Fumagalli Laura, Zhang Hong, Baruzzi Anna, Lowell Clifford A, Berton Giorgio
Department of Pathology, Section of General Pathology, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy.
J Immunol. 2007 Mar 15;178(6):3874-85. doi: 10.4049/jimmunol.178.6.3874.
The chemotactic peptide formyl-methionyl-leucyl-phenilalanine (fMLP) triggers intracellular protein tyrosine phosphorylation leading to neutrophil activation. Deficiency of the Src family kinases Hck and Fgr have previously been found to regulate fMLP-induced degranulation. In this study, we further investigate fMLP signaling in hck-/-fgr-/- neutrophils and find that they fail to activate a respiratory burst and display reduced F-actin polymerization in response to fMLP. Additionally, albeit migration of both hck-/-fgr-/-mouse neutrophils and human neutrophils incubated with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) through 3-microm pore size Transwells was normal, deficiency, or inhibition, of Src kinases resulted in a failure of neutrophils to migrate through 1-microm pore size Transwells. Among MAPKs, phosphorylation of ERK1/2 was not different, phosphorylation of p38 was only partially affected, and phosphorylation of JNK was markedly decreased in fMLP-stimulated hck-/-fgr-/- neutrophils and in human neutrophils incubated with PP2. An increase in intracellular Ca(2+) concentration and phosphorylation of Akt/PKB occurred normally in fMLP-stimulated hck-/-fgr-/- neutrophils, indicating that activation of both phosphoinositide-specific phospholipase C and PI3K is independent of Hck and Fgr. In contrast, phosphorylation of the Rho/Rac guanine nucleotide exchange factor Vav1 and the Rac target p21-activated kinases were markedly reduced in both hck-/-fgr-/- neutrophils and human neutrophils incubated with a PP2. Consistent with these findings, PP2 inhibited Rac2 activation in human neutrophils. We suggest that Hck and Fgr act within a signaling pathway triggered by fMLP receptors that involves Vav1 and p21-activated kinases, leading to respiratory burst and F-actin polymerization.
趋化肽甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）可触发细胞内蛋白酪氨酸磷酸化，从而导致中性粒细胞活化。此前发现，Src家族激酶Hck和Fgr的缺乏可调节fMLP诱导的脱颗粒作用。在本研究中，我们进一步研究了hck-/-fgr-/-中性粒细胞中的fMLP信号传导，发现它们无法激活呼吸爆发，并且在对fMLP的反应中F-肌动蛋白聚合减少。此外，尽管用Src家族激酶抑制剂4-氨基-5-（4-氯苯基）-7-（叔丁基）吡唑并[3,4-d]嘧啶（PP2）孵育的hck-/-fgr-/-小鼠中性粒细胞和人类中性粒细胞通过3微米孔径的Transwell迁移正常，但Src激酶的缺乏或抑制导致中性粒细胞无法通过1微米孔径的Transwell迁移。在丝裂原活化蛋白激酶（MAPK）中，fMLP刺激的hck-/-fgr-/-中性粒细胞和用PP2孵育的人类中性粒细胞中，细胞外调节蛋白激酶1/2（ERK1/2）的磷酸化没有差异，p38的磷酸化仅受到部分影响，而c-Jun氨基末端激酶（JNK）的磷酸化明显降低。在fMLP刺激的hck-/-fgr-/-中性粒细胞中，细胞内钙离子（Ca2+）浓度的增加和蛋白激酶B（Akt/PKB）的磷酸化正常发生，这表明磷酸肌醇特异性磷脂酶C和磷脂酰肌醇-3激酶（PI3K）的激活均独立于Hck和Fgr。相比之下，在hck-/-fgr-/-中性粒细胞和用PP2孵育的人类中性粒细胞中，Rho/Rac鸟嘌呤核苷酸交换因子Vav1和Rac靶标p21活化激酶的磷酸化均明显降低。与这些发现一致，PP2抑制了人类中性粒细胞中的Rac2活化。我们认为，Hck和Fgr在由fMLP受体触发的信号通路中起作用，该信号通路涉及Vav1和p21活化激酶，从而导致呼吸爆发和F-肌动蛋白聚合。
