Cytosolic Signaling Proteins Research Articles

Intertissue Mechanical Stress Affects Frizzled-Mediated Planar Cell Polarity in the Drosophila Notum Epidermis

Frizzled/planar cell polarity (Fz/PCP) signaling controls the orientation of sensory bristles and cellular hairs (trichomes) along the anteroposterior axis of the Drosophila thorax (notum). A subset of the trichome-producing notum cells differentiate as "tendon cells," serving as attachment sites for the indirect flight muscles (IFMs) to the exoskeleton. Through the analysis of chascon (chas), a gene identified by its ability to disrupt Fz/PCP signaling under overexpression conditions, and jitterbug (jbug)/filamin, we show that maintenance of anteroposterior planar polarization requires the notum epithelia to balance mechanical stress generated by the attachment of the IFMs. chas is expressed in notum tendon cells, and its loss of function disturbs cellular orientation at and near the regions where IFMs attach to the epidermis. This effect is independent of the Fz/PCP and fat/dachsous systems. The chas phenotype arises during normal shortening of the IFMs and is suppressed by genetic ablation of the IFMs. chas acts through jbug/filamin and cooperates with MyosinII to modulate the mechanoresponse of notum tendon cells. These observations support the notion that the ability of epithelia to respond to mechanical stress generated by one or more interactions with other tissues during development and organogenesis influences the maintenance of its shape and PCP features.

Immunohistochemical detection of tyrosine phosphatase SHP‐1 predicts outcome after radical prostatectomy for localized prostate cancer

The protein tyrosine kinase (PTK) receptors and cytosolic signaling proteins as well as the protein tyrosine phosphatases (PTPs) have important roles in regulation of growth of the benign and malignant prostate gland. Here, we studied expression of the protein tyrosine phosphatase SHP-1 in prostate cancer cell lines and in human prostatic tissues. SHP-1 is expressed at a high level in LNCaP prostate cancer cells compared with PC3 cells. Silencing of SHP-1 expression with siRNA in LNCaP cells led to an increased rate of proliferation, whereas overexpression of SHP-1 by means of transient and stable transfection in PC3 cells led to a decrease in proliferation. Corresponding changes were observed in cyclin D1 expression. We further demonstrate that LNCaP and PC3 cells respond differently to IL-6 stimulation. SHP-1 overexpression in PC3 cells reversed IL-6 stimulation of proliferation, whereas in SHP-1-silenced LNCaP cells, IL-6 inhibition of proliferation was not affected. In addition, IL-6 treatment led to higher levels of phosphorylated STAT3 in SHP-1-silenced LNCaP cells than in control cells. Next, SHP-1 expression in human prostate cancer was analyzed by immunohistochemical staining of tissue microarrays comprising tumor specimens from 100 prostate cancer patients. We found an inverse correlation between the tumor level of SHP-1 expression and time to biochemical recurrence and clinical progression among prostate cancer patients. In conclusion, our results suggest that a decreased level of SHP-1 expression in prostate cancer cells is associated with a high proliferation rate and an increased risk of recurrence or clinical progression after radical prostatectomy for localized prostate cancer.

Structure of three tandem filamin domains reveals auto-inhibition of ligand binding

Human filamins are large actin-crosslinking proteins composed of an N-terminal actin-binding domain followed by 24 Ig-like domains (IgFLNs), which interact with numerous transmembrane receptors and cytosolic signaling proteins. Here we report the 2.5 Å resolution structure of a three-domain fragment of human filamin A (IgFLNa19–21). The structure reveals an unexpected domain arrangement, with IgFLNa20 partially unfolded bringing IgFLNa21 into close proximity to IgFLNa19. Notably the N-terminus of IgFLNa20 forms a β-strand that associates with the CD face of IgFLNa21 and occupies the binding site for integrin adhesion receptors. Disruption of this IgFLNa20–IgFLNa21 interaction enhances filamin binding to integrin β-tails. Structural and functional analysis of other IgFLN domains suggests that auto-inhibition by adjacent IgFLN domains may be a general mechanism controlling filamin–ligand interactions. This can explain the increased integrin binding of filamin splice variants and provides a mechanism by which ligand binding might impact filamin structure.

