Arrest of cell growth by necdin, a nuclear protein expressed in postmitotic neurons

Necdin is a 325 amino acid residue protein localized to the nuclei of postmitotic neurons, which withdraw permanently from the cell cycle. To examine whether necdin confers the postmitotic phenotype, necdin cDNA was stably transfected into NIH3T3 cells, in which the protein was conditionally expressed using a eukaryotic lac repressor-operator expression system. When the transfectants were induced to express ectopic necdin, cell growth was arrested without appreciable reduction in cell viability. The expressed necdin molecule was localized to the nuclei of the transfectants. These results suggest that necdin is a nuclear factor that governs the permanent arrest of cell growth of postmitotic neurons during development of the nervous systems of vertebrates.     

Anisotropy of the electronic structure and superconducting gap in Bi2Sr2CaCu2O8

Simian virus 40 large T antigen interacts with three cellular proteins, pRb, p107, and p130, through a common binding site on the T antigen protein called the E1A conserved region 2-like (CR2-like) domain. Mutations in this domain inactivate the transforming activity of large T antigen. Since these mutations have been demonstrated to abolish binding to pRb and p107, and presumably therefore affect binding to p130, assessment of the relative roles of these three proteins in transformation of rodent fibroblasts by T antigen has been difficult. We have examined the role of T antigen-pRb interactions in transformation. We have introduced a mutant T antigen, which is unable to bind any of these three proteins, into primary mouse fibroblasts derived from the embryos of mice in which the Rb gene encoding the retinoblastoma protein had been disrupted. This mutant is unable to transform the Rb-negative fibroblasts, indicating that inactivation of pRb is not the sole function of the CR2-like domain in the induction of transformation of mouse fibroblasts by simian virus 40.     

The DnaJ domain of polyomavirus large T antigen is required to regulate Rb family tumor suppressor function

Tumor suppressors of the retinoblastoma susceptibility gene family regulate cell growth and differentiation. Polyomavirus large T antigens (large T) bind Rb family members and block their function. Mutations of large T sequences conserved with the DnaJ family affect large T binding to a cellular DnaK, heat shock protein 70. The same mutations abolish large T activation of E2F-containing promoters and Rb binding-dependent large T activation of cell cycle progression. Cotransfection of a cellular DnaJ domain blocks wild-type large T action, showing that the connection between the chaperone system and tumor suppressors is direct. Although they are inactive in assays dependent on Rb family binding, mutants in the J region retain the ability to associate with pRb, p107, and p130. This suggests that binding of Rb family members by large T is not sufficient for their inactivation and that a functional J domain is required as well. This work connects the DnaJ and DnaK molecular chaperones to regulation of tumor suppressors by polyomavirus large T.     

Regulation of E2F4 mitogenic activity during terminal differentiation by its heterodimerization partners for nuclear translocation

E2F/DP heterodimers play a pivotal role in the regulation of cell growth and differentiation. A decrease in E2F/DP activity occurs during cell cycle arrest and differentiation. However, very little is known about the specific role of the various E2F/DP members along the transition from proliferation to terminal differentiation. We have previously shown that E2F4 accounts for the vast majority of the endogenous E2F in differentiating muscle cells. Here, we show that E2F4, which lacks a nuclear localization signal (nls), is distributed in both the nucleus and the cytoplasm, in either asynchronously growing myoblasts or differentiated myotubes. E2F4 nuclear accumulation is induced by the binding in the cytoplasm with specific partners p107, pRb2/p130, and DP3delta, an nls-containing spliced form of DP3, which provide the nls. Although overexpression of E2F4/DP3delta reactivates the cell cycle in quiescent cells, the E2F4 nuclear accumulation induced by pRb2/p130 and p107 correlates with cell growth arrest Moreover, E2F4/DP3delta-induced cell cycle reactivation is efficiently counteracted by either p107 or pRb2/p130 overexpression. Reinduction in quiescent cells of DNA synthesis by E2F1/DP1 overexpression is abrogated by coexpression of pRb and is hampered by MyoD overexpression. Both pRb2/p130 and pRb, as well as MyoD, are up-regulated in myotubes. Accordingly, multinucleated myotubes, which are induced to reenter the S-phase by oncoviral proteins, are refractory to cell cycle reactivation by forced expression of E2F4/DP3delta or E2F1/DP1. Thus, E2F/DP repression represents only one of multiple redundant circuits that control the postmitotic state in terminally differentiated cells and that are targeted by adenovirus E1A and SV40 large T antigen.     

The role of a lymphoid-restricted, Grb2-like SH3-SH2-SH3 protein in T cell receptor signaling

We have characterized an SH3-SH2-SH3 linker protein that is prominently expressed in lymphoid tissues. This protein has 58% sequence identity to Grb2. An identical protein called Grap has been found in hematopoietic cells. In Jurkat cells, T cell receptor activation leads to the association of Grap with phosphoproteins p36/38 and, to a lesser degree, Shc. This interaction is mediated by the Grap SH2 domain, which has similar binding specificity to the Grb2 SH2 domain. Grap also associates via its SH3 domains with Sos, the Ras guanine nucleotide exchange factor; with dynamin, a GTPase involved in membrane protein trafficking; and with Sam68, a nuclear RNA-binding protein that serves as a substrate of Src kinases during mitosis. T cell activation effects an increase in Grap association with p36/38, Shc, Sos, and dynamin. Sam68 binding is constitutive. Phospholipase C-gamma1 and Fyn are also found in activated Grap signaling complexes, although these interactions may not be direct. We conclude that Grap is a prominent component of lymphocyte receptor signaling. Based on the known functions of bound effector molecules, Grap-mediated responses to antigen challenge may include endocytosis of the T cell receptor, cellular proliferation, and regulated entry into the cell cycle.     

