Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase.

Cyclin E binds and activates the cyclin-dependent kinase Cdk2 and catalyzes the transition from the G1 phase to the S phase of the cell cycle. The amount of cyclin E protein present in the cell is tightly controlled by ubiquitin-mediated proteolysis. Here we identify the ubiquitin ligase responsible for cyclin E ubiquitination as SCFFbw7 and demonstrate that it is functionally conserved in yeast, flies, and mammals. Fbw7 associates specifically with phosphorylated cyclin E, and SCFFbw7 catalyzes cyclin E ubiquitination in vitro. Depletion of Fbw7 leads to accumulation and stabilization of cyclin E in vivo in human and Drosophila melanogaster cells. Multiple F-box proteins contribute to cyclin E stability in yeast, suggesting an overlap in SCF E3 ligase specificity that allows combinatorial control of cyclin E degradation.

Functional interaction of Jun and homeodomain proteins.

We have used the yeast two-hybrid system to identify proteins that interact with the N-terminal region of c-Jun, which is known to be involved in regulatory interactions. One of the proteins identified is the homeodomain-containing protein Hex. The Hex homeodomain is sufficient for interaction; moreover, the homeodomains of several other transcription factors also interact. Mutations within helix III of the Hex homeodomain greatly reduce the interaction. In vitro, c-Jun/c-Fos, JunB/c-Fos, and JunD/c-Fos all interact with the Hex homeodomain more strongly than the respective Jun proteins (or c-Fos) alone, suggesting that heterodimerization exposes reactive regions in the N termini of the Jun proteins. In transfected cells, Hex expression inhibits Jun- or Jun/c-Fos-dependent transcription of a reporter gene; the presence of Hex-binding sites in the promoter enhances the inhibitory effect. Jun-dependent activation of transcription from the basic fibroblast growth factor gene, previously shown to be regulated by both Jun and homeodomain proteins, was also dramatically reduced by Hex expression. Furthermore, in contrast to the reduction of Jun-mediated transcription by Hex, we found that expression of the Drosophila ultrabithorax gene enhanced c-Jun-dependent transcription. We conclude that the functional interaction between members of the Jun and homeodomain families of transcription factors could play a critical role in regulating developmental and differentiation programs.

Oncostatin M activates stat DNA binding and transcriptional activity in primary human fetal astrocytes: low- and high-passage cells have distinct patterns of stat activation.

In this study we explored the activation of the JAK/Stat pathway by gp 130 family cytokines in primary human astrocytes. We report that of four gp 130 cytokines tested, only oncostatin M (OnM) resulted in the activation of Stat molecules. To test that the induced molecules were transcriptionally active, transcription from a Stat-responsive reporter plasmid (from the acute-phase gene alpha-2 macroglobulin) transiently transfected into astrocytes was assessed after activation by OnM and was blocked by cotransfection with dominant-negative Stat3 encoding plasmids strongly suggesting that the activation was Stat-mediated. While DNA binding complexes comprised of both Stat1 and Stat3 were induced in low-passage cells, only those containing Stat3 were formed by extracts from high-passage cells. Stat1 protein was detected in the cytoplasm of high-passage cells indicating that the inability to form SIF-B and -C complexes was due to a lack of activation of Stat1 rather than a lack of expression. These results indicate a fundamental difference between low- and high-passage astrocytes in response to cytokine treatment that might result in distinct patterns of gene expression through altered ratios of activated Stat3 and Stat1.

Dimer stability as a determinant of differential DNA binding activity of Stat3 isoforms.

Stat3alpha and Stat3beta are two Stat3 isoforms with marked quantitative differences in their DNA binding activities. To examine the molecular basis of the differential DNA binding activities, we measured DNA binding strength and dimer stability, two possible mechanisms responsible for these differences. Stat3alpha and Stat3beta showed no difference in DNA binding strength, i.e. they had similar association and dissociation rates for DNA binding. However, competition analyses performed with dissociating reagents including an anti-phosphotyrosine antibody, SH2 domain protein, and a phosphopeptide demonstrated that Stat3beta dimers are more stable than Stat3alpha dimers. We report here that dimer stability of activated forms plays a critical role in determining DNA binding activity of Stat3 isoforms. We found that C-terminal deletions of Stat3alpha increased both DNA binding activity and dimer stability of Stat3alpha. Our findings suggest that the acidic C-terminal region of Stat3alpha does not interfere with the DNA binding of activated Stat3alpha dimers, but destabilizes the dimeric forms of Stat3alpha. We propose that dimer stability described in vitro may be the underlying mechanism of in vivo stability of activated Stat3 proteins, regulating dephosphorylation of tyrosine 705.

Activation of stat3 and stat1 DNA binding and transcriptional activity in human brain tumour cell lines by gp130 cytokines.

In this study we examine the activation of the latent Stat family of transcription factors by the gp130 family of cytokines in cell lines derived from human brain tumours. Of the cytokines tested, oncostatin M resulted in the most dramatic induction of Stat1 and Stat3 in all cell lines analysed, as assessed by the formation of protein/DNA complexes. Interleukin-6, leukemia inhibitory factor, and ciliary neurotrophic factor also induced Stat complexes more selectively and to a lesser magnitude than oncostatin M. The kinetics of Stat1 and Stat3 activation was rapid and transient; the nuclear accumulation of DNA binding-proficient Stat protein was detected in the nucleus within minutes of cytokine induction. The transcriptional potential of the oncostatin M-activated Stat molecules was demonstrated in two glioma cell lines (U87-MG, SNB-19) by transient transfection experiments using a Stat-responsive reporter plasmid. Oncostatin M-dependent transcription from this reporter plasmid was reduced to uninduced levels by the inclusion of a dominant-negative Stat3 molecule, demonstrating that Stat molecules were responsible for the induction. These studies demonstrate that oncostatin M is the most potent activator of Stat molecules in a variety of brain tumour-derived cell lines, an observation that could have implications affecting the balance between proliferation/apoptosis of these cells.

c-Src activates the DNA binding and transcriptional activity of Stat3 molecules: serine 727 is not required for transcriptional activation under certain circumstances.

Stat3 proteins are constitutively activated in cells transformed by v-Src and the proteins have been shown to interact directly. Subsequent studies have shown that Stat3 is required for cellular transformation of NIH fibroblasts by v-Src, suggesting a potential role for Stat3 in aberrant cell growth. Stat3 is phosphorylated on a single tyrosine (tyrosine 705) which is required for effective dimer formation. An additional phosphorylation event (serine 727) is believed to be required for full transcriptional activity of Stat1 and Stat3 molecules. Here we report that c-Src activates the DNA binding activity of Stat3alpha, Stat3beta, and three Stat3 mutants, one in which serine 727 was replaced by alanine (Stat3alphaS727A) and C-terminal truncated molecules Delta48 and Delta55. Consistent with this finding is a general increase in the tyrosine 705-phosphorylated Stat3 in cells cotransfected with c-Src. Furthermore, transcription from an alpha-2 macroglobulin reporter gene is activated by Stat3alphaS727A to the same magnitude as compared to Stat3alpha and Stat3beta in the presence of c-Src. These results suggest that serine 727, contained in a consensus MAP kinase recognition site and shown to be the only serine in Stat3 phosphorylated in epidermal growth factor (EGF) treated cells, is not necessary for transcriptional activity comparable to wild-type Stat3alpha or Stat3beta when activated by c-Src in COS-7 cells.