Loss of PIP5KIbeta demonstrates that PIP5KI isoform-specific PIP2 synthesis is required for IP3 formation

Abstract

The three isoforms of PIP5KI (alpha, beta, and gamma) synthesize PI4,5P(2) (PIP(2)) by phosphorylating PI4P. Therefore, it is not clear why platelets, like all eukaryotic cells, have more than one isoform. To test the hypothesis that PIP5KI isoforms have nonoverlapping functions, we generated a murine line containing a null mutation of PIP5KIbeta and analyzed the effect on platelet signaling. PIP5KIbeta-null mice had normal platelet counts. In contrast to platelets lacking PIP5KIalpha, platelets lacking PIP5KIbeta exhibited impaired aggregation accompanied by disaggregation. Although platelets lacking PIP5KIbeta had only a moderate deficiency of PIP(2) under basal conditions, they had a striking deficiency in PIP(2) synthesis and IP(3) formation after thrombin stimulation. We have also observed that platelets lacking both PIP5KIalpha and PIP5KIbeta have a complete loss of thrombin-induced IP(3) synthesis even though they still contain PIP5KIgamma, the predominant PIP5KI isoform in platelets. These results demonstrate that PIP5KIbeta, like PIP5KIalpha, contributes to the rapid synthesis of a pool of PIP(2) that is required for second-messenger formation, whereas the pool of PIP(2) synthesized by PIP5KIgamma does not contribute to this process. Additionally, we found that PIP5KIbeta-null platelets failed to form arterial thrombi properly in vivo. Together, these data demonstrate that PIP5KIbeta is required for rapid PIP(2) synthesis, second-messenger production, and stable platelet adhesion under shear in vivo. These results also demonstrate that after stimulation of a G protein-coupled receptor, IP(3) is completely derived from a rapidly synthesized discrete pool of PIP(2) synthesized by PIP5KIalpha and PIP5KIbeta.

Fig. 1.

Targeting of PIP5KIβ produces complete null muation. (A) Genotypes of offspring derived from matings of PIP5KIβ^+/− mice. Compared with the number of wild-type embryos generated, the PIP5KIβ^−/− mice were born at slightly less than the expected frequency. (B) Micrographs demonstrating the normal morphology of organs lacking PIP5KIβ. H&E stain was used on all organs. (Magnification: ovary, ×100; testis, ×200; heart, ×200.) (C) RT-PCR using a sense primer from exon 1 and an antisense primer from exon 3. The absence of the PCR product demonstrates the loss of PIP5KIβ message beyond the gene trap. In contrast, using primers specific for β-actin demonstrated that targeting of PIP5KIβ had no effect on expression of an unrelated mRNA. (D) Anti-PIP5KIβ immunoblot shows complete loss of protein derived from PIP5KIβ^−/− murine muscle.

Fig. 2.

Platelets lacking PIP5KIβ have an aggregation defect. Murine platelets lacking PIP5KIβ were analyzed after agonist stimulation in a Lumi-Dual aggregometer. Compared with platelets derived from wild-type littermates, platelets derived from PIP5KIβ-knockout mice have a defect in aggregation in response to low doses of all analyzed agonists and exhibit disaggregation. Higher doses of agonists are able to overcome this defect. Results are representative of six experiments.

Fig. 3.

Loss of PIP5KIβ induces a defect in polyphosphoinositide synthesis. Thin-layer chromatography of extracts of radiolabeled platelets derived from wild-type (wt) and PIP5KIβ-knockout (ko) mice is shown. (A) Results of a representative experiment show effects of thrombin stimulation. (B) Pooled analysis of six experiments show the effect of loss of PIP5KIβ mutation on PIP and PIP₂ concentrations after thrombin stimulation.

Fig. 4.

PIP5KIα and PIP5KIβ are required for IP₃ formation. The effect on IP₃ production of loss-of-function mutations within the PIP5KIα and PIP5KIβ genes was analyzed. After stimulation by thrombin, the concentration of 1,4,5-IP₃ was determined by using methods similar to those described by Rittenhouse and Sasson (37). Although loss of PIP5KIα had no effect on thrombin-induced IP₃ production, loss of PIP5KIβ impaired production of IP₃ even at the earliest analyzed time point. Loss of both PIP5KIα and PIP5KIβ completely ablated thrombin-stimulated IP₃ production in platelets.

Fig. 5.

Lack of PIP5KIβ reduces ferric chloride-induced thrombus formation in the mouse carotid artery. Carotid arteries of PIP5KIβ^+/+ and PIP5KIβ^−/− mice were subjected to ferric chloride-induced injury, and blood flow was monitored as a measure of thrombus formation. (A) Representative experiment showing the Doppler tracings from a PIP5KIβ^+/+ mouse (Upper) and a PIP5KIβ^−/− littermate (Lower). The asterisk shows the time that the source of injury was removed from the blood vessel. After ≈1.5 min, an occlusion developed in the artery of the PIP5KIβ^+/+ mouse (note the downward reflection of the Doppler tracing). In contrast, there was no disruption of blood flow in the carotid artery of the PIP5KIβ^−/− mouse. (B) The percentage of mice that formed thrombi that resulted in greater than a 50% reduction in blood flow is reported (PIP5KIβ^+/+, n = 4; PIP5KIβ^−/−, n = 5).