Feedback Inhibition on Cell Wall Integrity Signaling by Zds1 Involves Gsk3 Phosphorylation of a cAMP-dependent Protein Kinase Regulatory Subunit

Abstract

We report here that budding yeast cAMP-dependent protein kinase (cAPK) is controlled by heat stress. A rise in temperature from 30 to 37°C was found to result in both a higher expression and an increased cytoplasmic localization of its regulatory subunit Bcy1. Both of these effects required phosphorylation of serines located in its localization domain. Surprisingly, classic cAPK-controlled processes were found to be independent of Bcy1 phosphorylation, indicating that these modifications do not affect cAPK activity as such. Alternatively, phosphorylation may recruit cAPK to, and thereby control, a specific subset of (perhaps novel) cAPK targets that are presumably localized extranuclearly. Zds1 and Zds2 may play a role in this process, since these were found required to retain hyperphosphorylated Bcy1 in the cytoplasm at 37°C. Mck1, a homologue of mammalian glycogen synthase kinase 3 and a downstream component of the heat-activated Pkc1-Slt2/Mpk1 cell wall integrity pathway, is partly responsible for hyperphosphorylations of Bcy1. Remarkably, Zds1 appears to act as a negative regulator of cell wall integrity signaling, and this activity is dependent in part on the phosphorylation status of Bcy1. Thus, Mck1 phosphorylation of Bcy1 and Zds1 may constitute an unprecedented negative feedback control on the cell wall integrity-signaling pathway.In eukaryotes from yeast to humans, cAMP-dependent protein kinases (cAPKs) 1 are ubiquitous signaling proteins. A structural characteristic of cAPKs is that the regulatory and catalytic activities are not covalently linked but are represented by two different subunits. The inactive cAPK holoenzyme consists of a regulatory (R) subunit homodimer with two catalytic subunits associated to it. Binding of the second messenger cAMP to the R-dimer leads to dissociation of the holoenzyme, and the released catalytic subunits are then able to phosphorylate protein substrates at serine or threonine residues comprising a defined consensus sequence.cAPK recognition sequences, however, are of low complexity and therefore insufficient to poise proteins as specific cAPK substrates. Moreover, in a single cell, cAPK can participate in several parallel pathways that control the phosphorylation status of specific substrates in response to different triggers. Compartmentalization of signaling molecules provides an important level of control to achieve additional signaling specificity. In multicellular organisms, protein kinase A anchor proteins (AKAPs) have been identified that target cAPK holoenzymes to specific subcellular locations (for a recent review, see Ref. 1 and references therein). These AKAPs function as adapters; one domain associates with an AKAP-binding surface created by dimerization of the R-subunit, whereas another distinct domain interacts with a cellular structure or organelle. AKAPmediated targeting of cAPK is thought to confer spatio-temporal control of cAPK signaling in order to phosphorylate substrates specifically.In the unicellular eukaryote, Saccharomyces cerevisiae cAPK i...

Pkc1 Acts Through Zds1 and Gic1 to Suppress Growth and Cell Polarity Defects of a Yeast eIF5A Mutant

“…Thus, Zds proteins may act as an integration point for distinct signaling pathways that helps maintain a balance among different signals. This model could explain why Zds1 downregulates both Pkc1-MAP kinase and Cdc42 activity (Bi and Pringle 1996;Griffioen et al 2003). In fact, high-copy ZDS1 does not suppress the temperature-sensitive phenotype of the PKC1 mutant stt1 (data not shown), as high-copy GIC1 does (discussed below).…”

“…In fact, high-copy ZDS1 does not suppress the temperature-sensitive phenotype of the PKC1 mutant stt1 (data not shown), as high-copy GIC1 does (discussed below). However, although overexpression of ZDS1 causes a cell integrity defect at elevated temperatures (Griffioen et al 2003), high-copy PKC1 is not toxic to zds1D zds2D cells at any temperature, and it does not exacerbate even the cold sensitivity of this double mutant (data not shown). Therefore, the inability of PKC1 to act as a suppressor in the strain tif51A-1 zds1D zds2D is not likely the result of Pkc1 overactivation and toxicity.…”

Saccharomyces cerevisiae Heat Shock Transcription Factor Regulates Cell Wall Remodeling in Response to Heat Shock

“…The ZDS1 gene, the most frequently isolated gene in this screen, and its paralog ZDS2 have been identified in numerous other screens designed to isolate genes that act as negative regulators of CDC42 (3), positive effectors of replication origin function (54), or stabilizers of linear centromeric plasmids (43). Other studies show that Zds1 has properties reminiscent of the PKA anchoring proteins (13). However, the exact functions of Zds1 and Zds2 have not been established.…”

LAS24/KOG1, a component of the TOR complex 1 (TORC1), is needed for resistance to local anesthetic tetracaine and normal distribution of actin cytoskeleton in yeast

“…For this reason, the relationship between Zds1p and Las24p function is hard to predict. However, Griffioen et al (2003) showed that Zds1p controlled the RAS/cAMP pathway via effects on Bcy1p localization and cell wall integrity. Moreover, zds1 mutant cells are more sensitive to rapamycin than wild-type cells (Xie et al, 2005).…”