Supplementary Material for: The Tyrosine Kinase Pyk2 Contributes to Complement-Mediated Phagocytosis in Murine Macrophages

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family and is mainly expressed in neuronal and hematopoietic cells. As FAK family members are involved in signaling connections downstream of integrins, we studied the role of Pyk2 in complement-receptor 3 (CR3, also known as Mac-1, integrin α_Mβ₂, CD11b/CD18)-mediated phagocytosis, a key process in innate immunity. Using 3 independent approaches, we observed that Pyk2 contributes to CR3-dependent phagocytosis by RAW 264.7 macrophages, but is dispensable for Fcγ receptor (FcγR)-mediated uptake. Reduction of Pyk2 expression levels via siRNA, the pharmacological inhibition of Pyk2 kinase activity as well as macrophage treatment with a cell permeable TAT fusion protein containing the C-terminus of Pyk2 (TAT-PRNK) significantly impaired CR3-mediated phagocytosis without affecting FcγR-mediated uptake. In addition, Pyk2 was strongly recruited to complement opsonized <i>Escherichia coli</i> and the pharmacological inhibition of Pyk2 significantly decreased uptake of the bacteria. Finally, CRISPR/Cas-mediated disruption of the <i>pyk2</i> gene in RAW 264.7 macrophages confirmed the role of this protein tyrosine kinase in CR3-mediated phagocytosis. Together, our data demonstrate that Pyk2 selectively contributes to the coordination of phagocytosis-promoting signals downstream of CR3, but is dispensable for FcγR-mediated phagocytosis.

富脯氨酸酪氨酸激酶2（Proline-rich tyrosine kinase 2, Pyk2）是黏着斑激酶（focal adhesion kinase, FAK）家族成员，主要在神经元与造血细胞中表达。鉴于FAK家族成员参与整合素下游的信号传导通路，本研究探究了Pyk2在补体受体3（complement-receptor 3, CR3，亦称Mac-1、整合素α_Mβ₂、CD11b/CD18）介导的吞噬作用中的功能——该过程是先天免疫的关键环节。我们采用3种独立实验方法，观察到Pyk2可促进RAW 264.7巨噬细胞的CR3依赖性吞噬作用，但对Fcγ受体（Fcγ receptor, FcγR）介导的细胞摄取并非必需。通过小干扰RNA（small interfering RNA, siRNA）下调Pyk2的表达水平、通过药理学手段抑制Pyk2的激酶活性，以及使用携带Pyk2羧基末端的细胞穿透性TAT融合蛋白（TAT-PRNK）处理巨噬细胞，均可显著削弱CR3介导的吞噬作用，却未对FcγR介导的细胞摄取产生影响。此外，Pyk2会被大量招募至补体调理的大肠杆菌（Escherichia coli）表面，而Pyk2的药理学抑制会显著降低该细菌的细胞摄取效率。最终，通过CRISPR/Cas介导的RAW 264.7巨噬细胞pyk2基因敲除实验，进一步证实了该蛋白酪氨酸激酶在CR3介导的吞噬作用中的核心作用。综上，本研究数据表明，Pyk2可选择性参与CR3下游吞噬促进信号的调控通路，而对FcγR介导的吞噬作用并非必需。