The PH Domain and the Polybasic c Domain of Cytohesin-1 Cooperate specifically in Plasma Membrane Association and Cellular Function

Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in ∼100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2–3 μM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the β-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation–isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton’s tyrosine kinase with the adjacent Bruton’s tyrosine kinase motif, a novel zinc-containing fold.

Binding of Insulin-like Growth Factor (IGF)–Binding Protein-5 to Smooth-Muscle Cell Extracellular Matrix Is a Major Determinant of the Cellular Response to IGF-I

Insulin-like growth factor–binding protein-5 (IGFBP-5) has been shown to bind to fibroblast extracellular matrix (ECM). Extracellular matrix binding of IGFBP-5 leads to a decrease in its affinity for insulin-like growth factor-I (IGF-I), which allows IGF-I to better equilibrate with IGF receptors. When the amount of IGFBP-5 that is bound to ECM is increased by exogenous addition, IGF-I’s effect on fibroblast growth is enhanced. In this study we identified the specific basic residues in IGFBP-5 that mediate its binding to porcine smooth-muscle cell (pSMC) ECM. An IGFBP-5 mutant containing alterations of basic residues at positions 211, 214, 217, and 218 had the greatest reduction in ECM binding, although three other mutants, R214A, R207A/K211N, and K202A/R206N/R207A, also had major decreases. In contrast, three other mutants, R201A/K202N/R206N/R208A, and K217N/R218A and K211N, had only minimal reductions in ECM binding. This suggested that residues R207 and R214 were the most important for binding, whereas alterations in K211 and R218, which align near them, had minimal effects. To determine the effect of a reduction in ECM binding on the cellular replication response to IGF-I, pSMCs were transfected with the mutant cDNAs that encoded the forms of IGFBPs with the greatest changes in ECM binding. The ECM content of IGFBP-5 from cultures expressing the K211N, R214A, R217A/R218A, and K202A/R206N/R207A mutants was reduced by 79.6 and 71.7%, respectively, compared with cells expressing the wild-type protein. In contrast, abundance of the R201A/K202N/R206N/R208A mutant was reduced by only 14%. Cells expressing the two mutants with reduced ECM binding had decreased DNA synthesis responses to IGF-I, but the cells expressing the R201A/K202N/R206N/R208A mutant responded well to IGF-I. The findings suggest that specific basic amino acids at positions 207 and 214 mediate the binding of IGFBP-5 to pSMC/ECM. Smooth-muscle cells that constitutively express the mutants that bind weakly to ECM are less responsive to IGF-I, suggesting that ECM binding of IGFBP-5 is an important variable that determines cellular responsiveness.

Activation and Cellular Localization of the Cyclosporine A-sensitive Transcription Factor NF-AT in Skeletal Muscle Cells

The widely used immunosuppressant cyclosporine A (CSA) blocks nuclear translocation of the transcription factor, NF-AT (nuclear factor of activated T cells), preventing its activity. mRNA for several NF-AT isoforms has been shown to exist in cells outside of the immune system, suggesting a possible mechanism for side effects associated with CSA treatment. In this study, we demonstrate that CSA inhibits biochemical and morphological differentiation of skeletal muscle cells while having a minimal effect on proliferation. Furthermore, in vivo treatment with CSA inhibits muscle regeneration after induced trauma in mice. These results suggest a role for NF-AT–mediated transcription outside of the immune system. In subsequent experiments, we examined the activation and cellular localization of NF-AT in skeletal muscle cells in vitro. Known pharmacological inducers of NF-AT in lymphoid cells also stimulate transcription from an NF-AT–responsive reporter gene in muscle cells. Three isoforms of NF-AT (NF-ATp, c, and 4/x/c3) are present in the cytoplasm of muscle cells at all stages of myogenesis tested. However, each isoform undergoes calcium-induced nuclear translocation from the cytoplasm at specific stages of muscle differentiation, suggesting specificity among NF-AT isoforms in gene regulation. Strikingly, one isoform (NF-ATc) can preferentially translocate to a subset of nuclei within a single multinucleated myotube. These results demonstrate that skeletal muscle cells express functionally active NF-AT proteins and that the nuclear translocation of individual NF-AT isoforms, which is essential for the ability to coordinate gene expression, is influenced markedly by the differentiation state of the muscle cell.