Differential regulation of the pocket domains of the retinoblastoma family proteins by the HPV16 E7 oncoprotein

The human papillomavirus E7 oncoprotein binds to the retinoblastoma (Rb) tumor suppressor protein, and the binding to Rb correlates with the oncogenic potential of E7. Recent studies from several laboratories indicated that the half-life of the Rb protein is reduced in cells that are stably transformed with E7, suggesting that E7 could induce the proteolytic degradation of Rb. To investigate whether the Rb degradation is a primary effect of E7 or a result of altered cell phenotype, we sought to develop assays that can distinguish between the two possibilities. Using recombinant adenovirus expressing the human papillomavirus type 16 E7 protein, we show that the expression of E7 leads to an increased rate of decay of the Rb protein. Moreover, Rb degradation immediately follows the expression of E7 suggesting that it is an early and primary effect. Consistent with a previous study, we observed that the E7-induced degradation of Rb can be blocked by the inhibitors of the 26S proteasome. We have also developed a transient transfection assay for the E7-induced degradation of Rb. Using this assay, we show that the pocket domain of Rb is necessary and sufficient for the E7-induced degradation. However, the proteolysis is relatively specific for Rb because the level of p107 or p130 was not significantly altered by the expression of E7. Thus, although E7 binds to all three members of the Rb family of proteins, the proteolysis is much more efficient in the case of Rb. In the transient transfection assays, adenovirus E1A and SV40 large T antigen failed to induce degradation of Rb, suggesting that the Rb degradation is a unique property of the E7 oncoprotein.     

The J domain of simian virus 40 large T antigen is required to functionally inactivate RB family proteins

Transformation by simian virus 40 large T antigen (TAg) is dependent on the inactivation of cellular tumor suppressors. Transformation minimally requires the following three domains: (i) a C-terminal domain that mediates binding to p53; (ii) the LXCXE domain (residues 103 to 107), necessary for binding to the retinoblastoma tumor suppressor protein, pRB, and the related p107 and p130; and (iii) an N-terminal domain that is homologous to the J domain of DnaJ molecular chaperone proteins. We have previously demonstrated that the N-terminal J domain of TAg affects the RB-related proteins by perturbing the phosphorylation status of p107 and p130 and promoting the degradation of p130 and that this domain is required for transformation of cells that express either p107 or p130. In this work, we demonstrate that the J domain of TAg is required to inactivate the ability of each member of the pRB family to induce a G1 arrest in Saos-2 cells. Furthermore, the J domain is required to override the repression of E2F activity mediated by p130 and pRB and to disrupt p130-E2F DNA binding complexes. These results imply that while the LXCXE domain serves as a binding site for the RB-related proteins, the J domain plays an important role in inactivating their function.     

The coxsackievirus-adenovirus receptor protein can function as a cellular attachment protein for adenovirus serotypes from subgroups A, C, D, E, and F

Attachment of an adenovirus (Ad) to a cell is mediated by the capsid fiber protein. To date, only the cellular fiber receptor for subgroup C serotypes 2 and 5, the so-called coxsackievirus-adenovirus receptor (CAR) protein, has been identified and cloned. Previous data suggested that the fiber of the subgroup D serotype Ad9 also recognizes CAR, since Ad9 and Ad2 fiber knobs cross-blocked each other's cellular binding. Recombinant fiber knobs and 3H-labeled Ad virions from serotypes representing all six subgroups (A to F) were used to determine whether the knobs cross-blocked the binding of virions from different subgroups. With the exception of subgroup B, all subgroup representatives cross-competed, suggesting that they use CAR as a cellular fiber receptor as well. This result was confirmed by showing that CAR, produced in a soluble recombinant form (sCAR), bound to nitrocellulose-immobilized virions from the different subgroups except subgroup B. Similar results were found for blotted fiber knob proteins. The subgroup F virus Ad41 has both short and long fibers, but only the long fiber bound sCAR. The sCAR protein blocked the attachment of all virus serotypes that bound CAR. Moreover, CHO cells expressing human CAR, in contrast to untransformed CHO cells, all specifically bound the sCAR-binding serotypes. We conclude therefore that Ad serotypes from subgroups A, C, D, E, and F all use CAR as a cellular fiber receptor.     

Adenoviral delivery of E2F-1 directs cell cycle reentry and p53-independent apoptosis in postmitotic adult myocardium in vivo

Irreversible exit from the cell cycle precludes the ability of cardiac muscle cells to increase cell number after infarction. Using adenoviral E1A, we previously demonstrated dual pocket protein- and p300-dependent pathways in neonatal rat cardiac myocytes, and have proven that E2F-1, which occupies the Rb pocket, suffices for these actions of E1A. By contrast, the susceptibility of adult ventricular cells to viral delivery of exogenous cell cycle regulators has not been tested, in vitro or in vivo. In cultured adult ventricular myocytes, adenoviral gene transfer of E2F-1 induced expression of proliferating cell nuclear antigen, cyclin-dependent protein kinase 4, cell division cycle 2 kinase, DNA synthesis, and apoptosis. In vivo, adenoviral delivery of E2F-1 by direct injection into myocardium induced DNA synthesis, shown by 5'-bromodeoxyuridine incorporation, and accumulation in G2/M, by image analysis of Feulgen-stained nuclei. In p53(-)/- mice, the prevalence of G1 exit was more than twofold greater; however, E2F-1 evoked apoptosis and rapid mortality comparably in both backgrounds. Thus, the differential effects of E2F-1 on G1 exit in wild-type versus p53-deficient mice illustrate the combinatorial power of viral gene delivery to genetically defined recipients: E2F-1 can override the G1/S checkpoint in postmitotic ventricular myocytes in vitro and in vivo, but leads to apoptosis even in p53(-)/- mice.