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PubMed2005

p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death.

Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and co-immunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Activation of Raf-mitogen-activated protein kinase signaling pathway by 1,25-dihydroxyvitamin D3 in normal human keratinocytes.

The biologic effects of the vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are believed to be mediated by an intracellular vitamin D receptor, which after ligand binding acts as a transcription factor modulating expression of a variety of genes. Besides having a well-known role in calcium metabolism, this hormone is an important regulator of proliferation in a majority of normal and neoplastic cells. Keratinocytes provide a convenient model for investigating the growth-related effects of vitamin D in normal cells. Growth of keratinocytes may be either stimulated or inhibited by 1,25(OH)2D3, depending on the degree of cell differentiation. We show here that 1,25(OH)2D3 stimulates DNA synthesis via sequential activation of Raf and the mitogen-activated protein kinase. Activation of these kinases is independent on protein and mRNA synthesis and is preceded by rapid tyrosine phosphorylation of an adaptor protein p66 (Shc) and formation of a complex between p66 Shc, a bridging molecule Grb2, and a Ras activator, mSos. Vitamin D receptor protein associates with Shc, indicating that this steroid hormone is able to signal through the transcription-independent pathways similar to those used by peptide hormones and cytokines.

Adaptor Proteins, Signal Transducing↗

PubMedDate not supplied

Detection of the p110 beta subunit of phosphatidylinositol 3-kinase complexed with neutral endopeptidase.

BACKGROUND: Neutral endopeptidase 24.11 (NEP) is a cell-surface peptidase that inactivates a variety of neuropeptide substrates. In addition to catalytic activity, NEP can exert biological effects through protein-protein interactions. We previously reported that NEP directly associated with tyrosine-phosphorylated Lyn kinase, and with the p85 subunit of the phosphatidylinositol 3-kinase (PI3 kinase) resulting in an NEP-Lyn-PI3 kinase protein complex. MATERIALS AND METHODS: In this report, we investigated the association of NEP with cytoplasmic proteins using ProteinChip Array, surface enhanced laser desorption/ionization (SELDI) technology combined with time-of-flight mass spectrometry, as well as immunoprecipitation and Western blottings. RESULTS: Using immunocapture on the ProteinChip surface, we identified a 122 kDa protein which associates with NEP derived from LNCaP cell lysates which had the identical molecular weight as the beta-subunit of p110 subunit of phosphatidylinositol 3-kinase. The identity of the p110 beta was confirmed by Western blot analysis of NEP and p110 beta immunoprecipitates using monoclonal antibodies specific for NEP and p110 beta. CONCLUSION: These data confirm the association of phosphatidylinositol 3-kinase (consisting of the p85 adaptor and p110 beta-subunit) with NEP. Furthermore, this work demonstrates the ability of mass spectrometry to identify proteins interacting with NEP and potentially other cell-surface peptidases.

Blotting, Western↗

PubMed2002

The translational regulation of lipoprotein lipase by epinephrine involves an RNA binding complex including the catalytic subunit of protein kinase A.

The balance of lipid flux in adipocytes is controlled by the opposing actions of lipolysis and lipogenesis, which are controlled primarily by hormone-sensitive lipase and lipoprotein lipase (LPL), respectively. Catecholamines stimulate adipocyte lipolysis through reversible phosphorylation of hormone-sensitive lipase, and simultaneously inhibit LPL activity. However, LPL regulation is complex and previous studies have described translational regulation of LPL in response to catecholamines because of an RNA-binding protein that interacts with the 3'-untranslated region of LPL mRNA. In this study, we identified several protein components of an LPL RNA binding complex. Using an LPL RNA affinity column, we identified two of the RNA-binding proteins as the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), and A kinase anchoring protein (AKAP) 121/149, one of the PKA anchoring proteins, which has known RNA binding activity. To determine whether the C subunit was involved in LPL translation inhibition, the C subunit was depleted from the cytoplasmic extract of epinephrine-stimulated adipocytes by immunoprecipitation. This resulted in the loss of LPL translation inhibition activity of the extract, along with decreased RNA binding activity in a gel shift assay. To demonstrate the importance of the AKAPs, inhibition of PKA-AKAP binding with a peptide competitor (HT31) prevented epinephrine-mediated inhibition of LPL translation. C subunit kinase activity was necessary for LPL RNA binding and translation inhibition, suggesting that the phosphorylation of AKAP121/149 or other proteins was an important part of RNA binding complex formation. The hormonal activation of PKA results in the reversible phosphorylation of hormone-sensitive lipase, which is the primary mediator of adipocyte lipolysis. These studies demonstrate a dual role for PKA to simultaneously inhibit LPL-mediated lipogenesis through inhibition of LPL translation.

