The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation.

We have identified a basic sequence in the N-terminal region of the 67-kDa serum response factor (p67SRF or SRF) responsible for its nuclear localization. A peptide containing this nuclear localization signal (NLS) translocates rabbit immunoglobulin G (IgG) into the nucleus as efficiently as a peptide encoding the simian virus 40 NLS. This effect is abolished by substituting any two of the four basic residues in this NLS. Overexpression of a modified form of SRF in which these basic residues have been mutated confirms the absolute requirement for this sequence, and not the other basic amino acid sequences adjacent to it, in the nuclear localization of SRF. Since this NLS is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase), we further investigated if A-kinase plays a role in the nuclear location of SRF. The nuclear transport of SRF proteins requires basal A-kinase activity, since inhibition of A-kinase by using either the specific inhibitory peptide PKIm or type II regulatory subunits (RII) completely prevents the nuclear localization of plasmid-expressed tagged SRF or an SRF-NLS-IgG conjugate. Direct phosphorylation of SRF by A-kinase can be discounted in this effect, since mutation of the putative phosphorylation sites in either the NLS peptide or the encoded full-length SRF protein had no effect on nuclear transport of the mutants. Finally, in support of an implication of A-kinase-dependent phosphorylation in a more general mechanism affecting nuclear import, we show that the nuclear transport of a simian virus 40-NLS-conjugated IgG or purified cyclin A protein is also blocked by inhibition of A-kinase, even though neither contains any potential sites for phosphorylation by A-kinase or can be phosphorylated by A-kinase in vitro

Solid-phase Synthesis and Cellular Localization of a C- and/or N-terminal Labelled Peptide

International audienc

Design of Carrier Peptide-Oligonucleotide Conjugates with Rapid Membrane Translocation and Nuclear Localization Properties

International audienc

Growth and differentiation of C2 myogenic cells are dependent on serum response factor.

In order to study to what extent and at which stage serum response factor (SRF) is indispensable for myogenesis, we stably transfected C2 myogenic cells with, successively, a glucocorticoid receptor expression vector and a construct allowing for the expression of an SRF antisense RNA under the direction of the mouse mammary tumor virus long terminal repeat. In the clones obtained, SRF synthesis is reversibly down-regulated by induction of SRF antisense RNA expression by dexamethasone, whose effect is antagonized by the anti-hormone RU486. Two kinds of proliferation and differentiation patterns have been obtained in the resulting clones. Some clones with a high level of constitutive SRF antisense RNA expression are unable to differentiate into myotubes; their growth can be blocked by further induction of SRF antisense RNA expression by dexamethasone. Other clones are able to differentiate and are able to synthesize SRF, MyoD, myogenin, and myosin heavy chain at confluency. When SRF antisense RNA expression is induced in proliferating myoblasts by dexamethasone treatment, cell growth is blocked and cyclin A concentration drops. When SRF antisense RNA synthesis is induced in arrested confluent myoblasts cultured in a differentiation medium, cell fusion is blocked and synthesis of not only SRF but also MyoD, myogenin, and myosin heavy chain is inhibited. Our results show, therefore, that SRF synthesis is indispensable for both myoblast proliferation and myogenic differentiation

Expression and activity of serum response factor is required for expression of the muscle-determining factor MyoD in both dividing and differentiating mouse C2C12 myoblasts.

To understand the mechanism by which the serum response factor (SRF) is involved in the process of skeletal muscle differentiation, we have assessed the effect of inhibiting SRF activity or synthesis on the expression of the muscle-determining factor MyoD. Inhibition of SRF activity in mouse myogenic C2C12 cells through microinjection of either the SRE oligonucleotide (which acts by displacing SRF proteins from the endogenous SRE sequences), purified SRF-DB (a 30-kDa portion of SRF containing the DNA-binding domain of SRF, which acts as a dominant negative mutant in vivo), or purified anti-SRF antibodies rapidly prevents the expression of MyoD. Moreover, the rapid shutdown of MyoD expression after in vivo inhibition of SRF activity is observed not only in proliferating myoblasts but also in myoblasts cultured under differentiating conditions. Additionally, by using a cellular system expressing a glucocorticoid-inducible antisense-SRF (from aa 74 to 244) we have shown that blocking SRF expression by dexamethasone induction of antisense SRF results in the lack of MyoD expression as probed by both immunofluorescence and Northern blot analysis. Taken together these data demonstrate that SRF expression and activity are required for the expression of the muscle-determining factor MyoD

Densovirus Crosses the Insect Midgut by Transcytosis and Disturbs the Epithelial Barrier Function

Densoviruses are parvoviruses that can be lethal for insects of different orders at larval stages. Although the horizontal transmission mechanisms are poorly known, densoviral pathogenesis usually starts with the ingestion of contaminated food by the host. Depending on the virus, this leads to replication restricted to the midgut or excluding it. In both cases the success of infection depends on the virus capacity to enter the intestinal epithelium. Using the Junonia coenia densovirus (JcDNV) as the prototype virus and the lepidopteran host Spodoptera frugiperda as an interaction model, we focused on the early mechanisms of infection during which JcDNV crosses the intestinal epithelium to reach and replicate in underlying target tissues. We studied the kinetics of interaction of JcDNV with the midgut epithelium and the transport mechanisms involved. Using several approaches, in vivo, ex vivo, and in vitro, at molecular and cellular levels, we show that JcDNV is specifically internalized by endocytosis in absorptive cells and then crosses the epithelium by transcytosis. As a consequence, viral entry disturbs the midgut function. Finally, we showed that four mutations on the capsid of JcDNV affect specific recognition by the epithelial cells but not their binding