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...