The Texture of Loyalty

This paper examines whether and how reforms in corporate governance structures and practices in the United States may reshape conventional notions of the fiduciary duties owed by independent directors of public companies. The paper identifies two focal points for the evolution of directors\u27 fiduciary duties. First, various reforms in corporate governance assign more specific responsibilities to directors, arguably reorienting directors\u27 loyalty to due discharge of a specified function along with ongoing or residual duties of loyalty owed in more general terms to the corporation and its shareholders. The relationships among these specific duties and more general ones may be complex, as may be the consequences of increased emphasis on work to be done by directors as members of committees in contrast to the board as a whole. Second, reforms in corporate governance imply that a director\u27s duty of loyalty to the corporation and its shareholders requires more than disinterest, narrowly defined. That is, a director\u27s duty is one of fidelity to the interests of the corporation that imposes more than an obligation to refrain from participating in board decisions in which the director has a material financial interest. The paper prefaces discussion of evolution of directors\u27 duties by addressing two more fundamental questions about contemporary corporate governance: what role precisely should be assigned to directors, distinct from a corporation\u27s officers and its other senior executives? And what implications follow for the powers of shareholders? To the extent that directors can reasonably be expected to serve only a relatively formal or vestigial function, an expansion in shareholders\u27 powers may be warranted. Overall, the paper is a study of interrelationships among legal and nonlegal mechanisms that shape expectations for directors\u27 conduct. Although distinct, none operates in a vacuum. Formal structures, definitions, and requirements may shape how directors discharge their responsibilities by focusing directors\u27 attention on their gravity and, by enabling independent directors to function more collegially, facilitating the development of institutions of corporate governance that function independently of senior management. Articulating the content of directors\u27 responsibilities with greater specificity heightens expectations that these responsibilities will be fulfilled. In turn, higher expectations for directors\u27 conduct may serve to legitimate directors\u27 capacity, once elected, to exercise discretion independent of intervention from shareholders

Adenovirus E4 Open Reading Frame 4-Induced Dephosphorylation Inhibits E1A Activation of the E2 Promoter and E2F-1-Mediated Transactivation Independently of the Retinoblastoma Tumor Suppressor Protein

AbstractPrevious studies have shown that the cell cycle-regulated E2F transcription factor is subjected to both positive and negative control by phosphorylation. Here we show that in transient transfection experiments, adenovirus E1A activation of the viral E2 promoter is abrogated by coexpression of the viral E4 open reading frame 4 (E4-ORF4) protein. This effect does not to require the retinoblastoma protein that previously has been shown to regulate E2F activity. The inhibitory activity of E4-ORF4 appears to be specific because E4-ORF4 had little effect on, for example, E4-ORF6/7 transactivation of the E2 promoter. We further show that the repressive effect of E4-ORF4 on E2 transcription works mainly through the E2F DNA-binding sites in the E2 promoter. In agreement with this, we find that E4-ORF4 inhibits E2F-1/DP-1-mediated transactivation. We also show that E4-ORF4 inhibits E2 mRNA expression during virus growth. E4-ORF4 has previously been shown to bind to and activate the cellular protein phosphatase 2A. The inhibitory effect of E4-ORF4 was relieved by okadaic acid, which inhibits protein phosphatase 2A activity, suggesting that E4-ORF4 represses E2 transcription by inducing transcription factor dephosphorylation. Interestingly, E4-ORF4 did not inhibit the transactivation capacity of a Gal4-E2F hybrid protein. Instead, E4-ORF4 expression appears to result in reduced stability of E2F/DNA complexes

Adenovirus E4orf4 Induces HPV-16 Late L1 mRNA Production

The adenovirus E4orf4 protein regulates the switch from early to late gene expression during the adenoviral replication cycle. Here we report that overexpression of adenovirus E4orf4 induces human papillomavirus type 16 (HPV-16) late gene expression from subgenomic expression plasmids. E4orf4 specifically overcomes the negative effects of two splicing silencers at the two late HPV-16 splice sites SD3632 and SA5639. This results in the production of HPV-16 spliced L1 mRNAs. We show that the interaction of E4orf4 with protein phosphatase 2A (PP2A) is necessary for induction of HPV-16 late gene expression. Also an E4orf4 mutant that fails to bind the cellular splicing factor ASF/SF2 fails to induce L1 mRNA production. Collectively, these results suggest that dephosphorylation of SR proteins by E4orf4 activates HPV-16 late gene expression. Indeed, a mutant ASF/SF2 protein in which the RS-domain had been deleted could itself induce HPV-16 late gene expression, whereas wild type ASF/SF2 could not

HAdV-2-suppressed growth of SV40 T antigen-transformed mouse mammary epithelial cell-induced tumours in SCID mice

