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PubMed1999

SHPS-1 is a scaffold for assembling distinct adhesion-regulated multi-protein complexes in macrophages.

Inhibitory immunoreceptors downregulate signaling by recruiting Src homology 2 (SH2) domain-containing tyrosine and/or lipid phosphatases to activating receptor complexes [1]. There are indications that some inhibitory receptors might also perform other functions [2] [3]. In adherent macrophages, two inhibitory receptors, SHPS-1 and PIR-B, are the major proteins binding to the tyrosine phosphatase SHP-1. SHPS-1 also associates with two tyrosine-phosphorylated proteins (pp55 and pp130) and a protein tyrosine kinase [4]. Here, we have identified pp55 and pp130 as the adaptor molecules SKAP55hom/R (Src-kinase-associated protein of 55 kDa homologue) and FYB/SLAP-130 (Fyn-binding protein/SLP-76-associated protein of 130 kDa), respectively, and the tyrosine kinase activity as PYK2. Two distinct SHPS-1 complexes were formed, one containing SKAP55hom/R and FYB/SLAP-130, and the other containing PYK2. Recruitment of FYB/SLAP-130 to SHPS-1 required SKAP55hom/R, whereas PYK2 associated with SHPS-1 independently. Formation of both complexes was independent of SHP-1 and tyrosine phosphorylation of SHPS-1. Finally, tyrosine phosphorylation of members of the SHPS-1 complexes was regulated by integrin-mediated adhesion. Thus, SHPS-1 provides a scaffold for the assembly of multi-protein complexes that might both transmit adhesion-regulated signals and help terminate such signals through SHP-1-directed dephosphorylation. Other inhibitory immunoreceptors might have similar scaffold-like functions.

Animals↗

PubMed2002

Identification of multiple binding partners for the amino-terminal domain of synapse-associated protein 97.

Multiprotein complexes mediate static and dynamic functions to establish and maintain cell polarity in both epithelial cells and neurons. Membrane-associated guanylate kinase (MAGUK) proteins are thought to be scaffolding molecules in these processes and bind multiple proteins via their obligate postsynaptic density (PSD)-95/Disc Large/Zona Occludens-1, Src homology 3, and guanylate kinase-like domains. Subsets of MAGUK proteins have additional protein-protein interaction domains. An additional domain we identified in SAP97 called the MAGUK recruitment (MRE) domain binds the LIN-2,7 amino-terminal (L27N) domain of mLIN-2/CASK, a MAGUK known to bind mLIN-7. Here we show that SAP97 binds two other mLIN-7 binding MAGUK proteins. One of these MAGUK proteins, DLG3, coimmunoprecipitates with SAP97 in lysates from rat brain and transfected Madin-Darby canine kidney cells. This interaction requires the MRE domain of SAP97 and surprisingly, both the L27N and L27 carboxyl-terminal (L27C) domains of DLG3. We also demonstrate that SAP97 can interact with the MAGUK protein, DLG2, but not the highly related protein, PALS2. The ability of SAP97 to interact with multiple MAGUK proteins is likely to be important for the targeting of specific protein complexes in polarized cells.

Adaptor Proteins, Signal Transducing↗

PubMed1989

Cloning of cDNAs encoding two related 100-kD coated vesicle proteins (alpha-adaptins).

Coat proteins of approximately 100-kD (adaptins) are components of the adaptor complexes which link clathrin to receptors in coated vesicles. The alpha-adaptins, which are found exclusively in endocytic coated vesicles, separate into two bands on SDS gels, designated A and C (Robinson, M. S., 1987. J. Cell Biol. 104:887-895). Two distinct cDNAs (sequences 1 and 2) encoding the two alpha-adaptins were cloned from a mouse brain cDNA library. Southern blotting indicates that there is one copy of each of the two alpha-adaptin genes, and that there are no additional closely related genes. Based on the size of the predicted protein products of the two genes (108 and 104 kD), the relative abundance of the two messages in brain and liver, and the reactivity of a sequence 1 fusion protein with different antibodies, it was possible to conclude that sequence 1 codes for A and sequence 2 for C. The two protein sequences are strikingly homologous to each other (84% identical amino acids), the major difference being an additional stretch of 41 amino acids, rich in prolines and acidic residues, inserted into the COOH-terminal half of A. In situ hybridization carried out on mouse brain sections indicates that the same cell type may express both transcripts, but that their relative expressions vary. Antipeptide antibodies are now being raised to find out whether the proteins are localized in functionally distinct populations of endocytic coated vesicles.

