Molecular analysis reveals localization of Saccharomyces cerevisiae protein kinase C to sites of polarized growth and Pkc1p targeting to the nucleus and mitotic spindle.
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.
Denis
Valérie
V
Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA.
Cyert
Martha S
MS
D020816:Amino Acid Motifs; D002384:Catalysis; D002467:Cell Nucleus / Q000378:metabolism*; D049109:Cell Proliferation; D005656:Fungal Proteins / Q000737:chemistry; D017353:Gene Deletion; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000648:ultrastructure; D019913:Nuclear Localization Signals; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000737:chemistry / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000502:physiology*; D008941:Spindle Apparatus / Q000378:metabolism*; D013696:Temperature
D005656:Fungal Proteins; D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; C490702:PKC1 protein, S cerevisiae; D011493:Protein Kinase C