3' Untranslated Regions↗

PubMed2003

Oligomerization controls in tissue-specific manner ligand binding of native, affinity-purified p42(IP4)/centaurin alpha1 and cytohesins-proteins with high affinity for the messengers D-inositol 1,3,4,5-tetrakisphosphate/phosphatidylinositol 3,4,5-trisphosphate.

Several distinct receptor proteins for the second messengers Ins(1,3,4,5)P(4) and PtdIns(3,4,5)P(3) are already known, such as the brain-specific p42(IP4), which we have previously cloned from different species, and cytohesins. However, it is still unclear whether proteins interacting with phosphoinositide and inositolpolyphosphate second messengers are regulated differently in different tissues. Here, we investigated these native proteins for comparison also from rat lung cytosol and purified them by PtdIns(3,4,5)P(3) affinity chromatography. Proteins selectively binding Ins(1,3,4,5)P(4) with high affinity also showed high affinity and specificity towards PtdIns(3,4,5)P(3). In lung cytosol, two prominent protein bands were found in the eluate from a PtdIns(3,4,5)P(3) affinity column. We identified these proteins by mass spectrometry as the cytohesin family of Arf guanosine nucleotide exchange factors (cytohesin 1, ARNO, GRP-1) and as Bruton's tyrosine kinase. Western blot analysis indicated that p42(IP4) was present in lung only at very low concentrations. Applying the affinity purification scheme established for rat lung cytosol to cytosol from rat brain, however, yielded only p42(IP4). We identified cytohesins in rat brain by Western blotting and PCR, but cytohesins surprisingly did not bind to the PtdIns(3,4,5)P(3)-affinity column. Gel filtration experiments of brain cytosol revealed that brain cytohesins are bound to large molecular weight complexes (150 to more than 500 kDa). Thus, we hypothesize that this finding explains why brain cytohesins apparently do not bind the inositolphosphate ligand. In lung cytosol, on the other hand, cytohesins occur as dimers. Gel filtration also showed that p42(IP4) in brain cytosol occurs as a monomer. Thus, oligomerization (homomeric or heteromeric) of InsP(4)/PtdInsP(3) binding proteins can modulate their function in a tissue-dependent manner because it can modify their ability to interact with the ligands.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Insulin receptor substrate 4 associates with the protein IRAS.

The insulin receptor substrates (IRSs) are key components in signaling from the insulin receptor, and consequently any proteins that interact with them are expected to participate in insulin signaling. In this study we have searched for proteins that interact with IRS-4 by identifying the proteins that coimmunoprecipitated with IRS-4 from human embryonic kidney 293 cells by microsequencing through mass spectrometry. A group of proteins was found. These included phosphatidylinositol 3-kinase, a protein previously identified as an IRS-4 interactor, and several proteins for which there was no previous evidence of IRS-4 association. One of these proteins, named IRAS, that had been found earlier in another context was examined in detail. The results from the overexpression of IRAS, where its amount was about the same as that of IRS-4, indicated that IRAS associated directly with IRS-4 and showed that the increased complexation of IRS-4 with IRAS did not alter the insulin-stimulated tyrosine phosphorylation of IRS-4 or the association of IRS-4 with phosphatidylinositol 3-kinase or Grb2. On the other hand, overexpression of IRAS enhanced IRS-4-dependent insulin stimulation of the extracellularly regulated kinase. The domains of IRAS and IRS-4 responsible for the association of these two proteins were identified, and it was shown that IRAS also associates with IRS-1, IRS-2, and IRS-3.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Mouse p56 blocks a distinct function of eukaryotic initiation factor 3 in translation initiation.

