Martin C. Schmidt

Global analysis of protein phosphorylation in yeast

Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes. Until recently, our biochemical understanding of protein phosphorylation on a global scale has been extremely limited; only one half of the yeast kinases have known in vivo substrates and the phosphorylating kinase is known for less than 160 phosphoproteins. Here we describe, with the use of proteome chip technology, the in vitro substrates recognized by most yeast protein kinases: we identified over 4,000 phosphorylation events involving 1,325 different proteins. These substrates represent a broad spectrum of different biochemical functions and cellular roles. Distinct sets of substrates were recognized by each protein kinase, including closely related kinases of the protein kinase A family and four cyclin-dependent kinases that vary only in their cyclin subunits. Although many substrates reside in the same cellular compartment or belong to the same functional category as their phosphorylating kinase, many others do not, indicating possible new roles for several kinases. Furthermore, integration of the phosphorylation results with protein–protein interaction and transcription factor binding data revealed novel regulatory modules. Our phosphorylation results have been assembled into a first-generation phosphorylation map for yeast. Because many yeast proteins and pathways are conserved, these results will provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.

Globular amyloid β-peptide1−42 oligomer − a homogenous and stable neuropathological protein in Alzheimer's disease

Amyloid β‐peptide (Aβ)1−42 oligomers have recently been discussed as intermediate toxic species in Alzheimers disease (AD) pathology. Here we describe a new and highly stable Aβ1−42 oligomer species which can easily be prepared in vitro and is present in the brains of patients with AD and Aβ1−42‐overproducing transgenic mice. Physicochemical characterization reveals a pure, highly water‐soluble globular 60‐kDa oligomer which we named ‘Aβ1−42 globulomer’. Our data indicate that Aβ1−42 globulomer is a persistent structural entity formed independently of the fibrillar aggregation pathway. It is a potent antigen in mice and rabbits eliciting generation of Aβ1−42 globulomer‐specific antibodies that do not cross‐react with amyloid precursor protein, Aβ1−40 and Aβ1−42 monomers and Aβ fibrils. Aβ1−42 globulomer binds specifically to dendritic processes of neurons but not glia in hippocampal cell cultures and completely blocks long‐term potentiation in rat hippocampal slices. Our data suggest that Aβ1−42 globulomer represents a basic pathogenic structural principle also present to a minor extent in previously described oligomer preparations and that its formation is an early pathological event in AD. Selective neutralization of the Aβ globulomer structure epitope is expected to have a high potential for treatment of AD.

Molecular characterization of human and bovine endothelin converting enzyme (ECE‐1)

A membrane‐bound protease activity that specifically converts Big endothelin‐1 has been purified from bovine endothelial cells (FBHE) The enzyme was cleaved with trypsin and the peptide sequencing analysis confirmed it to be a zinc chelating metalloprotease containing the typical HEXXH (HELTH) motif. RT‐PCR and cDNA screens were employed to isolate the complete cDNAs of the bovine and human enzymes. This human metalloprotease was expressed heterologously in cell culture oocytes. The catalytic activity of the recombinant enzyme is the same as that determined for the natural enzyme. The data suggest that the characterized enzyme represents the functional human endothelin converting enzyme ECE‐1.

Yeast Pak1 Kinase Associates with and Activates Snf1

ABSTRACT Members of the Snf1/AMP-activated protein kinase family are activated under conditions of nutrient stress by a distinct upstream kinase. Here we present evidence that the yeast Pak1 kinase functions as a Snf1-activating kinase. Pak1 associates with the Snf1 kinase in vivo, and the association is greatly enhanced under glucose-limiting conditions when Snf1 is active. Snf1 kinase complexes isolated from pak1Δ mutant strains show reduced specific activity in vitro, and affinity-purified Pak1 kinase is able to activate the Snf1-dependent phosphorylation of Mig1 in vitro. Purified Pak1 kinase promotes the phosphorylation of the Snf1 polypeptide on threonine 210 within the activation loop in vitro, and an increased dosage of the PAK1 gene causes increased Snf1 threonine 210 phosphorylation in vivo. Deletion of the PAK1 gene does not produce a Snf phenotype, suggesting that one or more additional protein kinases is able to activate Snf1 in vivo. However, deletion of the PAK1 gene suppresses many of the phenotypes associated with the deletion of the REG1 gene, providing genetic evidence that Pak1 activates Snf1 in vivo. The closest mammalian homologue of yeast Pak1 kinase, calcium-calmodulin-dependent protein kinase kinase beta, may play a similar role in mammalian nutrient stress signaling.

