In a previous study, we found that the SHIP2 protein became tyrosine phosphorylated and associated with the Shc adapter protein in response to the treatment of cells with growth factors and insulin (T. Habib, J. A. Hejna, R. E. Moses, and S. J. Decker, J. Biol. Chem. 273:18605-18609, 1998). We describe here a novel interaction between SHIP2 and the p130Cas adapter protein, a mediator of actin cytoskeleton organization. SHIP2 and p130Cas association was detected in anti-SHIP2 immunoprecipitates from several cell types. Reattachment of trypsinized cells stimulated tyrosine phosphorylation of SHIP2 and increased the formation of a complex containing SHIP2 and a faster-migrating tyrosine-phosphorylated form of p130Cas . The fastermigrating form of p130Cas was no longer recognized by antibodies to the amino terminus of p130 Cas and appeared to be generated through proteolysis. Interaction of the SHIP2 protein with the various forms of p130Cas was mediated primarily through the SH2 domain of SHIP2. Immunofluorescence studies indicated that SHIP2 localized to focal contacts and to lamellipodia. Increased adhesion was observed in HeLa cells transiently expressing exogenous WT-SHIP2. These effects were not seen with SHIP2 possessing a mutation in the SH2 domain (R47G). Transfection of a catalytic domain deletion mutant of SHIP2 (⌬RV) inhibited cell spreading. Taken together, our studies suggest an important role for SHIP2 in adhesion and spreading.Products of phosphatidylinositol (PI) metabolism are important second messengers in cellular signaling pathways (1,11,70,76). Activation of PI 3Ј-kinase, which phosphorylates the 3Ј position of the inositol ring of PI, is a critical event in growth factor, insulin, and G protein-mediated signal transduction (14,25,49). In addition, PI 3Ј-kinase plays an important role in the regulation of adhesion and migration (68). PI 3Ј-kinase activation localizes to cell-cell and cell-matrix adhesion sites in epithelial cells, as well as to membrane ruffles in fibroblasts (78). Inhibition of PI 3Ј-kinase attenuated integrin-mediated adhesion and migration in several cell types, while expression of the catalytic p110 subunit of PI 3Ј-kinase enhanced cell adhesion (18,22,29,31,33,34,52,80). Moreover, p85, the regulatory subunit of PI 3Ј-kinase, interacted with proteins regulating adhesion and migration such as focal adhesion kinase (FAK) and p130Crk -associated substrate (p130 Cas ) (3,8,43).In vivo, the major substrate for PI 3Ј-kinase is phosphatidylinositol-4,5-bisphosphate [PI-(4,5)-P2] leading to the formation of phosphatidylinositol-3,4,5-trisphosphate [PI-(3,4,5)-P3] (63). Significant pools of phosphatidylinositol-3,4-bisphosphate [PI-(3,4)-P2] are also generated following PI 3Ј-kinase activation primarily through dephosphorylation of PI-(3,4,5)-P3 by 5Ј inositol phosphatases (27). PI-(3,4,5)-P3 and PI-(3,4)-P2 specifically interact with pleckstrin homology (PH) domains of proteins, regulating activity or intracellular localization of cellular enzymes such as Akt/PKB and its upstream k...
Phosphoinositide-dependent kinase (PDK1) regulates a number of pathways involved in responses to stress and in growth factor signaling; however, little is known concerning the mechanisms governing the activity of PDK1. In this report, we find that oxidative stress (H(2)O(2)) and vanadate induce tyrosine phosphorylation of PDK1. These effects of H(2)O(2) and vanadate were found in 293T cells and CH310T1/2 cells expressing exogenous PDK1 and in A20 lymphoma cells expressing endogenous PDK1. Exogenously expressed PDK1 was also tyrosine-phosphorylated in response to NGF treatment of 293T expressing TrkA. H(2)O(2) induced a more rapid tyrosine phosphorylation of PDK1 relative to vanadate, and only vanadate-induced tyrosine phosphorylation of PDK1 was sensitive to pretreatment of cells with wortmannin. In vitro, PDK1 could be tyrosine-phosphorylated by both the c-Src and Abl tyrosine kinases. Both H(2)O(2) and vanadate treatments increased the activity of PDK1 when the serum/glucocorticoid regulated kinase (SGK) was used as substrate. Vanadate treatment appeared to bypass the requirement for phosphatidylinositol 3,4,5-trisphosphate when Akt was used as substrate for PDK1. Tyrosine phosphorylation of PDK1 by the Abl tyrosine kinase also increased the activity of PDK1 toward SGK and Akt. These data suggest a novel mechanism through which PDK1 activity may be regulated.