Adaptor Protein Complex alpha Subunits↗

PubMed2004

A multiprotein trafficking complex composed of SAP97, CASK, Veli, and Mint1 is associated with inward rectifier Kir2 potassium channels.

Strong inward rectifier potassium (Kir2) channels are important in the control of cell excitability, and their functions are modulated by interactions with intracellular proteins. Here we identified a complex of scaffolding/trafficking proteins in brain that associate with Kir2.1, Kir2.2, and Kir2.3 channels. By using a combination of affinity interaction pulldown assays and co-immunoprecipitations from brain and transfected cells, we demonstrated that a complex composed of SAP97, CASK, Veli, and Mint1 associates with Kir2 channels via the C-terminal PDZ-binding motif. We further demonstrated by using in vitro protein interaction assays that SAP97, Veli-1, or Veli-3 binds directly to the Kir2.2 C terminus and recruits CASK. Co-immunoprecipitations indicated that specific Veli isoforms participate in forming distinct protein complexes in brain, where Veli-1 stably associates with CASK and SAP97, Veli-2 associates with CASK and Mint1, and Veli-3 associates with CASK, SAP97, and Mint1. Additionally, immunocytochemistry of rat cerebellum revealed overlapping expression of Kir2.2, SAP97, CASK, Mint1, with Veli-1 in the granule cell layer and Veli-3 in the molecular layer. We propose a model whereby Kir2.2 associates with distinct SAP97-CASK-Veli-Mint1 complexes. In one complex, SAP97 interacts directly with the Kir2 channels and recruits CASK, Veli, and Mint1. Alternatively, Veli-1 or Veli-3 interacts directly with the Kir2 channels and recruits CASK and SAP97; association of Mint1 with the complex requires Veli-3. Expression of Kir2.2 in polarized epithelial cells resulted in targeting of the channels to the basolateral membrane and co-localization with SAP97 and CASK, whereas a dominant interfering form of CASK caused the channels to mislocalize. Therefore, CASK appears to be a central protein of a macromolecular complex that participates in trafficking and plasma membrane localization of Kir2 channels.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Role of Crk-associated substrate lymphocyte type in the pathophysiology of rheumatoid arthritis in tax transgenic mice and in humans.

OBJECTIVE: To investigate the role of Crk-associated substrate lymphocyte type (Cas-L), a downstream signaling molecule of beta1 integrins, in the pathophysiology of rheumatoid arthritis (RA). METHODS: We analyzed human T lymphotropic virus type I (HTLV-I) tax transgenic mice as well as samples from human RA patients. Splenocytes from tax transgenic mice were cultured on mouse endothelial cell-covered Transwell inserts, and cells migrating through the endothelial monolayer were counted. Biochemical studies were performed to analyze the protein expression and tyrosine phosphorylation of Cas-L. Immunohistochemical analysis was performed to detect Cas-L-positive cells that had infiltrated into the joints. RESULTS: Migratory activity of splenocytes from tax transgenic mice with arthritis (ATg) was much higher than that of tax transgenic mice without arthritis (NTg) and littermate control mice. The expression of Cas-L protein and its tyrosine phosphorylation were increased in ATg mice compared with NTg and control mice, and this was accompanied by enhanced autophosphorylation of Fyn and Lck. Immunohistochemical analysis demonstrated a large number of Cas-L-positive lymphocytes migrating into the affected joints. Furthermore, in human RA, Cas-L-positive lymphocytes were shown to infiltrate to the inflammatory lesions. CONCLUSION: These results strongly suggest that Cas-L plays an important role in the pathophysiology of RA.

