The AMP-activated protein kinase (AMPK) family is a group of Serine/Threonine kinases highly conserved in eukaryotes, from yeast and insects to plants and mammals. Their primary role is the integration of signals regarding nutrient availability and environmental stresses, ensuring the adaptation to those conditions and cell survival (Hardie G., 2007; Ghillebert R. et al., 2011). As its homologue AMPK, in Saccharomyces cerevisiae Snf1 exists as a heterotrimeric complex. Core of this enzyme is the catalytic α subunit (Snf1), made up of a canonical catalytic domain in its N-terminus and of an autoinhibitory C-terminal domain which mediates the interaction with the regulatory subunits of this kinase (Rudolph M.J. et al., 2005). These subunits are: the β subunit (Sip1, Sip2 and Gal83, alternatively), which regulates Snf1 localization (Vincent O. et al., 2000) and the γ subunit (Snf4) that, interacting with the autoinhibitory domain of Snf1, guarantees the complete activation of the kinase (Momcilovic M. et al., 2008). Beyond the interaction with Snf4, the activation of the protein kinase Snf1 is determined by the phosphorylation of Thr210 residue in the α subunit (McCartney R.R. and Schmidt M.C., 2001). Three upstream kinases (Sak1, Tos3, Elm1) are responsible for such a phosphorylation. Those kinases are constitutively active, but metabolic signals, such as high glucose concentrations, promote the activity of the phosphatase complex Reg1/Glc7 which dephosphorylates and hence inactivates Snf1 (Huang D. et al.,1996; Sanz P. et al., 2000; Sutherland C. et al., 2003; Hong S. et al., 2003). In budding yeast, Snf1 is required for adaptation to glucose limitation and for growth on non-fermentable carbon sources. In those conditions Snf1 controls the expression of more than 400 genes. Apart from carbon metabolism, Snf1 affects several other processes; in fact, this kinase controls the expression of some important genes involved in the resistance to different environmental stresses (osmotic and alkaline stresses) or in the regulation of different cellular processes such as sporulation, aging, filamentous and invasive growth (Portillo F. et al., 2005; Ashrafi K. et al., 2000; Vyas V.K. et al., 2003). As a transcriptional regulator, Snf1 exerts its role modulating gene transcription at different levels. This kinase regulates different transcription factors, such as the transcription inhibitor Mig1 (Treitel M.A. et al., 1998; Papamichos-Choronaris M. et al., 2004) or some other transcription factors like Adr1, Sit4, Cat8 and Gcn4 which regulate the expression of genes involved in central metabolic functions, such as gluconeogenesis and respiration (Hedbacker K. and Carlson M., 2008; Smets B. et al., 2010; Kacherovsky N. et al., 2008). Moreover, protein kinase Snf1 is even able to positively regulate the transcription of some metabolic genes influencing the chromatin remodelling process and the recruitment of some Pre-Initiation Complex (PIC) components at promoters. In fact, Snf1 promotes acetylation of histone H3 by either the direct phosphorylation of Ser10 of histone H3 and the phosphorylation of acetyl-tranferase Gcn5 (Lo W. et al., 2005; van Oevelen C.J. et al., 2006; Liu Y. et al., 2010). Moreover, Snf1 is involved in the recruitment to some promoters of Mediator complex (Young E.T. et al., 2002), SAGA complex (van Oevelen C.J. et al., 2006), TATA-binding protein (TBP) (Shirra M.K. et al., 2005) and RNA Pol II (Tachibana C. et al., 2007; Young R.T. et al., 2012). My PhD research activity was focused on the role of Snf1 in the regulation of the expression of G1-specific genes, and thus in its function as modulator of cell cycle progression. Data obtained in our laboratory showed that, in cells grown in 2% glucose, deletion of SNF1 gene caused a delayed G1/S phase transition, consistently with a decreased expression of CLB5 gene. In keeping with that defective expression, the snf1Δ strain showed a severe reduction of Clb5 protein levels and a consequent decrease of phosphorylation of Clb5/Cdk1 complex targets, such as Sld2, which are responsible for the onset of DNA replication. Moreover, our co-immunoprecipitation assays highlighted that Snf1 interacts with Swi6, the common subunit of SBF (Swi4-Swi6) and MBF (Mbp1-Swi6) transcription complexes which regulate the expression of G1-specific genes (Nasmyth, K. and Dirick, L., 1991; Koch C. et al., 1993). Remarkably, the phenotype of the snf1 null mutant was complemented by a glucose concentration higher than 2% (5%), suggesting that the role of Snf1 in the modulation of cell cycle progression could depend on the nutritional status of cells. Those data, published in Pessina S. et al., 2010, newly indicated that Snf1 was involved in the regulation of G1/S transition and pointed to a role for this kinase in the modulation of G1-specific gene expression. To gain further insight into