Diacylglycerol Kinase γ Serves as an Upstream Suppressor of Rac1 and Lamellipodium Formation*
Diacylglycerol Kinase γ Serves as an Upstream Suppressor of Rac1 and Lamellipodium Formation*
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DOI：
10.1074/jbc.m314031200
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发表时间：
2004-07
影响因子：
4.8
通讯作者：
Shuichi Tsushima;M. Kai;Keiko Yamada;S. Imai;K. Houkin;H. Kanoh;F. Sakane
中科院分区：
文献类型：
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作者：
Shuichi Tsushima;M. Kai;Keiko Yamada;S. Imai;K. Houkin;H. Kanoh;F. Sakane
已鉴定出九种二酰基甘油激酶（DGK）同工酶。然而，我们对它们各自功能的了解仍然有限。在此，我们证明了DGKγ在调节受Rac1调控的细胞形态方面的作用。我们发现，作为显性负性突变体的激酶失活型DGKγ的表达，以及用R59949抑制内源性DGKγ活性，在没有生长因子刺激的情况下，会诱导NIH3T3成纤维细胞形成板状伪足和膜皱褶。相反，在表达组成型活性DGKγ时，血小板衍生生长因子诱导的板状伪足形成受到显著抑制。此外，组成型活性DGKγ突变体抑制整合素介导的细胞铺展。这些效应具有同工酶特异性，因为在相同的实验中，密切相关的同工酶DGKα和DGKβ的相应突变体均不影响细胞形态。这些结果表明，DGKγ特异性地参与导致细胞骨架重排的Rac1介导的信号通路。支持这一点的是，DGKγ与显性活性Rac1共定位，特别是在板状伪足中。此外，我们发现内源性DGKγ与细胞内的Rac1存在物理关联。显性负性Rac1表达阻断了激酶失活型DGKγ诱导的板状伪足形成，表明DGKγ在Rac1的上游起作用。这一模型得到了一些研究的支持，这些研究表明激酶失活型DGKγ选择性地激活Rac1，而不激活Cdc42。综上所述，这些结果有力地表明，DGKγ通过其催化作用作为Rac1的上游抑制因子发挥功能，从而影响板状伪足/皱褶的形成。
Nine diacylglycerol kinase (DGK) isozymes have been identified. However, our knowledge of their individual functions is still limited. Here, we demonstrate the role of DGKγ in regulating Rac1-governed cell morphology. We found that the expression of kinase-dead DGKγ, which acts as a dominant-negative mutant, and inhibition of endogenous DGKγ activity with R59949 induced lamellipodium and membrane ruffle formation in NIH3T3 fibroblasts in the absence of growth factor stimulation. Reciprocally, lamellipodium formation induced by platelet-derived growth factor was significantly inhibited upon expression of constitutively active DGKγ. Moreover, the constitutively active DGKγ mutant suppressed integrin-mediated cell spreading. These effects are isoform-specific because, in the same experiments, none of the corresponding mutants of DGKα and DGKβ, closely related isoforms, affected cell morphology. These results suggest that DGKγ specifically participates in the Rac1-mediated signaling pathway leading to cytoskeletal reorganization. In support of this, DGKγ co-localized with dominant-active Rac1 especially in lamellipodia. Moreover, we found that endogenous DGKγ was physically associated with cellular Rac1. Dominant-negative Rac1 expression blocked the lamellipodium formation induced by kinase-dead DGKγ, indicating that DGKγ acts upstream of Rac1. This model is supported by studies demonstrating that kinase-dead DGKγ selectively activated Rac1, but not Cdc42. Taken together, these results strongly suggest that DGKγ functions through its catalytic action as an upstream suppressor of Rac1 and, consequently, lamellipodium/ruffle formation.