Selection of Gβ Subunits with Point Mutations That Fail to Activate Specific Signaling Pathways In Vivo: Dissecting Cellular Responses Mediated by a Heterotrimeric G Protein in Dictyostelium discoideum

In Dictyostelium discoideum, a unique Gβ subunit is required for a G protein–coupled receptor system that mediates a variety of cellular responses. Binding of cAMP to cAR1, the receptor linked to the G protein G2, triggers a cascade of responses, including activation of adenylyl cyclase, gene induction, actin polymerization, and chemotaxis. Null mutations of the cAR1, Gα2, and Gβ genes completely impair all these responses. To dissect specificity in Gβγ signaling to downstream effectors in living cells, we screened a randomly mutagenized library of Gβ genes and isolated Gβ alleles that lacked the capacity to activate some effectors but retained the ability to regulate others. These mutant Gβ subunits were able to link cAR1 to G2, to support gene expression, and to mediate cAMP-induced actin polymerization, and some were able to mediate to chemotaxis toward cAMP. None was able to activate adenylyl cyclase, and some did not support chemotaxis. Thus, we separated in vivo functions of Gβγ by making point mutations on Gβ. Using the structure of the heterotrimeric G protein displayed in the computer program CHAIN, we examined the positions and the molecular interactions of the amino acids substituted in each of the mutant Gβs and analyzed the possible effects of each replacement. We identified several residues that are crucial for activation of the adenylyl cyclase. These residues formed an area that overlaps but is not identical to regions where bovine Gtβγ interacts with its regulators, Gα and phosducin.

Dissociation of Oct-1 from the Nuclear Peripheral Structure Induces the Cellular Aging-associated Collagenase Gene Expression

The cellular aging-associated transcriptional repressor that we previously named as Orpheus was identical to Oct-1, a member of the POU domain family. Oct-1 represses the collagenase gene, one of the cellular aging-associated genes, by interacting with an AT-rich cis-element in the upstream of the gene in preimmortalized cells at earlier population-doubling levels and in immortalized cells. In these stages of cells, considerable fractions of the Oct-1 protein were prominently localized in the nuclear periphery and colocalized with lamin B. During the cellular aging process, however, this subspecies of Oct-1 disappeared from the nuclear periphery. The cells lacking the nuclear peripheral Oct-1 protein exhibited strong collagenase expression and carried typical senescent morphologies. Concomitantly, the binding activity and the amount of nuclear Oct-1 protein were reduced in the aging process and resumed after immortalization. However, the whole cellular amounts of Oct-1 protein were not significantly changed during either process. Thus, the cellular aging-associated genes including the collagenase gene seemed to be derepressed by the dissociation of Oct-1 protein from the nuclear peripheral structure. Oct-1 may form a transcriptional repressive apparatus by anchoring nuclear matrix attachment regions onto the nuclear lamina in the nuclear periphery.

SET1, A Yeast Member of the Trithorax Family, Functions in Transcriptional Silencing and Diverse Cellular Processes

The trithorax gene family contains members implicated in the control of transcription, development, chromosome structure, and human leukemia. A feature shared by some family members, and by other proteins that function in chromatin-mediated transcriptional regulation, is the presence of a 130- to 140-amino acid motif dubbed the SET or Tromo domain. Here we present analysis of SET1, a yeast member of the trithorax gene family that was identified by sequence inspection to encode a 1080-amino acid protein with a C-terminal SET domain. In addition to its SET domain, which is 40–50% identical to those previously characterized, SET1 also shares dispersed but significant similarity to Drosophila and human trithorax homologues. To understand SET1 function(s), we created a null mutant. Mutant strains, although viable, are defective in transcriptional silencing of the silent mating-type loci and telomeres. The telomeric silencing defect is rescued not only by full-length episomal SET1 but also by the conserved SET domain of SET1. set1 mutant strains display other phenotypes including morphological abnormalities, stationary phase defects, and growth and sporulation defects. Candidate genes that may interact with SET1 include those with functions in transcription, growth, and cell cycle control. These data suggest that yeast SET1, like its SET domain counterparts in other organisms, functions in diverse biological processes including transcription and chromatin structure.

Phosphorylated Human Keratinocyte Ornithine Decarboxylase Is Preferentially Associated with Insoluble Cellular Proteins

Ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, is highly regulated by many trophic stimuli, and changes in its levels and organization correlate with cytoskeletal changes in normal human epidermal keratinocytes (NHEK). NHEK ODC exhibits a filamentous perinuclear/nuclear localization that becomes more diffuse under conditions that alter actin architecture. We have thus asked whether ODC colocalizes with a component of the NHEK cytoskeleton. Confocal immunofluorescence showed that ODC distribution in NHEK was primarily perinuclear; upon disruption of the actin cytoskeleton with cytochalasin D, ODC distribution was diffuse. The ODC distribution in untreated NHEK overlapped with that of keratin in the perinuclear but not cytoplasmic area; after treatment with cytochalasin D, overlap between staining for ODC and for keratin was extensive. No significant overlap with actin and minimal overlap with tubulin filament systems were observed. Subcellular fractionation by sequential homogenizations and centrifugations of NHEK lysates or detergent and salt extractions of NHEK in situ revealed that ODC protein and activity were detectable in both soluble and insoluble fractions, with mechanical disruption causing additional solubilization of ODC activity (three- to sevenfold above controls). Fractionation and ODC immunoprecipitation from [32P]orthophosphate-labeled NHEK lysates showed that a phosphorylated form of ODC was present in the insoluble fractions. Taken together, these data suggest that two pools of ODC exist in NHEK. The first is the previously described soluble pool, and the second is enriched in phospho-ODC and associated with insoluble cellular material that by immunohistochemistry appears to be organized in conjunction with the keratin cytoskeleton.