Adaptor Proteins, Signal Transducing↗

PubMed2004

COP and clathrin-coated vesicle budding: different pathways, common approaches.

Vesicle and tubule transport containers move proteins and lipids from one membrane system to another. Newly forming transport containers frequently have electron-dense coats. Coats coordinate the accumulation of cargo and sculpt the membrane. Recent advances have shown that components of both COP1 and clathrin-adaptor coats share the same structure and the same motif-based cargo recognition and accessory factor recruitment mechanisms, which leads to insights on conserved aspects of coat recruitment, polymerisation and membrane deformation. These themes point to the way in which evolutionarily conserved features underpin these diverse pathways.

ADP-Ribosylation Factor 1↗

PubMed2006

Apoptosome: a platform for the activation of initiator caspases.

Apoptosome refers to the adaptor protein complex that mediates the activation of an initiator caspase at the onset of apoptosis. In mammalian cells, caspase-9, caspase-8, and caspase-2 rely on the apoptotic protease-activating factor 1 (Apaf-1)-apoptosome, death-inducing signaling complex (DISC), and PIDDosome, respectively, for activation. In Drosophila, activation of the caspase-9 homolog Dronc requires assembly of an apoptosome comprised of Dark/Hac-1/Dapaf-1. In Caenorhabditis elegans, activation of the caspase CED-3 is facilitated by the CED-4-apoptosome. Recent biochemical and structural investigation revealed significant insights into the assembly and function of the various apoptosomes. Nonetheless, conclusive mechanisms by which the initiator caspases are activated by the apoptosomes remain elusive. Several models have been proposed to explain the activation process. The induced proximity model summarizes the general process of initiator caspase activation. The proximity-driven dimerization model describes how initiator caspases respond to induced proximity and offers an explanation for their activation. Regardless of how initiator caspases are activated, enhanced activity must be correlated with altered active site conformation. The induced conformation model posits that the activated conformation for the active site of a given initiator caspase is attained through direct interaction with the apoptosome or through homo-oligomerization facilitated by the apoptosome.

Animals↗

PubMed2003

Identification of a family of endocytic proteins that define a new alpha-adaptin ear-binding motif.

Endocytosis by clathrin-coated vesicles (CCVs) is an important mechanism mediating protein internalization. Here, we show that two proteins identified through a proteomics analysis of CCVs are new components of the endocytic machinery. The proteins, named NECAP (adaptin-ear-binding coat-associated protein) 1 and 2, are paralogues that display no sequence similarity or common domains with any known protein. Both are enriched in CCV coats, and further analysis of the brain-enriched isoform, NECAP 1, shows its partial localization to clathrin-coated pits and direct binding to the globular ear domain of the alpha-adaptin subunit (alpha-ear) of the adaptor protein 2 (AP-2) complex. Intriguingly, this interaction is mediated by a new motif, WVQF, that uses a distinct alpha-ear interface relative to known alpha-ear-binding partners. Disruption of this interaction blocks clathrin-mediated endocytosis. Together, our studies identify a new family of endocytic proteins that define a unique AP-2-binding motif.

Adaptor Protein Complex alpha Subunits↗

PubMed2001

Interactions between adaptor protein-1 of the clathrin coat and microtubules via type 1a microtubule-associated proteins.

