Recent work has defined a class of transcriptional activators, members of which activate transcription in yeast, plant, insect and mammalian cells. These proteins contain two parts: one directs DNA binding and the other, called the activating region, presumably interacts with some component of the transcriptional machinery. Activating regions are typically acidic and require some poorly-understood aspect of structure, probably at least in part an alpha-helix. Here we describe a new member of this class, formed by fusing a DNA-binding fragment of the yeast activator GAL4 to a highly acidic portion of the herpes simplex virus protein VP16 (ref. 11; also called Vmw65). VP16 activates transcription of immediate early viral genes by using its amino-terminal sequences to attach to one or more host-encoded proteins that recognise DNA sequences in their promoters. We show that the hybrid protein (GAL4-VP16) activates transcription unusually efficiently in mammalian cells when bound close to, or at large distances from the gene. We suggest that the activating region of VP16 may be near-maximally potent and that it is not coincidental that such a strong activator is encoded by a virus.

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Proteins encoded by oncogenes such as v-fpslfes, v-src, v-yes, v-abl, and v-fgr are cytoplasmic protein tyrosine kinases which, unlike transmembrane receptors, are localized to the inside of the cell. These proteins possess two contiguous regions of sequence identity: a C-terminal catalytic domain of 260 residues with homology to other tyrosine-specific and serine-threonine-specific protein kinases, and a unique domain of approximately 100 residues which is located N terminal to the kinase region and is absent from kinases that span the plasma membrane. In-frame linker insertion mutations in Fujinami avian sarcoma virus which introduced dipeptide insertions into the most stringently conserved segment of this N-terminal domain in P130"w-lps impaired the ability of Fujinami avian sarcoma virus to transform rat-2 cells. The P130gag-s proteins encoded by these transformation-defective mutants were deficient in protein-tyrosine kinase activity in rat cells. However v-fps polypeptides derived from the mutant Fujinami avian sarcoma virus genonles and expressed in Escherichia coli as trpE-v-fps fusion proteins displayed essentially wild-type enzymatic activity, even though they contained the mutated sites. Deletion of the N-terminal domain from wild-type and mutant v-frs bacterial proteins had little effect on autophosphorylating activity. The conserved N-terminal domain of P130EagPS is therefore not required for catalytic activity, but can profoundly influence the adjacent kinase region. The presence of this noncatalytic domain in all known cytoplasmic tyrosine kinases of higher and lower eucaryotes argues for an important biological function. The relative inactivity of the mutant proteins in rat-2 cells compared with bacteria suggests that the noncatalytic domain may direct specific interactions of the enzymatic region with cellular components that regulate or mediate tyrosine kinase function.

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The budding yeast Saccharomyces cerevisiae differentiates into filamentous invasively growing forms under conditions of nutrient limitation. This response is dependent on the transcription factor Ste12 and on the mating pheromone-response mitogen-activated protein (MAP) kinase cascade, but a mechanism for regulation of Ste12 by nutrient limitation has not been defined. Here we show that Ste12 function in filamentous growth is regulated by the cyclin-dependent kinase Srb10 (also known as Cdk8), which is associated with the RNA polymerase II holoenzyme. Srb10 inhibits filamentous growth in cells growing in rich medium by phosphorylating Ste12 and decreasing its stability. Under conditions of limiting nitrogen, loss of Srb10 protein and kinase activity occurs, with a corresponding loss of Ste12 phosphorylation. Mutation of the Srb10-dependent phosphorylation sites increases pseudohyphal development but has no effect on the pheromone response of haploid yeast. Srb10 kinase activity is also regulated independently of the mating pheromone-response pathway. This indicates that Srb10 controls Ste12 activity for filamentous growth in response to nitrogen limitation and is consistent with the hypothesis that Srb10 regulates gene-specific activators in response to physiological signals to coordinate gene expression with growth potential.

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Phosphorylation of the yeast transcription factor GAL4 at S699 is required for efficient galactose-inducible transcription. We demonstrate that this site is a substrate for the RNA polymerase holoenzyme-associated CDK SRB10. S699 phosphorylation requires SRB10 in vivo, and this site is phosphorylated by purified SRB10/ SRB11 CDK/cyclin in vitro. RNA Pol II holoenzymes purified from WT yeast phosphorylate GAL4 at sites observed in vivo whereas holoenzymes from srb10 yeast are incapable of phosphorylating GAL4 at S699. Mutations at GAL4 S699 and srb10 are epistatic for GAL induction, demonstrating that SRB10 regulates GAL4 activity through this phosphorylation in vivo. These results demonstrate a function for the SRB10/ CDK8 holoenzyme-associated CDK that involves regulation of transactivators by phosphorylation during transcriptional activation.

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