Protein binding and signaling properties of RIN1 suggest a unique effector function

Human RIN1 was first characterized as a RAS binding protein based on the properties of its carboxyl-terminal domain. We now show that full-length RIN1 interacts with activated RAS in mammalian cells and defines a minimum region of 434 aa required for efficient RAS binding. RIN1 interacts with the “effector domain” of RAS and employs some RAS determinants that are common to, and others that are distinct from, those required for the binding of RAF1, a known RAS effector. The same domain of RIN1 that binds RAS also interacts with 14-3-3 proteins, extending the similarity between RIN1 and other RAS effectors. When expressed in mammalian cells, the RAS binding domain of RIN1 can act as a dominant negative signal transduction blocker. The amino-terminal domain of RIN1 contains a proline-rich sequence similar to consensus Src homology 3 (SH3) binding regions. This RIN1 sequence shows preferential binding to the ABL–SH3 domain in vitro. Moreover, the amino-terminal domain of RIN1 directly associates with, and is tyrosine phosphorylated by, c-ABL. In addition, RIN1 encodes a functional SH2 domain that has the potential to activate downstream signals. These data suggest that RIN1 is able to mediate multiple signals. A differential pattern of expression and alternate splicing indicate several levels of RIN1 regulation.

Constitutive signaling by the phototaxis receptor sensory rhodopsin II from disruption of its protonated Schiff base–Asp-73 interhelical salt bridge

Sensory rhodopsin II (SRII) is a repellent phototaxis receptor in the archaeon Halobacterium salinarum, similar to visual pigments in its seven-helix structure and linkage of retinal to the protein by a protonated Schiff base in helix G. Asp-73 in helix C is shown by spectroscopic analysis to be a counterion to the protonated Schiff base in the unphotolyzed SRII and to be the proton acceptor from the Schiff base during photoconversion to the receptor signaling state. Coexpression of the genes encoding mutated SRII with Asn substituted for Asp-73 (D73N) and the SRII transducer HtrII in H. salinarum cells results in a 3-fold higher swimming reversal frequency accompanied by demethylation of HtrII in the dark, showing that D73N SRII produces repellent signals in its unphotostimulated state. Analogous constitutive signaling has been shown to be produced by the similar neutral residue substitution of the Schiff base counterion and proton acceptor Glu-113 in human rod rhodopsin. The interpretation for both seven-helix receptors is that light activation of the wild-type protein is caused primarily by photoisomerization-induced transfer of the Schiff base proton on helix G to its primary carboxylate counterion on helix C. Therefore receptor activation by helix C–G salt-bridge disruption in the photoactive site is a general mechanism in retinylidene proteins spanning the vast evolutionary distance between archaea and humans.

RGS-GAIP, a GTPase-activating Protein for Gαi Heterotrimeric G Proteins, Is Located on Clathrin-coated Vesicles

RGS-GAIP (Gα-interacting protein) is a member of the RGS (regulator of G protein signaling) family of proteins that functions to down-regulate Gαi/Gαq-linked signaling. GAIP is a GAP or guanosine triphosphatase-activating protein that was initially discovered by virtue of its ability to bind to the heterotrimeric G protein Gαi3, which is found on both the plasma membrane (PM) and Golgi membranes. Previously, we demonstrated that, in contrast to most other GAPs, GAIP is membrane anchored and palmitoylated. In this work we used cell fractionation and immunocytochemistry to determine with what particular membranes GAIP is associated. In pituitary cells we found that GAIP fractionated with intracellular membranes, not the PM; by immunogold labeling GAIP was found on clathrin-coated buds or vesicles (CCVs) in the Golgi region. In rat liver GAIP was concentrated in vesicular carrier fractions; it was not found in either Golgi- or PM-enriched fractions. By immunogold labeling it was detected on clathrin-coated pits or CCVs located near the sinusoidal PM. These results suggest that GAIP may be associated with both TGN-derived and PM-derived CCVs. GAIP represents the first GAP found on CCVs or any other intracellular membranes. The presence of GAIP on CCVs suggests a model whereby a GAP is separated in space from its target G protein with the two coming into contact at the time of vesicle fusion.

