Jean Dahl

Phosphorylation of middle T by pp60c-src: A switch for binding of phosphatidylinositol 3-kinase and optimal tumorigenesis

Substitution of phenylalanine for tyrosine 315 of the polyoma virus middle T (mT) protein lowers the incidence and limits the spectrum of tumors induced following inoculation of the virus into newborn mice. This substitution removes the major site of phosphorylation by pp60c-src without altering the ability of mT to associate with or to activate pp60c-src. The mutant mT fails to show binding of a phosphatidylinositol 3-kinase (Ptdlns 3-kinase) activity that is normally present in wild-type mT complexes. Furthermore, an anti-peptide antiserum that specifically recognizes mT lacking phosphate at tyrosine 315 precipitates binary (mT-pp60c-src) but not ternary (mT-pp60c-src-Ptdlns 3-kinase) complexes from wild-type infected cell extracts. Reprecipitation with either anti-pp60c-src or anti-mT serum brings down ternary complexes containing mT phosphorylated on tyrosine 315. Phosphorylation of mT by pp60c-src in vivo is therefore a critical event for binding of Ptdlns 3-kinase and for expression of the full tumorigenic potential of the virus.

Induction and Utilization of an ATM Signaling Pathway by Polyomavirus

ABSTRACT Progression from G1 to S is essential for polyomavirus DNA replication and depends on the interaction of large T with the retinoblastoma gene product pRb. This virus-induced replication pathway is accompanied by p53 activation resembling a DNA damage response (12). We sought to determine whether this pathway depends in part on activation of the ATM (ataxia telangiectasia mutated) kinase and whether the virus gains advantages from this pathway beyond that of entry into S. We show that polyomavirus infection activates the S- and G2-phase checkpoints in primary as well as established mouse cells. Infected cells undergo a prolonged S phase compared to uninfected serum-stimulated cells and show no evidence of a G2→M transition before lytic death ensues. Infection is accompanied by increases in ATM activity in vitro and in the level of ATM-S1981-P in vivo. The incubation of infected cells with caffeine, a known ATM inhibitor, did not block entry into S but reduced the rate of viral compared to cellular DNA synthesis. Importantly, caffeine lowered the yields of viral DNA an average of 3- to 6-fold and those of infectious virus by as much as 10-fold. Virus yields were 10-fold lower in ATM −/− p53−/− than in ATM+/+ p53−/− mouse embryo fibroblasts, indicating a p53-independent role of ATM in productive infection. Replacement of the normal SMC1 (structural maintenance of chromosomes, or cohesin) protein, a critical ATM substrate in the DNA repair pathway, with its phosphorylation mutant SMC1S957AS966A also lowered virus yields by roughly 90%. We suggest that polyomavirus activates and utilizes a component(s) of an ATM pathway of DNA repair to prolong S phase and aid its own replication.

Ganglioside GD1a Restores Infectibility to Mouse Cells Lacking Functional Receptors for Polyomavirus

ABSTRACT Recent investigations on the pathway of cell entry by polyomavirus (Py) and simian virus 40 (SV40) have defined specific gangliosides as functional receptors mediating virus binding and transport from the plasma membrane to the endoplasmic reticulum (B. Tsai et al., EMBO J. 22:4346-4355, 2003; Gilbert and Benjamin, in press). These studies were carried out with C6 rat glioma cells, a heterologous host chosen for its known deficiency in ganglioside biosynthesis. Here, a cell genetic approach was undertaken to identify components required for the early steps of infection using mouse cells as the natural host for Py. Receptor-negative (R−) mouse cells, screened based on resistance to Py infection, were shown to bind Py but failed to allow entry of the virus. R− cells were also found to be resistant to SV40. Infectibility was restored or enhanced by the addition of the same specific gangliosides found in earlier studies with C6 cells. In one R− line, overexpression of caveolin-1 also increased infectibility. These results support and extend findings on gangliosides in lipid rafts as functional receptors and mediators of internalization for Py and SV40.

