Ascertaining the biochemical function of an essential pectin methylesterase in the gut microbe Bacteroides thetaiotaomicron.

Pectins are a major dietary nutrient source for the human gut microbiota. The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II-derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR approaches revealed that it is a bimethylated nonaoligosaccharide (GlcA-ß1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-ß1,3)-Rha-α1,3-Api-ß1,2-(Araf-α1,3)-(GalA-α1,4)-GalA) containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an α/ß-hydrolase fold, consisting of a central twisted 10-stranded ß-sheet sandwiched by several α-helices. This constitutes a new fold for pectin methylesterases, which are predominantly right-handed ß-helical proteins. Bioinformatic analyses revealed that the family is dominated by sequences from prominent genera of the human gut microbiota, including Bacteroides and Prevotella Our re-sults not only highlight the critical role played by this family of enzymes in pectin metabolism but also provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut.

Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover.

The collagenase subfamily of matrix metalloproteinases (MMPs) have important roles in the remodeling of collagenous matrices. The proteinase-activated receptor (PAR) family has a unique mechanism of activation requiring proteolysis of an extracellular domain forming a neo-N terminus that acts as a tethered ligand, a process that has been associated with the development of arthritis. Canonical PAR2 activation typically occurs via a serine proteinase at Arg36-Ser37, but other proteinases can cleave PARs downstream of the tethered ligand and "disarm" the receptor. To identify additional cleavage sites within PAR2, we synthesized a 42-amino-acid peptide corresponding to the extracellular region. We observed that all three soluble MMP collagenases, MMP-1, MMP-8, and MMP-13, cleave PAR2 and discovered a novel cleavage site (Ser37-Leu38). Metalloproteinases from resorbing bovine nasal cartilage and recombinant human collagenases could cleave a quenched fluorescent peptide mimicking the canonical PAR2 activation region, and kinetic constants were determined. In PAR2-overexpressing SW1353 chondrocytes, we demonstrated that the activator peptide SLIGKV-NH2 induces rapid calcium flux, inflammatory gene expression (including MMP1 and MMP13), and the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase. The corresponding MMP cleavage-derived peptide (LIGKVD-NH2) exhibited no canonical activation; however, we observed phosphorylation of ERK, providing evidence of biased agonism. Importantly, we demonstrated that preincubation with active MMP-1 reduced downstream PAR2 activation by a canonical activator, matriptase, but not SLIGKV-NH2 These results support a role for collagenases as proteinases capable of disarming PAR2, revealing a mechanism that suppresses PAR2-mediated inflammatory responses.

The WNT10B Network Is Associated with Survival and Metastases in Chemoresistant Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) commonly develops resistance to chemotherapy, yet markers predictive of chemoresistance in this disease are lacking. Here, we define WNT10B-dependent biomarkers for ß-CATENIN/HMGA2/EZH2 signaling predictive of reduced relapse-free survival. Concordant expression of HMGA2 and EZH2 proteins is observed in MMTV-Wnt10bLacZ transgenic mice during metastasis, and Hmga2 haploinsufficiency decreased EZH2 protein expression, repressing lung metastasis. A novel autoregulatory loop interdependent on HMGA2 and EZH2 expression is essential for ß-CATENIN/TCF-4/LEF-1 transcription. Mechanistically, both HMGA2 and EZH2 displaced Groucho/TLE1 from TCF-4 and served as gatekeepers for K49 acetylation on ß-CATENIN, which is essential for transcription. In addition, we discovered that HMGA2-EZH2 interacts with the PRC2 complex. Absence of HMGA2 or EZH2 expression or chemical inhibition of Wnt signaling in a chemoresistant patient-derived xenograft (PDX) model of TNBC abolished visceral metastasis, repressing AXIN2, MYC, EZH2, and HMGA2 expression in vivo. Combinatorial therapy of a WNT inhibitor with doxorubicin synergistically activated apoptosis in vitro, resensitized PDX-derived cells to doxorubicin, and repressed lung metastasis in vivo. We propose that targeting the WNT10B biomarker network will provide improved outcomes for TNBC. SIGNIFICANCE: These findings reveal targeting the WNT signaling pathway as a potential therapeutic strategy in triple-negative breast cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/5/982/F1.large.jpg.