Protein Kinase Activity and Identification of a Toxic Effector Domain of the Target of Rapamycin TOR Proteins in Yeast

In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM.

Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.

Testosterone Signaling through Internalizable Surface Receptors in Androgen Receptor-free Macrophages

Testosterone acts on cells through intracellular transcription-regulating androgen receptors (ARs). Here, we show that mouse IC-21 macrophages lack the classical AR yet exhibit specific nongenomic responses to testosterone. These manifest themselves as testosterone-induced rapid increase in intracellular free [Ca2+], which is due to release of Ca2+ from intracellular Ca2+ stores. This Ca2+ mobilization is also inducible by plasma membrane-impermeable testosterone-BSA. It is not affected by the AR blockers cyproterone and flutamide, whereas it is completely inhibited by the phospholipase C inhibitor U-73122 and pertussis toxin. Binding sites for testosterone are detectable on the surface of intact IC-21 cells, which become selectively internalized independent on caveolae and clathrin-coated vesicles upon agonist stimulation. Internalization is dependent on temperature, ATP, cytoskeletal elements, phospholipase C, and G-proteins. Collectively, our data provide evidence for the existence of G-protein-coupled, agonist-sequestrable receptors for testosterone in plasma membranes, which initiate a transcription-independent signaling pathway of testosterone.

Membrane-anchored Plakoglobins Have Multiple Mechanisms of Action in Wnt Signaling

In Wnt signaling, β-catenin and plakoglobin transduce signals to the nucleus through interactions with TCF-type transcription factors. However, when plakoglobin is artificially engineered to restrict it to the cytoplasm by fusion with the transmembrane domain of connexin (cnxPg), it efficiently induces a Wnt-like axis duplication phenotype in Xenopus. In Xenopus embryos, maternal XTCF3 normally represses ventral expression of the dorsalizing gene Siamois. Two models have been proposed to explain the Wnt-like activity of cnxPg: 1) that cnxPg inhibits the machinery involved in the turnover of cytosolic β-catenin, which then accumulates and inhibits maternal XTCF3, and 2) that cnxPg directly acts to inhibit XTCF3 activity. To distinguish between these models, we created a series of N-terminal deletion mutations of cnxPg and examined their ability to induce an ectopic axis in Xenopus, activate a TCF-responsive reporter (OT), stabilize β-catenin, and colocalize with components of the Wnt signaling pathway. cnxPg does not colocalize with the Wnt pathway component Dishevelled, but it does lead to the redistribution of APC and Axin, two proteins involved in the regulation of β-catenin turnover. Expression of cnxPg increases levels of cytosolic β-catenin; however, this effect does not completely explain its signaling activity. Although cnxPg and Wnt-1 stabilize β-catenin to similar extents, cnxPg activates OT to 10- to 20-fold higher levels than Wnt-1. Moreover, although LEF1 and TCF4 synergize with β-catenin and plakoglobin to activate OT, both suppress the signaling activity of cnxPg. In contrast, XTCF3 suppresses the signaling activity of both β-catenin and cnxPg. Both exogenous XLEF1 and XTCF3 are sequestered in the cytoplasm of Xenopus cells by cnxPg. Based on these data, we conclude that, in addition to its effects on β-catenin, cnxPg interacts with other components of the Wnt pathway, perhaps TCFs, and that these interactions contribute to its signaling activity.