Std1 and Mth1 Proteins Interact with the Glucose Sensors To Control Glucose-Regulated Gene Expression in Saccharomyces cerevisiae

ABSTRACT The Std1 protein modulates the expression of glucose-regulated genes, but its exact molecular role in this process is unclear. A two-hybrid screen for Std1-interacting proteins identified the hydrophilic C-terminal domains of the glucose sensors, Snf3 and Rgt2. The homologue of Std1, Mth1, behaves differently from Std1 in this assay by interacting with Snf3 but not Rgt2. Genetic interactions between STD1, MTH1, SNF3, andRGT2 suggest that the glucose signaling is mediated, at least in part, through interactions of the products of these four genes. Mutations in MTH1 can suppress the raffinose growth defect of a snf3 mutant as well as the glucose fermentation defect present in cells lacking both glucose sensors (snf3 rgt2). Genetic suppression by mutations in MTH1 is likely to be due to the increased and unregulated expression of hexose transporter genes. In media lacking glucose or with low levels of glucose, the hexose transporter genes are subject to repression by a mechanism that requires the Std1 and Mth1 proteins. An additional mechanism for glucose sensing must exist since a strain lacking all four genes (snf3 rgt2 std1 mth1) is still able to regulateSUC2 gene expression in response to changes in glucose concentration. Finally, studies with green fluorescent protein fusions indicate that Std1 is localized to the cell periphery and the cell nucleus, supporting the idea that it may transduce signals from the plasma membrane to the nucleus.

β-subunits of Snf1 kinase are required for kinase function and substrate definition

The Snf1 kinase and its mammalian homolog, the AMP‐activated protein kinase, are heterotrimeric enzymes composed of a catalytic α‐subunit, a regulatory γ‐subunit and a β‐subunit that mediates heterotrimer formation. Saccharomyces cerevisiae encodes three β‐subunit genes, SIP1, SIP2 and GAL83. Earlier studies suggested that these subunits may not be required for Snf1 kinase function. We show here that complete and precise deletion of all three β‐subunit genes inactivates the Snf1 kinase. The sip1Δ sip2Δ gal83Δ strain is unable to derepress invertase, grows poorly on alternative carbon sources and fails to direct the phosphorylation of the Mig1 and Sip4 proteins in vivo. The SIP1 sip2Δ gal83Δ strain manifests a subset of Snf phenotypes (Raf+, Gly−) observed in the snf1Δ 10 strain (Raf−, Gly−), suggesting that individual β‐subunits direct the Snf1 kinase to a subset of its targets in vivo. Indeed, deletion of individual β‐subunit genes causes distinct differences in the induction and phosphorylation of Sip4, strongly suggesting that the β‐subunits play an important role in substrate definition.

ADP Regulates SNF1, the Saccharomyces cerevisiae Homolog of AMP-Activated Protein Kinase

Summary The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.

