All these results led us to the conclusion that Sch9 regulated localization of Bcy1 via Zds1. Bcy1 modification was found to be dependent on Yak1 (Griffioen et al., 2001). It was reported that faster-migrating iso-form of Bcy1-HA was detected in exponential phase wild-type cells, whereas a predominant slower-migrating iso-form of Bcy1-HA was

detected in stationary phase wild-type cells. But a predominant faster-migrating iso-form of Bcy1-HA was detected SCH772984 in yak1Δ mutant in either exponential phase or stationary phase. Recently, we reported that the faster-migrating iso-form of Bcy1-HA was detected in sch9Δ mutant cells, either in exponential phase or in stationary phase (Zhang et al., 2011). To investigate whether Sch9 regulated Bcy1 phosphorylation via Yak1, we tested whether Sch9 affected the protein level of Yak1. Figure 7a, shows that the protein level of Yak1 in log-phase glucose-grown sch9Δ cells (Line 2) was markedly lower than in log phase glucose-grown W303-1A (Line 1). The protein level of Yak1 in stationary phase W303-1A (Line 3) was dramatically lower than in the log-phase W303-1A (Line 1), whereas a predominant slower-migrating iso-form AZD6244 of Yak1-HA was detected in stationary phase sch9Δ (Line 4). As shown in Fig. 7b, the protein level of Yak1 in glycerol-grown sch9Δ cells was markedly lower than in glycerol-grown W303-1A (Line 1). Phosphatase treatment of this

slower-migrating iso-form of Yak1-HA resulted in the fast-migrating iso-form of Yak1-HA (Fig. 7b). These results suggest that Sch9 is involved in the regulation of Yak1 phosphorylation. In multicellular organisms, AKAPs targeted PKA holoenzyme to specific subcellular locations (Griffioen & Thevelein, 2002). AKAP-mediated targeting Tobramycin of PKA was thought to confer spatio-temporal control of PKA signaling to phosphorylate specific localized substrates. Compartmentalization of signal transduction pathways is an important spatio-temporal control of PKA signaling. In this way, signaling molecules of the same pathway are

brought into close vicinity, thereby increasing the probability that they only affect each other appropriately. Recently, we reported that Bcy1 was predominately localized in nucleus in sch9Δ cells, whereas a large part of catalytic subunits of PKA transferred from nucleus into cytoplasm in sch9Δ cells (Zhang et al., 2011). Thus the liberated catalytic subunits were not restricted by the regulatory subunits and consequently able to phosphorylate preferentially substrates located nearby (e.g. fructose-1,6-bisphosphatase, trehalase), all leading to a high PKA activity phenotype of sch9Δ cells. Our research indicated that Sch9 regulated localization of Bcy1 via Zds1. In yeast, Zds1 may act as the anchor protein of PKA. We report for the first time that Sch9 and Zds1 interact physically. However, the mechanisms of Sch9 regulating Zds1 still need to be clarified.