the function of Snf1, we then analyzed the expression profile of G1-specific genes in cells synchronized in G1 phase by α-factor treatment and released into fresh medium. Our analyses showed that loss of Snf1 (snf1Δ strain) severely affected the expression of CLN2, PCL1 (SBF-dependent) and CLB5, RNR1 (SBF-dependent) genes, suggesting that Snf1 regulates the expression of both SBF- and MBF-dependent genes. Although protein Snf1 was not detectable at promoters of G1-specific genes, we investigated whether it could modulate the activity of SBF and MBF complexes and we found that in a snf1Δ strain the recruitment of Swi6 to G1-specific promoters was affected. Moreover, our Chromatin ImmunoPrecipitation (ChIP) assays also showed that in a snf1Δ strain the defective association of Swi6 to promoters led to a decreased recruitment of both the FACT complex, which is involved in the chromatin remodelling at G1-specific promoters, and of the RNA Pol II. Since it is known that the subcellular localization of Swi6 influences its interaction with promoters, then we analyzed its localization in G1 synchronized cells. In keeping with literature data (Sidorova J.M. et al., 1995; Taberner, F.J. and Igual, J.C., 2010), our analyses showed that in wild type cells synchronized in G1 phase by α-factor treatment Swi6 was essentially nuclear. Instead, in a snf1Δ strain Swi6 was localized in the nucleus only in the 60% of the G1-arrested cells, consistently with the reduced binding of Swi6 to G1-specific promoters. It is well known that the Swi6 interaction to DNA is mediated by the DNA binding-proteins Swi4 and Mbp1 (Andrews B.J. and Moore L.A., 1992; Moll T. et al., 1992). Therefore we extended our analyses to those proteins and we found that also the nuclear localization and the subsequent binding to DNA of Swi4 and Mbp1 were affected in a snf1Δ strain. Therefore, our data provide a representative snapshot of what occurs in vivo in a snf1 null mutant, supporting the notion that Snf1 promotes the expression of G1-specific genes modulating the nuclear localization of SBF and MBF components and thus promoting the formation of a complete Pre-initiation Complex (PIC) at G1-specific promoters. It is well known that phosphorylation of Snf1 at Thr210 leads to the full activation of the kinase (Hong S.P. et al., 2003; Sutherland C.M. et al., 2003). Then, in order to obtain insight into the Snf1 molecular mechanism in cell cycle regulation, we investigated its phosphorylation on Thr210 during cell cycle progression. Snf1 was slightly phosphorylated on the Thr210 residue during all the cell cycle, suggesting that this kinase was partially active. To determine whether the activation of Snf1 was involved in its function as regulator of G1 transcription, we analyzed the expression of SBF- and MBF-dependent genes in the SNF1-T210A mutant and we found that in this mutant the expression of those genes was reduced. In keeping with those data, the expression of G1-specific genes resulted affected also in a SNF1-K84R mutant, in which the ATP binding site has been destroyed causing a severe reduction of Snf1 kinase activity. On the base of those findings, we investigated whether Snf1 could exert its role in G1 phase through the phosphorylation of specific substrates and we found that Snf1 phosphorylates in vitro Swi6 on Ser760. Nevertheless, analyses of site-specific mutants (SWI6-S760A or SWI6-S760E) did not show any alteration of G1/S transition, suggesting that this phosphorylation was not involved in the role of Snf1 as regulator of cell cycle. The ChIP analyses of Swi6 binding to CLN2 and RNR1 promoters, then, showed that in the SNF1-K84R mutant the recruitment of Swi6 was slightly affected; nevertheless, that alteration was not severe as that of a snf1Δ strain. Consistently, neither the recruitment of FACT complex nor the binding of RNA Pol II to G1-specific promoters was affected in the SNF1-K84R mutant. Since this last finding seemed to disagree with the severe reduction of mRNA expression of SBF- and MBF-dependent genes observed in the SNF1-K84R mutant, we wondered whether defects in transcriptional elongation might occur in that strain. Thus, we analyzed the occupancy of FACT complex and of RNA Pol II at the internal regions of CLN2 and RNR1 genes by ChIP analyses; in the SNF1-K84R mutant the occupancy of both those complexes was decreased, suggesting that the kinase activity of Snf1 promotes the transcriptional elongation across G1-specific genes. In conclusion, the sum of data here presented indicates that protein kinase Snf1 is involved at different levels in the modulation of the G1-specific gene expression, thus highlighting a new function for Snf1 in the regulation of G1/S transition.

(2013). Protein Kinase Snf1/AMPK: a new regulator of G1/S transition in Saccharomyces cerevisiae. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2013).

Protein Kinase Snf1/AMPK: a new regulator of G1/S transition in Saccharomyces cerevisiae

BUSNELLI, SARA
2013

Abstract

The AMP-activated protein kinase (AMPK) family is a group of Serine/Threonine kinases highly conserved in eukaryotes, from yeast and insects to plants and mammals. Their primary role is the integration of signals regarding nutrient availability and environmental stresses, ensuring the adaptation to those conditions and cell survival (Hardie G., 2007; Ghillebert R. et al., 2011). As its homologue AMPK, in Saccharomyces cerevisiae Snf1 exists as a heterotrimeric complex. Core of this enzyme is the catalytic α subunit (Snf1), made up of a canonical catalytic domain in its N-terminus and of an autoinhibitory C-terminal domain which mediates the interaction with the regulatory subunits of this kinase (Rudolph M.J. et al., 2005). These subunits are: the β subunit (Sip1, Sip2 and Gal83, alternatively), which regulates Snf1 localization (Vincent O. et al., 2000) and the γ subunit (Snf4) that, interacting with the autoinhibitory domain of Snf1, guarantees the complete activation of the kinase (Momcilovic M. et al., 2008). Beyond the interaction with Snf4, the activation of the protein kinase Snf1 is determined by the phosphorylation of Thr210 residue in the α subunit (McCartney R.R. and Schmidt M.C., 2001). Three upstream kinases (Sak1, Tos3, Elm1) are responsible for such a phosphorylation. Those kinases are constitutively active, but metabolic signals, such as high glucose concentrations, promote the activity of the phosphatase complex Reg1/Glc7 which dephosphorylates and hence inactivates Snf1 (Huang D. et al.,1996; Sanz P. et al., 2000; Sutherland C. et al., 2003; Hong S. et al., 2003). In budding yeast, Snf1 is required for adaptation to glucose limitation and for growth on non-fermentable carbon sources. In those conditions Snf1 controls the expression of more than 400 genes. Apart from carbon metabolism, Snf1 affects several other processes; in fact, this kinase controls the expression of some important genes involved in the resistance to different environmental stresses (osmotic and alkaline stresses) or in the regulation of different cellular processes such as sporulation, aging, filamentous and invasive growth (Portillo F. et al., 2005; Ashrafi K. et al., 2000; Vyas V.K. et al., 2003). As a transcriptional regulator, Snf1 exerts its role modulating gene transcription at different levels. This kinase regulates different transcription factors, such as the transcription inhibitor Mig1 (Treitel M.A. et al., 1998; Papamichos-Choronaris M. et al., 2004) or some other transcription factors like Adr1, Sit4, Cat8 and Gcn4 which regulate the expression of genes involved in central metabolic functions, such as gluconeogenesis and respiration (Hedbacker K. and Carlson M., 2008; Smets B. et al., 2010; Kacherovsky N. et al., 2008). Moreover, protein kinase Snf1 is even able to positively regulate the transcription of some metabolic genes influencing the chromatin remodelling process and the recruitment of some Pre-Initiation Complex (PIC) components at promoters. In fact, Snf1 promotes acetylation of histone H3 by either the direct phosphorylation of Ser10 of histone H3 and the phosphorylation of acetyl-tranferase Gcn5 (Lo W. et al., 2005; van Oevelen C.J. et al., 2006; Liu Y. et al., 2010). Moreover, Snf1 is involved in the recruitment to some promoters of Mediator complex (Young E.T. et al., 2002), SAGA complex (van Oevelen C.J. et al., 2006), TATA-binding protein (TBP) (Shirra M.K. et al., 2005) and RNA Pol II (Tachibana C. et al., 2007; Young R.T. et al., 2012). My PhD research activity was focused on the role of Snf1 in the regulation of the expression of G1-specific genes, and thus in its function as modulator of cell cycle progression. Data obtained in our laboratory showed that, in cells grown in 2% glucose, deletion of SNF1 gene caused a delayed G1/S phase transition, consistently with a decreased expression of CLB5 gene. In keeping with that defective expression, the snf1Δ strain showed a severe reduction of Clb5 protein levels and a consequent decrease of phosphorylation of Clb5/Cdk1 complex targets, such as Sld2, which are responsible for the onset of DNA replication. Moreover, our co-immunoprecipitation assays highlighted that Snf1 interacts with Swi6, the common subunit of SBF (Swi4-Swi6) and MBF (Mbp1-Swi6) transcription complexes which regulate the expression of G1-specific genes (Nasmyth, K. and Dirick, L., 1991; Koch C. et al., 1993). Remarkably, the phenotype of the snf1 null mutant was complemented by a glucose concentration higher than 2% (5%), suggesting that the role of Snf1 in the modulation of cell cycle progression could depend on the nutritional status of cells. Those data, published in Pessina S. et al., 2010, newly indicated that Snf1 was involved in the regulation of G1/S transition and pointed to a role for this kinase in the modulation of G1-specific gene expression. To gain further insight into the function of Snf1, we then