Okabe Seiichi, Fukuda Seiji, Kim Young-June, Niki Masaru, Pelus Louis M, Ohyashiki Kazuma, Pandolfi Pier Paolo, Broxmeyer Hal E
Department of Microbiology/Immunology and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Blood. 2005 Jan 15;105(2):474-80. doi: 10.1182/blood-2004-03-0843. Epub 2004 Sep 2.
Events mediating stromal cell-derived factor-1 (SDF-1alpha/CXCL12) chemotaxis of lymphocytes are not completely known. We evaluated intracellular signaling through RasGAP-associated protein p62Dok-1 (downstream of tyrosine kinase [Dok-1]) and associated proteins. SDF-1alpha/CXCL12 stimulated Dok-1 tyrosine phosphorylation and association with RasGAP, adaptor protein p46Nck, and Crk-L in Jurkat T cells. The phosphorylation of Dok-1 was blocked by pretreatment of cells with the src kinase inhibitor PP2. Src kinase family member Lck was implicated. SDF-1alpha/CXCL12 did not phosphorylate Dok-1 in J.CaM1.6 cells, a Jurkat derivative not expressing Lck, but did phosphorylate Dok-1 in J.CaM1.6 cells expressing Lck. SDF-1alpha/CXCL12 induced the tyrosine phosphorylation of Pyk2 and the association of Pyk2 with zeta chain-associated protein-70 kilodaltons (Zap-70) and Vav. SDF-1alpha/CXCL12 enhanced the association of RasGAP with Pyk2. CXCR4-expressing NIH3T3 and Baf3 cells transfected with full-length Dok-1 cDNA were suppressed in their responses to SDF-1alpha/CXCL12-induced chemotaxis; mitogen-activated protein (MAP) kinase activity was also decreased. Chemotaxis to SDF-1/CXCL12 was significantly enhanced in Dok-1(-/-) CD4+ and CD8+ splenic T cells. These results implicate Dok-1, Nck, Crk-L, and Src kinases-especially Lck, Pyk2, Zap-70, Vav, and Ras-GAP-in intracellular signaling by SDF-1alpha/CXCL12, and they suggest that Dok-1 plays an important role in SDF-1alpha/CXCL12-induced chemotaxis in T cells.
介导淋巴细胞基质细胞衍生因子-1（SDF-1α/CXCL12）趋化作用的事件尚不完全清楚。我们评估了通过RasGAP相关蛋白p62Dok-1（酪氨酸激酶下游[Dok-1]）及相关蛋白的细胞内信号传导。SDF-1α/CXCL12刺激Jurkat T细胞中Dok-1的酪氨酸磷酸化以及与RasGAP、衔接蛋白p46Nck和Crk-L的结合。用src激酶抑制剂PP2预处理细胞可阻断Dok-1的磷酸化。src激酶家族成员Lck与之相关。SDF-1α/CXCL12在不表达Lck的Jurkat衍生物J.CaM1.6细胞中不使Dok-1磷酸化，但在表达Lck的J.CaM1.6细胞中可使Dok-1磷酸化。SDF-1α/CXCL12诱导Pyk2的酪氨酸磷酸化以及Pyk2与70千道尔顿ζ链相关蛋白（Zap-70）和Vav的结合。SDF-1α/CXCL12增强了RasGAP与Pyk2的结合。用全长Dok-1 cDNA转染的表达CXCR4的NIH3T3和Baf3细胞对SDF-1α/CXCL12诱导的趋化反应受到抑制；丝裂原活化蛋白（MAP）激酶活性也降低。在Dok-1基因敲除（-/-）的CD4 +和CD8 +脾T细胞中，对SDF-1/CXCL12的趋化作用显著增强。这些结果表明Dok-1、Nck、Crk-L和src激酶——尤其是Lck、Pyk2、Zap-70、Vav和Ras-GAP——参与了SDF-1α/CXCL12的细胞内信号传导，并且提示Dok-1在T细胞中SDF-1α/CXCL12诱导的趋化作用中起重要作用。