(Coffin, 1996). Reverse transcriptase must perform two specializedThe prevailing model for reverse transcription suggests template switches during retroviral DNA synthesis.that the first strand transfer occurs only after -ssDNA Here, we used Moloney murine leukemia virus-based synthesis is completed when reverse transcriptase reaches vectors to examine the site of one of these switches the 5Ј end of genomic RNA (Gilboa et al., 1979). This during intracellular reverse transcription. Consistent model was based in part on the observation that fullwith original models for reverse transcription, but in length -ssDNA is a prominent product in so-called 'endocontrast to previous experimental data, we observed genous reactions', which involve studying DNA synthethat this first strand transfer nearly always occurred sized after the addition of nucleotide substrates to precisely at the 5Ј end of genomic RNA. This finding detergent-permeabilized purified virions (Haseltine et al., allowed us to use first strand transfer to study the 1979). However, -ssDNA is not detectable in infected classes of errors that reverse transcriptase can and/or cells unless the infecting virus is defective in RNase H does make when it switches templates at a defined activity (Coffin, 1979;Blain and Goff, 1995). The apparent position during viral DNA synthesis. We found that absence of -ssDNA from infected cells suggests either errors occurred at the site of first strand transfer that full-length -ssDNA is short lived and all -ssDNÃ 1000-fold more frequently than reported average that is synthesized performs the first jump, or else that reverse transcriptase error rates for template-internal discrete-length -ssDNA is not formed during intracellular positions. We then analyzed replication products of reverse transcription, possibly because the first strand specialized vectors that were designed to test possible transfer occurs before reverse transcriptase reaches the 5Ј origins for the switch-associated errors. Our results end of the RNA. Whether the first strand transfer occurs suggest that at least some errors arose via nonfrom the 5Ј end of the genome or instead takes place from templated nucleotide addition followed by mismatch an earlier, internal position has different implications extension at the point of strand transfer. We discuss regarding which contacts between enzyme and primerthe significance of our findings as they relate to the template are important during template switching. Footpossible contribution that template switch-associated printing of reverse transcriptase on a simple template has errors may make to retroviral mutation rates.revealed that the enzyme makes extensive contacts with Keywords: fidelity/retrovirus/reverse transcriptase the template strand both in front of and behind the growing point for DNA synthesis, but what contacts are made in the template switch intermediate is unknown (Woehrl et al., 1995a,b).
We have probed the interaction of human erythropoietin (EPO) with its receptor (EPO-R) by analyzing a panel of 17 EPO mutants in a variety of in vitro assays. Mutant proteins were expressed in 293s cells and quantified by using an N-terminal epitope tag in conjunction with a surface plasmon resonance assay. Receptor binding was studied using both a soluble form of the EPO-R extracellular domain in an ELISA-format binding competition assay and full-length EPO-R in transfected BaF3 cells. Proliferative activity of the mutants was also determined in the BaF3-derived cell line and was correlated with the results from binding assays. Based on the results of these assays, we identified two distinct receptor binding sites on the EPO molecule. We propose that one site, containing residues Arg-150 and Lys-152, binds initially to EPO receptor on the cell surface. A second site, containing Arg-103 and , is involved in binding a second EPO-R at the cell surface, thus forming a homodimeric receptor complex. Furthermore, we demonstrate that one EPO mutant (R103A), which has previously been shown to lack proliferative function, is in fact an EPO antagonist. Taken together, these data support a sequential dimerization mechanism of EPO-R activation.Human erythropoietin (EPO) is a 166-aa glycoprotein that is involved in the proliferation and differentiation of erythroid progenitor cells (1). These cellular responses are mediated by the EPO receptor (EPO-R), a 508-aa glycoprotein containing a single transmembrane domain (2) that has been classified as a member of the growth hormone subfamily of class I cytokine receptors (3). Several studies have implicated receptor dimerization in the EPO signal transduction mechanism. For example, a constitutively active variant of EPO-R has been isolated, containing an arginine to cysteine mutation at position 129 in the receptor extracellular domain (4). The R129C mutant receptors have been shown to form disulfide-linked dimers in plasma membranes (5), and several similar cysteine mutations have been discovered that render the receptor constitutively active (6). EPO-R has also been coexpressed with inactive EPO-R analogs that lack most of the cytoplasmic domain, revealing a dominant inhibitory effect of the inactive receptor on proliferative activity of the wild-type (WT) receptor (6, 7). Recent biophysical studies (8) have also suggested that EPO binds to two receptor molecules, one with high affinity ("1 nM) and one with low affinity (-1 ,uM Although dimerization of EPO-R at the cell surface has not been directly observed, we sought to explore the EPO-R activation mechanism in terms of a sequential dimerization model, as has been described for human growth hormone (hGH) receptor. X-ray crystallography (9) and other biophysical techniques (10) have demonstrated that one hGH molecule binds to two receptor molecules, and mutagenesis studies (11-13) have revealed important receptor binding determinants on hGH. hGH binds initially to one receptor via residues comprising "site 1" on the ho...