Access Denied: Snf1 Activation Loop Phosphorylation Is Controlled by Availability of the Phosphorylated Threonine 210 to the PP1 Phosphatase

Phosphorylation of the Saccharomyces cerevisiae Snf1 kinase activation loop is determined by the integration of two reaction rates: the rate of phosphorylation by upstream kinases and the rate of dephosphorylation by Glc7. The activities of the Snf1-activating kinases do not appear to be glucose-regulated, since immune complex kinase assays with each of the three Snf1-activating kinases show similar levels of activity when prepared from cells grown in either high or low glucose. In contrast, the dephosphorylation of the Snf1 activation loop was strongly regulated by glucose. When de novo phosphorylation of Snf1 was inhibited, phosphorylation of the Snf1 activation loop was found to be stable in low glucose but rapidly lost upon the addition of glucose. A greater than 10-fold difference in the rates of Snf1 activation loop dephosphorylation was detected. However, the activity of the Glc7-Reg1 phosphatase may not itself be directly regulated by glucose, since the Glc7-Reg1 enzyme was active in low glucose toward another substrate, the transcription factor Mig1. Glucose-mediated regulation of Snf1 activation loop dephosphorylation is controlled by changes in the ability of the Snf1 activation loop to act as a substrate for Glc7.

Purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae

Members of the Snf1/AMPK family of protein kinases are activated by distinct upstream kinases that phosphorylate a conserved threonine residue in the Snf1/AMPK activation loop. Recently, the identities of the Snf1- and AMPK-activating kinases have been determined. Here we describe the purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. The identities of proteins associated with the Snf1-activating kinases were determined by peptide mass fingerprinting. These kinases, Sak1, Tos3 and Elm2 do not appear to require the presence of additional subunits for activity. Sak1 and Snf1 co-purify and co-elute in size exclusion chromatography, demonstrating that these two proteins form a stable complex. The Snf1-activating kinases phosphorylate the activation loop threonine of Snf1 in vitro with great specificity and are able to do so in the absence of beta and gamma subunits of the Snf1 heterotrimer. Finally, we showed that the Snf1 kinase domain isolated from bacteria as a GST fusion protein can be activated in vitro and shows substrate specificity in the absence of its beta and gamma subunits.

Evidence of alternative promoters directing isoform-specific expression of human endothelin-converting enzyme-1 mRNA in cultured endothelial cells.

Abstract The endothelins, a family of closely related vasoactive and mitogenic peptides, are thought to play an important role in cardiovascular pathophysiology. The conversion of the inactive precursor ”big endothelin” to the biologically active peptide is catalyzed in vitro and in vivo by endothelin-converting enzymes (ECE). Recently the cDNA cloning of two homologous proteins, termed ECE-1 and ECE-2, has been reported. ECE-1 may play a key role in the activation and regulation of the cardiovascular endothelin proteolytic cascade. ECE-1 mRNA is expressed in two isoforms, termed α and β, which are identical except for the 5′-terminal regions. To investigate the transcriptional regulation of isoform-specific ECE-1 mRNA expression we isolated phage clones from a human genomic library and identified the α- and β-specific exons of ECE-1. The exon/intron organization of the 5′-terminal region of the human ECE-1 gene in conjunction with putative transcription initiation start sites suggests the existence of two alternative promoters, each directing the expression of either isoform. A reverse transcription/polymerase chain reaction assay indicated differential mRNA expression of ECE-1 isoforms. Using a luciferase reporter gene assay, we found that the genomic region upstream of exon 1α confers strong promoter activity in the human endothelial cell line ECV 304, which was previously shown to express predominantly ECE-1α mRNA. Transfection of serial deletion mutants in ECV304 cells indicated the existence of three positive and also one negative regulating element within 2 kb of the α-promoter region. Luciferase reporter gene studies also revealed that the genomic region upstream of exon 3, which encodes the putative ECE-1β specific N-terminus, was able to direct luciferase expression in primary cultured bovine aortic endothelial cells, indicating the existence of an alternative promoter. Transfection of nested deletions spanning 1.2 kb upstream of the putative translation initiation codon of ECE-1β suggested the existence of three positive regulating regions within the β-specific promoter. Both ECE-1 promoters lack TATA or CAAT boxes, and the two show different patterns of consensus sequences for transcription factors, suggesting a differential transcriptional regulation of isoform-specific ECE-1 mRNA expression.