Members of the p56 family of mammalian proteins are strongly induced in virus-infected cells and in cells treated with interferons or double-stranded RNA. Previously, we have reported that human p56 inhibits initiation of translation by binding to the "e" subunit of eukaryotic initiation factor 3 (eIF3) and subsequently interfering with the eIF3/eIF2.GTP.Met-tRNAi (ternary complex) interaction. Here we report that mouse p56 also interferes with eIF3 functions and inhibits translation. However, the murine protein binds to the "c" subunit, not the "e" subunit, of eIF3. Consequently, it has only a marginal effect on eIF3.ternary complex interaction. Instead, the major inhibitory effect of mouse p56 is manifested at a different step of translation initiation, namely the binding of eIF4F to the 40 S ribosomal subunit.eIF3.ternary complex. Thus, mouse and human p56 proteins block different functions of eIF3 by binding to its different subunits.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The Crk signaling pathway contributes to the bombesin-induced activation of the small GTPase Rap1 in Swiss 3T3 cells.

Rap1 is a small GTPase implicated in cell proliferation and differentiation. The mechanisms how endogenous Rap1 is activated by many mitogenic stimuli including the neuropeptide bombesin remained unclear. Here we analyse which signaling pathways are necessary for Rap1 activation. Bombesin-mediated Rap1 activation in Swiss 3T3 and primary mouse embryo fibroblasts requires signaling components similar to those being essential for complex formation between p130Cas and Crk adapter proteins. The Crk/CRKL-binding region of the Rap1-specific exchange factor C3G (CBR) inhibits the bombesin-stimulated Rap1 activity in transfected Swiss 3T3 cells. Further characterization in COS cells showed that the CBR or a c-Crk I SH3 mutant specifically reduces both the basal as well as the stimulated Rap1 activity in a dose-dependent manner, whereas Ras is not affected. The CBR is complexed with endogenous c-Crk II and CRKL and blocks the protein association with catalytically active C3G. Such suppressors of Crk signaling do not affect Erk-phosphorylation induced by bombesin. Embryonic fibroblasts from b-raf knockout mice showed a bombesin-inducible Erk-phosphorylation, providing evidence that B-Raf does not link Rap1 to Erk-activation in bombesin-stimulated fibroblasts. We conclude that cellular Crk/CRKL complexes, recruited to upstream signaling components, contribute to basal and bombesin-induced Rap1 activity, which is independent from the Ras-Raf-Erk pathway under these circumstances.

3T3 Cells↗

PubMed2005

Modulation of the interleukin-6 receptor subunit glycoprotein 130 complex and its signaling by LMO4 interaction.

The interleukin (IL)-6-type cytokines play major roles in a variety of biological processes by signaling through a common receptor subunit, glycoprotein (gp) 130. We performed yeast two-hybrid screening to identify new binding partners of the activated gp130 and the associated Janus kinases. LMO4, a LIM domain-containing protein that belongs to a family of oncogenes, was identified in this assay. Further studies show that LMO4 associates with gp130 and Janus kinase1 in several mammalian cell types. It also interacts with protein-tyrosine phosphatase 2 (SHP2) and suppressor of cytokine signaling 3 (SOCS3). The binding domains involved in these interactions were mapped, and the interactions were shown to be in a direct manner by in vitro binding assays. It is likely that LMO4 exists in the gp130 complex. The cellular localization of LMO4 was detected primarily in the nucleus with a substantial amount also detected in the cytoplasm in several cell types. The effect of LMO4 in IL-6 signaling was subsequently examined. Overexpression of LMO4 enhanced the transcriptional activity and target gene expression of Stat 3 (signal transducers and activators of transcription 3). Consistent with this, silencing LMO4 expression in stable cell lines expressing the small interfering RNA of LMO4 decreased Stat3 activity. Furthermore, the half-life of gp130 was shortened, and the production of acute phase proteins induced by IL-6 was reduced. Together, our data reveal a positive regulatory role of LMO4 in IL-6 signaling, possibly by acting as a scaffold for stabilization of the gp130 complex. These studies may open up a link between the oncogenic effect of LMO proteins and their regulatory role in cytokine signaling in general.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Molecular alterations of monophasic synovial sarcoma: loss of chromosome 3p does not alter RASSF1 and MLH1 transcriptional activity.

Differential diagnosis of monophasic synovial sarcoma requires the detection of specific biological markers. In this study we evaluated the presence of molecular alterations in 15 monophasic synovial sarcomas. Multiple changes affecting chromosome arms were detected by CGH-array in all microdissected cases available, and an association between gain or loss of specific regions harbouring cancer progression-associated genes and aneuploid status was found. The most frequent alteration was loss of 3p including 3p21.3-p23 region that, however, did not involve the promoter regions of the corresponding genes, RASSF1 and MLH1. Using Real-Time PCR, mRNA levels of both resulted moderately high compared to normal tissue; however, the weak to absent protein expression suggests RASSF1 and MLH1 post-transcription deregulation. Moreover, immunohistochemical analysis revealed that both mesenchymal and epithelial antigens were present in diploid tumours. These findings confirm the genetic complexity of monophasic synovial sarcoma and underline the need to integrate different analyses for a better knowledge of this tumour, essential to investigate new diagnostic and prognostic markers.

