LAMMER Kinase Governs the Expression and Cellular Localization of Gas2, a Key Regulator of Flocculation in Schizosaccharomyces pombe.

It was reported that LAMMER kinase in Schizosaccharomyces pombe plays an important role in cation-dependent and galactose-specific flocculation. Analogous to other flocculating yeasts, when cell wall extracts of the Δlkh1 strain were treated to the wild-type strain, it displayed flocculation. Gas2, a 1,3-ß-glucanosyl transferase, was isolated from the EDTA-extracted cell-surface proteins in the Δlkh1 strain. While disruption of the gas2+ gene was not lethal and reduced the flocculation activity of the ∆lkh1 strain, the expression of a secreted form of Gas2, in which the GPI anchor addition sequences had been removed, conferred the ability to flocculate upon the WT strain. The Gas2-mediated flocculation was strongly inhibited by galactose but not by glucose. Immunostaining analysis showed that the cell surface localization of Gas2 was crucial for the flocculation of fission yeast. In addition, we identified the regulation of mbx2+ expression by Lkh1 using RT-qPCR. Taken together, we found that Lkh1 induces asexual flocculation by regulating not only the localization of Gas2 but also the transcription of gas2+ through Mbx2.

LAMMER Kinase Modulates Cell Cycle by Phosphorylating the MBF Repressor, Yox1, in Schizosaccharomyces pombe.

Lkh1, a LAMMER kinase homolog in the fission yeast Schizosaccharomyces pombe, acts as a negative regulator of filamentous growth and flocculation. It is also involved in the response to oxidative stress. The lkh1-deletion mutant displays slower cell growth, shorter cell size, and abnormal DNA content compared to the wild type. These phenotypes suggest that Lkh1 controls cell size and cell cycle progression. When we performed microarray analysis using the lkh1-deletion mutant, we found that only four of the up-regulated genes in the lkh1-deletion were associated with the cell cycle. Interestingly, all of these genes are regulated by the Mlu1 cell cycle box binding factor (MBF), which is a transcription complex responsible for regulating the expression of cell cycle genes during the G1/S phase. Transcription analyses of the MBF-dependent cell-cycle genes, including negative feedback regulators, confirmed the up-regulation of these genes by the deletion of lkh1. Pull-down assay confirmed the interaction between Lkh1 and Yox1, which is a negative feedback regulator of MBF. This result supports the involvement of LAMMER kinase in cell cycle regulation by modulating MBF activity. In vitro kinase assay and NetPhosK 2.0 analysis with the Yox1T40,41A mutant allele revealed that T40 and T41 residues are the phosphorylation sites mediated by Lkh1. These sites affect the G1/S cell cycle progression of fission yeast by modulating the activity of the MBF complex.

LAMMER Kinase Lkh1 Is an Upstream Regulator of Prk1-Mediated Non-Sexual Flocculation in Fission Yeast.

The cation-dependent galactose-specific flocculation activity of the Schizosaccharomyces pombe null mutant of lkh1 +, the gene encoding LAMMER kinase homolog, has previously been reported by our group. Here, we show that disruption of prk1 +, another flocculation associated regulatory kinase encoding gene, also resulted in cation-dependent galactose-specific flocculation. Deletion of prk1 increased the flocculation phenotype of the lkh1 + null mutant and its overexpression reversed the flocculation of cells caused by lkh1 deletion. Transcript levels of prk1 + were also decreased by lkh1 + deletion. Cumulatively, these results indicate that Lkh1 is one of the negative regulators acting upstream of Prk1, regulating non-sexual flocculation in fission yeast.

Possible Roles of LAMMER Kinase Lkh1 in Fission Yeast by Comparative Proteome Analysis.

To investigate the possible roles of LAMMER kinase homologue, Lkh1, in Schizosaccharomyces pombe, whole proteins were extracted from wild type and lkh1-deletion mutant cells and subjected to polyacrylamide gel electrophoresis. Differentially expressed proteins were identified by tandem mass spectrometry (MS/MS) and were compared with a protein database. In whole-cell extracts, 10 proteins were up-regulated and 9 proteins were down-regulated in the mutant. In extracellular preparations, 6 proteins were up-regulated in the lkh1 (+) null mutant and 4 proteins successfully identified: glycolipid anchored surface precursor, ß-glucosidase (Psu1), cell surface protein, glucan 1,3-ß-glucosidase (Bgl2), and exo-1,3 ß-glucanase (Exg1). These results suggest that Lkh1 is involved in regulating cell wall assembly.