Nucleocytoplasmic Distribution of Budding Yeast Protein Kinase A Regulatory Subunit Bcy1 Requires Zds1 and Is Regulated by Yak1-Dependent Phosphorylation of Its Targeting Domain

Abstract

In Saccharomyces cerevisiae the subcellular distribution of Bcy1 is carbon source dependent. In glucose-grown cells, Bcy1 is almost exclusively nuclear, while it appears more evenly distributed between nucleus and cytoplasm in carbon source-derepressed cells. Here we show that phosphorylation of its N-terminal domain directs Bcy1 to the cytoplasm. Biochemical fractionation revealed that the cytoplasmic fraction contains mostly phosphorylated Bcy1, whereas unmodified Bcy1 is predominantly present in the nuclear fraction. Site-directed mutagenesis of two clusters (I and II) of serines near the N terminus to alanine resulted in an enhanced nuclear accumulation of Bcy1 in ethanol-grown cells. In contrast, substitutions to Asp led to a dramatic increase of cytoplasmic localization in glucose-grown cells. Bcy1 modification was found to be dependent on Yak1 kinase and, consequently, in ethanol-grown yak1 cells the Bcy1 remained nuclear. A two-hybrid screen aimed to isolate genes encoding proteins that interact with the Bcy1 N-terminal domain identified Zds1. In ethanol-grown zds1 cells, cytoplasmic localization of Bcy1 was largely absent, while overexpression of ZDS1 led to increased cytoplasmic Bcy1 localization. Zds1 does not regulate Bcy1 modification since this was found to be unaffected in zds1 cells. However, in zds1 cells cluster II-mediated, but not cluster I-mediated, cytoplasmic localization of Bcy1 was found to be absent. Altogether, these results suggest that Zds1-mediated cytoplasmic localization of Bcy1 is regulated by carbon source-dependent phosphorylation of cluster II serines, while cluster I acts in a Zds1-independent manner.Throughout the eukaryotic kingdom cyclic AMP (cAMP)-dependent protein kinases (PKAs) play important and diverse roles in signal transduction (for reviews, see references 2, 7, and 28 and references therein). Structurally, PKAs are conserved, consisting of two catalytic subunits that bind, in their inactive configuration, to a regulatory subunit homodimer. Binding of cAMP to the regulatory subunit results in dissociation, and thereby activation, of the catalytic subunits (7, 28). The multitude of intracellular PKA substrates and their different subcellular distribution raises important questions about the specificity, timing, and substrate targeting of PKA-mediated signaling. One regulatory level to ensure proper signal transduction is specific targeting of signaling components to subcellular compartments. In multicellular eukaryotes A-kinase anchor proteins (AKAPs) have been identified that target type I or type II (RI or RII) PKA-regulatory subunits to their effector substrates localized in various subcellular compartments (for recent reviews, see references 5 and 6 and references therein). AKAPs possess a site for constitutive avid binding of RI or RII and a targeting domain that complexes with subcellular structures. Directing PKA to specific microenvironments facilitates phosphorylation of colocalized effector molecules.In contrast to cells from multicellular organisms, yeast...

Systematic identification of signal integration by protein kinase A

“…This targeting implies that the AKAP-PKA complexes in mammals can integrate locally and modulate distinct intracellular signaling pathways (9,17,18). No ortholog has been identified for AKAPs in Saccharomyces cerevisiae, but PKA localization appears to be regulated in a condition-dependent manner (19,20), at least partly through the phosphorylation of its N terminus (20).…”

The Yak1 Protein Kinase Lies at the Center of a Regulatory Cascade Affecting Adhesive Growth and Stress Resistance inSaccharomyces cerevisiae

“…Several targets of Yak1 have been identified. In response to glucose starvation, the regulatory PKA subunit Bcy1 is phosphorylated and restricted to the cytoplasm in an Yak1-dependent manner (Griffioen et al 2001), but the physiological role of this regulation is not clear. Glucose starvation further stimulates Yak1-mediated phosphorylation of Pop2/Caf1, which forms part of the Ccr4-Caf1-Not deadenylation complex that controls expression of numerous genes involved in stress response and carbohydrate metabolism (Moriya et al 2001).…”

zds1, a Novel Gene Encoding an Ortholog of Zds1 and Zds2, Controls Sexual Differentiation, Cell Wall Integrity and Cell Morphology in Fission Yeast

“…Intriguingly, Zds1 localizes to the bud neck, bud tip, and cytoplasm while Zds2p localizes to the nucleus of S. cerevisiae (Bi and Pringle 1996). Since the Zds1p and Zds2p proteins display some common roles, such as the suppression of various mutants (Bi and Pringle 1996;Heo et al 1999;Schwer and Shuman 1996;Griffioen et al 2001;Bandhakavi et al 2003) but also have distinct roles, such as in silencing (Roy and Runge 2000), it is plausible that the different localization patterns of S. cerevisiae Zds1p and Zds2p may reflect their different functions. Thus, the disparate localizations of the truncated S. pombe Zds1 proteins may reflect various functions of the protein that we do not yet understand.…”

“…ZDS2 was isolated as a multicopy suppressor of sin4, a gene that confers resistance to the anticancer agent cisplatin (Burger et al 2000). ZDS1 and ZDS2 were also isolated as (i) multicopy suppressors of temperature-sensitive mutations of a yeast mRNA-capping enzyme (CEG1) (Schwer and Shuman 1996), (ii) negative regulators of Cdc42p (Bi and Pringle 1996), (iii) multicopy suppressors of a cka2 mutant (cka2 encodes the a9-subunit of casein kinase II) (Bandhakavi et al 2003), (iv) suppressors of the camptothecin-hypersensitive trf4 mutant (Walowsky et al 1999), (v) genes that stabilize short linear centromeric plasmids (Roy and Runge 1999), (vi) multicopy suppressors of an rhc21 mutant (rhc21 encodes a component of the cohesin complex) (Heo et al 1999), (vii) genes that interact with the Bcy1p N-terminal domain (Griffioen et al 2001), and (viii) multicopy suppressors of a 1, 3-b-glucan synthase mutant (Sekiya-Kawasaki et al 2002). In addition, Zds1p and Zds2p were reported to be involved in transcriptional silencing and longevity (Roy and Runge 2000).…”