Crk-associated substrate (Cas) signaling protein functions with integrins to specify axon guidance during development

Members of the Cas family of Src homology 3 (SH3)-domain-containing cytosolic signaling proteins are crucial regulators of actin cytoskeletal dynamics in non-neuronal cells; however, their neuronal functions are poorly understood. Here, we identify a Drosophila Cas (DCas), find that Cas proteins are highly expressed in neurons and show that DCas is required for correct axon guidance during development. Functional analyses reveal that Cas specifies axon guidance by regulating the degree of fasciculation among axons. These guidance defects are similar to those observed in integrin mutants, and genetic analysis shows that integrins function together with Cas to facilitate axonal defasciculation. These results strongly support Cas proteins working together with integrins in vivo to direct axon guidance events.

Cytosolic signaling protein Ecsit also localizes to mitochondria where it interacts with chaperone NDUFAF1 and functions in complex I assembly

Ecsit is a cytosolic adaptor protein essential for inflammatory response and embryonic development via the Toll-like and BMP (bone morphogenetic protein) signal transduction pathways, respectively. Here, we demonstrate a mitochondrial function for Ecsit (an evolutionary conserved signaling intermediate in Toll pathways) in the assembly of mitochondrial complex I (NADH:ubiquinone oxidoreductase). An N-terminal targeting signal directs Ecsit to mitochondria, where it interacts with assembly chaperone NDUFAF1 in 500- to 850-kDa complexes as demonstrated by affinity purification and vice versa RNA interference (RNAi) knockdowns. In addition, Ecsit knockdown results in severely impaired complex I assembly and disturbed mitochondrial function. These findings support a function for Ecsit in the assembly or stability of mitochondrial complex I, possibly linking assembly of oxidative phosphorylation complexes to inflammatory response and embryonic development.

Docosahexaenoic acid selectively inhibits plasma membrane targeting of lipidated proteins

Membrane localization of lipidated cytosolic signaling proteins is mediated by interactions between specific lipid anchors and membranes, but little is known about the regulatory role of membrane composition in lipidated protein membrane targeting. Here, using green fluorescent protein (GFP) chimeras and quantitative fluorescence microscopy in living mouse colonocytes, we show that docosahexaenoic acid (DHA), a dietary polyunsaturated fatty acid (PUFA) with membrane lipid-modifying properties, selectively inhibits plasma membrane (PM) targeting and increases the endomembrane localization of lipidated proteins that are cytoplasmic cargo in the exocytic pathway, without affecting the exocytic pathway itself. DHA selectivity seems to be dictated by the protein trafficking route, independent of the functional state of proteins and the location and composition of membrane anchors. DHA enrichment in cell membranes was required to elicit the inhibitory effect. These data reveal that membrane lipid composition influences cell signaling by modulating intracellular trafficking and localization of membrane proteins, providing a potential molecular mechanism for the documented health benefits of DHA.

The Protein-tyrosine Phosphatase SHP-1 Associates with the Phosphorylated Immunoreceptor Tyrosine-based Activation Motif of FcγRIIa to Modulate Signaling Events in Myeloid Cells

Fc gamma RIIa is a low affinity IgG receptor uniquely expressed in human cells that promotes phagocytosis of immune complexes and induces inflammatory cytokine gene transcription. Recent studies have revealed that phagocytosis initiated by Fc gamma RIIa is tightly controlled by the inositol phosphatase SHIP-1, and the protein-tyrosine phosphatase SHP-1. Whereas the molecular nature of SHIP-1 involvement with Fc gamma RIIa has been well studied, it is not clear how SHP-1 is activated by Fc gamma RIIa to mediate its regulatory effect. Here we report that Fc gamma RIIa clustering induces SHP-1 phosphatase activity in THP-1 cells. Using synthetic phosphopeptides, and stable transfectants expressing immunoreceptor tyrosine-based activation motif (ITAM) tyrosine mutants of Fc gamma RIIa, we demonstrate that SHP-1 associates with the phosphorylated amino-terminal ITAM tyrosine of Fc gamma RIIa, whereas the tyrosine kinase Syk associates with the carboxyl-terminal ITAM tyrosine. Association of SHP-1 with Fc gamma RIIa ITAM appears to suppress total cellular tyrosine phosphorylation. Furthermore, Fc gamma RIIa clustering results in the association of SHP-1 with key signaling molecules such as Syk, p85 subunit of PtdIns 3-kinase, and p62dok, suggesting that these molecules may be substrates of SHP-1 in this system. Finally, overexpression of wild-type SHP-1 but not catalytically deficient SHP-1 led to a down-regulation of NF kappa B-dependent gene transcription in THP-1 cells activated by clustering Fc gamma RIIa.