The classical view suggests that adaptor proteins of the clathrin coat mediate the sorting of cargo protein passengers into clathrin-coated pits and the recruitment of clathrin into budding areas in the donor membrane. In the present study, we provide biochemical and morphological evidence that the adaptor protein 1 (AP-1) adaptor of the trans-Golgi network clathrin interacts with microtubules. AP-1 in cytosolic extracts interacted with in vitro assembled microtubules, and these interactions were inhibited by ATP depletion of the extracts or in the presence of 5'-adenylylimidodiphosphate. An overexpressed gamma-subunit of the AP-1 complex associated with microtubules, suggesting that this subunit may mediate the interaction of AP-1 with the cytoskeleton. Purified AP-1 did not interact with purified microtubules, but interaction occurred when an isolated microtubule-associated protein fraction was added to the reaction mix. The gamma-adaptin subunit of AP-1 specifically co-immunoprecipitated with a microtubule-associated protein of type 1a from rat brain cytosol. This suggests that type 1a microtubule-associated protein may mediate the association of AP-1 with microtubules in the cytoplasm. The microtubule binding activity of AP-1 was markedly inhibited in cytosol of mitotic cells. By means of its interaction with microtubule-associated proteins, we propose novel roles for AP-1 adaptors in modulating the dynamics of the cytoskeleton, the stability and shape of coated organelles, and the loading of nascent AP-1-coated vesicles onto appropriate microtubular tracks.

Adaptor Protein Complex gamma Subunits↗

PubMed2004

Tumor necrosis factor receptor 1 and its signaling intermediates are recruited to lipid rafts in the traumatized brain.

The tumor necrosis factor (TNF) ligand-receptor system plays an essential role in apoptosis that contributes to secondary damage after traumatic brain injury (TBI). TNF also stimulates inflammation by activation of gene transcription through the IkappaB kinase (IKK)/NF-kappaB and JNK (c-Jun N-terminal protein kinase)/AP-1 signaling cascades. The mechanism by which TNF signals between cell death and survival and the role of receptor localization in the activation of downstream signaling events are not fully understood. Here, TNF receptor 1 (TNFR1) signaling complexes in lipid rafts were investigated in the cerebral cortex of adult male Sprague Dawley rats subjected to moderate (1.8-2.2 atmospheres) fluid-percussion TBI and naive controls. In the normal rat cortex, a portion of TNFR1 was present in lipid raft microdomains, where it associated with the adaptor proteins TRADD (TNF receptor-associated death domain), TNF receptor-associated factor-2 (TRAF-2), the Ser/Thr kinase RIP (receptor-interacting protein), TRAF1, and cIAP-1 (cellular inhibitor of apoptosis protein-1), forming a survival signaling complex. Moderate TBI resulted in rapid recruitment of TNFR1, but not TNFR2 or Fas, to lipid rafts and induced alterations in the composition of signaling intermediates. TNFR1 and TRAF1 were polyubiquitinated in lipid rafts after TBI. Subsequently, the signaling complex contained activated caspase-8, thus initiating apoptosis. In addition, TBI caused a transient activation of NF-kappaB, but receptor signaling interacting proteins IKKalpha and IKKbeta were not detected in raft-containing fractions. Thus, redistribution of TNFR1 in lipid rafts and nonraft regions of the plasma membrane may regulate the diversity of signaling responses initiated by these receptors in the normal brain and after TBI.

Animals↗

PubMed2006

Abnormal progression through meiosis in men with nonobstructive azoospermia.

OBJECTIVE: To study meiotic abnormalities in men with nonobstructive azoospermia. DESIGN: Analysis of synaptonemal complex and recombination in testicular tissue. SETTING: Research laboratory. PATIENT(S): Twenty-nine men with nonobstructive azoospermia and 12 men with normal spermatogenesis. INTERVENTION(S): Testicular tissues were processed with immunofluorescent staining using antibodies against proteins associated with synaptonemal complex and recombination events. MAIN OUTCOME MEASURE(S): Synaptonemal complex configuration and recombination in meiosis I. RESULT(S): In patients with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: nearly half of them had a complete absence of meiotic cells, one case had germ cells arrested at the zygotene stage of meiotic prophase, and, in general, the rest had impaired fidelity of chromosome synapsis and recombination in pachytene cells. Compared with controls, these patients had significantly more cells in leptotene/zygotene and higher frequencies of unpaired chromosome regions in pachytene. Significantly reduced recombination, an increased frequency of achiasmate autosome bivalents, and sex univalents in pachytene were also observed in these patients with nonobstructive azoospermia. CONCLUSION(S): Defects in chromosome synapsis and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to the unexplained infertility in these patients.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Proteasome-mediated regulation of the hDlg tumour suppressor protein.

