Podosomes play crucial roles in macrophage adhesion and migration. Wiskott–Aldrich syndrome protein (WASP; also known as WAS)-mediated actin polymerization is one of the key events initiating podosome formation. Nevertheless, membrane signals to trigger WASP activation at macrophage podosomes remain unclear. Here, we show that phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] lipids are enriched at the podosome and stably recruit WASP rather than the WASP-5KE mutant. Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit β (PIK3CB) is spatially located at the podosome core. Inhibition of PIK3CB and overexpression of phosphatase and tensin homolog (PTEN) impede F-actin polymerization of the podosome. PIK3CB activation is regulated by Abl1 and Src family kinases. At the podosome core, Src and Hck promote the phosphorylation of Tyr488 in the consensus Y-x-x-M motif of Abl1, which enables the association of phosphoinositide 3-kinase (PI3K) regulatory subunits. Knockdown of Abl1 rather than Abl2 suppresses the PI3K/Akt pathway, regardless of Src and Hck activities. Reintroduction of wild-type Abl1 rather than the Abl1-Y488F mutant rescues PI3KR1 recruitment and PI3K activation. When PIK3CB, Abl1 or Src/Hck is suppressed, macrophage podosome formation, matrix degradation and chemotactic migration are inhibited. Thus, Src/Hck-mediated phosphorylation of Abl1 Tyr488 triggers PIK3CB-dependent PI(3,4,5)P3 production and orchestrates the assembly and function of macrophage podosomes.

足小体在巨噬细胞粘附和迁移中起着至关重要的作用。维斯科特-奥尔德里奇综合征蛋白（WASP；也称为 WAS）介导的肌动蛋白聚合是启动足小体形成的关键事件之一。然而，触发巨噬细胞足体 WASP 激活的膜信号仍不清楚。在这里，我们发现磷脂酰肌醇 (3,4,5)-三磷酸 [PI(3,4,5)P3] 脂质在足体处富集，并稳定地招募 WASP 而不是 WASP-5KE 突变体。磷脂酰肌醇 4,5-二磷酸 3-激酶催化亚基 β (PIK3CB) 在空间上位于足小体核心。PIK3CB 的抑制以及磷酸酶和张力蛋白同源物 (PTEN) 的过度表达会阻碍足小体的 F-肌动蛋白聚合。PIK3CB 激活由 Abl1 和 Src 家族激酶调节。在足小体核心，Src 和 Hck 促进 Abl1 共有 YxxM 基序中 Tyr488 的磷酸化，从而实现磷酸肌醇 3 激酶 (PI3K) 调节亚基的关联。无论 Src 和 Hck 活性如何，敲低 Abl1 而不是 Abl2 都会抑制 PI3K/Akt 通路。重新引入野生型 Abl1 而不是 Abl1-Y488F 突变体可挽救 PI3KR1 募集和 PI3K 激活。当 PIK3CB、Abl1 或 Src/Hck 受到抑制时，巨噬细胞足小体形成、基质降解和趋化迁移都会受到抑制。因此，Src/Hck 介导的 Abl1 Tyr488 磷酸化触发 PIK3CB 依赖性 PI(3,4,5)P3 的产生并协调巨噬细胞足小体的组装和功能。