The human gut microbe Bacteroides thetaiotaomicron encodes the founding member of a novel glycosaminoglycan-degrading polysaccharide lyase family PL29.

Glycosaminoglycans (GAGs) and GAG-degrading enzymes have wide-ranging applications in the medical and biotechnological industries. The former are also an important nutrient source for select species of the human gut microbiota (HGM), a key player in host-microbial interactions. How GAGs are metabolized by the HGM is therefore of interest and has been extensively investigated in the model human gut microbe Bacteroides thetaiotaomicron. The presence of as-yet uncharacterized GAG-inducible genes in its genome and of related species, however, is testament to our incomplete understanding of this process. Nevertheless, it presents a potential opportunity for the discovery of additional GAG-degrading enzymes. Here, we investigated a gene of unknown function (BT_3328) from the chondroitin sulfate (CS) utilization locus of B. thetaiotaomicron NMR and UV spectroscopic assays revealed that it encodes a novel polysaccharide lyase (PL), hereafter referred to as BtCDH, reflecting its source (B. thetaiotaomicron (Bt)) and its ability to degrade the GAGs CS, dermatan sulfate (DS), and hyaluronic acid (HA). When incubated with HA, BtCDH generated a series of unsaturated HA sugars, including Δ4,5UA-GlcNAc, Δ4,5UA-GlcNAc-GlcA-GlcNac, Δ4,5UA-[GlcNAc-GlcA]2-GlcNac, and Δ4,5UA-[GlcNAc-GlcA]3-GlcNac, as end products and hence was classed as endo-acting. A combination of genetic and biochemical assays revealed that BtCDH localizes to the cell surface of B. thetaiotaomicron where it enables extracellular GAG degradation. BtCDH homologs were also detected in several other HGM species, and we therefore propose that it represents the founding member of a new polysaccharide lyase family (PL29). The current discovery also contributes new insights into CS metabolism by the HGM.

Complex pectin metabolism by gut bacteria reveals novel catalytic functions.

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degradation involves previously undiscovered enzyme families and catalytic activities. The degradation system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet.

Corrigendum: Complex pectin metabolism by gut bacteria reveals novel catalytic functions.

This corrects the article DOI: 10.1038/nature21725.

The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans.

The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of ß-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans. The mechanism of substrate recognition displayed by the enzyme, however, remains unclear. Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain. The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility. The structure of the enzyme in complex with Xylp-ß-1,4-Xylp-ß-1,4-Xylp-[α-1,3-Araf]-ß-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (-2* subsite) that abuts onto the catalytic center. The -2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants. Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the -2* subsite, abrogates catalytic activity. Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed. This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.

Recovery from rapamycin: drug-insensitive activity of yeast target of rapamycin complex 1 (TORC1) supports residual proliferation that dilutes rapamycin among progeny cells.

The target of rapamycin complex 1 (TORC1) is a key conserved regulator of eukaryotic cell growth. The xenobiotic rapamycin is a potent inhibitor of the yeast complex. Surprisingly, the EGO complex, a nonessential in vivo activator of TORC1, is somehow required for yeast cells to recover efficiently from a period of treatment with rapamycin. Why? Here, we found that rapamycin is only a partial inhibitor of TORC1. We confirmed that saturating amounts of rapamycin do not fully inhibit proliferation of wild-type cells, and we found that the residual proliferation in the presence of the drug is dependent on the EGO complex and on the activity of TORC1. We found that this residual TORC1-dependent proliferation is key to recovery from rapamycin treatment. First, the residual proliferation rate correlates with the ability of cells to recover from treatment. Second, the residual proliferation rate persists long after washout of the drug and until cells recover. Third, the total observable pool of cell-associated rapamycin is extremely stable and decreases only with increasing cell number after washout of the drug. Finally, consideration of the residual proliferation rate alone accurately and quantitatively accounts for the kinetics of recovery of wild-type cells and for the nature and severity of the ego- mutant defect. Overall, our results revealed that rapamycin is a partial inhibitor of yeast TORC1, that persistence of the drug limits recovery, and that rapamycin is not detoxified by yeast but is passively diluted among progeny cells because of residual proliferation.

Variation in scrotal color among widely distributed vervet monkey populations (Chlorocebus aethiops pygerythrus and Chlorocebus aethiops sabaeus).

Vervet monkeys (Chlorocebus aethiops) exhibit bright blue scrotal skin which may function to mediate social interactions by acting as a socio-sexual signal. Previous research on scrotal coloration among vervet monkeys was limited to experimental work on captive Ch. a. sabaeus, the least colorful vervet subspecies, and two field studies of the more colorful Ch. a. pygerythrus. In a study of free-ranging and captive vervet monkeys in South Africa (Ch. pygerythrus), West Africa (Ch. a. sabaeus) and the Caribbean (Ch. a. sabaeus), we examined scrotal color variation across geographically distant subspecies. We provide an exploration of how digital photographs may be used to quantify and analyze blue and green skin coloration by examining the blue-yellow opponency channel and luminance channel as color measures. We found that that at all ages the scrotal color of Ch. a. pygerythrus males was always bluer and darker than that of Ch. a. sabaeus males. Among Ch. a. pygerythrus scrotal color becomes bluer and lightens with increasing age, while the color of Ch. a. sabaeus males also lightens, but becomes less blue with increasing age. We suggest that color variation is related to maturation and may function as an age-related signal among Ch. a. pygerythrus and Ch. a. sabaeus. We also found color was related to three morphological features among adults. For Ch. a. pygerythrus, higher body weight is associated with more blue color and longer canine length is associated with lighter color. Lighter color was associated with longer body lengths among Ch. a. sabaeus. Future studies focused on color variation within age classes are needed to examine the potential signal content of color in this species.

Functional specialisation of yeast Rho1 GTP exchange factors.

Rho GTPases are regulated in complex spatiotemporal patterns that might be dependent, in part at least, on the multiplicity of their GTP exchange factors (GEFs). Here, we examine the extent of and basis for functional specialisation of the Rom2 and Tus1 GEFs that activate the yeast Rho1 GTPase, the orthologue of mammalian RhoA. First, we find that these GEFs selectively activate different Rho1-effector branches. Second, the synthetic genetic networks around ROM2 and TUS1 confirm very different global in vivo roles for these GEFs. Third, the GEFs are not functionally interchangeable: Tus1 cannot replace the essential role of Rom2, even when overexpressed. Fourth, we find that Rom2 and Tus1 localise differently: Rom2 to the growing bud surface and to the bud neck at cytokinesis; Tus1 only to the bud neck, but in a distinct pattern. Finally, we find that these GEFs are dependent on different protein co-factors: Rom2 function and localisation is largely dependent on Ack1, a SEL1-domain-containing protein; Tus1 function and localisation is largely dependent on the Tus1-interacting protein Ypl066w (which we name Rgl1). We have revealed a surprising level of diversity among the Rho1 GEFs that contributes another level of complexity to the spatiotemporal control of Rho1.

Synthetic genetic interactions allele dependence, uses, and conservation.

Genetic interactions occur between a pair of genes when the phenotype of the double mutant leads to an unexpected phenotype, one that is not predicted from the phenotypes of the single mutants alone. Here, we focus on genetic enhancements, otherwise known as synthetic genetic interactions, where the double mutant phenotype is more severe than expected. Such interactions are rife in natural populations and underlie complex traits, variable penetrance, variable expressivity, and genetic predisposition. Such interactions can also contribute valuable information for functional genomics analysis. Pairwise synthetic genetic interactions are now being systematically uncovered for some simple model genomes. These data are affording us an unparalleled opportunity to examine, understand and exploit genetic enhancements. Here we focus on some key lessons, insights, and confusions arising from these large-scale datasets. We consider if genome-wide datasets support traditional assumptions about the functional relationships between gene products that underlie genetic enhancements. We argue that the genetic enhancement network of an organism is not uniform in nature and is highly dependent on the nature of the interacting alleles. We consider how such genetic networks can be exploited to inform gene product function. Finally, we consider the extent to which genetic enhancement networks are conserved between species.

The functional relationships underlying a synthetic genetic network.

Here, we focus on synthetic lethal genetic interactions, examples of genetic enhancements, where mutations in two different genes result in lethality but only when present together. We recently identified the synthetic lethal network around the PKC1 gene encoding the essential protein kinase C of yeast. We found that this network is heavily enriched for interactions with genes whose products are closely linked to Pkc1 signaling in vivo. Here, we show that: the PKC1 gene elicits a distinct spectrum of genetic interactions to SLT2, encoding a non-essential component of the very same signaling pathway. We also show that the terminal phenotype underlying the synthetic lethal network around PKC1 is not uniform. Synthetic lethal genetic networks thus appear to be very heterogeneous in nature with important implications for what functional relationships can be discovered from them.

The synthetic genetic network around PKC1 identifies novel modulators and components of protein kinase C signaling in Saccharomyces cerevisiae.

Budding yeast Saccharomyces cerevisiae contains one protein kinase C (PKC) isozyme encoded by the essential gene PKC1. Pkc1 is activated by the small GTPase Rho1 and plays a central role in the cell wall integrity (CWI) signaling pathway. This pathway acts primarily to remodel the cell surface throughout the normal life cycle and upon various environmental stresses. The pathway is heavily branched, with multiple nonessential branches feeding into and out of the central essential Rho1-Pkc1 module. In an attempt to identify novel components and modifiers of CWI signaling, we determined the synthetic lethal genetic network around PKC1 by using dominant-negative synthetic genetic array analysis. The resulting mutants are hypersensitive to lowered Pkc1 activity. The corresponding 21 nonessential genes are closely related to CWI function: 14 behave in a chemical-genetic epistasis test as acting in the pathway, and 6 of these genes encode known components. Twelve of the 21 null mutants display elevated CWI reporter activity, consistent with the idea that the pathway is activated by and compensates for loss of the gene products. Four of the 21 mutants display low CWI reporter activity, consistent with the idea that the pathway is compromised in these mutants. One of the latter group of mutants lacks Ack1(Ydl203c), an uncharacterized SEL-1 domain-containing protein that we find modulates pathway activity. Epistasis analysis places Ack1 upstream of Pkc1 in the CWI pathway and dependent on the upstream Rho1 GTP exchange factors Rom2 and Tus1. Overall, the synthetic genetic network around PKC1 directly and efficiently identifies known and novel components of PKC signaling in yeast.

Mpt5p, a stress tolerance- and lifespan-promoting PUF protein in Saccharomyces cerevisiae, acts upstream of the cell wall integrity pathway.

Pumilio family (PUF) proteins affect specific genes by binding to, and inhibiting the translation or stability of, their transcripts. The PUF domain is required and sufficient for this function. One Saccharomyces cerevisiae PUF protein, Mpt5p (also called Puf5p or Uth4p), promotes stress tolerance and replicative life span (the maximum number of doublings a mother cell can undergo before entering into senescence) by an unknown mechanism thought to partly overlap with, but to be independent of, the cell wall integrity (CWI) pathway. Here, we found that mpt5Delta mutants also display a short chronological life span (the time cells stay alive in saturated cultures in synthetic medium), a defect that is suppressed by activation of CWI signaling. We found that Mpt5p is an upstream activator of the CWI pathway: mpt5Delta mutants display the appropriate phenotypes and genetic interactions, display low basal activity of the pathway, and are defective in activation of the pathway upon thermal stress. A set of mRNAs that specifically bind to Mpt5p was recently reported. One such putative target, LRG1, encodes a GTPase-activating protein for Rho1p that directly links Mpt5p to CWI signaling: Lrg1p inhibits CWI signaling, LRG1 mRNA contains a consensus Mpt5p-binding site in its putative 3' untranslated region, loss of Lrg1p suppresses the temperature sensitivity and CWI signaling defects of mpt5Delta mutants, and LRG1 mRNA abundance is inhibited by Mpt5p. We conclude that Mpt5p is required for normal replicative and chronological life spans and that the CWI pathway is a key and direct downstream target of this PUF protein.