The Dlg tumour suppressor protein is intimately involved in the control of cell contact and polarity. Previous studies have shown that hDlg is a target for a number of viral transforming proteins. In particular, the high risk human papillomavirus (HPV) E6 proteins target hDlg for proteasome-mediated degradation, an activity that appears to contribute to HPV-induced malignancy. However, little information is available concerning the normal regulation of hDlg. In this study we have investigated the role of the proteasome in the regulation of endogenous hDlg protein levels in epithelial cell lines. We demonstrate that hDlg is, indeed, degraded via the proteasome both in the presence and absence of HPV, in a fashion that is dependent on the ability of the cells to form cell junctions. By western blot and immunofluorescence analysis we show that hDlg is efficiently degraded in isolated cells; however, upon cell-cell contact, hDlg is both hyper-phosphorylated and stabilised. Strikingly, in both transformed rodent cells and undifferentiated cervical cancer cells, this ability to stabilise Dlg upon increased cell density is lost. These results demonstrate a complex pattern of hDlg regulation by phosphorylation and proteasome degradation in response to cell contact. Loss of this regulation probably represents a significant step in the development of malignancy.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Generation of high curvature membranes mediated by direct endophilin bilayer interactions.

Endophilin 1 is a presynaptically enriched protein which binds the GTPase dynamin and the polyphosphoinositide phosphatase synptojanin. Perturbation of endophilin function in cell-free systems and in a living synapse has implicated endophilin in endocytic vesicle budding (Ringstad, N., H. Gad, P. Low, G. Di Paolo, L. Brodin, O. Shupliakov, and P. De Camilli. 1999. Neuron. 24:143-154; Schmidt, A., M. Wolde, C. Thiele, W. Fest, H. Kratzin, A.V. Podtelejnikov, W. Witke, W.B. Huttner, and H.D. Soling. 1999. Nature. 401:133-141; Gad, H., N. Ringstad, P. Low, O. Kjaerulff, J. Gustafsson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crun, M.H. Ellisman, et al. 2000. Neuron. 27:301-312). Here, we show that purified endophilin can directly bind and evaginate lipid bilayers into narrow tubules similar in diameter to the neck of a clathrin-coated bud, providing new insight into the mechanisms through which endophilin may participate in membrane deformation and vesicle budding. This property of endophilin is independent of its putative lysophosphatydic acid acyl transferase activity, is mediated by its NH2-terminal region, and requires an amino acid stretch homologous to a corresponding region in amphiphysin, a protein previously shown to have similar effects on lipid bilayers (Takei, K., V.I. Slepnev, V. Haucke, and P. De Camilli. 1999. Nat. Cell Biol. 1:33-39). Endophilin cooligomerizes with dynamin rings on lipid tubules and inhibits dynamin's GTP-dependent vesiculating activity. Endophilin B, a protein with homology to endophilin 1, partially localizes to the Golgi complex and also deforms lipid bilayers into tubules, underscoring a potential role of endophilin family members in diverse tubulovesicular membrane-trafficking events in the cell.