Cytoskeletal remodeling of the airway smooth muscle cell: a mechanism for adaptation to mechanical forces in the lung.

Airway smooth muscle is continuously subjected to mechanical forces caused by changes in lung volume during breathing. These mechanical oscillations have profound effects on airway smooth muscle contractility both in vivo and in vitro. Alterations in airway smooth muscle properties in response to mechanical forces may result from adaptive changes in the organization of the actin cytoskeleton. Recent advances suggest that in airway smooth muscle, two cytosolic signaling proteins that associate with focal adhesion complexes, focal adhesion kinase (FAK) and paxillin, are involved in transducing external mechanical signals. FAK and paxillin regulate changes in the organization of the actin cytoskeleton and the activation of contractile proteins. Actin is in a dynamic state in airway smooth muscle and undergoes polymerization and depolymerization during the contraction-relaxation cycle. The organization of the cytoskeletal proteins, vinculin, talin, and alpha-actinin, which mediate linkages between actin filaments and transmembrane integrins, is also regulated by contractile stimulation in airway smooth muscle. The fluidity of the cytoskeletal structure of the airway smooth muscle cell may be fundamental to its ability to adapt and respond to the mechanical forces imposed on it in the lung during breathing.

Death by Integrins, Again

A programmed cell death pathway has been proposed by Cardo-Vila that appears to connect the regulation of protein kinase C (PKC), the cytosolic protein annexin V, and α v β 5 , a cell adhesion receptor that binds to extracellular matrix. A screen involving phage display led to the identification of a peptide that interacted specifically with the cytoplasmic tail of the β 5 integrin subunit. With a peptide-specific antibody, the authors purified and identified annexin V from endothelial cell extracts. Annexin V is a cytosolic signaling protein that associates with and inhibits PKC. Intracellular delivery of the peptide into cultured endothelial cells caused apoptosis while the cells were still attached to extracellular matrix (in contrast, anoikis is a type of cell death that results from cell detachment). This effect was not observed in the absence of β5 expression. The peptide disrupted the β 5 interaction with annexin V, but not the association of annexin V with PKC. The authors postulate that the peptide induces apoptosis by breaking a survival signaling pathway between integrins and PKC, thus allowing annexin V to inhibit PKC, which results in cell death. M. Cardo-Vila, W. Arap, R. Pasqaulini, αvβ5 integrin-dependent programmed cell death triggered by a peptide mimic of annexin V. Mol. Cell 11 , 1151-1162 (2003). [Online Journal]

Death by Integrins, Again

A programmed cell death pathway has been proposed by Cardo-Vila that appears to connect the regulation of protein kinase C (PKC), the cytosolic protein annexin V, and αvβ5, a cell adhesion receptor that binds to extracellular matrix. A screen involving phage display led to the identification of a peptide that interacted specifically with the cytoplasmic tail of the β5 integrin subunit. With a peptide-specific antibody, the authors purified and identified annexin V from endothelial cell extracts. Annexin V is a cytosolic signaling protein that associates with and inhibits PKC. Intracellular delivery of the peptide into cultured endothelial cells caused apoptosis while the cells were still attached to extracellular matrix (in contrast, anoikis is a type of cell death that results from cell detachment). This effect was not observed in the absence of β5 expression. The peptide disrupted the β5 interaction with annexin V, but not the association of annexin V with PKC. The authors postulate that the peptide induces apoptosis by breaking a survival signaling pathway between integrins and PKC, thus allowing annexin V to inhibit PKC, which results in cell death. M. Cardo-Vila, W. Arap, R. Pasqaulini, αvβ5 integrin-dependent programmed cell death triggered by a peptide mimic of annexin V. Mol. Cell 11, 1151-1162 (2003). [Online Journal]

Cytoplasmic transport of Stat3 by receptor-mediated endocytosis.

Signal transducer and activator of transcription (STAT) proteins are cytoplasmic transcription factors that translocate to the nucleus and regulate gene expression upon activation of cytokine or growth factor receptors. While this translocation event is essential for gene regulation by STATs, their mechanism of transport through the cytoplasm to the nucleus has remained elusive. We now report that cytoplasmic transport of Stat3 is an active process that requires receptor-mediated endocytosis. Stat3 co-localizes with endocytic vesicles in transit from the cell membrane to the perinuclear region in response to growth factor stimulation. Consistent with a role for receptor endocytosis in growth factor signaling, disruption of endocytosis with specific inhibitors blocks Stat3 nuclear translocation and Stat3-dependent gene regulation. These results indicate that receptor-mediated endocytosis may be a general mechanism of transport through the cytoplasm for a subset of cytoplasmic signaling proteins destined for the nucleus.

