Molecular Analysis Reveals Localization ofSaccharomyces cerevisiaeProtein Kinase C to Sites of Polarized Growth and Pkc1p Targeting to the Nucleus and Mitotic Spindle

Abstract

The catalytic activity and intracellular localization of protein kinase C (PKC) are both highly regulated in vivo. This family of kinases contains conserved regulatory motifs, i.e., the C1, C2, and HR1 domains, which target PKC isoforms to specific subcellular compartments and restrict their activity spatially. Saccharomyces cerevisiae contains a single PKC isozyme, Pkc1p, which contains all of the regulatory motifs found in mammalian PKCs. Pkc1p localizes to sites of polarized growth, consistent with its main function in maintaining cell integrity. We dissected the molecular basis of Pkc1p localization by expressing each of its domains individually and in combinations as green fluorescent protein fusions. We find that the Rho1p-binding domains, HR1 and C1, are responsible for targeting Pkc1p to the bud tip and cell periphery, respectively. We demonstrate that Pkc1p activity is required for its normal localization to the bud neck, which also depends on the integrity of the septin ring. In addition, we show for the first time that yeast protein kinase C can accumulate in the nucleus, and we identify a nuclear exit signal as well as nuclear localization signals within the Pkc1p sequence. Thus, we propose that Pkc1p shuttles in and out of the nucleus and consequently has access to nuclear substrates. Surprisingly, we find that deletion of the HR1 domain results in Pkc1p localization to the mitotic spindle and that the C2 domain is responsible for this targeting. This novel nuclear and spindle localization of Pkc1p may provide a molecular explanation for previous observations that suggest a role for Pkc1p in regulating microtubule function.

Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

“…Mutants in the MAPK cascade do not share these pkc1 phenotypes. Finally, the demonstration that the N-terminal C2-like domain of Pkc1 localizes to the mitotic spindle (Denis and Cyert 2005) supports the notion that this kinase has a target at the mitotic apparatus.…”

“…Pkc1 becomes delocalized during G2 phase and finally relocalized at the mother-bud neck during mitosis, a transition that requires an intact septin ring (Denis and Cyert 2005). A molecular dissection of Pkc1 suggested that each regulatory subdomain was responsible for localizing a pool of Pkc1 to specific subcellular sites (Denis and Cyert 2005), knowledge that may contribute to our understanding of its functions beyond activation of the MAPK cascade.…”

Cell Wall Integrity Signaling inSaccharomyces cerevisiae

“…Later in the cell cycle, it becomes delocalized and finally relocalized at the mother-bud neck. The neck localization of Pkc1 requires an intact septin ring (64). A recent molecular dissection of Pkc1 suggested that each domain was responsible for localizing a pool of Pkc1 to various subcellular sites (64).…”

Microcompartments within the yeast plasma membrane

“…These include the GEF Rom2 and its target GTPase Rho1 (Figure 2) (see Yoshida et al , 2009 and references therein). The latter organizes the actin cytoskeleton via its interaction with formins and also functions as an activator of the central CWI component Pkc1, which also localizes to the bud neck during cytokinesis (Andrews and Stark , 2000 ;Denis and Cyert , 2005 Actomyosin ring…”