces the expression of TbetaRII in human pancreatic cancer cell lines by modulating the transcriptional components that bind a specific DNA region of the TbetaRII promoter. This region of the TbetaRII promoter possesses Sp1 and NF-Y binding sites in close proximity (located at -102 and -83, respectively). Treatment of cells with TSA activates the TbetaRII promoter in a time-dependent manner through the recruitment of p300 and PCAF into a Sp1.NF-Y.HDAC complex that binds this DNA element. The recruitment of p300 and PCAF into the complex is associated with a concomitant acetylation of Sp1 and an overall decrease in the amount of HDAC associated with the complex. Transient overexpression of p300 or PCAF potentiated TSA-induced TbetaRII promoter activity. The effect of PCAF was dependent on its histone acetyltransferase activity, whereas that of p300 was independent. Stable transfection of PCAF caused an increase in TbetaRII mRNA expression, the association of PCAF with TbetaRII promoter, and the acetylation of Sp1. Taken together, these results showed that TSA treatment of pancreatic cancer cells leads to transcriptional activation of the TbetaRII promoter through modulation of the components of a Sp1.NF-Y.p300.PCAF.HDAC-1 multiprotein complex. Moreover, the interaction of NF-Y with the Sp1-associated complex may further explain why this specific Sp1 site mediates transcriptional responsiveness to TSA.

Acetyltransferases↗

PubMed2003

sup-9, sup-10, and unc-93 may encode components of a two-pore K+ channel that coordinates muscle contraction in Caenorhabditis elegans.

Genetic studies of sup-9, unc-93, and sup-10 strongly suggest that these genes encode components of a multi-subunit protein complex that coordinates muscle contraction in Caenorhabditis elegans. We cloned sup-9 and sup-10 and found that they encode a two-pore K+ channel and a novel transmembrane protein, respectively. We also found that UNC-93 and SUP-10 colocalize with SUP-9 within muscle cells, and that UNC-93 is a member of a novel multigene family that is conserved among C. elegans, Drosophila, and humans. Our results indicate that SUP-9 and perhaps other two-pore K+ channels function as multiprotein complexes, and that UNC-93 and SUP-10 likely define new classes of ion channel regulatory proteins.

Amino Acid Sequence↗

PubMed2006

Calpain involvement in the remodeling of cytoskeletal anchorage complexes.

Cells offer different types of cytoskeletal anchorages: transitory structures such as focal contacts and perennial ones such as the sarcomeric cytoskeleton of muscle cells. The turnover of these structures is controlled with different timing by a family of cysteine proteases activated by calcium, the calpains. The large number of potential substrates present in each of these structures imposes fine tuning of the activity of the proteases to avoid excessive action. This phenomenon is thus guaranteed by various types of regulation, ranging from a relatively high calcium concentration necessary for activation, phosphorylation of substrates or the proteases themselves with either a favorable or inhibitory effect, possible intervention of phospholipids, and the presence of a specific inhibitor and its possible degradation before activation. Finally, formation of multiprotein complexes containing calpains offers a new method of regulation.

Animals↗

PubMed1993

Nuclear localization of vertebrate cyclin A correlates with its ability to form complexes with cdk catalytic subunits.

Cyclins control the activities of cyclin-dependent protein kinases (cdks) and hence play a key role in cell cycle regulation. While B-type cyclins associate with p34cdc2 to trigger entry into mitosis, progression through S phase requires cyclin A, presumably in association with p33cdk2. Vertebrate A- and B-type cyclins display strikingly distinct subcellular localizations, but the mechanisms underlying these differential distributions are unknown. Here, we have begun to study the requirements for nuclear localization of cyclin A. We have isolated a cDNA coding for chicken cyclin A and constructed a series of deletion mutants. These were then transfected into HeLa cells, and the subcellular distribution of the mutant cyclin A proteins was determined by indirect immunofluorescence microscopy. In parallel, the cyclin A mutants were assayed for their ability to form complexes with cdk subunits. We found that deletion of more than 100 residues from the N terminus of cyclin A did not impair nuclear localization or cdk subunit binding and kinase activation. In contrast, removal of as few as 15 residues from the C terminus, or deletion of part of the internal cyclin box domain, abolished nuclear localization of cyclin A as well as its ability to bind to and activate cdk subunits. These results suggest that nuclear transport of cyclin A may depend on the formation of multiprotein complexes comprising cdk catalytic subunits.

Amino Acid Sequence↗

PubMed2002

Involvement of subcomplexes of 32 and 14 kDa subunits in RPA's DNA binding activity through redox change.