Differential expression of endophilin 1 and 2 dimers at central nervous system synapses.

Endophilin 1 is proposed to participate in synaptic vesicle biogenesis through SH3 domain-mediated interactions with the polyphosphoinositide phosphatase synaptojanin and the GTPase dynamin. Endophilin family members have also been identified as binding partners for a number of diverse cellular proteins. We define here the endophilin 1-binding site within synaptojanin 1 and show that this sequence independently and selectively purifies from brain extracts endophilin 1 and a closely related protein, endophilin 2. Endophilin 2, like endophilin 1, is highly expressed in brain, concentrated in nerve terminals, and found in complexes with synaptojanin and dynamin. Although a fraction of endophilins 1 and 2 coexist in the same complex, the distribution of these endophilin isoforms among central synapses only partially overlaps. Endophilins 1 and 2 are found predominantly as stable dimers through a predicted coiled-coil domain in their conserved NH2-terminal moiety. Dimerization may allow endophilins to link a number of different cellular targets to the endocytic machinery.

A Functional Nuclear Localization Sequence in the C-terminal Domain of SHP-1

The Src homology 2 domain-containing protein tyrosine phosphatases SHP-1 and SHP-2 play an important role in many intracellular signaling pathways. Both SHP-1 and SHP-2 have been shown to interact with a diverse range of cytosolic and membrane-bound signaling proteins. Generally, SHP-1 and SHP-2 perform opposing roles in signaling processes; SHP-1 acts as a negative regulator of transduction in hemopoietic cells, whereas SHP-2 acts as a positive regulator. Intriguingly, SHP-1 has been proposed to play a positive regulating role in nonhemopoietic cells, although the mechanisms for this are not understood. Here we show that green fluorescent protein-tagged SHP-1 is unexpectedly localized within the nucleus of transfected HEK293 cells. In contrast, the highly related SHP-2 protein is more abundant within the cytoplasm of transfected cells. In accordance with this, endogenous SHP-1 is localized within the nucleus of several other nonhemopoietic cell types, whereas SHP-2 is distributed throughout the cytoplasm. In contrast, SHP-1 is confined to the cytoplasm of hemopoietic cells, with very little nuclear SHP-1 evident. Using chimeric SHP proteins and mutagenesis studies, the nuclear localization signal of SHP-1 was identified within the C-terminal domain of SHP-1 and found to consist of a short cluster of basic amino acids (KRK). Although the KRK motif resembles half of a bipartite nuclear localization signal, it appears to function independently and is absolutely required for nuclear import. Our findings show that SHP-1 and SHP-2 are distinctly localized within nonhemopoietic cells, with the localization of SHP-1 differing dramatically between nonhemopoietic and hemopoietic cell lineages. This implies that SHP-1 nuclear import is a tightly regulated process and indicates that SHP-1 may possess novel nuclear targets.

Protein modules as organizers of membrane structure

Investigations conducted over the past 18 months have shed new light on how modular protein-binding domains, in particular PDZ domains, co-ordinate the assembly of functional plasma membrane domains. Members of the MAGUK (membrane-associated guanylate kinase) protein family, like PSD-95, use multiple domains to cluster ion channels, receptors, adhesion molecules and cytosolic signaling proteins at synapses, cellular junctions, and polarized membrane domains. Other PDZ proteins, like the Drosophila protein INAD and the epithelial Na(+)/H(+) regulatory factor (NHERF), organize cellular signaling by localizing transmembrane and cytosolic components to specific membrane domains and assembling these components into functional complexes. The organization of these proteins into discreet structures has functional consequences for downstream signaling.

