Showing papers by "Marian Carlson published in 2004"

Cyclic AMP-dependent protein kinase regulates the subcellular localization of Snf1-Sip1 protein kinase.

Abstract: The Snf1/AMP-activated protein kinase family has diverse roles in cellular responses to metabolic stress. In Saccharomyces cerevisiae, Snf1 protein kinase has three isoforms of the beta subunit that confer versatility on the kinase and that exhibit distinct patterns of subcellular localization. The Sip1 beta subunit resides in the cytosol in glucose-grown cells and relocalizes to the vacuolar membrane in response to carbon stress. We show that translation of Sip1 initiates at the second ATG of the open reading frame, yielding a potential site for N myristoylation, and that mutation of the critical glycine abolishes relocalization. We further show that the cyclic AMP-dependent protein kinase (protein kinase A [PKA]) pathway maintains the cytoplasmic localization of Sip1 in glucose-grown cells. The Snf1 catalytic subunit also exhibits aberrant localization to the vacuolar membrane in PKA-deficient cells, indicating that PKA regulates the localization of Snf1-Sip1 protein kinase. These findings establish a novel mechanism of regulation of Snf1 protein kinase by the PKA pathway.

Pak1 Protein Kinase Regulates Activation and Nuclear Localization of Snf1-Gal83 Protein Kinase

Abstract: The Snf1/AMP-activated protein kinase (AMPK) family is important for responses to metabolic stress (for reviews, see references 10 and 16). In mammals, AMPK is activated by increases in AMP during metabolic stress, by the hormones leptin and adiponectin (24, 44), and by drugs used in the treatment of type 2 diabetes (46). AMPK coordinates energy homeostasis, regulating lipid and glucose metabolism. In the yeast Saccharomyces cerevisiae, Snf1 kinase is also activated by stresses, notably glucose limitation (14, 23, 41, 43). Snf1 regulates the transcription of many genes and the activity of metabolic enzymes in response to carbon stress, and Snf1 is required for the utilization of alternate carbon sources (2, 8). Snf1 also affects meiosis and sporulation, aging (1), haploid invasive growth (4), and diploid pseudohyphal growth (18). Snf1 is regulated by three upstream kinases, Pak1, Tos3, and Elm1, which phosphorylate the activation loop threonine of the catalytic subunit (Thr210) and activate Snf1 protein kinase (13, 25, 33). Any one of these three upstream kinases suffices for Snf1 function in vivo. A mammalian ortholog, the LKB1 tumor suppressor kinase, similarly phosphorylates and activates AMPK (11, 13, 42). Snf1 catalytic activity is also regulated by at least two other mechanisms. Protein phosphatase 1 (Reg1-Glc7) controls the phosphorylation and activity of Snf1 kinase (22, 23, 29), and the Std1 (Msn3) protein, which interacts with glucose sensors (31), plays a modest role in regulating Snf1 catalytic activity (19). Snf1 protein kinase, like AMPK, is heterotrimeric, and the kinase comprises the Snf1 catalytic subunit, the Snf4 activating subunit, and one of three β-subunit isoforms, Gal83, Sip1, or Sip2. The different forms of Snf1 kinase are here designated, according to their β subunit, as Snf1-Gal83, Snf1-Sip1, and Snf1-Sip2. The three β subunits exhibit considerable functional redundancy, as all three must be mutated to confer a Snf− phenotype, but they also play distinct roles (30, 45). Gal83 mediates the interaction of Snf1 kinase with Sip4, a transcriptional activator of gluconeogenic genes (30, 36), and with the transcriptional apparatus (38); Sip2 has been implicated in aging (1, 20); and each of the three β subunits has a distinct role in haploid invasive growth (39). A major function of the β subunits is to regulate the subcellular localization of the kinase (38), thereby governing access to different substrates. All three β subunits are cytoplasmic when cells are grown in abundant glucose, but when cells are shifted to low glucose or a nonfermentable carbon source, Snf1-Gal83 becomes enriched in the nucleus, Snf1-Sip1 relocalizes around the vacuole, and Snf1-Sip2 remains cytoplasmic. The cyclic AMP-dependent protein kinase (protein kinase A) regulates the localization of Snf1-Sip1 (12) but does not affect Snf1-Gal83 (38). The existence of three functionally distinct β-subunit isoforms with different, and differently regulated, subcellular localizations contributes to the functional versatility of Snf1 kinase in regulating multiple cellular processes and responses to stress. Why do yeast cells have three upstream activating kinases for Snf1? Pak1, Tos3, and Elm1 appear to exhibit considerable functional redundancy, as all three cognate genes must be deleted to confer a Snf− mutant phenotype (13, 33). However, this finding does not exclude the possibility that the kinases indeed have distinct functions. The existence of three different forms of Snf1 protein kinase raised the possibility that each upstream kinase exhibits specificity for Snf1 protein that is associated with a particular β subunit. We have examined the functional correspondence between the three upstream kinases and the three forms of Snf1 protein kinase. We show that Pak1 is the most critical kinase for activation of Snf1-Gal83 but that Elm1 also has a significant role; moreover, we show that Pak1 also affects Snf1-Sip2, as does Elm1. These findings rule out the possibility of a one-to-one correspondence between upstream kinases and forms of Snf1 protein kinase with respect to activation. We further identify a second, unexpected role for Pak1 in regulating Snf1-Gal83: phosphorylation of Snf1 by Pak1 is required for the relocalization of Snf1-Gal83 to the nucleus in response to carbon stress. Thus, Pak1 not only activates Snf1-Gal83 but also controls its localization and thereby regulates nuclear Snf1 protein kinase activity.

