Protein Kinase A Regulates 3-Phosphatidylinositide Dynamics during Platelet-derived Growth Factor-induced Membrane Ruffling and Chemotaxis
Protein Kinase A Regulates 3-Phosphatidylinositide Dynamics during Platelet-derived Growth Factor-induced Membrane Ruffling and Chemotaxis
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DOI：
10.1074/jbc.m804448200
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发表时间：
2008-12-12
影响因子：
4.8
通讯作者：
Howe, Alan K.
中科院分区：
文献类型：
--
作者：
Deming, Paula B.;Campbell, Shirley L.;Howe, Alan K.
cAMP依赖性蛋白激酶（PKA）的空间调节是成纤维细胞趋化性所必需的;然而，PKA调节细胞迁移机制的机制在很大程度上仍然未知。在这里，我们报告的PKA在血小板衍生生长因子（PDGF）诱导的趋化性的功能之一是通过调节磷脂酰肌醇3，4，5-三磷酸（PIP 3）的动力学，以促进膜皱褶。PKA活性的抑制显着改变膜动力学和衰减的外周膜皱褶的形成响应PDGF。PKA抑制也显着减少了响应于均匀刺激和PDGF梯度的富含PIP 3的膜皱褶的数量和大小。这种皱褶缺陷使用一种新开发的方法进行量化，基于计算机视觉边缘检测算法。PKA抑制引起PDGF刺激后PIP 3大量积累的显著衰减，而对PI 3-激酶（PI 3 K）活性无影响。PIP 3动力学的缺陷与生长因子诱导的内源性Akt和Rac激活的PKA抑制细胞的膜募集的显着抑制相关。同时抑制PKA和Rac对生长因子诱导的皱褶动力学具有相加的抑制作用。相反，组成型活性Rac等位基因的表达能够挽救膜皱褶中的缺陷，并恢复荧光PIP 3标记物在PKA抑制细胞中的膜皱褶的定位，即使在没有PI 3 K活性的情况下。这些数据表明，像Rac一样，PKA独立于PI 3 K活性的直接调节而促进PIP 3和膜动力学，并表明PIP 3/3-磷脂酰肌醇（3-PI）脂质的调节代表了PKA在PDGF诱导的趋化事件调节中的主要靶点。
Spatial regulation of the cAMP-dependent protein kinase (PKA) is required for chemotaxis in fibroblasts; however, the mechanism(s)by which PKA regulates the cell migration machinery remain largely unknown. Here we report that one function of PKA during platelet-derived growth factor (PDGF)-induced chemotaxis was to promote membrane ruffling by regulating phosphatidylinositol 3,4,5-trisphosphate (PIP3) dynamics. Inhibition of PKA activity dramatically altered membrane dynamics and attenuated formation of peripheral membrane ruffles in response to PDGF. PKA inhibition also significantly decreased the number and size of PIP3-rich membrane ruffles in response to uniform stimulation and to gradients of PDGF. This ruffling defect was quantified using a newly developed method, based on computer vision edge-detection algorithms. PKA inhibition caused a marked attenuation in the bulk accumulation of PIP3 following PDGF stimulation, without effects on PI3-kinase (PI3K) activity. The deficits in PIP3 dynamics correlated with a significant inhibition of growth factor-induced membrane recruitment of endogenous Akt and Rac activation in PKA-inhibited cells. Simultaneous inhibition of PKA and Rac had an additive inhibitory effect on growth factor-induced ruffling dynamics. Conversely, the expression of a constitutively active Rac allele was able to rescue the defect in membrane ruffling and restore the localization of a fluorescent PIP3 marker to membrane ruffles in PKA-inhibited cells, even in the absence of PI3K activity. These data demonstrate that, like Rac, PKA contributes to PIP3 and membrane dynamics independently of direct regulation of PI3K activity and suggest that modulation of PIP3/3-phosphatidylinositol (3-PI) lipids represents a major target for PKA in the regulation of PDGF-induced chemotactic events.