Adaptor Proteins, Signal Transducing↗

PubMed2004

SH2-B promotes insulin receptor substrate 1 (IRS1)- and IRS2-mediated activation of the phosphatidylinositol 3-kinase pathway in response to leptin.

Leptin regulates energy homeostasis primarily by binding and activating its long form receptor (LRb). Deficiency of either leptin or LRb causes morbid obesity. Leptin stimulates LRb-associated JAK2, thus initiating multiple pathways including the Stat3 and phosphatidylinositol (PI) 3-kinase pathways that mediate leptin biological actions. Here we report that SH2-B, a JAK2-interacting protein, promotes activation of the PI 3-kinase pathway by recruiting insulin receptor substrate 1 (IRS1) and IRS2 in response to leptin. SH2-B directly bound, via its PH and SH2 domain, to both IRS1 and IRS2 both in vitro and in intact cells and mediated formation of a JAK2/SH2-B/IRS1 or IRS2 tertiary complex. Consequently, SH2-B dramatically enhanced leptin-stimulated tyrosine phosphorylation of IRS1 and IRS2 in HEK293 cells stably expressing LRb, thus promoting association of IRS1 and IRS2 with the p85 regulatory subunit of PI 3-kinase and phosphorylation and activation of Akt. SH2-B mutants with lower affinity for IRS1 and IRS2 exhibited reduced ability to promote association of JAK2 with IRS1, tyrosine phosphorylation of IRS1, and association of IRS1 with p85 in response to leptin. Moreover, deletion of the SH2-B gene impaired leptin-stimulated tyrosine phosphorylation of endogenous IRS1 in mouse embryonic fibroblasts (MEF), which was reversed by reintroduction of SH2-B. Similarly, SH2-B promoted growth hormone-stimulated tyrosine phosphorylation of IRS1 in both HEK293 and MEF cells. Our data suggest that SH2-B is a novel mediator of the PI 3-kinase pathway in response to leptin or other hormones and cytokines that activate JAK2.

Adaptor Proteins, Signal Transducing↗

PubMed2002

The alpha5beta1 integrin selectively enhances epidermal growth factor signaling to the phosphatidylinositol-3-kinase/Akt pathway in intestinal epithelial cells.

We have investigated EGF-driven signaling processes in rat intestinal epithelial cell lines that overexpress either the alpha5beta1 integrin or the alpha2beta1 integrin. Both cell types display efficient activation of Erk/MAP kinase, but only the alpha5beta1 expressing cells display a strong activation of Akt. A complex is formed between activated EGFR and alpha5beta1, but not with alpha2beta1; this complex also contains ErbB3 and p85. Thus alpha5beta1 can support efficient activation of both the Erk and the phosphatidylinositol-3-kinase/Akt branches of the EGFR signaling cascade, whereas alpha2beta1 can support only the Erk branch.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Synaptic scaffolding molecule interacts with axin.

Synaptic scaffolding molecule (S-SCAM) is a synaptic protein that consists of PDZ domains, a guanylate kinase domain, and WW domains. It interacts with N-methyl-d-aspartate receptor subunits, neuroligin, and beta-catenin. Here, we identified Axin as a novel binding partner of S-SCAM. Axin was co-immunoprecipitated with S-SCAM from rat brain, detected in the post-synaptic density fraction in rat brain subcellular fractionation, and partially co-localized with S-SCAM in neurons. The guanylate kinase domain of S-SCAM directly bound to the GSK3beta-binding region of Axin. S-SCAM formed a complex with beta-catenin and Axin, but competed with GSK3beta for Axin-binding. Thereby, S-SCAM inhibited the Axin-mediated phosphorylation of beta-catenin by GSK3beta.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Inhibitory tyrosine protein kinase p50csk is associated with protein-tyrosine phosphatase PTP-PEST in hemopoietic and non-hemopoietic cells.

