SH2-containing inositol 5'-phosphatase SHIP2 associates with the p130(Cas) adapter protein and regulates cellular adhesion and spreading.

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 p130(Cas) adapter protein, a mediator of actin cytoskeleton organization. SHIP2 and p130(Cas) 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 p130(Cas). The faster-migrating form of p130(Cas) 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 p130(Cas) 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 (DeltaRV) inhibited cell spreading. Taken together, our studies suggest an important role for SHIP2 in adhesion and spreading.

Oxidative stress and vanadate induce tyrosine phosphorylation of phosphoinositide-dependent kinase 1 (PDK1).

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.

Altering the intracellular environment increases the frequency of tandem repeat deletion during Moloney murine leukemia virus reverse transcription.

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.

Stable transformation of xeroderma pigmentosum group A cells with an XPA minigene restores normal DNA repair and mutagenesis of UV-treated plasmids.

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.

A sequential dimerization mechanism for erythropoietin receptor activation.

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 Ser-104 (and possibly Arg-14), 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.

Characterization of the human XPA promoter.

We cloned the human xeroderma pigmentosum group A gene (XPA) and characterized the XPA promoter (pXPA) by transient cat expression. The pXPA is extraordinarily weak in human fibroblasts (1% of RSV-LTR) and appears to function without any of the usual promoter elements. Regions containing positive and negative control elements were localized.