Phosphatidylinositol-4-phosphate 5-Kinase Regulates Fission Yeast Cell Integrity through a Phospholipase C-mediated Protein Kinase C-independent Pathway

Abstract

Fission yeast its3-1 mutant is an allele of the essential gene its3؉ that encodes a phosphatidylinositol-4-phosphate 5-kinase (PIP5K) that produces phosphatidylinositol 4,5-bisphosphate. We found that the its3-1 mutant is sensitive to micafungin, a (1,3)-␤-D-glucan synthase inhibitor, suggesting a cell wall integrity defect. Consistently, its3-1 mutation caused synthetic lethality with a (1,3)-␤-Dglucan synthase mutant, bgs1-i2, and its3-1 mutant cells showed aberrant localization of green fluorescent protein-Bgs1. Similar aberrant localization of green fluorescent protein-tagged Rgf1, a putative phosphatidylinositol 4,5-bisphosphate-binding guanine nucleotide exchange factor for Rho protein, in its3-1 mutants was observed, suggesting a defective Rgf1/Rho pathway. To unravel the molecular mechanism(s), putative downstream components of PIP5K signaling were analyzed. Unexpectedly, overexpression of phospholipase C (Plc1), but not that of protein kinase C (PKC; Pck1 and Pck2), suppressed the phenotypes of the its3-1 mutant. These findings indicate that PKCs are not involved in the suppression, and further analysis revealed that PKCs are not downstream of Plc1 in fission yeast. Also, the enzymatic activity of Plc1 is essential for the suppression of the phenotypes and for the viability of the its3-1 mutant. These findings suggest that Its3 PIP5K regulates cell integrity through a Plc1-mediated PKC-independent pathway, in addition to the Rho/PKC pathway.

Eisosomes Regulate Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Cortical Clusters and Mitogen-activated Protein (MAP) Kinase Signaling upon Osmotic Stress

“…3B). Thus, Pmk1 activity correlates with cellular PI(4,5)P 2 levels, consistent with previously demonstrated role for PI(4,5)P 2 in activating this pathway in budding yeast (22,23,37).…”

“…We found that overexpression of the PI 5-kinase increased MAPK activation, whereas mutations in the PI 5-kinase inhibited MAPK activation. These and other data indicate that PI(4,5)P 2 participates in activation of the MAPK cell integrity pathway in both budding yeast and fission yeast (22,23,37). The recent discovery that eisosomes act with synaptojanin-related lipid phosphatases to decrease PI(4,5)P 2 levels in both budding yeast and fission yeast thus raised the possibility that eisosomes inhibit cell integrity signaling through lipids.…”

“…Importantly, MAPK activation under osmotic stress is completely dependent on the activity of both Rho2 and Pck2 (20). Rgf1, a Rho GEF, is also required for Pmk1 activation upon osmotic stress (21), and its membrane localization is altered in cells expressing the hypomorphic allele of PI 5-kinase its3-1 (22). This raises the possibility that PI(4,5)P 2 might regulate the fission yeast cell integrity activity, but the connection has remained unclear.…”

Valproic Acid Affects Membrane Trafficking and Cell-Wall Integrity in Fission Yeast

“…Intriguingly, all the membrane trafficking mutants that we have thus far identified displayed defects in cellwall integrity, including the hypersensitivity to micafungin, an inhibitor of 1,3-b-d-glucan synthase (Carver 2004;Deng et al 2005). We then examined the effect of micafungin on the Dvps45 cells as compared with other membrane trafficking mutants.…”

Identification and Characterization of Novel Immunomodulatory Bursal-derived Pentapeptide-II (BPP-II)

“…Key steps during T cell activation are the activation of Ras and Rho family GTPase, which are also important targets for the products of PI3K. Pip5k1c regulates the adhesion through facilitating RhoA GTPase and integrin activation by a Rho guanine nucleotide exchange factor 12 (Arhgef12) (24,25), which was up-regulated after BPP-II treatment (supplemental Table S4). Furthermore, Pip5k1c(r) contributes to the critical second messenger inositol trisphosphate and diacylglycerol (26).…”