Somatic polyploidization and cellular proliferation drive body size evolution in nematodes

Most of the hypodermis of a rhabditid nematode such as Caenorhabditis elegans is a single syncytium. The size of this syncytium (as measured by body size) has evolved repeatedly in the rhabditid nematodes. Two cellular mechanisms are important in the evolution of body size: changes in the numbers of cells that fuse with the syncytium, and the extent of its acellular growth. Thus nematodes differ from mammals and other invertebrates in which body size evolution is caused by changes in cell number alone. The evolution of acellular syncytial growth in nematodes is also associated with changes in the ploidy of hypodermal nuclei. These nuclei are polyploid as a consequence of iterative rounds of endoreduplication, and this endocycle has evolved repeatedly. The association between acellular growth and endoreduplication is also seen in C. elegans mutations that interrupt transforming growth factor-β signaling and that result in dwarfism and deficiencies in hypodermal ploidy. The transforming growth factor-β pathway is a candidate for being involved in nematode body size evolution.

Cellular responses to psychomotor stimulant and neuroleptic drugs are abnormal in mice lacking the D1 dopamine receptor

Stimulation of dopamine D1 receptors has profound effects on addictive behavior, movement control, and working memory. Many of these functions depend on dopaminergic systems in the striatum and D1–D2 dopamine receptor synergies have been implicated as well. We show here that deletion of the D1 dopamine receptor produces a neural phenotype in which amphetamine and cocaine, two addictive psychomotor stimulants, can no longer stimulate neurons in the striatum to express cFos or JunB or to regulate dynorphin. By contrast, haloperidol, a typical neuroleptic that acts preferentially at D2-class receptors, remains effective in inducing catalepsy and striatal Fos/Jun expression in the D1 mutants, and these behavioral and neural effects can be blocked by D2 dopamine receptor agonists. These findings demonstrate that D2 dopamine receptors can function without the enabling role of D1 receptors but that D1 dopamine receptors are essential for the control of gene expression and motor behavior by psychomotor stimulants.

Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains

Results of transgenetic studies argue that the scrapie isoform of the prion protein (PrPSc) interacts with the substrate cellular PrP (PrPC) during conversion into nascent PrPSc. While PrPSc appears to accumulate primarily in lysosomes, caveolae-like domains (CLDs) have been suggested to be the site where PrPC is converted into PrPSc. We report herein that CLDs isolated from scrapie-infected neuroblastoma (ScN2a) cells contain PrPC and PrPSc. After lysis of ScN2a cells in ice-cold Triton X-100, both PrP isoforms and an N-terminally truncated form of PrPC (PrPC-II) were found concentrated in detergent-insoluble complexes resembling CLDs that were isolated by flotation in sucrose gradients. Similar results were obtained when CLDs were purified from plasma membranes by sonication and gradient centrifugation; with this procedure no detergents are used, which minimizes artifacts that might arise from redistribution of proteins among subcellular fractions. The caveolar markers ganglioside GM1 and H-ras were found concentrated in the CLD fractions. When plasma membrane proteins were labeled with the impermeant reagent sulfo-N-hydroxysuccinimide-biotin, both PrPC and PrPSc were found biotinylated in CLD fractions. Similar results on the colocalization of PrPC and PrPSc were obtained when CLDs were isolated from Syrian hamster brains. Our findings demonstrate that both PrPC and PrPSc are present in CLDs and, thus, support the hypothesis that the PrPSc formation occurs within this subcellular compartment.

On the molecular basis and regulation of cellular capacitative calcium entry: Roles for Trp proteins

During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular Ca2+ homeostasis and the physiological roles of CCE.