p50(csk) is a cytosolic tyrosine protein kinase expressed in all cell types. Accumulating data show that it inhibits multiple cellular processes, as a consequence of its ability to repress the enzymatic activity of Src family tyrosine protein kinases. We previously demonstrated that, via its Src homology 3 (SH3) domain, Csk is tightly bound to PEP, a protein-tyrosine phosphatase (PTP) exclusively expressed in hemopoietic cells. In this report, we have tested the possibility that Csk also interacts with PTP-PEST, a ubiquitous PTP sharing structural homology with PEP. Our studies revealed that Csk was associated with PTP-PEST in a variety of cell types, including non-hemopoietic cells. This interaction involved the SH3 region of p50(csk) and a proline-rich region (PPPLPERTPESFVLADM) outside the catalytic region of PTP-PEST. Even though both PTP-PEST and PEP were associated with Csk, significant differences were noted between these two PTPs. PTP-PEST, but not PEP, was also complexed with Shc, an adaptor molecule implicated in the Ras pathway. Moreover, PTP-PEST and PEP were found to accumulate primarily in distinct intracellular compartments in cell fractionation studies. In combination, these findings indicated that, like PEP, PTP-PEST is probably involved in Csk-mediated functions in mammalian cells. Moreover, they suggested that the roles of Csk-PTP-PEST and Csk-PEP are likely to be different.

Amino Acid Sequence↗

PubMed2003

p62 overexpression in breast tumors and regulation by prostate-derived Ets factor in breast cancer cells.

p62 is a multifunctional cytoplasmic protein able to noncovalently bind ubiquitin and several signaling proteins, suggesting a regulatory role connected to the ubiquitin-proteasome pathway. No studies to date have linked p62 protein expression with pathological states. Here we demonstrate the overabundance of p62 protein in malignant breast tissue relative to normal breast tissue. The proteasome inhibitor PSI increased p62 mRNA and protein; however, PSI treatment of breast epithelial cells transfected with the p62 promoter did not affect promoter activity. High levels of prostate-derived Ets factor (PDEF) mRNA have been identified in breast cancer compared to normal breast. Only the PSA and maspin promoters have been identified as targets of this transcription factor. Here we show that PDEF stimulates the p62 promoter through at least two sites, and likely acts as a coactivator. PSI treatment abrogates the PDEF-stimulated increase of p62 promoter activity by 50%. Thus, multiple mechanisms for the induction of p62 exist. We conclude that (1) p62 protein is overexpressed in breast cancer; (2) p62 mRNA and protein increase in response to PSI, with no change of basal promoter activity; (3) PDEF upregulates p62 promoter activity through at least two sites; and (4) PSI downregulates PDEF-induced p62 promoter activation through one of these sites.

Acetylcysteine↗

PubMed2004

RNAi-mediated Hip1R silencing results in stable association between the endocytic machinery and the actin assembly machinery.

Actin filaments transiently associate with the endocytic machinery during clathrin-coated vesicle formation. Although several proteins that might mediate or regulate this association have been identified, in vivo demonstration of such an activity has not been achieved. Huntingtin interacting protein 1R (Hip1R) is a candidate cytoskeletal-endocytic linker or regulator because it binds to clathrin and actin. Here, Hip1R levels were lowered by RNA interference (RNAi). Surprisingly, rather than disrupting the transient association between endocytic and cytoskeletal proteins, clathrin-coated structures (CCSs) and their endocytic cargo became stably associated with dynamin, actin, the Arp2/3 complex, and its activator, cortactin. RNAi double-depletion experiments demonstrated that accumulation of the cortical actin-endocytic complexes depended on cortactin. Fluorescence recovery after photobleaching showed that dynamic actin filament assembly can occur at CCSs. Our results provide evidence that Hip1R helps to make the interaction between actin and the endocytic machinery functional and transient.

Actin Cytoskeleton↗

PubMed2001

Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP.

Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.

Actins↗

PubMed2002

Identification of a basic surface area of the FADD death effector domain critical for apoptotic signaling.

Death effector domains (DEDs) are protein-protein interaction domains found in the death inducing signaling complex (DISC). Performing a structure-based alignment of all DED sequences we identified a region of high diversity in alpha-helix 3 and propose a classification of DEDs into class I DEDs typically containing a stretch of basic residues in the alpha-helix 3 region whereas DEDs of class II do not. Functional assays using mutants of Fas-associated death domain revealed that this basic region influences binding and recruitment of caspase-8 and cellular FLICE inhibitor protein to the DISC.

Adaptor Proteins, Signal Transducing↗