Abstract
Following thrombin stimulation, platelet PIP5KI synthesizes phosphatidylinositol 4,5-bisphosphate (PIP2), which can be hydrolyzed by phospholipase C to generate second messengers such as IP3. PIP2 also regulates cytoskeletal dynamics by directly interacting with actin-binding proteins. Three isoforms of PIP5KI (α, β, and γ) are all capable of phosphorylating PI4P to synthesize PIP2. However, these isoforms have different primary structures, expression levels in various tissues, and intracellular localization. We have generated and characterized murine lines lacking PIP5KIβ or PIP5KIγ, which are the two predominant platelet isoforms. We also phenotyped platelets lacking PIP5KIα, which is the least abundant isoform. PIP5KIβ-null mice appeared developmentally normal and had normal platelet counts, however they had small defects in aggregation following exposure to all agonists. In contrast, platelets lacking PIP5KIα aggregated normally. Although platelets lacking PIP5KIβ have only a moderate deficiency of PIP2 under basal conditions, they have a striking deficiency in PIP2 synthesis and IP3 formation following thrombin stimulation. We have also observed that platelets lacking both PIP5KIα and PIP5KIβ have a complete loss of thrombin-induced IP3 synthesis, even though they still contain PIP5KIγ, which is the predominant PIP5KI isoform in platelets. Additionally, we found when using a carotid injury model that PIP5KIβ-null platelets failed to properly form arterial thrombi in vivo. This demonstrates that PIP5KIβ, like PIP5KIα, contributes to the rapid synthesis of a pool of PIP2 that is required for second messenger formation and in vivo platelet adhesion. This contrasts the PIP5KIγ-synthesized pool of PIP2 that does not contribute to these processes. We have found that loss of PIP5KIγ null mutation impairs cardiac development and leads to embryonic lethality. PIP5KIγ null megakaryocytes derived from yolk sac progenitor cells have a defect in anchoring their cell membranes to the underlying actin cytoskeleton. To understand the role of this PIP5KI isoform in platelet biology, we conditionally rescued the PIP5KIγ null mutation within myocardiocytes allowing us to obtain living mice. Platelets from these animals lacked PIP5KIγ, yet aggregated normally when exposed to all agonists. To analyze these cells for a failure to anchor their cell membranes, we used laser tweezers to pull the cell membrane apart from the cytoskeleton. Wild type cells had rigid membranes that resisted stretching by trapped fibrinogen-coated beads that were pulled by laser tweezers. In contrast, the PIP5KIγ-null platelets had flexible membranes that were easily stretched, and ultimately allowed membrane tethers to form. Together, these results demonstrate that following stimulation of a G-protein coupled receptor, IP3 is completely derived from a rapidly synthesized discrete pool of PIP2 that is synthesized by PIP5KIα and PIP5KIβ. In contrast, the pool of PIP2 synthesized by PIP5KIγ contributes to preserving the integrity of the membrane cytoskeleton. In conclusion, this work demonstrates that spatially restricted PIP2 synthesis by individual PIP5KI isoforms differentially controls second messenger formation and the integrity of the actin cytoskeleton.
Isoform-specific complementation of the yeast sac6 null mutation by human fimbrin.