The elasticity and failure of fluid-filled cellular solids: Theory and experiment

We extend and apply theories of filled foam elasticity and failure to recently available data on foods. The predictions of elastic modulus and failure mode dependence on internal pressure and on wall integrity are borne out by photographic evidence of distortion and failure under compressive loading and under the localized stress applied by a knife blade, and by mechanical data on vegetables differing only in their turgor pressure. We calculate the dry modulus of plate-like cellular solids and the cross over between dry-like and fully fluid-filled elastic response. The bulk elastic properties of limp and aging cellular solids are calculated for model systems and compared with our mechanical data, which also show two regimes of response. The mechanics of an aged, limp beam is calculated, thus offering a practical procedure for comparing experiment and theory. This investigation also thereby offers explanations of the connection between turgor pressure and crispness and limpness of cellular materials.

A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity

This study addresses the properties of a newly identified internal ribosome entry site (IRES) contained within the mRNA of the homeodomain protein Gtx. Sequential deletions of the 5′ untranslated region (UTR) from either end did not define distinct IRES boundaries; when five nonoverlapping UTR fragments were tested, four had IRES activity. These observations are consistent with other cellular IRES analyses suggesting that some cellular IRESes are composed of segments (IRES modules) that independently and combinatorially contribute to overall IRES activity. We characterize a 9-nt IRES module from the Gtx 5′ UTR that is 100% complementary to the 18S rRNA at nucleotides 1132–1124. In previous work, we demonstrated that this mRNA segment could be crosslinked to its complement within intact 40S subunits. Here we show that increasing the number of copies of this IRES module in the intercistronic region of a dicistronic mRNA strongly enhances IRES activity in various cell lines. Ten linked copies increased IRES activity up to 570-fold in Neuro 2a cells. This level of IRES activity is up to 63-fold greater than that obtained by using the well characterized encephalomyocarditis virus IRES when tested in the same assay system. When the number of nucleotides between two of the 9-nt Gtx IRES modules was increased, the synergy between them decreased. In light of these findings, we discuss possible mechanisms of ribosome recruitment by cellular mRNAs, address the proposed role of higher order RNA structures on cellular IRES activity, and suggest parallels between IRES modules and transcriptional enhancer elements.

Interaction of 5-lipoxygenase with cellular proteins

5-Lipoxygenase (5LO) plays a pivotal role in cellular leukotriene synthesis. To identify proteins interacting with human 5LO, we used a two-hybrid approach to screen a human lung cDNA library. From a total of 1.5 × 107 yeast transformants, nine independent clones representing three different proteins were isolated and found to specifically interact with 5LO. Four 1.7- to 1.8-kb clones represented a 16-kDa protein named coactosin-like protein for its significant homology with coactosin, a protein found to be associated with actin in Dictyostelium discoideum. Coactosin-like protein thus may provide a link between 5LO and the cytoskeleton. Two other yeast clones of 1.5 kb encoded transforming growth factor (TGF) type β receptor-I-associated protein 1 partial cDNA. TGF type β receptor-I-associated protein 1 recently has been reported to associate with the activated form of the TGF β receptor I and may be involved in the TGF β-induced up-regulation of 5LO expression and activity observed in HL-60 and Mono Mac 6 cells. Finally, three identical 2.1-kb clones contained the partial cDNA of a human protein with high homology to a hypothetical helicase K12H4.8 from Caenorhabditis elegans and consequently was named ΔK12H4.8 homologue. Analysis of the predicted amino acid sequence revealed the presence of a RNase III motif and a double-stranded RNA binding domain, indicative of a protein of nuclear origin. The identification of these 5LO-interacting proteins provides additional approaches to studies of the cellular functions of 5LO.

The cellular ecology of progressive neoplastic transformation: A clonal analysis

A comparison was made of the competence for neoplastic transformation in three different sublines of NIH 3T3 cells and multiple clonal derivatives of each. Over 90% of the neoplastic foci produced by an uncloned transformed (t-SA′) subline on a confluent background of nontransformed cells were of the dense, multilayered type, but about half of the t-SA′ clones produced only light foci in assays without background. This asymmetry apparently arose from the failure of the light focus formers to register on a background of nontransformed cells. Comparison was made of the capacity for confluence-mediated transformation between uncloned parental cultures and their clonal derivatives by using two nontransformed sublines, one of which was highly sensitive and the other relatively refractory to confluence-mediated transformation. Transformation was more frequent in the clones than in the uncloned parental cultures for both sublines. This was dramatically so in the refractory subline, where the uncloned culture showed no overt sign of transformation in serially repeated assays but increasing numbers of its clones exhibited progressive transformation. The reason for the greater susceptibility of the pure clones is apparently the suppression of transformation among the diverse membership that makes up the uncloned parental culture. Progressive selection toward increasing degrees of transformation in confluent cultures plays a major role in the development of dense focus formers, but direct induction by the constraint of confluence may contribute by heritably damaging cells. In view of our finding of increased susceptibility to transformation in clonal versus uncloned populations, expansion of some clones at the expense of others during the aging process would contribute to the marked increase of cancer with age.