Characterization Of The Localization, Cellular Function And Regulation Of The Protein Kinase Kin2 In Budding Yeast

Yeast budding is a process of highly polarized growth. It relies on the asymmetric organization of the cytoskeleton and directed vesicular transport. Polarized growth in most eukaryotes serves as the foundation for the differentiation and the execution of specific functions. It is also key to the evolution of eukaryotes from single-celled organism to multicellular life forms. A number of proteins play roles in the regulation of polarized growth including the MARK/PAR-1 protein kinases in animals. These two kinases play crucial roles in the establishment of body axis during embryonic development in the nematode and fruitfly and the establishment of cell polarity in the epithelial cells and neurons in mammals. The budding yeast Saccharomyces cerevisiae contains two homologs of animal MARK/PAR-1 protein kinases—Kin1 and Kin2. The two yeast kinases share similar amino acid sequence and cellular functions. They all regulate vesicular transport and play an important role in the unfolded protein response in the endoplasmic reticulum. However, the subcellular localization of Kinl and Kin2 is not clear. A previous study showed that Kin2 localizes to some punctated spots located in the cytoplasm. This localization does not suit the role of Kin2 in the regulation of polarized growth. In addition, it is not known if Kin2 may play roles in other cellular processes and how its kinase activity is controlled. This study will investigate these questions.First, we examined the subcellular localization of Kin2 and its targeting domain. We expressed GFP-Kin2 fusion protein in wild-type cells under the control of KIN2's own promoter on single-copy plasmid. We found that the GFP fluorescence was too faint to be detected. We then switched to high-copy plasmids and detected GFP fluorescence in the cells. Kin2 localized to the sites that undergo polarized growth. In small-budded cells, Kin2 was enriched on the entire bud cortex. When the bud grows to a medium size, Kin2 was no longer enriched on the bud cortex. In large-budded cells, Kin2 localized to the mother-bud neck. This pattern of localization suits the role of Kin2 in the regulation of polarized growth. In addition, we observed that Kin2 also localized to the entire plasma membrane. We also examined the localization of YlKin1, a homolog of the dimorphic yeast Yarrowia lipolytica, a yeast species distantly related to S. cerevisiae. YlKinl also showed a localization identical to that of Kin2.Which domains in Kin2 mediate its localization? We identified two targeting domains in Kin2. One locates at the N-terminal region spanning the kinase domain. We named it TD1 (targeting domain 1). The other one locates at the C-terminus that covers the KA1 domain (Kinase-Associated domain 1). We named it TD2. TD1 or TD2 alone is sufficient to target to the polarity sites. Moreover, TD2 also localized to the plasma membrane. We found that TD1 is crucial for Kin2's function in vesicular transport as the N-terminal Kin2 segments that did not localize to the polarity sites failed to rescue the growth defect of sec1-1, sec2-41, and see15-1 mutants. This finding indicates that the localization to the sites of polarized growth is critical for Kin2's function in exocytosis. Since the TD2 domain negatively regulates Kin2's function in secretion and the removal of this domain enhanced Kin2's activity, it is likely that TD2-mediated plasma membrane localization could be involved in the regulation of other cellular processes such as cell wall biosynthesis (see the following section).Next, we examined the possible function of Kin2 in other cellular processes. We constructed a kin10? kin2? strain in our strain background (S288c derived). However, no any defect in growth or cell morphology was detected. We then overexpressed Kin2 in the cells. Kin2 overexpression did not impair growth. However, cell morphology became defective. The buds became elongated and the cells formed clusters. This phenotype was more pronounced in the gin4? strain, which is mildly defective in the organization of the septin cytoskeleton. Cells overexpressing Kin2 displayed defective septin organization and chitin deposition in the cell wall. The cells were sensitive to the cell wall-damaging agent calcofluor and the detergent SDS that might damage the plasma membrane. These findings suggest that Kin2 may be involved in septin organization and cell wall organization. TD1 and TD2 are both involved in these processes. Overexpression of Kin2-N segments could also impair growth. The cells became large and round, indicative of a partial loss of cell polarity. From a yeast two-hybrid screen, we identified the interaction of Kin2 with Tosl, Cdc11 (a septin subunit), and Pea2 (a polarisome component). Kin2-C also interacted with Pea2 in bimolecular fluorescence complementation (BiFC) assay. Tosl is a cell wall protein. We speculate that Kin2 may regulate cell wall synthesis via Tosl. Because the polarisome is involved in septin organization, we speculate that Kin2 may regulate septin organization via Pea2 and Cdc11.Last, we identified the interaction between a Kin2-C segment with Rho3, a Rho GTPase, by two-hybrid screen. The Kin2-C segment preferentially interacted with the GTP-bound form of Rho3. We also detected the interaction between Kin2 and Rho3 in GST pull-down and BiFC assays. BiFC showed that Kin2 interacted with Rho3 in vivo and the interaction occurred at the polarity sites. Rho3 is likely an upstream regulator of Kin2 and helps to relieve Kin2's intramolecular inhibition. We observed that a higher dose of Rho3Q74L enhanced the effect of Kin2 on the impairment of septin organization upon overexpression, implying that Rho3 may promote Kin2 function. However, we failed to detect the promotion of Kin2 function in the suppression of sec3-2, sec1-1, and sec2-41 mutants by high-copy RH03. The possibility remains to be tested by other methods. We also isolated the 14-3-3 protein, Bmh1, as a Kin2-interacting protein by two-hybrid screen. The interaction domain was mapped to the N-terminal region of Kin2. We speculate that Bmhl may play a role in the maintenance of the active state of Kin2.Kin2 homologs are widespread in fungi. Particularly, Kin2 homologs in the human pathogen Cryptoccocus neoformans, the rice blast fungus Magnaporthe oryzae, and the wheat blight fungus Fusarium graminearum have been identified as important virulence factors. Our study will shed light on the investigation of these Kin2 orthologs in pathogenesis.