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ABSTRACTThe yeast Saccharomyces cerevisiaetranscription factor Ste12p is responsible for activating genes in response to MAP kinase cascades controlling mating and filamentous growth. Ste12p is negatively regulated by two inhibitor proteins, Dig1p (also called Rst1p) and Dig2p (also called Rst2p). The expression of a C-terminal Ste12p fragment (residues 216 to 688) [Ste12p(216–688)] from aGALpromoter causes FUS1induction in a strain expressing wild-type STE12, suggesting that this region can cause the activation of endogenous Ste12p. Residues 262 to 594 are sufficient to cause STE12-dependent FUS1induction when overexpressed, and this region of Ste12p was found to bind Dig1p but not Dig2p in yeast extracts. In contrast, recombinant glutathione S-transferase–Dig2p binds to the Ste12p DNA-binding domain (DBD). Expression of DIG2, but notDIG1, from a GALpromoter inhibits transcriptional activation by an Ste12p DBD-VP16 fusion. Furthermore, disruption of dig1, but not dig2, causes elevated transcriptional activation by a LexA–Ste12p(216–688) fusion. Ste12p has multiple regions within the C terminus (flanking residue 474) that can promote multimerization in vitro, and we demonstrate that these interactions can contribute to the activation of endogenous Ste12p by overproduced C-terminal fragments. These results demonstrate that Dig1p and Dig2p do not function by redundant mechanisms but rather inhibit pheromone-responsive transcription through interactions with separate regions of Ste12p.

Gal4p activates transcription of the Saccharomyces GAL genes in response to galactose and is phosphorylated during interaction with the RNA polymerase II (Pol II) holoenzyme. One phosphorylation at S699 is necessary for full GAL induction and is mediated by Srb10p/CDK8 of the RNA Pol II holoenzyme mediator subcomplex. Gal4p S699 phosphorylation is necessary for sensitive response to inducer, and its requirement for GAL induction can be abrogated by high concentrations of galactose in strains expressing wild-type GAL2 and GAL3. Gal4p S699 phosphorylation occurs independently of Gal3p and is responsible for the long-term adaptation response observed in gal3 yeast. SRB10 and GAL3 are shown to represent parallel mechanisms for GAL gene induction. These results demonstrate that Gal4p activity is controlled by two independent signals: one that acts through Gal3p-galactose and a second that is mediated by the holoenzyme-associated cyclin-dependent kinase Srb10p. Since Srb10p is regulated independently of galactose, our results suggest a function for CDK8 in coordinating responses to specific inducers with the environment through the phosphorylation of gene-specific activators.

ABSTRACTGal4p activates transcription of the Saccharomyces GALgenes in response to galactose and is phosphorylated during interaction with the RNA polymerase II (Pol II) holoenzyme. One phosphorylation at S699 is necessary for full GALinduction and is mediated by Srb10p/CDK8 of the RNA Pol II holoenzyme mediator subcomplex. Gal4p S699 phosphorylation is necessary for sensitive response to inducer, and its requirement for GALinduction can be abrogated by high concentrations of galactose in strains expressing wild-typeGAL2and GAL3. Gal4p S699 phosphorylation occurs independently of Gal3p and is responsible for the long-term adaptation response observed in gal3yeast.SRB10and GAL3are shown to represent parallel mechanisms for GALgene induction. These results demonstrate that Gal4p activity is controlled by two independent signals: one that acts through Gal3p-galactose and a second that is mediated by the holoenzyme-associated cyclin-dependent kinase Srb10p. Since Srb10p is regulated independently of galactose, our results suggest a function for CDK8 in coordinating responses to specific inducers with the environment through the phosphorylation of gene-specific activators.

Proteins encoded by oncogenes such as v-fps/fes, 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 P130gag-fps impaired the ability of Fujinami avian sarcoma virus to transform rat-2 cells. The P130gag-fps 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 genomes 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-fps bacterial proteins had little effect on autophosphorylating activity. The conserved N-terminal domain of P130gag-fps 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.

The 2 micron plasmid of Saccharomyces cerevisiae codes for a site-specific recombinase, the FLP protein, that catalyzes efficient recombination across two 599-base-pair (bp) inverted repeats of the plasmid DNA both in vivo and in vitro. We analyzed the interaction of the purified FLP protein with the target sequences of two point mutants that exhibit impaired FLP-mediated recombination in vivo. One mutation lies in one of the 13-bp repeat elements that had been previously shown to be protected from DNase digestion by the FLP protein. This mutation dramatically reduces FLP-mediated recombination in vitro and appears to act by reducing the binding of FLP protein to its target sequence. The second mutation lies within the 8-bp core region of the FLP target sequence. The FLP protein introduces staggered nicks surrounding this 8-bp region, and these nicks are thought to define the sites of strand exchange. The mutation in the core region abolishes recombination with a wild-type site. However, recombination between two mutated sites is very efficient. This result suggests that proper base pairing between the two recombining sites is an important feature of FLP-mediated recombination.