analyzed the expression profile of G1-specific genes in cells synchronized in G1 phase by α-factor treatment and released into fresh medium. Our analyses showed that loss of Snf1 (snf1Δ strain) severely affected the expression of CLN2, PCL1 (SBF-dependent) and CLB5, RNR1 (SBF-dependent) genes, suggesting that Snf1 regulates the expression of both SBF- and MBF-dependent genes. Although protein Snf1 was not detectable at promoters of G1-specific genes, we investigated whether it could modulate the activity of SBF and MBF complexes and we found that in a snf1Δ strain the recruitment of Swi6 to G1-specific promoters was affected. Moreover, our Chromatin ImmunoPrecipitation (ChIP) assays also showed that in a snf1Δ strain the defective association of Swi6 to promoters led to a decreased recruitment of both the FACT complex, which is involved in the chromatin remodelling at G1-specific promoters, and of the RNA Pol II. Since it is known that the subcellular localization of Swi6 influences its interaction with promoters, then we analyzed its localization in G1 synchronized cells. In keeping with literature data (Sidorova J.M. et al., 1995; Taberner, F.J. and Igual, J.C., 2010), our analyses showed that in wild type cells synchronized in G1 phase by α-factor treatment Swi6 was essentially nuclear. Instead, in a snf1Δ strain Swi6 was localized in the nucleus only in the 60% of the G1-arrested cells, consistently with the reduced binding of Swi6 to G1-specific promoters. It is well known that the Swi6 interaction to DNA is mediated by the DNA binding-proteins Swi4 and Mbp1 (Andrews B.J. and Moore L.A., 1992; Moll T. et al., 1992). Therefore we extended our analyses to those proteins and we found that also the nuclear localization and the subsequent binding to DNA of Swi4 and Mbp1 were affected in a snf1Δ strain. Therefore, our data provide a representative snapshot of what occurs in vivo in a snf1 null mutant, supporting the notion that Snf1 promotes the expression of G1-specific genes modulating the nuclear localization of SBF and MBF components and thus promoting the formation of a complete Pre-initiation Complex (PIC) at G1-specific promoters. It is well known that phosphorylation of Snf1 at Thr210 leads to the full activation of the kinase (Hong S.P. et al., 2003; Sutherland C.M. et al., 2003). Then, in order to obtain insight into the Snf1 molecular mechanism in cell cycle regulation, we investigated its phosphorylation on Thr210 during cell cycle progression. Snf1 was slightly phosphorylated on the Thr210 residue during all the cell cycle, suggesting that this kinase was partially active. To determine whether the activation of Snf1 was involved in its function as regulator of G1 transcription, we analyzed the expression of SBF- and MBF-dependent genes in the SNF1-T210A mutant and we found that in this mutant the expression of those genes was reduced. In keeping with those data, the expression of G1-specific genes resulted affected also in a SNF1-K84R mutant, in which the ATP binding site has been destroyed causing a severe reduction of Snf1 kinase activity. On the base of those findings, we investigated whether Snf1 could exert its role in G1 phase through the phosphorylation of specific substrates and we found that Snf1 phosphorylates in vitro Swi6 on Ser760. Nevertheless, analyses of site-specific mutants (SWI6-S760A or SWI6-S760E) did not show any alteration of G1/S transition, suggesting that this phosphorylation was not involved in the role of Snf1 as regulator of cell cycle. The ChIP analyses of Swi6 binding to CLN2 and RNR1 promoters, then, showed that in the SNF1-K84R mutant the recruitment of Swi6 was slightly affected; nevertheless, that alteration was not severe as that of a snf1Δ strain. Consistently, neither the recruitment of FACT complex nor the binding of RNA Pol II to G1-specific promoters was affected in the SNF1-K84R mutant. Since this last finding seemed to disagree with the severe reduction of mRNA expression of SBF- and MBF-dependent genes observed in the SNF1-K84R mutant, we wondered whether defects in transcriptional elongation might occur in that strain. Thus, we analyzed the occupancy of FACT complex and of RNA Pol II at the internal regions of CLN2 and RNR1 genes by ChIP analyses; in the SNF1-K84R mutant the occupancy of both those complexes was decreased, suggesting that the kinase activity of Snf1 promotes the transcriptional elongation across G1-specific genes. In conclusion, the sum of data here presented indicates that protein kinase Snf1 is involved at different levels in the modulation of the G1-specific gene expression, thus highlighting a new function for Snf1 in the regulation of G1/S transition.
COCCETTI, PAOLA
Saccharomyces cerevisiae, cell cycle, Snf1
BIO/10 - BIOCHIMICA
English
BIOTECNOLOGIE INDUSTRIALI - 15R
25
2011/2012
(2013). Protein Kinase Snf1/AMPK: a new regulator of G1/S transition in Saccharomyces cerevisiae. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2013).
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