The eukaryotic replication protein A (RPA) is a heterotrimeric protein complex. It consists of 70, 32, and 14 kDa subunits that are involved in DNA replication, repair, and genetic recombination. RPA is a 4-cysteine type zinc-finger protein. RPA's zinc-finger domain is not essential for DNA binding activity, but it is involved in the regulation of RPA's DNA binding activity through reduction-oxidation (redox). In this study, we show that yeast RPA's ssDNA binding activity is regulated by redox potential through its subcomplexes of 32 and 14 kDa subunits. In contrast, the subunits' complex, RPA70, formed a stable complex with ssDNA, even under non-reducing conditions. The addition of DTT and H202 had no effect on its DNA binding activity. In RPA70, since the addition of the subcomplexes of the 32 and 14 kDa subunits, it restored the modulating ssDNA binding activity to native RPA's DNA binding activity. These results suggest that the subcomplexes of the 32 and 14 kDa subunits may be involved in the modulating RPA's DNA binding activity through redox change. These studies, therefore, show the novel structure and function relationship of a multiprotein complex in that the role of a specific domain (or one subunit) is regulated by the other subunits.

DNA↗

PubMed1994

Spreading the silence: epigenetic transcriptional regulation during Drosophila development.

In early Drosophila development a complex cascade of diffusible transcription factors generates an intricate expression pattern of developmental regulators such as the homeotic genes. The mechanism which subsequently maintains the pattern during the rest of development is mainly using epigenetic features for its function. Evidence comes from the analysis of the Polycomb-group (Pc-G), a class of genes which is responsible for maintaining the inactive state of expression. The Pc-G was found to share many parallels to genes involved in heterochromatin formation. Different members of the Pc-G interact in large multiprotein complexes, which apparently can cover and inactivate large chromosomal domains. Specific DNA elements have been identified that are used by the Pc-G proteins to nucleate these specialized domains of silent chromatin. Thus, the Pc-G proteins appear to permanently inactivate genes by generating heterochromatin-like structures which could then be inherited by the daughter cells in an epigenetic manner. Heritable gene silencing is an important but little understood mechanism in pattern formation. Phenomenologically related effects have been observed in many organisms. These range from the transcriptional silencing of the inactive mating type loci in yeast to parental imprinting phenomena and X-chromosome inactivation in mammals. Analysis of these functions in Drosophila provides an excellent model system for studying the molecular basis of such epigenetic mechanisms that use higher order chromatin structures for transcriptional repression.

Animals↗

PubMed2003

Purification and identification of a novel complex which is involved in androgen receptor-dependent transcription.

The androgen receptor (AR) binds to and activates transcription of target genes in response to androgens. In an attempt to isolate cofactors capable of influencing AR transcriptional activity, we used an immunoprecipitation method and identified a 44-kDa protein, designated p44, as a new AR-interacting protein. p44 interacts with AR in the nucleus and with an androgen-regulated homeobox protein (NKX3.1) in the cytoplasm of LNCaP cells. Transient-transfection assays revealed that p44 enhances AR-, glucocorticoid receptor-, and progesterone receptor-dependent transcription but not estrogen receptor- or thyroid hormone receptor-dependent transcription. p44 was recruited onto the promoter of the prostate-specific antigen gene in the presence of the androgen in LNCaP cells. p44 exists as a multiprotein complex in the nuclei of HeLa cells. This complex, but not p44 alone, enhances AR-driven transcription in vitro in a cell-free transcriptional system and contains the protein arginine methyltransferase 5, which acts synergistically with p44 to enhance AR-driven gene expression in a methyltransferase-independent manner. Our data suggest a novel mechanism by which the protein arginine methyltransferase is involved in the control of AR-driven transcription. p44 expression is dramatically enhanced in prostate cancer tissue compared with adjacent benign prostate tissue.

Cell Nucleus↗

PubMed2001

The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes.

During oxygenic photosynthesis in cyanobacteria and chloroplasts of plants and eukaryotic algae, conversion of light energy to biologically useful chemical energy occurs in the specialized thylakoid membranes. Light-induced charge separation at the reaction centers of photosystems I and II, two multisubunit pigment-protein complexes in the thylakoid membranes, energetically drive sequential photosynthetic electron transfer reactions in this membrane system. In general, in the prokaryotic cyanobacterial cells, the thylakoid membrane is distinctly different from the plasma membrane. We have recently developed a two-dimensional separation procedure to purify thylakoid and plasma membranes from the genetically widely studied cyanobacterium Synechocystis sp. PCC 6803. Immunoblotting analysis demonstrated that the purified plasma membrane contained a number of protein components closely associated with the reaction centers of both photosystems. Moreover, these proteins were assembled in the plasma membrane as chlorophyll-containing multiprotein complexes, as evidenced from nondenaturing green gel and low-temperature fluorescence spectroscopy data. Furthermore, electron paramagnetic resonance spectroscopic analysis showed that in the partially assembled photosystem I core complex in the plasma membrane, the P700 reaction center was capable of undergoing light-induced charge separation. Based on these data, we propose that the plasma membrane, and not the thylakoid membrane, is the site for a number of the early steps of biogenesis of the photosynthetic reaction center complexes in these cyanobacterial cells.

