The Raf-1 serine/threonine protein kinase.

Raf-1 belongs to a family of serine/threonine protein kinases which are highly conserved through evolution in multicellular organisms. Raf-1 kinase has gained much attention due to its function as a critical shuttle enzyme that connects stimulation of growth factor receptors and protein kinase C at the cell membrane to changes in the expression of genes involved in the control of cell growth, differentiation and survival. Regulation of Raf-1 activity is complex and involves Ras, as well as several serine/threonine and tyrosine kinases. Through a series of phosphorylation events, extracellular signals are connected through the Raf-1/MAP kinase pathway to activity-regulation of several oncogene-class transcription factors via phosphorylation of specific serine residues. Under ordinary circumstances, the cascade involving Raf-1 eventually leads to changes in gene expression and protein synthesis. Upon constitutive activation of Raf-1 kinase, as a result of genetic changes, a variety of cell types acquire a transformed phenotype.

Protein kinase C alpha activates RAF-1 by direct phosphorylation.

The kinase Raf-1 can be activated by treatment of cells with mitogens and by the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (reviewed in refs 1,2). Activated Raf-1 triggers a protein kinase cascade by direct phosphorylation of MAP kinase kinase, resulting in phosphorylation of ternary complex factor and Jun by MAP kinase. Here we investigate the molecular mechanism and biological consequences of PKC alpha-mediated Raf-1 activation in NIH3T3 fibroblasts. PKC alpha directly phosphorylates and activates Raf-1 both in vitro and in vivo. PKC alpha induces Raf-1 phosphorylation at several sites, including a serine residue at position 499. Mutation of serine at this position or at residue 259 does not abrogate Raf-1 stimulation by a combination of Ras plus the src tyrosine kinase Lck, but severely impedes Raf-1 activation by PKC alpha. Consistent with such a direct interaction is the observation that Raf-1 and PKC alpha cooperate in the transformation of NIH3T3 cells. The Ser499 phosphorylation site is necessary for this synergism.

Characterization of potentially functional 5S rRNA-encoding genes within ribosomal DNA repeats of the nematode Meloidogyne arenaria.

In the plant-parasitic nematode Meloidogyne arenaria, isolated rDNA repeats show length heterogeneity, and are unusual in that they contain putative 5S ribosomal RNA pseudogenes [Vahidi et al., J. Mol. Evol. 27 (1988) 222-227]. Potentially functional 5S rRNA-encoding genes can also be identified in various rDNA repeats, which appear to be tandemly organized in the genome.

Repeats and subrepeats in the intergenic spacer of rDNA from the nematode Meloidogyne arenaria.

Ribosomal DNA (rDNA) repeats of the plant-parasitic nematode Meloidogyne arenaria are heterogeneous in size and appear to contain 5S rRNA gene sequences. Moreover, in a recA+ bacterial host, plasmid clones of a 9 kb rDNA repeat show deletion events within a 2 kb intergenic spacer (IGS), between 28S and 5S DNA sequences. These deletions appear to result from a reduction in the number of tandem 129 bp repeats in the IGS. The loss of such repeats might explain how rDNA length heterogeneity, observed in the Meloidogyne genome, could have arisen. Each 129 bp repeat also contains three copies of an 8 bp subrepeat, which has sequence similarity to an element found in the IGS repeats of some plant rDNAs.

Unusual sequences, homologous to 5S RNA, in ribosomal DNA repeats of the nematode Meloidogyne arenaria.

There are sequences homologous to 5S ribosomal RNA in the ribosomal DNA (rDNA) repeats of the plant-parasitic nematode Meloidogyne arenaria. This is surprising, because in all other higher eukaryotes studied to date, the genes for 5S RNA are unlinked to and distinct from a tandem rDNA repeat containing the genes for 18S, 5.8S, and 28S ribosomal RNA. Previously, only prokaryotes and certain "lower eukaryotes" (protozoa and fungi) had been found to have both the larger rRNAs and 5S rRNA represented within a single DNA repeat. This has raised questions on the organization of these repeats in the earliest cell (progenote), and on subsequent evolutionary relationships between pro- and eukaryotes. Evidence is presented for rearrangements and deletions within Meloidogyne rDNA. The unusual life cycles (different levels of ploidy, reproduction by meiotic and mitotic parthenogenesis) of members of this genus might allow rapid fixation of any variants with introduced 5S RNA sequences. The 5S RNA sequences in Meloidogyne rDNA may not be expressed, but their presence raises important questions as to the evolutionary origins and stability of repeat gene families.