A conserved noncatalytic domain SH2 (for src homology region 2) is located immediately N terminal to the kinase domains of all cytoplasmic protein-tyrosine kinases. We found that the wild-type v-fps SH2 domain stimulated the enzymatic activity of the adjacent kinase domain 10-fold and functioned as a powerful positive effector of catalytic and transforming activities within the v-fps oncoprotein (P130gag-fps). Partial proteolysis of P130gag-fps and supporting genetic data indicated that the v-fps SH2 domain exerts its effect on catalytic activity through an intramolecular interaction with the kinase domain. Amino acid alterations in the SH2 domain that impaired kinase function interfered with association of the SH2 domain with the kinase domain. Deletion of a conserved octapeptide motif converted the v-fps SH2 domain from an activator to an inhibitor of tyrosine kinase activity. This latent inhibitory activity of v-fps SH2 has functional implications for phospholipase C-gamma and p21ras GTPase-activating protein, both of which have two distinct SH2 domains suggestive of complex regulation. In addition to regulating the specific activity of the kinase domain, the SH2 domain of P130gag-fps was also found to be required for the tyrosine phosphorylation of specific cellular proteins, notably polypeptides of 124 and 62 kilodaltons. The SH2 domain therefore appears to play a dual role in regulation of kinase activity and recognition of cellular substrates.

Gal4p regulates expression of genes necessary for galactose catabolism in Saccharomyces cerevisiae. We have previously shown that phosphorylation of Gal4p requires both its DNA binding and transcriptional-activation functions and have suggested that phosphorylation occurs as a consequence of interaction with general transcription factors. In this study, we show that phosphorylation occurs rapidly on a limited fraction of overexpressed Gal4p present in a sodium dodecyl sulfate-extractable subcellular fraction while a significant fraction remains stably unphosphorylated. Taken together with our previous observations, we conclude that Gal4p is phosphorylated only if it becomes localized to the nucleus and is capable of both DNA binding and transcriptional activation. We demonstrate that Gal4p is multiply phosphorylated at both the C and N termini, and we identify the precise locations of three sites of phosphorylation at serines 691, 696, and 699. Of these sites, only serine 699 must be phosphorylated for galactose-inducible transcription to occur. Mutation of S-699 to alanine significantly impairs GAL induction by galactose in GAL80+ cells but does not affect transcriptional activation by Gal4p in gal80- cells. In gal80- cells, Gal4p phosphorylation, including that of serine 699, is stimulated by the presence of both galactose and glucose, indicating that phosphorylation at this site is not specifically activated by galactose. Serine 699 phosphorylation requires Gal4p's DNA binding function and is influenced by the function of the RNA polymerase II holoenzyme component Gal11p. These results suggest that a phosphorylation on Gal4p, likely resulting from interaction with the holoenzyme, modulates the induction process by regulating interaction between Gal4p and Gal80p.

Receptors for interferons and other cytokines signal through the action of associated protein tyrosine kinases of the JAK family and latent cytoplasmic transcription factors of the STAT family. Genetic and biochemical analysis of interferon signaling indicates that activation of STATs by interferons requires two distinct JAK family kinases. Loss of either of the required JAKs prevents activation of the other JAK and extinguishes STAT activation. These observations suggest that JAKs provide interferon receptors with a critical catalytic signaling function and that at least two JAKs must be incorporated into an active receptor complex. JAK and STAT proteins are also activated by ligands such as platelet-derived growth factor (PDGF), which act through receptors that possess intrinsic protein tyrosine kinase activity, raising questions about the role of JAKs in signal transduction by this class of receptors. Here, we show that all three of the ubiquitously expressed JAKs--JAK1, JAK2, and Tyk2--become phosphorylated on tyrosine in both mouse BALB/c 3T3 cells and human fibroblasts engineered to express the PDGF-beta receptor. All three proteins are also associated with the activated receptor. Through the use of cell lines each lacking an individual JAK, we find that in contrast to interferon signaling, PDGF-induced JAK phosphorylation and activation of STAT1 and STAT3 is independent of the presence of any other single JAK but does require receptor tyrosine kinase activity. These results suggests that the mechanism of JAK activation and JAK function in signaling differs between receptor tyrosine kinases and interferon receptors.

The FLP recombinase, encoded by the 2 micron plasmid of Saccharomyces cerevisiae, promotes efficient recombination in vivo and in vitro between its specific target sites (FLP sites). It was previously determined that FLP interacts with DNA sequences within its target site (B. J. Andrews, G. A. Proteau, L. G. Beatty, and P. D. Sadowski. Cell 40:795-803, 1985), generates a single-stranded break on both DNA strands within the FLP site, and remains covalently attached to the 3' end of each break. We now show that the FLP protein is bound to the 3' side of each break by an O-phosphotyrosyl residue and that it appears that the same tyrosyl residue(s) is used to attach to either DNA strand within the FLP site.

We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed.