The structure of the catalytically inactive mutant (C215S) of the human protein-tyrosine phosphatase 1B (PTP1B) has been solved to high resolution in two complexes. In the first, crystals were grown in the presence of bis-(para-phosphophenyl) methane (BPPM), a synthetic highaffinity low-molecular weight nonpeptidic substrate (K m ‫؍‬ 16 M), and the structure was refined to an R-factor of 18.2% at 1.9 Å resolution. In the second, crystals were grown in a saturating concentration of phosphotyrosine (pTyr), and the structure was refined to an R-factor of 18.1% at 1.85 Å. Difference Fourier maps showed that BPPM binds PTP1B in two mutually exclusive modes, one in which it occupies the canonical pTyr-binding site (the active site), and another in which a phosphophenyl moiety interacts with a set of residues not previously observed to bind aryl phosphates. The identification of a second pTyr molecule at the same site in the PTP1B͞C215S-pTyr complex confirms that these residues constitute a low-affinity noncatalytic aryl phosphate-binding site. Identification of a second aryl phosphate binding site adjacent to the active site provides a paradigm for the design of tight-binding, highly specific PTP1B inhibitors that can span both the active site and the adjacent noncatalytic site. This design can be achieved by tethering together two small ligands that are individually targeted to the active site and the proximal noncatalytic site.Protein-tyrosine phosphatases (PTPases), working in concert with protein-tyrosine kinases, regulate a vast array of cellular events, including passage through the cell cycle, proliferation and differentiation, metabolism, cytoskeletal organization, neuronal development, and the immune response (1, 2). PTPases constitute a large family of enzymes that parallel protein-tyrosine kinases in their structural diversity and complexity. A recent estimate suggests that as many as 500 PTPase genes may be encoded within the human genome (2). In vivo, PTPases catalyze the removal of the phosphoryl group from phosphotyrosine (pTyr) residue(s) in protein substrates. However, the K m value for free pTyr is three to four orders of magnitude higher than the best protein͞peptide substrates (3, 4). Several groups have demonstrated that PTPases display a range of k cat ͞K m values for pTyr-containing peptides. This sequence specificity has been exploited in the design of potent and selective PTPase inhibitors by incorporating a nonhydrolyzable pTyr analog into optimal peptide templates (5). However, owing to proteolytic susceptibility and weak partitioning across the plasma membrane, peptide-based compounds are not highly desirable for the development of medicinally effective drugs.As an initial step toward the development of selective, low-molecular weight nonpeptidic PTPase inhibitors, we investigated the active site substrate specificity of PTP1, the rat structural homologue of human protein-tyrosine phosphatase 1B (PTP1B). PTP1B (3) is the prototypical intracellular PTPase and is found in a wide varie...

The 138 genes encoding the 79 ribosomal proteins (RPs) of Saccharomyces cerevisiae form the tightest cluster of coordinately regulated genes in nearly all transcriptome experiments. The basis for this observation remains unknown. We now provide evidence that two factors, Fhl1p and Ifh1p, are key players in the transcription of RP genes. Both are found at transcribing RP genes in vivo. Ifh1p, but not Fhl1p, leaves the RP genes when transcription is repressed. The occupancy of the RP genes by Ifh1p depends on its interaction with the phospho-peptide recognizing forkhead-associated domain of Fhl1p. Disruption of this interaction is severely deleterious to ribosome synthesis and cell growth. Loss of functional Fhl1p leads to cells that have only 20% the normal amount of RNA and that synthesize ribosomes at only 5-10% the normal rate. Homeostatic mechanisms within the cell respond by reducing the transcription of rRNA to match the output of RPs, and by reducing the global transcription of mRNA to match the capacity of the translational apparatus.

The extracellular signal-regulated protein kinase 2 (ERK2) is the founding member of a family of mitogenactivated protein kinases (MAPKs) that are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. The MAPKs are unique among the Ser/Thr protein kinases in that they require both Thr and Tyr phosphorylation for full activation. The dual phosphorylation of Thr-183 and Tyr-185 in ERK2 is catalyzed by MAPK/ERK kinase 1 (MEK1). However, the identity and relative activity of protein phosphatases that inactivate ERK2 are less well established. In this study, we performed a kinetic analysis of ERK2 dephosphorylation by protein phosphatases using a continuous spectrophotometric enzymecoupled assay that measures the inorganic phosphate produced in the reaction. Eleven different protein phosphatases, many previously suggested to be involved in ERK2 regulation, were compared, including tyrosinespecific phosphatases (PTP1B, CD45, and HePTP), dual specificity MAPK phosphatases (VHR, MKP3, and MKP5), and Ser/Thr protein phosphatases (PP1, PP2A, PP2B, PP2C␣, and PP). The results provide biochemical evidence that protein phosphatases display exquisite specificity in their substrate recognition and implicate HePTP, MKP3, and PP2A as ERK2 phosphatases. The fact that ERK2 inactivation could be carried out by multiple specific phosphatases shows that signals can be integrated into the pathway at the phosphatase level to determine the cellular response to external stimuli. Important insights into the roles of various protein phosphatases in ERK2 kinase signaling are obtained, and further analysis of the mechanism by which different protein phosphatases recognize and inactivate MAPKs will increase our understanding of how this kinase family is regulated.

The mitogen-activated protein (MAP) kinase phosphatase-3 (MKP3) is a dual specificity phosphatase that specifically inactivates one subfamily of MAP kinases, the extracellular signal-regulated kinases (ERKs). Inactivation of MAP kinases occurs by dephosphorylation of Thr(P) and Tyr(P) in the TXY kinase activation motif. To gain insight into the mechanism of ERK2 inactivation by MKP3, we have carried out an analysis of the MKP3-catalyzed dephosphorylation of the phosphorylated ERK2. We find that ERK2/pTpY dephosphorylation by MKP3 involves an ordered, distributive mechanism in which MKP3 binds the bisphosphorylated ERK2/pTpY, dephosphorylates Tyr(P) first, dissociates and releases the monophosphorylated ERK2/pT, which is then subjected to dephosphorylation by a second MKP3, yielding the fully dephosphorylated ERK2. The bisphosphorylated ERK2 is a highly specific substrate for MKP3 with a k cat /K m of 3.8 ؋ 10 6 M ؊1 s ؊1 , which is more than 6 orders of magnitude higher than that for small molecule aryl phosphates and an ERK2-derived phosphopeptide encompassing the pTEpY motif. This strikingly high substrate specificity displayed by MKP3 may result from a combination of high affinity binding interactions between the N-terminal domain of MKP3 and ERK2 and specific ERK2-induced allosteric activation of the MKP3 C-terminal phosphatase domain.

The ribosomal protein genes of Saccharomyces cerevisiae, responsible for nearly 40% of the polymerase II transcription initiation events, are characterized by the constitutive tight binding of the transcription factor Rap1. Rap1 binds at many places in the yeast genome, including glycolytic enzyme genes, the silent MAT loci, and telomeres, its specificity arising from specific cofactors recruited at the appropriate genes. At the ribosomal protein genes two such cofactors have recently been identified as Fhl1 and Ifh1. We have now characterized the interaction of these factors at a bidirectional ribosomal protein promoter by replacing the Rap1 sites with LexA operator sites. LexA-Gal4(AD) drives active transcription at this modified promoter, although not always at the correct initiation site. Tethering Rap1 to the promoter neither drives transcription nor recruits Fhl1 or Ifh1, showing that Rap1 function requires direct DNA binding. Tethering Fhl1 also fails to activate transcription, even though it does recruit Ifh1, suggesting that Fhl1 does more than simply provide a platform for Ifh1. Tethering Ifh1 to the promoter leads to low-level transcription, at the correct initiation sites. Remarkably, activation by tethered LexA-Gal4(AD) is strongly reduced when TOR kinase is inhibited by rapamycin. Thus, TOR can act independently of Fhl1/Ifh1 at ribosomal protein promoters. We also show that, in our strain background, the response of ribosomal protein promoters to TOR inhibition is independent of the Ifh1-related protein Crf1, indicating that the role of this corepressor is strain specific. Fine-structure chromatin mapping of several ribosomal protein promoters revealed that histones are essentially absent from the Rap1 sites, while Fhl1 and Ifh1 are coincident with each other but distinct from Rap1.The 138 genes encoding the 79 ribosomal proteins (RP) of Saccharomyces cerevisiae are arguably the most coordinately regulated cluster of genes, spread throughout the yeast genome (7, 11). It was originally thought that the basis for much of this regulation lay in the presence of binding sites for the protein Rap1 upstream of a large majority of the RP genes (17, 21).However, Rap1 is a protein of many functions (reviewed by references 28 and 29). It is the primary transcription factor for the glycolytic genes and several translation factor genes. It acts as the major duplex DNA binding protein of telomeres. It nucleates the silencing of the HML and HMR mating-type loci. Genome-wide chromatin immunoprecipitation (ChIP) analysis revealed that Rap1 binds to about 5% of yeast genes and participates in the activation of 37% of RNA polymerase II transcripts in exponentially growing yeast cells (21). There is good evidence that the initial step of Rap1 is to clear nucleosomes from a patch of DNA (28,40) and that the second step is to recruit specific factors to carry out the appropriate function. It is now clear that for the RP genes these factors are Fhl1 and Ifh1, which are found almost exclusively at RP genes. Gcr1 and Gcr2 ...