Emerging role of RAB GTPases in cancer and human disease.

Emerging evidence implicates alterations in the RAB small GTPases and their associated regulatory proteins and effectors in multiple human diseases including cancer. We have recently shown that RAB25, located at chromosome 1q22, is amplified at the DNA level and overexpressed at the RNA level in ovarian and breast cancer. These changes correlated with a worsened outcome in both diseases. In addition, enforced expression of RAB25 in both breast and ovarian cancer cells decreased apoptosis and increased proliferation and aggressiveness in vivo, potentially explaining the worsened prognosis. A better understanding of genetic alterations as well as the physiologic and pathophysiologic roles of RAB GTPases may open new opportunities for therapeutic intervention and better outcomes.

"Sleeping beauty": quiescence in Saccharomyces cerevisiae.

The cells of organisms as diverse as bacteria and humans can enter stable, nonproliferating quiescent states. Quiescent cells of eukaryotic and prokaryotic microorganisms can survive for long periods without nutrients. This alternative state of cells is still poorly understood, yet much benefit is to be gained by understanding it both scientifically and with reference to human health. Here, we review our knowledge of one "model" quiescent cell population, in cultures of yeast grown to stationary phase in rich media. We outline the importance of understanding quiescence, summarize the properties of quiescent yeast cells, and clarify some definitions of the state. We propose that the processes by which a cell enters into, maintains viability in, and exits from quiescence are best viewed as an environmentally triggered cycle: the cell quiescence cycle. We synthesize what is known about the mechanisms by which yeast cells enter into quiescence, including the possible roles of the protein kinase A, TOR, protein kinase C, and Snf1p pathways. We also discuss selected mechanisms by which quiescent cells maintain viability, including metabolism, protein modification, and redox homeostasis. Finally, we outline what is known about the process by which cells exit from quiescence when nutrients again become available.

Misfolded proteins are competent to mediate a subset of the responses to heat shock in Saccharomyces cerevisiae.

Cells may sense heat shock via the accumulation of thermally misfolded proteins. To explore this possibility, we determined the effect of protein misfolding on gene expression in the absence of temperature changes. The imino acid analog azetidine-2-carboxylic acid (AZC) is incorporated into protein competitively with proline and causes reduced thermal stability or misfolding. We found that adding AZC to yeast at sublethal concentrations sufficient to arrest proliferation selectively induced expression of heat shock factor-regulated genes to a maximum of 27-fold and that these inductions were dependent on heat shock factor. AZC treatment also selectively repressed expression of the ribosomal protein genes, another heat shock factor-dependent process, to a maximum of 20-fold. AZC treatment thus strongly and selectively activates heat shock factor. AZC treatment causes this activation by misfolding proteins. Induction of HSP42 by AZC treatment required protein synthesis; treatment with ethanol, which can also misfold proteins, activated heat shock factor, but treatment with canavanine, an arginine analog less potent than AZC at misfolding proteins, did not. However, misfolded proteins did not strongly induce the stress response element regulon. We conclude that misfolded proteins are competent to specifically trigger activation of heat shock factor in response to heat shock.

The protein kinase C pathway is required for viability in quiescence in Saccharomyces cerevisiae.

