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  • Two nutrient sensing pathways the

    2019-11-06

    Two nutrient-sensing pathways, the cAMP-protein kinase A (PKA) pathway and the target of rapamycin complex 1 (TORC1) pathway, are implicated in Msn2/4 regulation. Under non-stress conditions, Msn2 localizes in the cytoplasm and is inactivated by both PKA-dependent phosphorylation of the nuclear localization signal (NLS) and association with Bmh2, a yeast 1-3-3 protein, in a TORC1-dependent manner [9], [10]. When Protease Inhibitor Cocktail (EDTA-Free, 100X in DMSO) face acute glucose starvation, both PKA and TORC1 are inactivated and Msn2 rapidly enters the nucleus through dephosphorylation by the protein phosphatase 1 (PP1) and release from Bmh2, leading to transcriptional activation of many STRE-controlled genes [10], [11]. In addition, both Yak1 and Rim15, whose activity is restrained by PKA and TORC1, are also involved in the activation of Msn2 by direct phosphorylation [12], [13]. Apart from nutrient-sensing pathways, the mitogen-activated protein kinase (MAPK) Hog1 and protein phosphatase 2A (PP2A) associated with Cdc55, a regulatory B subunit, regulate the Msn2/4-dependent transcriptional activation through modulating chromatin association and nuclear accumulation of Msn2/4 in response to osmotic stress [14], [15], [16]. Msn2/4 and Hsf1 are two primary stress-responsive transcription factors cooperatively regulating gene expression under multiple stress conditions. These transcription factors share some common regulatory mechanisms such as activation by direct phosphorylation by Yak1 and Rim15 [12], [13]. In addition, we recently reported that Hsf1 activity is negatively regulated by CK2-dependent phosphorylation for ethanol stress-specific activation [17], raising the possibility that Msn2/4 might also be regulated by CK2. CK2 is a highly conserved and constitutively active Ser/Thr protein kinase. CK2 is a tetrameric enzyme, composed of two catalytic subunits (encoded by CKA1 and CKA2) and two regulatory subunits (encoded by CKB1 and CKB2) in S. cerevisiae[18]. CK2 plays a role in a large number of fundamental cellular processes for cell growth and viability [18], [19], [20]. In addition, several reports have recently demonstrated that CK2 is linked to stress-related responses including autophagy, osmotic stress, ethanol stress, and DNA damage [17], [21], [22], [23], [24]. Interestingly, protein stability and phosphorylation status of CK2 regulatory subunits, considered a modulator of substrate selectivity, were changed under stress condition [25], [26], implying that CK2 activity toward the substrate might also be regulated in response to stress conditions. Nevertheless, little is known about the function of CK2 engaged in stress-related responses, in contrast to numerous findings on the growth-related roles for CK2.
    Materials and methods
    Results
    Discussion Msn2 and Msn4 mediate transcriptional activation of numerous genes in response to multiple different environmental signals as a central regulator [31]. In this study, we presented compelling evidence that CK2 positively regulates transcriptional activity of Msn2 in response to multiple environmental stress conditions including glucose starvation, H2O2, and lactic acid stress. Msn2 is known to be secluded in the cytoplasm by physical interaction with Bmh2, a 14-3-3 protein [10]. The 14-3-3 family proteins are highly conserved dimeric proteins participating in a wide range of cellular processes through binding to hundreds of proteins containing the phosphorylated 14-3-3 binding motif [32]. 14-3-3 proteins are also phosphorylated at multiple residues, inhibiting the interaction with their partner proteins [32], [33]. Particularly, it has been purposed that phosphorylation of 14-3-3 protein by CK2 may disrupt interaction with their partner proteins in mammals [34], raising the possibility that CK2 might repress the Bmh2-mediated cytoplasmic sequestration of Msn2 through Bmh2 phosphorylation. In addition, nuclear export of Msn2 is mediated by Msn5, a yeast karyopherin [9]. Disruption of MSN5 leads to permanent nuclear accumulation of Msn2, even under normal condition, but could not elicit induction of its target genes [8], implying that another regulatory steps, such as phosphorylation, are require for Msn2 activation apart from its nuclear accumulation. Taken together, these results support the notion that CK2 synergistically regulates transcriptional activation of Msn2-dependent genes through phosphorylation of not only Msn2 but also other factors regulating accumulation of Msn2 in the nucleus. In fact, disruption of a catalytic subunit CKA2, even with a partial CK2 activity by intact CKA1, more successfully reduced transcriptional activity of Msn2/4, compared with the mutation of the CK2-dependent phosphorylation sites in Msn2 (Fig. 3, Fig. 5). In addition, CK2 might affect transcription of Msn2 targets by regulating other players. For example, it has been shown that CK2 regulates transcriptional elongation and gene expression of a number of heat-shock induced genes through tyrosine phosphorylation of histone H2A [35].