Stimulation of cell proliferation and polyphosphoinositide metabolism in Saccharomycescerevisiae GL7 by ergosterol

The effect of ergosterol on cell division and phospholipid metabolism was investigated in Saccharomyces cerevisiae strain GL7, a sterol and unsaturated fatty acid auxotroph. Cells growing poorly on cholesterol were stimulated to grow more rapidly by supplementing the medium with 100 ng of ergosterol per ml. Within 10 min after ergosterol addition to cells prelabeled with 32Pi or [3H]inositol the isotope content of the polyphosphoinositides increases markedly followed by an equally striking and rapid decrease. Subsequently upon continuous labeling, 32P incorporation into phosphatidylinositol and, to a lesser degree, other phospholipids increased. Finally 3h after ergosterol addition the growth rate increased. Only stimulation of the first process, i.e. polyphosphoinositide metabolism, upon ergosterol addition is resistant to inhibition by cycloheximide.

Effects of cycloartenol and lanosterol on artificial and natural membranes

Abstract Cycloartenol, a 9,19-cyclopropane sterol which is isomeric with lanosterol, showed an ability intermediate between lanosterol and cholesterol to increase the microviscosity of lecithin vesicles, to serve as a growth factor for the sterol auxotroph Mycoplasma capricolum , and to increase the microviscosity of M. capricolum membranes. The corresponding membrane effects of cyclolaudenol which contains a methyl group added to C-24 of the isooctenyl side chain of cycloartenol are more like those shown by lanosterol. We propose that the enhanced effectiveness of cycloartenol over lanosterol is due to a more favorable spatial disposition of the angular 14α-methyl group on the α-face of the molecule promoting more effective van der Waals contacts between the phospholipid fatty acyl chains and the sterol α-face. Side chain alkylation appears to perturb such contacts, reducing the effectiveness of cyclolaudenol for competent membrane function in M. capricolum .

Identification of TAZ as a Binding Partner of the Polyomavirus T Antigens

ABSTRACT A polyomavirus mutant isolated by the tumor host range selection procedure (19) has a three-amino-acid deletion (Δ2-4) in the common N terminus of the T antigens. To search for a cellular protein bound by wild-type but not the mutant T antigen(s), a yeast two-hybrid screen of a mouse embryo cDNA library was carried out with a bait of wild-type small T antigen (sT) fused N terminally to the DNA-binding domain of Gal4. TAZ, a transcriptional coactivator with a WW domain and PDZ-binding motif (17), was identified as a binding partner. TAZ bound in vivo to all three T antigens with different apparent affinities estimated as 1:7:100 (large T antigen [lT]:middle T antigen [mT]:sT). The Δ2-4 mutant T antigens showed no detectable binding. The sT and mT of the host range transformation-defective (hr-t) mutant NG59 with an alteration in the common sT/mT region (179 D→NI) and a normal N terminus also failed to bind TAZ, while the unaltered lT bound but with reduced affinity compared to that seen in a wild-type virus infection. The WW domain but not the PDZ-binding motif of TAZ was essential for T antigen binding. The Δ2-4 mutant was defective in viral DNA replication. Forced overexpression of TAZ blocked wild-type DNA replication in a manner dependent on the binding site for the polyomavirus enhancer-binding protein 2α. Wild-type polyomavirus T antigens effectively block transactivation by TAZ. The functional significance of TAZ interactions with polyomavirus T antigens is discussed.

Studies of Partially Transforming Polyomavirus Mutants Establish a Role for Phosphatidylinositol 3-Kinase in Activation of pp70 S6 Kinase

Infection of mouse fibroblasts by wild-type polyomavirus results in increased phosphorylation of ribosomal protein S6 (D.A. Talmage, J. Blenis, and T.L. Benjamin, Mol. Cell. Biol. 8:2309-2315, 1988). Here we identify pp70 S6 kinase (pp70S6K) as a target for signal transduction events leading from polyomavirus middle tumor antigen (mT). Two partially transforming virus mutants altered in different mT signalling pathways have been studied to elucidate the pathway leading to S6 phosphorylation. An upstream role for mT-phosphatidylinositol 3-kinase (PI3K) complexes in pp70S6K activation is implicated by the failure of 315YF, a mutant unable to promote PI3K binding, to elicit a response. This conclusion is supported by studies using wortmannin, a known inhibitor of PI3K. In contrast, stable interaction of mT with Shc, a protein thought to be involved upstream of Ras, is dispensable for pp70S6K activation. 250YS, a mutant mT which retains a binding site for PI3K but lacks one for Shc, stimulates pp70S6K to wild-type levels. Mutants 315YF and 250YS induce partial transformation of rats fibroblasts with distinct phenotypes, as judged from morphological and growth criteria. Neither mutant induces growth in soft agar, indicating that an increase in S6 phosphorylation, while necessary for cell cycle progression in normal mitogenesis, is not sufficient for anchorage-independent cell growth. In the polyomavirus systems, the latter requires integration of signals from mT involving both Shc and PI3K.