Cell Membrane↗

PubMed2005

Evidence for direct roles of two additional factors, SECp43 and soluble liver antigen, in the selenoprotein synthesis machinery.

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA([Ser]Sec), but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2'-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA([Ser]Sec), and consequently reduced glutathione peroxidase 1 expression. Double knockdown of SECp43 and SLA resulted in decreased selenoprotein expression. SECp43 formed a complex with Sec tRNA([Ser]Sec) and SLA, and the targeted removal of one of these proteins affected the binding of the other to Sec tRNA([Ser]Sec). SECp43 was located primarily in the nucleus, whereas SLA was found in the cytoplasm. Co-transfection of both proteins resulted in the nuclear translocation of SLA suggesting that SECp43 may also promote shuttling of SLA and Sec tRNA([Ser]Sec) between different cellular compartments. Taken together, these data establish the role of SECp43 and SLA in selenoprotein biosynthesis through interaction with tRNA([Ser]Sec) in a multiprotein complex. The data also reveal a role of SECp43 in regulation of selenoprotein expression by affecting the synthesis of Um34 on tRNA([Ser]Sec) and the intracellular location of SLA.

Animals↗

PubMed2006

Mixed-lineage kinase 3 regulates B-Raf through maintenance of the B-Raf/Raf-1 complex and inhibition by the NF2 tumor suppressor protein.

The Ras --> Raf --> MEK1/2 --> extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway couples mitogenic signals to cell proliferation. B-Raf and Raf-1 function within an oligomer wherein they are regulated in part by mutual transactivation. The MAPK kinase kinase (MAP3K) mixed-lineage kinase 3 (MLK3) is required for mitogen activation of B-Raf and cell proliferation. Here we show that the kinase activity of MLK3 is not required for support of B-Raf activation. Instead, MLK3 is a component of the B-Raf/Raf-1 complex and is required for maintenance of the integrity of this complex. We show that the activation of ERK and the proliferation of human schwannoma cells bearing a loss-of-function mutation in the neurofibromatosis 2 (NF2) gene require MLK3. We find that merlin, the product of NF2, blunts the activation of both ERK and c-Jun N-terminal kinase (JNK). Finally, we demonstrate that merlin and MLK3 can interact in situ and that merlin can disrupt the interactions between B-Raf and Raf-1 or those between MLK3 and either B-Raf or Raf-1. Thus, MLK3 is part of a multiprotein complex and is required for ERK activation. The levels of this complex may be negatively regulated by merlin.

Animals↗

PubMed1996

Hormonally regulated double- and single-stranded DNA-binding complexes involved in mouse beta-casein gene transcription.

Transcription of the 252-base pair-long mouse beta-casein gene promoter is induced by the synergistic action of insulin, prolactin, and glucocorticoid in a primary mammary epithelial cell culture. The promoter contains a region termed block C having a highly conserved sequence and position among many casein genes. Mutation of block C reduced the response of the promoter to lactogenic hormones 84%. Nuclear extracts from lactating mouse mammary glands contained both a double-stranded and a single-stranded DNA binding protein complex (DS1 and SS), which specifically bind to the sequences AAATTAGCATGT and CCACAA of block C, respectively. The DS1 and the SS protein complexes were approximately 400 and 280 kDa, respectively. Each complex contained a DNA-binding component(s) having a molecular mass of approximately 120 kDa for DS1 and 80 and 65 kDa for SS. Deoxycholate, which interferes with the protein-protein interactions, inhibited the binding activities of DS1 and SS. The maximal increase in the binding activity of DS1 and SS in the mammary gland occurred during pregnancy and during lactation, respectively. In organ culture, the DS1 activity is increased by epidermal growth factor or prolactin in combination with insulin, whereas the SS activity is enhanced by insulin, prolactin, and glucocorticoid. These results suggest that multiprotein complexes binding to the double- and single-stranded DNA of block C mediate hormonal induction of beta-casein gene transcription.

Animals↗

PubMed2005

DYRK1A enhances the mitogen-activated protein kinase cascade in PC12 cells by forming a complex with Ras, B-Raf, and MEK1.

