Sympathetic neurons depend on nerve growth factor (NGF) for survival and die by apoptosis in its absence. We have investigated the pattern of expression of the Jun and Fos family of transcription factors in dying sympathetic neurons using antibodies specific for each family member. When sympathetic neurons are deprived of NGF, the level of c-Jun protein significantly increases, whereas the levels of the other members of the Jun and Fos family remain relatively constant. c-Jun also becomes more phosphorylated, probably on its amino terminal transactivation domain. When microinjected into sympathetic neurons, an expression vector for a c-Jun dominant negative mutant protects them against NGF withdrawal-induced death, indicating that AP-1 activity is essential for neuronal cell death. Furthermore, overexpression of the full-length c-Jun protein is, in itself, sufficient to induce apoptosis in sympathetic neurons.

Recent evidence suggests that the force for poleward movement of chromosomes during mitosis is generated at or close to the kinetochores. Chromosome movement depends on motion relative to microtubules, but the identities of the motors remain uncertain. One candidate for a mitotic motor is dynein, a large multimeric enzyme which can move along microtubules toward their slow growing end. Dyneins were originally found in axonemes of cilia and flagella where they power microtubule sliding. Recently, cytoplasmic dyneins have also been found, and specific antibodies have been raised against them. The cellular localization of dynein has previously been studied with several antibodies raised against flagellar dynein, but the relevance of these data to the distribution of cytoplasmic dynein is not known. Antibodies raised against cytoplasmic dyneins have shown localization of dynein antigens to the mitotic spindles in Caenorhabditis elegans embryos (Lye et al., personal communication) and punctate cytoplasmic structures in Dictyostelium amoebae. Using antibodies that recognize subunits of cytoplasmic dyneins, we show here that during mitosis, cytoplasmic dynein antigens concentrate near the kinetochores, centrosomes and spindle fibres of HeLa and PtK1 cells, whereas at interphase they are distributed throughout the cytoplasm. This is consistent with the hypothesis that cytoplasmic dynein is a mitotic motor.

No abstract

The Ras proteins play a central role in regulating cell growth and their mutation can lead to abnormal proliferation. To analyse the potential link betwen AP1 activity, encoded by members of the jun and fos gene families, and Ras-mediated cellular transformation, we have studied several NIH3T3 clones which overexpress the Ha-Ras or Ki-Ras oncogenes. These transformed ®broblasts accumulated higher levels of cJun, JunB, Fra1 and Fra2 proteins relative to their normal counterparts. They also displayed increased AP1 DNA binding activity which was predominantly composed of cJun and Fra1 containing dimers. Following serum stimulation of Ras clones, the elevated levels of cJun and Fra1 remained steady, while the induction of JunB and Fra2 was partially attenuated. Moreover, deregulated Ras signaling resulted in a complete loss of the serum inducibility of cFos and FosB. Ectopic co-expression of cJun and Fra1 in NIH3T3 ®broblasts led to a transformed phenotype, attenuation of cFos serum inducibility, increased AP1 activity and Cyclin D1 accumulation, all characteristics of oncogenic Ras expressing cells. These results demonstrate that cJun and Fra1 are crucial mediators of the Ras-transformation process.

Long tracts of CCG trinucleotide or CCGNN pentanucleotide repeats in DNA have previously been shown to resist assembly into nucleosomes. This may provide a molecular explanation for the nature of certain rare, folate-sensitive fragile sites in human chromosomes that contain expanded CCG triplet tracts. Further, it is known that methylation of CpG dinucleotides at or near these fragile sites enhances the fragile phenotype. Here DNAs containing 76 tandem CCG triplets or 48 CCGNN pentanucleotide repeats were methylated with SssI methylase at three different levels of methylation. Using competitive nucleosome reconstitution/gel shift assays, the ability of these DNAs and a mixed sequence DNA from the pUC19 plasmid were compared in their ability to assemble into nucleosomes. DNA methylation had no significant effect on nucleosome formation over the pUC 19 fragment. However, the highly methylated DNAs containing 76 CCG triplets or 48 CCGNN pentanucleotide repeats were 2.0 +/- 0. 2-fold and 2.1 +/- 0.3-fold less efficient in nucleosome assembly than the respective unmethylated forms, and 4.4 +/- 0.4-fold and 12. 6 +/- 1.6-fold less efficient than a pUC19 fragment of similar length.

CCAAT/enhancer-binding proteins (C/EBPs) are basic region/leucine zipper transcription factors that function as regulators of cell growth and differentiation in numerous cell types. We previously localized transcriptional activation and inhibitory regions in one family member, C/EBP⑀. Here we describe the further characterization of a C/EBP⑀ inhibitory domain termed regulatory domain I. We show that functionally related domains are present in C/EBP␣, C/EBP␤, and C/EBP␦. These domains contain an evolutionarily conserved five-amino acid motif (the regulatory domain motif (RDM)) that conforms to the consensus sequence (I/V/ L)KXEP. Mutagenesis studies revealed that the residues at positions 1, 2, and 4 of the RDM are critical for inhibitory domain function. Data base searches identified RDM-like sequences in a number of nuclear proteins. We found that small regions from c-Jun, JunB, and JunD containing this sequence also function as transcriptional inhibitory domains. Importantly, the RDM is similar to the recognition sequence for attachment of the ubiquitin-like protein, small ubiquitin-like modifier-1 (SUMO-1), and the conserved lysine residue of each C/EBP RDM served as an attachment site for SUMO-1. SUMO-1 attachment decreased the inhibitory effect of the C/EBP⑀ regulatory domain, suggesting that sumoylation may play an important role in modulating C/EBP⑀ activity as well as that of the other C/EBP family members.

We have analysed the different Jun and Fos proteins as NIH3T3 fibroblasts pass from exponential growth to quiescence and during the first 24 h after their re-entry into the cell cycle following serum stimulation. We show that these proteins can be divided into 3 subgroups based on their pattern of expression. The first contains c-Jun, Jun-D and Fra-2 which are expressed at high level in cycling cells and are only mildly induced by serum. The second contains Jun-B, c-Fos, Fos-B and deltaFos-B whose levels are low in cycling cells but increase strongly and rapidly after stimulation by serum. The third group contains only Fra-1, which is absent from cycling cells and behaves as a delayed early response protein after serum stimulation. AP-1 binding activity is low both in cycling and quiescent fibroblasts but increases after stimulation by serum with kinetics matching the induction of the various Jun and Fos proteins. Antibody supershift analyses demonstrate that the composition of AP-1 binding activity reflects the relative abundance of each Jun and Fos protein. Furthermore, the state of post-translational modification varies continuously for all of the AP-1 proteins as growth conditions change. These data indicate that AP-1 activity during the G0-G1 transition is finely regulated and complex, involving changes both in protein expression and in posttranslational modification.

No abstract