Loss of PIP5KIγ, unlike other PIP5KI isoforms, impairs the integrity of the membrane cytoskeleton in murine megakaryocytes

Phosphatidylinositol-4,5-bisphosphate (PIP2) is an abundant phospholipid that contributes to second messenger formation and has also been shown to contribute to the regulation of cytoskeletal dynamics in all eukaryotic cells. Although the alpha, beta, and gamma isoforms of phosphatidylinositol-4-phosphate-5-kinase I (PIP5KI) all synthesize PIP2, mammalian cells usually contain more than one PIP5KI isoform. This raises the question of whether different isoforms of PIP5KI fulfill different functions. Given the speculated role of PIP2 in platelet and megakaryocyte actin dynamics, we analyzed murine megakaryocytes lacking individual PIP5KI isoforms. PIP5KI gamma(-/-) megakaryocytes exhibited plasma membrane blebbing accompanied by a decreased association of the membrane with the cytoskeleton. This membrane defect was rescued by adding back wildtype PIP5KI gamma, but not by adding a catalytically inactive mutant or a splice variant lacking the talin-binding motif. Notably, both PIP5KI beta- and PIP5KI gamma(-/-) cells had impaired PIP2 synthesis. However, PIP5KI beta-null cells lacked the membrane-cytoskeleton defect. Furthermore, overexpressing PIP5KI beta in PIP5KI gamma(-/-) cells failed to revert this defect. Megakaryocytes lacking the PIP5KI gamma-binding partner, talin1, mimicked the membrane-cytoskeleton defect phenotype seen in PIP5KI gamma(-/-) cells. These findings demonstrate a unique role for PIPS KI gamma in the anchoring of the cell membrane to the cytoskeleton in megakaryocytes, probably through a pathway involving talin. These observations further demonstrate that individual PIP5KI isoforms fulfill distinct functions within cells.