AbstractHuman adenovirus (HAdV) vectors are promising tools for cancer therapy, but the shortage of efficient animal models for productive HAdV infections has restricted the evaluation of systemic effects to mainly immunodeficient mice. Previously, we reported a highly efficient replication of HAdV-2 in a non-tumorigenic mouse mammary epithelial cell line, NMuMG. Here we show that HAdV-2 gene expression and progeny formation in NMuMG cells transformed with the SV40 T antigen (NMuMG-T cells) were as efficient as in the parental NMuMG cells. Injection of HAdV-2 into tumours established by NMuMG-T in SCID mice caused reduced tumour growth and signs of intratumoural lesions. HAdV-2 replicated within the NMuMG-T-established tumours, but not in interspersed host-derived tissues within the tumours. The specific infection of NMuMG-T-derived tumours was verified by the lack of viral DNA in kidney, lung or spleen although low levels of viral DNA was occasionally found in liver

The 5′-end heterogeneity of adenovirus virus-associated RNAI contributes to the asymmetric guide strand incorporation into the RNA-induced silencing complex

Human Adenovirus type 5 encodes two short RNA polymerase III transcripts, the virus-associated (VA) RNAI and VA RNAII, which can adopt stable hairpin structures that resemble micro-RNA precursors. The terminal stems of the VA RNAs are processed into small RNAs (mivaRNAs) that are incorporated into RISC. It has been reported that VA RNAI has two transcription initiation sites, which produce two VA RNAI species; a major species, VA RNAI(G), which accounts for 75% of the VA RNAI pool, and a minor species, VA RNAI(A), which initiates transcription three nucleotides upstream compared to VA RNAI(G). We show that this 5′-heterogeneity results in a dramatic difference in RISC assembly. Thus, both VA RNAI(G) and VA RNAI(A) are processed by Dicer at the same position in the terminal stem generating the same 3′-strand mivaRNA. This mivaRNA is incorporated into RISC with 200-fold higher efficiency compared to the 5′-strand of mivaRNAI. Of the small number of 5′-strands used in RISC assembly only VA RNAI(A) generated active RISC complexes. We also show that the 3′-strand of mivaRNAI, although being the preferred substrate for RISC assembly, generates unstable RISC complexes with a low in vitro cleavage activity, only around 2% compared to RISC assembled on the VA RNAI(A) 5′-strand

Single molecule analysis of combinatorial splicing

Alternative splicing creates diverse mRNA isoforms from single genes and thereby enhances complexity of transcript structure and of gene function. We describe a method called spliceotyping, which translates combinatorial mRNA splicing patterns along transcripts into a library of binary strings of nucleic acid tags that encode the exon composition of individual mRNA molecules. The exon inclusion pattern of each analyzed transcript is thus represented as binary data, and the abundance of different splice variants is registered by counts of individual molecules. The technique is illustrated in a model experiment by analyzing the splicing patterns of the adenovirus early 1A gene and the beta actin reference transcript. The method permits many genes to be analyzed in parallel and it will be valuable for elucidating the complex effects of combinatorial splicing

Two Cellular Protein Kinases, DNA-PK and PKA, Phosphorylate the Adenoviral L4-33K Protein and Have Opposite Effects on L1 Alternative RNA Splicing

Accumulation of the complex set of alternatively processed mRNA from the adenovirus major late transcription unit (MLTU) is subjected to a temporal regulation involving both changes in poly (A) site choice and alternative 3′ splice site usage. We have previously shown that the adenovirus L4-33K protein functions as an alternative splicing factor involved in activating the shift from L1-52,55K to L1-IIIa mRNA. Here we show that L4-33K specifically associates with the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) in uninfected and adenovirus-infected nuclear extracts. Further, we show that L4-33K is highly phosphorylated by DNA-PK in vitro in a double stranded DNA-independent manner. Importantly, DNA-PK deficient cells show an enhanced production of the L1-IIIa mRNA suggesting an inhibitory role of DNA-PK on the temporal switch in L1 alternative RNA splicing. Moreover, we show that L4-33K also is phosphorylated by protein kinase A (PKA), and that PKA has an enhancer effect on L4-33K-stimulated L1-IIIa splicing. Hence, we demonstrate that these kinases have opposite effects on L4-33K function; DNA-PK as an inhibitor and PKA as an activator of L1-IIIa mRNA splicing. Taken together, this is the first report identifying protein kinases that phosphorylate L4-33K and to suggest novel regulatory roles for DNA-PK and PKA in adenovirus alternative RNA splicing

Regulation of Human Adenovirus Alternative RNA Splicing by the Adenoviral L4-33K and L4-22K Proteins

Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced viral mRNAs. Studies aimed at characterizing the interactions between the virus and the host cell RNA splicing machinery have identified three viral proteins of special significance for the control of late viral gene expression: L4-33K, L4-22K, and E4-ORF4. L4-33K is a viral alternative RNA splicing factor that controls L1 alternative splicing via an interaction with the cellular protein kinases Protein Kinase A (PKA) and DNA-dependent protein kinase (DNA-PK). L4-22K is a viral transcription factor that also has been implicated in the splicing of a subset of late viral mRNAs. E4-ORF4 is a viral protein that binds the cellular protein phosphatase IIA (PP2A) and controls Serine/Arginine (SR)-rich protein activity by inducing SR protein dephosphorylation. The L4-33K, and most likely also the L4-22K protein, are highly phosphorylated in vivo. Here we will review the function of these viral proteins in the post-transcriptional control of adenoviral gene expression and further discuss the significance of potential protein kinases phosphorylating the L4-33K and/or L4-22K proteins