Inositol phosphatases play an important role in regulation of cellular levels of lipid second messengers. Recently we have reported a novel function for SHIP2 in cell adhesion and spreading. In this study, we further characterize the adhesion-dependent tyrosine phosphorylation of SHIP2 and examine the role of Src family tyrosine kinases in the regulation of SHIP2 function. SHIP2 was tyrosine phosphorylated during cell attachment and spreading on collagen I, but not on fibronectin, collagen IV, laminin or poly-L-lysine. SHIP2 tyrosine phosphorylation, induced by plating on a collagen-I-coated surface but not by epidermal growth factor or insulin treatment of cells, was completely blocked by small molecule inhibitors of Src family kinases. SHIP2 could be phosphorylated in vitro by recombinant Src kinase and tyrosines 986-987 in the NPXY motif of SHIP2 appear to be the major sites of phosphorylation for Src both in vitro and in vivo. An activated form of Src induced strong tyrosine phosphorylation of SHIP2 while a dominant-negative form decreased collagen-I-dependent SHIP2 phosphorylation. SHIP2 associated with the adapter protein Shc via its NPXY motif during cell spreading on collagen I in a Src activity-dependent manner. Expression of SHIP2 with mutated NPXY motif caused deregulation of lamellipodia formation during spreading on collagen I. These observations indicate that SHIP2 is regulated by Src family kinases during cell attachment and spreading on collagen I and suggest an important role for SHIP2 as a part of a signaling pathway that regulates actin cytoskeleton remodeling.
During retroviral DNA synthesis reverse transcriptase frequently performs nonrequired template switches that can lead to genetic rearrangements or recombination. It has been postulated that template switching occurs after pauses in the action of reverse transcriptase. Hence factors which affect pausing, such as polymerization rate, may affect the frequency of template switching. To address the hypothesis that increasing the time required to complete reverse transcription increases the frequency of template switching, we established conditions that lengthened the time required to complete a single round of intracellular Moloney murine leukemia virus reverse transcription approximately threefold. Under these conditions, which resulted from intracellular nucleotide pool imbalances generated with hydroxyurea, we examined template switching frequency using a lacZ-based tandem repeat deletion assay. We observed that the frequency of deletion during reverse transcription in hydroxyurea-treated cells was approximately threefold higher than that in untreated control cells. These findings suggest that rates of retroviral recombination may vary when the intracellular environment is altered.
The ability of an XPA minigene construct to complement the DNA repair defect in xeroderma pigmentosum group A (XP-A) cells was demonstrated. XP-A cells (XP12BE-SV) were stably transformed with an XPA minigene linked to a neomycin resistance (neor) expression cassette. The G418-resistant clone XAN1 was isolated and its DNA repair phenotype compared with XP12BE-SV cells transformed with a cosmid containing a human chromosome 8 gene and a neo(r) cassette and selected for G418 resistance (2-0-A2), DNA repair-normal human fibroblasts and untransfected XP12BE-SV cells. Colony forming ability after UV-irradiated reactivation of a UV-irradiated chloramphenicol acetyltransferase (CAT) expression vector and UV-induced mutagenesis in a supF tRNA shuttle vector (pSP189) were all restored to normal levels in XAN1 cells. In addition, mutation spectra in the supF gene of pSP189 after replication in all four cell lines were compiled at low (100 J/m2) and high (1000 J/m2) UV doses. The majority of mutations were point mutations and these were predominately G:C-->A:T transitions regardless of dose for all cell lines. Dose-dependent differences were observed in the positions of mutation hot spots in pSP189 mutation spectra after replication in all four cell lines. Mutation spectra for XAN1 and GM0637 cells had only minor differences. An increase in the proportion of transversions was observed only in plasmids irradiated with a low UV dose and replicated in XAN1 cells. 2-0-A2 cells were reported to have partial restoration of DNA repair that was later suggested to be caused by a reversion. 2-0-A2 cells were nearly identical to XP12BE-SV cells in all aspects investigated, indicating that transformation to neor had no effect on DNA repair in these cells.
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