Protein kinase C, encoded by PKC1, regulates construction of the cell surface in vegetatively growing yeast cells. Pkc1 in part acts by regulating Mpk1, a MAP kinase. Mutants lacking Bck1, a component of the MAP kinase branch of the pathway, fail to respond normally to nitrogen starvation, which causes entry into quiescence. Given that the Tor1 and Tor2 proteins are key inhibitors of entry into quiescence, the Pkc1 pathway may regulate these proteins. We find that pkc1Delta and mpk1Delta mutants rapidly die by cell lysis upon carbon or nitrogen starvation. The Pkc1 pathway does not regulate the TOR proteins: transcriptional changes dependent on inhibition of the TORs occur normally in pkc1Delta and mpk1Delta mutants when starved for nitrogen; pkc1Delta and mpk1Delta mutants die rapidly upon treatment with rapamycin, an inhibitor of the TORs. We find that Mpk1 is transiently activated by rapamycin treatment via a novel mechanism. Finally, we find that rapamycin treatment or nitrogen starvation induces resistance to the cell wall-digesting enzyme zymolyase by a Pkc1-dependent mechanism. Thus, the Pkc1 pathway is not a nutrient sensor but acts downstream of TOR inhibition to maintain cell integrity in quiescence.

Identification of LBM180, a lamellar body limiting membrane protein of alveolar type II cells, as the ABC transporter protein ABCA3.

Lamellar bodies are the specialized secretory organelles of alveolar type II (ATII) epithelial cells through which the cell packages pulmonary surfactant and regulates its secretion. Surfactant within lamellar bodies is densely packed as circular arrays of lipid membranes and appears to be the product of several trafficking and biosynthetic processes. To elucidate these processes, we reported previously on the generation of a monoclonal antibody (3C9) that recognizes a unique protein of the lamellar body membrane of 180 kDa, which we named LBM180. We report that mass spectrometry of the protein precipitated by this antibody generated a partial sequence that is identical to the ATP-binding cassette protein, ABCA3. Homology analysis of partial sequences suggests that this protein is highly conserved among species. The ABCA3 gene transcript was found in cell lines of human lung origin, in ATII cells of human, rat, and mouse, as well as different tissues of rat, but the highest expression of ABCA3 was observed in ATII cells. Expression of this transcript was at its maximum prior to birth, and hormonal induction of ABCA3 transcript was observed in human fetal lung at the same time as other surfactant protein transcripts were induced, suggesting that ABCA3 is developmentally regulated. Molecular and biochemical studies show that ABCA3 is targeted to vesicle membranes and is found in the limiting membrane of lamellar bodies. Because ABCA3 is a member of a subfamily of ABC transporters that are predominantly known to be involved in the regulation of lipid transport and membrane trafficking, we speculate that this protein may play a key role in lipid organization during the formation of lamellar bodies.

Direct and novel regulation of cAMP-dependent protein kinase by Mck1p, a yeast glycogen synthase kinase-3.

The MCK1 gene of Saccharomyces cerevisiae encodes a protein kinase homologous to metazoan glycogen synthase kinase-3. Previous studies implicated Mck1p in negative regulation of pyruvate kinase. In this study we find that purified Mck1p does not phosphorylate pyruvate kinase, suggesting that the link is indirect. We find that purified Tpk1p, a cAMP-dependent protein kinase catalytic subunit, phosphorylates purified pyruvate kinase in vitro, and that loss of the cAMP-dependent protein kinase regulatory subunit, Bcy1p, increases pyruvate kinase activity in vivo. We find that purified Mck1p inhibits purified Tpk1p in vitro, in the presence or absence of Bcy1p. Mck1p must be catalytically active to inhibit Tpk1p, but Mck1p does not phosphorylate this target. We find that abolition of Mck1p autophosphorylation on tyrosine prevents the kinase from efficiently phosphorylating exogenous substrates, but does not block its ability to inhibit Tpk1p in vitro. We find that this mutant form of Mck1p appears to retain the ability to negatively regulate cAMP-dependent protein kinase in vivo. We propose that Mck1p, in addition to phosphorylating some target proteins, also acts by a separate, novel mechanism: autophosphorylated Mck1p binds to and directly inhibits, but does not phosphorylate, the catalytic subunits of cAMP-dependent protein kinase.