Induction and Bypass of p53 during Productive Infection by Polyomavirus

ABSTRACT Lytic infection by polyomavirus leads to elevated levels of p53 and induction of p53 target genes p21Cip1/WAF1 (p21) and BAX. This is seen both in polyomavirus-infected primary mouse cell cultures and in kidney tissue of infected mice. Stabilization of p53 and induction of a p53 response are accompanied by phosphorylation of p53 on serine 18, mimicking a DNA damage response. Stabilization of p53 does not depend on p19Arf interaction with mdm2. Cells infected by a mutant virus defective in binding pRb and in inducing G1-to-S progression show a greatly diminished p53 response. However, cells infected by wild-type virus and blocked from entering S phase by addition of mimosine still show a p53 response. These results suggest a role of E2F target genes in inducing a p53 response. Polyomavirus large T antigen coprecipitates with p53 phosphorylated on serine 18 and also with p21Cip1/WAF1. Implications of these and other findings on possible mechanisms of induction and override of p53 functions during productive infection by polyomavirus are discussed.

Functional asymmetry of the regions juxtaposed to the membrane-binding sequence of polyomavirus middle T antigen.

The functional importance of the two clusters of positively charged amino acids which flank the hydrophobic membrane-anchoring sequence of polyomavirus middle T (mT) protein has been investigated by using site-directed mutagenesis. A clear asymmetry was apparent. No effect on transformation was seen following multiple alterations or complete removal of the cluster at the carboxyl end of the protein. In contrast, a single substitution replacing the first arginine amino terminal to the hydrophobic stretch with glutamic acid, but not with lysine, histidine, or methionine, produced a partially transformation-defective mutant with a novel phenotype. This mutant failed to confer anchorage-independent growth on F111 established rat embryo fibroblasts but induced foci with altered morphology compared with wild-type mT. Biochemical studies on this mutant revealed that F111 clones expressing levels of mutant mT equivalent to those of wild-type controls showed a 65% reduction in pp60c-src activation and an 87% reduction in mT-associated phosphatidylinositol 3-kinase activity. However, F111 clones expressing seven times more mutant mT than did wild-type controls showed equal or greater levels of kinase activities yet remained incompletely transformed. Possible mechanisms involving this transformation-sensitive region of mT are discussed.

Transcriptional and post-translational regulation of the quiescence factor and putative tumor suppressor p150Sal2

The evolutionarily conserved SALL genes encode transcription factors with roles in embryonic development. The product of the SALL2 gene was first identified as a binding partner of the mouse polyoma virus large T antigen and later shown to possess tumor suppressor‐like functions. Independent studies identified SALL2 as a factor regulating the quiescent state in human fibroblasts. Here, we investigate factors that regulate the expression of SALL2 and turnover of p150Sal2 in growing vs. resting cells. The transcription factor AP4 increases along with SALL2 in quiescent cells and positively regulates SALL2 expression. TGFβ effectively inhibits expression of SALL2 and its regulator AP4 when added to quiescent fibroblasts. TGFβ repression of SALL2 and AP4 is independent of the induction of connective tissue growth factor (CTGF) by TGFβ. p150Sal2 disappears rapidly on restoration of serum. In both growing fibroblasts and established ovarian surface epithelial cells, p150Sal2 undergoes polyubiquitination and proteosomal degradation. A CUL4/DDB1 E3 ligase containing RBBP7 as the p150Sal2 receptor has been identified as mediating the destruction of p150Sal2 as cells transition from a quiescent to an actively growing state.—Sung, C. K., Dahl, J., Yim, H., Rodig, S., Benjamin, T. L. Transcriptional and post‐translational regulation of the quiescence factor and putative tumor suppressor p150Sal2. FASEB J. 25, 1275–1283 (2011). www.fasebj.org