The Gab1 Protein Is a Docking Site for Multiple Proteins Involved in Signaling by the B Cell Antigen Receptor

Gab1 is a member of the docking/scaffolding protein family which includes IRS-1, IRS-2, c-Cbl, p130(cas), and p62(dok). These proteins contain a variety of protein-protein interaction motifs including multiple tyrosine residues that when phosphorylated can act as binding sites for Src homology 2 (SH2) domain-containing signaling proteins. We show in the RAMOS human B cell line that Gab1 is tyrosine-phosphorylated in response to B cell antigen receptor (BCR) engagement. Moreover, tyrosine phosphorylation of Gab1 correlated with the binding of several SH2-containing signaling proteins to Gab1 including Shc, Grb2, phosphatidylinositol 3-kinase, and the SHP-2 tyrosine phosphatase. Far Western analysis showed that the SH2 domains of Shc, SHP-2, and the p85 subunit of phosphatidylinositol 3-kinase could bind directly to tyrosine-phosphorylated Gab1 isolated from activated RAMOS cells. In contrast, the Grb2 SH2 domain did not bind directly to Gab1 but instead to the Shc and SHP-2 associated with Gab1. We also show that Gab1 is present in the membrane-enriched particulate fraction of RAMOS cells and that Gab1/signaling protein complexes are found in this fraction after BCR engagement. Thus, tyrosine-phosphorylated Gab1 may recruit cytosolic signaling proteins to cellular membranes where they can act on membrane-bound targets. This may be a critical step in the activation of multiple BCR signaling pathways.

Alterations in the Growth Factor Signal Transduction Pathways and Modulators of the Cell Cycle in Endocervical Cells from Macaques Exposed to TCDD

After more than a year had elapsed since a single oral exposure to 2 and 4 μg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg, there was an apparent dose-related increased incidence of significant endocervical squamous metaplasia in a group of cynomolgus macaques (Scottet al.,1998). In the present experiments we investigated the mechanisms by which chemicals like TCDD could induce epithelial cell transdifferentiation in the primate endocervix. One focus of investigation was epidermal growth factor receptor (EGFR) and the key cytosolic signaling kinases, c-Src and protein tyrosine kinase (PTK), whose responses to TCDD are well characterized. A second focus was the distal kinase Erk2 that transduces the cytosolic signal into a nuclear signal, and which in combination with nuclear casein kinase II (CKII), can lead to activation of p53. Finally, we studied three key target proteins of activated p53 (wafl/p21, Cdc2 p34, and Cdk4), whose modulation could produce cell cycle effects.The studies were carried out using primary cell cultures prepared from endocervical epithelium recovered at necropsy from TCDD-treated (2 and 4 μg TCDD/kg) and untreated macaques. There was a significant decrease in EGFR binding activity in cells from TCDD-treated animals as compared to controls. A marked increase in the protein amount of H-Ras and a significant increase in the activity of c-Src kinase, PTK, and Erk2 were found in cells from TCDD-treated animals. A significant decrease in the activity of CKII and in the protein amount of p53, wafl/p21, and Cdc2 p34 was found. On the other hand, a substantial increase in the protein amount of Cdk4 and DNA binding activity of AP-1 was found in cells from TCDD-treated animals.In vitroexperiments using primary cultures of endocervical cells from untreated macaques revealed that these cells have AhR, and that c-Src protein is functionally attached to the AhR and is specifically activated upon ligand binding as judged by the following criteria. (1) A structure-activity relationship study with TCDD and three dioxin congeners revealed a rank order for their potency in activation of AhR-associated c-Src kinase from cervical cells which was identical to that of previously determined toxicity indices. (2) TCDD-induced, AhR-associated c-Src kinase activity was abolished when an AhR immunoprecipitate from cervical cells was preincubated with α-naphthoflavone (AhR blocker) or geldanamycin (Src kinase inhibitor) prior to the addition of TCDD. (3) The analysis of the AhR complex showed three proteins of molecular weights of 100 (AhR), 90, and 60 kDa. (4) The same protein with molecular weight 60 kDa was found when the immunoprecipitate with anti AhR-antibody was analyzed by SDS-PAGE, then transferred into nitrocellulose membrane followed by immunobloting the membrane with anti c-Src-antibody. Our data suggest that TCDD induced pathology in endocervical cells through changes in growth factor receptor signaling, other cytosolic signaling proteins, tumor suppressor proteins, and cell cycle proteins.

Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins.

Extracellular growth and differentiation factors induce changes in gene expression in the nucleus by initiating a series of protein associations that alter the subcellular localization of intracellular signaling proteins. Initial events involve receptor homo- or heterodimerization and subsequent recruitment of cytosolic signaling proteins to the inner leaflet of the plasma membrane. Intermediate events involve the translocation of proteins into the nucleus. Late events involve the recruitment of transcriptional activators to the vicinity of specific genes in the nucleus, resulting in increased gene transcription. The ability to induce signals at each of these three phases of signaling pathways is illustrated by the use of a heterodimeric chemical inducer of dimerization that causes a proximal relationship between two different target proteins.