Opposite Effects of Insulin on Focal Adhesion Proteins in 3T3-L1 Adipocytes and in Cells Overexpressing the Insulin Receptor

Insulin can regulate the abundance and organization of filamentous actin within cells in culture. Early studies using cell lines that overexpress the insulin receptor demonstrated that insulin caused a rapid reversible disassembly of actin filaments that coincided with the rapid tyrosine dephosphorylation of focal adhesion kinase. We have extended these studies by demonstrating that paxillin, another focal adhesion protein, and Src undergo tyrosine dephosphorylation in response to insulin in Chinese hamster ovary (CHO) and rat hepatoma (HTC) cells that overexpress the insulin receptor. This contrasted with the effect of insulin in parental CHO and HTC cells in which focal adhesion proteins were not dephosphorylated in response to the hormone. In addition, insulin caused a dispersion of focal adhesion proteins and disruption of actin filament bundles only in cells that overexpressed the insulin receptor. Moreover, in 3T3-L1 adipocytes, which are considered prototypic insulin-responsive cells, actin filament assembly was stimulated, and focal adhesion protein tyrosine phosphorylation was not altered. 3T3-L1 cells have more insulin receptors than either parental CHO or HTC cells but have fivefold less insulin receptors than the overexpressing cell lines. We hypothesize that a threshold may exist in which the overexpression of insulin receptors determines how insulin signaling pathways regulate the actin cytoskeleton.

Direct Involvement of N-Cadherin–mediated Signaling in Muscle Differentiation

Cell–cell interactions, mediated by members of the cadherin family of Ca2+-dependent adhesion molecules, play key roles in morphogenetic processes as well as in the transduction of long-range growth and differentiation signals. In muscle differentiation cell adhesion is involved in both early stages of myogenic induction and in later stages of myoblast interaction and fusion. In this study we have explored the involvement of a specific cadherin, namely N-cadherin, in myogenic differentiation. For that purpose we have treated different established lines of cultured myoblasts with beads coated with N-cadherin–specific ligands, including a recombinant N-cadherin extracellular domain, and anti-N-cadherin antibodies. Immunofluorescent labeling for cadherins and catenins indicated that treatment with the cadherin-reactive beads for several hours enhances the assembly of cell–cell adherens-type junctions. Moreover, immunofluorescence and immunoblotting analyses indicated that treatment with the beads for 12–24 h induces myogenin expression and growth arrest, which are largely independent of cell plating density. Upon longer incubation with the beads (2–3 d) a major facilitation in the expression of several muscle-specific sarcomeric proteins and in cell fusion into myotubes was observed. These results suggest that surface clustering or immobilization of N-cadherin can directly trigger signaling events, which promote the activation of a myogenic differentiation program.

Nck-2, a Novel Src Homology2/3-containing Adaptor Protein That Interacts with the LIM-only Protein PINCH and Components of Growth Factor Receptor Kinase-signaling Pathways

Many of the protein–protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3- and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. We report here the identification, cloning, and characterization of a widely expressed, Nck-related adaptor protein termed Nck-2. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We show that Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCH-Nck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, we show that Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-β, and IRS-1. The association of Nck-2 with EGF receptors was regulated by EGF stimulation and involved largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributed to the complex formation. The association of Nck-2 with PDGF receptor-β was dependent on PDGF activation and was mediated solely by the SH2 domain of Nck-2. Additionally, we have detected a stable association between Nck-2 and IRS-1 that was mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, we provide evidence indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2- and SH3-domain–containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways.

Roles of Bone Morphogenetic Protein Type I Receptors and Smad Proteins in Osteoblast and Chondroblast Differentiation

The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor–like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.

Organization of G Proteins and Adenylyl Cyclase at the Plasma Membrane

There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein α and β subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein α subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.

Aberrant B cell receptor signaling from B29 (Igβ, CD79b) gene mutations of chronic lymphocytic leukemia B cells

Chronic lymphocytic leukemia (CLL) B cells characteristically exhibit low or undetectable surface B cell receptor (BCR) and diminished responses to BCR-mediated signaling. These features suggest that CLL cells may have sustained mutations affecting one or more of the BCR proteins required for receptor surface assembly and signal transduction. Loss of expression and mutations in the critical BCR protein B29 (Igβ, CD79b), are prevalent in CLL and could produce the hallmark features of these leukemic B cells. Because patient CLL cells are intractable to manipulation, we developed a model system to analyze B29 mutations. Jurkat T cells stably expressing μ, κ, and mb1 efficiently assembled a functional BCR when infected with recombinant vaccinia virus bearing wild-type B29. In contrast, a B29 CLL mutant protein truncated in the transmembrane domain did not associate with μ or mb1 at the cell surface. Another B29 CLL mutant lacking the C-terminal immunoreceptor tyrosine activation motif tyrosine and distal residues brought the receptor to the surface as well as wild-type B29 but showed significant impairment in anti-IgM-stimulated signaling events including mitogen-activated protein kinase activation. These findings demonstrate that B29 mutations previously identified in CLL patients can affect BCR-dependent signaling and may contribute to the unresponsive B cell phenotype in CLL. Finally, the features of the B29 mutations in CLL predict that they may be generated by somatic hypermutation.