Acyltransferases↗

PubMed2004

Nef-induced alteration of the early/recycling endosomal compartment correlates with enhancement of HIV-1 infectivity.

human immunodeficiency virus type 1 (HIV-1) Nef interacts with the clathrin-associated AP-1 and AP-3 adaptor complexes, stabilizing their association with endosomal membranes. These findings led us to hypothesize a general impact of this viral protein on the endosomal system. Here, we have shown that Nef specifically disturbs the morphology of the early/recycling compartment, inducing a redistribution of early endosomal markers and a shortening of the tubular recycling endosomal structures. Furthermore, Nef modulates the trafficking of the transferrin receptor (TfR), the prototypical recycling surface protein, indicating that it also disturbs the function of this compartment. Nef reduces the rate of recycling of TfR to the plasma membrane, causing TfR to accumulate in early endosomes and reducing its expression at the cell surface. These effects depend on the leucine-based motif of Nef, which is required for the membrane stabilization of AP-1 and AP-3 complexes. Since we show that this motif is also required for the full infectivity of HIV-1 virions, these results indicate that the positive influence of Nef on viral infectivity may be related to its general effects on early/recycling endosomal compartments.

Amino Acid Motifs↗

PubMed1994

Role of p85 subunit of phosphatidylinositol-3-kinase as an adaptor molecule linking the insulin receptor, p62, and GTPase-activating protein.

After insulin stimulation of rat HTC hepatoma cells overexpressing normal human insulin receptors (IR), an antiserum to the p85 subunit of phosphatidylinositol-3-kinase (PIK) (alpha-p85) immunoprecipitated three major tyrosine-phosphorylated proteins: IR, insulin receptor substrate-1 (IRS-1), and a new 62-kDa protein (p62). Studies with antibodies to GTPase activating protein (alpha-GAP) and p62 GAP-associated protein suggested that p62 was the same as (or closely related to) p62 GAP-associated protein. In order to understand how p62 interacts with p85, we employed: 1) antibodies to the p110 subunit of PIK (alpha-p110); and 2) antiserum to IRS-1. To determine which subunit of PIK (p110 or p85) p62 associates with, we first immunoprecipitated insulin-treated cell lysates with alpha-p110 and subsequently immunoprecipitated with alpha-p85 followed by Western blotting analysis with anti-phosphotyrosine antibody (alpha-PY). In response to insulin, most of the tyrosine-phosphorylated p62 was complexed to p85 alone rather than with the PIK heterodimer. Moreover, p62 was absent in alpha-IRS-1 immunoprecipitates. These data suggest that: 1) p62 GAP-associated protein is tyrosine phosphorylated after insulin stimulation of cells; 2) p62 and IRS-1 form separate complexes with p85; 3) p62-GAP complex may be linked to p85 that is not bound to p110; 4) p85 may serve as an adaptor molecule in insulin receptor signaling, interacting with and regulating other intracellular proteins via SH2 domains.

Animals↗

PubMedDate not supplied

Zyxin, axin, and Wiskott-Aldrich syndrome protein are adaptors that link the cadherin/catenin protein complex to the cytoskeleton at adherens junctions in the seminiferous epithelium of the rat testis.

During spermatogenesis, the movement of germ cells across the seminiferous epithelium is associated with extensive junction restructuring. Yet the underlying mechanism (or mechanisms) that regulates these events is largely unknown. If the molecular architecture of the cell-cell actin-based adherens junction (AJ), such as ectoplasmic specialization (ES) and tubulobulbar complex- two testis-specific AJ types, is known, many functional mechanistic studies can be designed. We thus undertook an investigation to study 3 adaptors in the seminiferous epithelium: zyxin, axin, and Wiskott-Aldrich syndrome protein (WASP). All 3 adaptors were shown to be products of Sertoli and germ cells. Zyxin was shown to be a stage-specific protein that was most prominent during stages V-VII and restricted mostly to pachytene spermatocytes, but it could also be detected at the site of basal and apical ectoplasmic specialization (ES). Zyxin, axin, and WASP were shown to be structurally linked to the N-cadherin/beta-catenin/alpha-actinin/actin complex but not to the nectin-3/afadin or the beta 1-integrin-mediated protein complexes. Interestingly, zyxin, axin, and WASP are also structurally linked to vimentin (an intermediate filament protein) and alpha-tubulin (the subunit of a microtubule), which suggests that they have a role (or roles) in the regulation of the dynamics of the desmosome-like junction and microtubule. These results illustrate that zyxin, axin, and WASP are adaptors in both AJs and intermediate filament-based desmosome-like junctions. This raises the possibility that classic cadherins are also associated with vimentin-based intermediate filaments via these adaptors in the testis. While virtually no N-cadherin was found to associate with vimentin in the seminiferous tubules, it did associate with vimentin when testis lysates were used. Interestingly, about 5% of the E-cadherin associated with vimentin in isolated seminiferous tubules, and about 50% of the E-cadherin in the testis used vimentin as its attachment site. These data suggest that cadherins in the testis, unlike those in other epithelia, use different attachment sites to anchor the cadherin/catenin complex to the cytoskeleton. The levels of zyxin, axin, and WASP were also assessed during AF-2364-mediated AJ disruption of the testis, which illustrated a time-dependent protein reduction that was similar to the trends observed in nectin-3 and afadin but was the opposite of those observed for N-cadherin and beta-catenin, which were induced. Collectively, these results illustrate that while these adaptors are structurally associated with the cadherin/catenin complex in the testis, they are regulated differently.