Nrg1 and nrg2 transcriptional repressors are differently regulated in response to carbon source.

Abstract: The Nrg1 and Nrg2 repressors of Saccharomyces cerevisiae have highly similar zinc fingers and closely related functions in the regulation of glucose-repressed genes. We show that NRG1 and NRG2 are differently regulated in response to carbon source at both the RNA and protein levels. Expression of NRG1 RNA is glucose repressed, whereas NRG2 RNA levels are nearly constant. Nrg1 protein levels are elevated in response to glucose limitation or growth in nonfermentable carbon sources, whereas Nrg2 levels are diminished. Chromatin immunoprecipitation assays showed that Nrg1 and Nrg2 bind DNA both in the presence and absence of glucose. In mutant cells lacking the corepressor Ssn6(Cyc8)-Tup1, promoter-bound Nrg1, but not Nrg2, functions as an activator in a reporter assay, providing evidence that the two Nrg proteins have distinct properties. We suggest that the differences in expression and function of these two repressors, in combination with their similar DNA-binding domains, contribute to the complex regulation of the large set of glucose-repressed genes.

Mutations in the Gal83 Glycogen-Binding Domain Activate the Snf1/Gal83 Kinase Pathway by a Glycogen-Independent Mechanism

Abstract: The yeast Snf1 kinase and its mammalian ortholog, AMP-activated protein kinase (AMPK), regulate responses to metabolic stress. Previous studies identified a glycogen-binding domain in the AMPK beta1 subunit, and the sequence is conserved in the Snf1 kinase beta subunits Gal83 and Sip2. Here we use genetic analysis to assess the role of this domain in vivo. Alteration of Gal83 at residues that are important for glycogen binding of AMPK beta1 abolished glycogen binding in vitro and caused diverse phenotypes in vivo. Various Snf1/Gal83-dependent processes were upregulated, including glycogen accumulation, expression of RNAs encoding glycogen synthase, haploid invasive growth, the transcriptional activator function of Sip4, and activation of the carbon source-responsive promoter element. Moreover, the glycogen-binding domain mutations conferred transcriptional regulatory phenotypes even in the absence of glycogen, as determined by analysis of a mutant strain lacking glycogen synthase. Thus, mutation of the glycogen-binding domain of Gal83 positively affects Snf1/Gal83 kinase function by a mechanism that is independent of glycogen binding.