A regulator of G protein signaling interaction surface linked to effector specificity

Proteins of the regulator of G protein signaling (RGS) family accelerate GTP hydrolysis by the α subunits (Gα) of G proteins, leading to rapid recovery of signaling cascades. Many different RGS proteins can accelerate GTP hydrolysis by an individual Gα, and GTP hydrolysis rates of different Gαs can be enhanced by the same RGS protein. Consequently, the mechanisms for specificity in RGS regulation and the residues involved remain unclear. Using the evolutionary trace (ET) method, we have identified a cluster of residues in the RGS domain that includes the RGS-Gα binding interface and extends to include additional functionally important residues on the surface. One of these is within helix α3, two are in α5, and three are in the loop connecting α5 and α6. A cluster of surface residues on Gα previously identified by ET, and composed predominantly of residues from the switch III region and helix α3, is spatially contiguous with the ET-identified residues in the RGS domain. This cluster includes residues proposed to interact with the γ subunit of Gtα's effector, cGMP phosphodiesterase (PDEγ). The proximity of these clusters suggests that they form part of an interface between the effector and the RGS-Gα complex. Sequence variations in these residues correlate with PDEγ effects on GTPase acceleration. Because ET identifies residues important for all members of a protein family, these residues likely form a general site for regulation of G protein-coupled signaling cascades, possibly by means of effector interactions.

Exercise-induced changes in expression and activity of proteins involved in insulin signal transduction in skeletal muscle: Differential effects on insulin-receptor substrates 1 and 2

Level of physical activity is linked to improved glucose homeostasis. We determined whether exercise alters the expression and/or activity of proteins involved in insulin-signal transduction in skeletal muscle. Wistar rats swam 6 h per day for 1 or 5 days. Epitrochlearis muscles were excised 16 h after the last exercise bout, and were incubated with or without insulin (120 nM). Insulin-stimulated glucose transport increased 30% and 50% after 1 and 5 days of exercise, respectively. Glycogen content increased 2- and 4-fold after 1 and 5 days of exercise, with no change in glycogen synthase expression. Protein expression of the glucose transporter GLUT4 and the insulin receptor increased 2-fold after 1 day, with no further change after 5 days of exercise. Insulin-stimulated receptor tyrosine phosphorylation increased 2-fold after 5 days of exercise. Insulin-stimulated tyrosine phosphorylation of insulin-receptor substrate (IRS) 1 and associated phosphatidylinositol (PI) 3-kinase activity increased 2.5- and 3.5-fold after 1 and 5 days of exercise, despite reduced (50%) IRS-1 protein content after 5 days of exercise. After 1 day of exercise, IRS-2 protein expression increased 2.6-fold and basal and insulin-stimulated IRS-2 associated PI 3-kinase activity increased 2.8-fold and 9-fold, respectively. In contrast to IRS-1, IRS-2 expression and associated PI 3-kinase activity normalized to sedentary levels after 5 days of exercise. Insulin-stimulated Akt phosphorylation increased 5-fold after 5 days of exercise. In conclusion, increased insulin-stimulated glucose transport after exercise is not limited to increased GLUT4 expression. Exercise leads to increased expression and function of several proteins involved in insulin-signal transduction. Furthermore, the differential response of IRS-1 and IRS-2 to exercise suggests that these molecules have specialized, rather than redundant, roles in insulin signaling in skeletal muscle.