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.

Elm1p, Pak1p, and Tos3p are upstream kinases for the SNF1 complex that have partially redundant functions.

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, and RGT2 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 regulate SUC2 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.

cThe glucose analog 2-deoxyglucose (2DG) inhibits the growth of Saccharomyces cerevisiae and human tumor cells, but its modes of action have not been fully elucidated. Yeast cells lacking Snf1 (AMP-activated protein kinase) are hypersensitive to 2DG. Overexpression of either of two low-affinity, high-capacity glucose transporters, Hxt1 and Hxt3, suppresses the 2DG hypersensitivity of snf1⌬ cells. The addition of 2DG or the loss of Snf1 reduces HXT1 and HXT3 expression levels and stimulates transporter endocytosis and degradation in the vacuole. 2DG-stimulated trafficking of Hxt1 and Hxt3 requires Rod1/Art4 and Rog3/ Art7, two members of the ␣-arrestin trafficking adaptor family. Mutations in ROD1 and ROG3 that block binding to the ubiquitin ligase Rsp5 eliminate Rod1-and Rog3-mediated trafficking of Hxt1 and Hxt3. Genetic analysis suggests that Snf1 negatively regulates both Rod1 and Rog3, but via different mechanisms. Snf1 activated by 2DG phosphorylates Rod1 but fails to phosphorylate other known targets, such as the transcriptional repressor Mig1. We propose a novel mechanism for 2DG-induced toxicity whereby 2DG stimulates the modification of ␣-arrestins, which promote glucose transporter internalization and degradation, causing glucose starvation even when cells are in a glucose-rich environment.C ells sense and respond to changes in the nutrient supply to ensure optimal cell growth and survival. To achieve this adaptation, cell-signaling cues dictate compensatory alterations in the transcriptome and proteome (1-5). The addition of the glucose analog 2-deoxyglucose (2DG) to cells causes a glucose starvation-like response, inhibiting growth and reducing viability even in the presence of abundant glucose (6, 7). 2DG is taken up and converted to 2-deoxyglucose-6-phosphate (2DG-6P) (8, 9); however, the absence of a hydroxyl group on C-2 prevents the further catabolism of 2DG-6P by phosphoglucose isomerase. Accumulation of 2DG-6P may result in product inhibition of hexokinase, thereby inhibiting glycolysis (10). In Saccharomyces cerevisiae, 2DG reportedly inhibits the biosynthesis of both cell wall polysaccharide and glycoprotein, causing cells to become osmotically fragile (11,12). Whether these are the only means by which 2DG short-circuits normal glucose utilization remains unclear.Addressing this question is of significant clinical importance, because 2DG is a potent inhibitor of cancer cell proliferation. 2DG impedes cancer progression in animal models and continues to be assessed as an anticancer therapeutic (13-16). 2DG selectively inhibits cancerous cells as a result of a key metabolic shift that distinguishes many malignant cells from the surrounding normal tissues. Many tumor cells shunt glucose through the glycolytic pathway and use lactic acid fermentation to generate ATP, a phenomenon first recognized by Otto Warburg (17,18). In spite of the widespread use of 2DG, the mechanism by which it inhibits cell growth remains controversial; it has been reported to generate a dead-end metabolite in 2DG-6P tha...

The Snf1 kinase and its mammalian homolog, the AMP-activated protein kinase, are heterotrimeric enzymes composed of a catalytic a-subunit, a regulatory g-subunit and a b-subunit that mediates heterotrimer formation. Saccharomyces cerevisiae encodes three b-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 b-subunit genes inactivates the Snf1 kinase. The sip1D sip2D gal83D 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 sip2D gal83D strain manifests a subset of Snf phenotypes (Raf + , Gly ± ) observed in the snf1D 10 strain (Raf ± , Gly ± ), suggesting that individual b-subunits direct the Snf1 kinase to a subset of its targets in vivo. Indeed, deletion of individual b-subunit genes causes distinct differences in the induction and phosphorylation of Sip4, strongly suggesting that the b-subunits play an important role in substrate de®nition.