Adherens Junctions↗

PubMed1994

Involvement of Janus kinases, p52shc, Raf-1, and MEK-1 in the IL-6-induced mitogen-activated protein kinase cascade of a growth-responsive B cell line.

We have previously shown that the IL-6R in a growth-responsive B cell line, AF10, induces activation of mitogen-activated protein (MAP) kinase. Here we demonstrate the activation of Raf-1 and MEK-1, which act as a MAP kinase kinase kinase and a MAP kinase kinase, respectively, in the MAP kinase cascade induced by IL-6 in AF10 cells. IL-6 also induced tyrosine phosphorylation of the signaling transducing subunit of the IL-6R in AF10 cells, along with tyrosine phosphorylation of the gp130-associated tyrosine protein kinase JAK1 and the adaptor molecule p52shc. Although induction of tyrosine phosphorylation and activation of MAP kinase by IL-6 in a differentiation-responsive B cell line, SKW 6.4, were below the limits of detection, the phorbol ester PMA did activate Raf-1, MEK-1, and MAP kinase without inducing the phosphorylation of gp130, JAKs, or p52shc. These results suggest that JAK kinase family members associated with the IL-6R may participate in the activation of MAP kinase in AF10 cells by way of an adaptor protein and Ras-dependent kinase cascade.

Adaptor Proteins, Signal Transducing↗

PubMed1996

NADPH oxidase activity is independent of p47phox in vitro.

The neutrophil superoxide generating NADPH oxidase is activated by the assembly of cytosolic protein components with a membrane-associated flavocytochrome. The activity can be reconstituted in vitro using purified cytosolic factors p47(phox), p67(phox), and Rac plus the phospholipid-reconstituted flavocytochrome b558. Here, we demonstrate that activity is reconstituted in the absence of p47(phox) when high concentrations of p67(phox) and Rac are used. Vmax values were the same in the presence or absence of p47(phox), yet p47(phox) increases the affinity of both p67(phox) and Rac for the oxidase complex by nearly 2 orders of magnitude. p67(phox)-(1-246), a truncated form of the protein which eliminates SH3 domains involved in binding to p47(phox), also supports superoxide generation, both in the presence and absence of p47(phox), providing further evidence for p47(phox) independent activity. In the absence of p47(phox), p67(phox)-(1-246) binds to the NADPH oxidase complex 3-fold more tightly than does native p67(phox), indicating that the C terminus contains a region which masks binding to the oxidase complex. Results indicate that p47(phox) does not play a direct role in regulating electron transfer. Rather, its function is to serve as an adaptor protein to enhance the assembly of the other cytosolic components with the flavocytochrome and possibly to unmask a binding region in the N terminus of p67(phox) by binding to its C-terminal domains. p67(phox) and/or Rac play a more direct role in regulating electron transfer.

Arachidonic Acid↗