Dual-specificity tyrosine-phosphorylated and regulated kinase 1A (Dyrk1A) is the human homologue of the Drosophila mnb (minibrain) gene. In Drosophila, mnb is involved in postembryonic neurogenesis. In human, DYRK1A maps within the Down syndrome critical region of chromosome 21 and is overexpressed in Down syndrome embryonic brain. Despite its potential involvement in the neurobiological alterations observed in Down syndrome patients, the biological functions of the serine/threonine kinase DYRK1A have not been identified yet. Here, we report that DYRK1A overexpression potentiates nerve growth factor (NGF)-mediated PC12 neuronal differentiation by up-regulating the Ras/MAP kinase signaling pathway independently of its kinase activity. Furthermore, we show that DYRK1A prolongs the kinetics of ERK activation by interacting with Ras, B-Raf, and MEK1 to facilitate the formation of a Ras/B-Raf/MEK1 multiprotein complex. These data indicate that DYRK1A may play a critical role in Ras-dependent transducing signals that are required for promoting or maintaining neuronal differentiation and suggest that overexpression of DYRK1A may contribute to the neurological abnormalities observed in Down syndrome patients.

Animals↗

PubMed2002

Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex.

Plus-strand RNA viruses characteristically replicate their genome in association with altered cellular membranes. In the present study, the capacity of hepatitis C virus (HCV) proteins to elicit intracellular membrane alterations was investigated by expressing, in tetracycline-regulated cell lines, a comprehensive panel of HCV proteins individually as well as in the context of the entire HCV polyprotein. As visualized by electron microscopy (EM), expression of the combined structural proteins core-E1-E2-p7, the NS3-4A complex, and protein NS4B induced distinct membrane alterations. By immunogold EM (IEM), the membrane-altering proteins were always found to localize to the respective altered membranes. NS4B, a protein of hitherto unknown function, induced a tight structure, designated membranous web, consisting of vesicles in a membranous matrix. Expression of the entire HCV polyprotein gave rise to membrane budding into rough endoplasmic reticulum vacuoles, to the membranous web, and to tightly associated vesicles often surrounding the membranous web. By IEM, all HCV proteins were found to be associated with the NS4B-induced membranous web, forming a membrane-associated multiprotein complex. A similar web-like structure in livers of HCV-infected chimpanzees was previously described (Pfeifer et al., Virchows Arch. B., 33:233-243, 1980). In view of this finding and the observation that all HCV proteins accumulate on the membranous web, we propose that the membranous web forms the viral replication complex in HCV-infected cells.

Animals↗

PubMed1996

A kinase anchor protein 75 targets regulatory (RII) subunits of cAMP-dependent protein kinase II to the cortical actin cytoskeleton in non-neuronal cells.

Neuronal A kinase anchor protein (AKAP) homologs, such as AKAPs 75 and 150, tether cAMP-dependent protein kinase II (PKAII) isoforms to the postsynaptic cytoskeleton, thereby creating target sites for cAMP action. These AKAPs, which bind regulatory subunits (RIIs) of PKAII, are also expressed in certain non-neuronal cells. Non-neuronal cell lines that stably express wild type and mutant AKAP75 transgenes were generated to investigate the extraneuronal function of AKAPs. In non-neuronal cells, AKAP75 accumulates selectively in the actin-rich, cortical cytoskeleton in close proximity with the plasma membrane. AKAP75 efficiently sequesters cytoplasmic RIIalpha and RIIbeta (PKAII isoforms) and translocates these polypeptides to the cell cortex. Two structural modules in AKAP75, T1 (residues 27-48), and T2 (residues 77-100), are essential for targeting AKAP75.RII complexes to the cortical cytoskeleton. Deletions or amino acid substitutions in T1 and/or T2 result in the dispersion of both AKAP75 and RII subunits throughout the cytoplasm. AKAP75 is co-localized with F-actin and fodrin in the cortical cytoskeleton. Incubation of cells with 5 microM cytochalasin D disrupts actin filaments and dissociates actin from the cell cortex. In contrast, the bulk of AKAP75 and fodrin remain associated with the cortical region of cytochalasin D-treated cells. Thus, targeting of AKAP75 does not depend upon direct binding with F-actin. Rather, AKAP75 (like fodrin) may be associated with a multiprotein complex that interacts with integral plasma membrane proteins.

A Kinase Anchor Proteins↗

PubMed1998

TolB protein of Escherichia coli K-12 interacts with the outer membrane peptidoglycan-associated proteins Pal, Lpp and OmpA.

The Tol-Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. Transmembrane domains of TolQ, TolR and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. TolB and the central domain of TolA interact in vitro with the outer membrane porins. In this study, both genetic and biochemical analyses were carried out to analyse the links between TolB, Pal and other components of the cell envelope. It was shown that TolB could be cross-linked in vivo with Pal, OmpA and Lpp, while Pal was associated with TolB and OmpA. The isolation of pal and tolB mutants disrupting some interactions between these proteins represents at first approach to characterizing the residues contributing to the interactions. We propose that TolB and Pal are part of a multiprotein complex that links the peptidoglycan to the outer membrane. The Tol-Pal proteins might form transenvelope complexes that bring the two membranes into close proximity and help some outer membrane components to reach their final destination.

Amino Acid Sequence↗