Schaller M D, Sasaki T
Department of Cell Biology and Anatomy and the Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 1997 Oct 3;272(40):25319-25. doi: 10.1074/jbc.272.40.25319.
pp125(FAK) and CAKbeta/Pyk2/CadTK/RAFTK are related protein-tyrosine kinases. It is therefore of interest whether CAKbeta shares some of the properties of pp125(FAK). Using recombinant glutathione S-transferase fusion proteins, we show that the C-terminal domains of both proteins bind paxillin in vitro. The C-terminal domain of CAKbeta was engineered to be autonomously expressed in chicken embryo cells and, like pp125(FAK) and p41/43(FRNK) (the C-terminal noncatalytic domain of pp125(FAK)), was found to localize to cellular focal adhesions. In contrast, full-length CAKbeta was generally found diffusely distributed throughout the cell, although a fraction of the cells exhibited focal adhesion localization. Vanadate treatment of pp125(FAK)- and CAKbeta-overexpressing CE cells induced a dramatic increase in the phosphotyrosine content of a common set of proteins including tensin, paxillin, and p130(Cas), but some of these substrates, particularly p130(Cas), appeared to be differentially phosphorylated by pp125(FAK) and CAKbeta. Levels of tyrosine phosphorylation were higher in CAKbeta-overexpressing cells, and additional phosphotyrosine-containing species were specifically immunoprecipitated. In addition, vanadate treatment of CE cells overexpressing CAKbeta, but not pp125(FAK) overexpressors, induced a profound morphological change, which could be a consequence of the observed differences in substrate phosphorylation.
pp125（粘着斑激酶）和CAKβ/Pyk2/CadTK/RAFTK是相关的蛋白酪氨酸激酶。因此，CAKβ是否具有pp125（粘着斑激酶）的某些特性是一个值得关注的问题。利用重组谷胱甘肽S-转移酶融合蛋白，我们发现这两种蛋白的C末端结构域在体外均能结合桩蛋白。CAKβ的C末端结构域经改造后可在鸡胚细胞中自主表达，并且与pp125（粘着斑激酶）和p41/43（FRNK）（pp125（粘着斑激酶）的C末端非催化结构域）一样，定位于细胞粘着斑。相比之下，全长CAKβ通常在整个细胞中呈弥散分布，不过有一部分细胞表现出粘着斑定位。用钒酸盐处理过表达pp125（粘着斑激酶）和CAKβ的鸡胚细胞，会使包括张力蛋白、桩蛋白和p130（Cas）在内的一组常见蛋白的磷酸酪氨酸含量显著增加，但其中一些底物，特别是p130（Cas），似乎被pp125（粘着斑激酶）和CAKβ进行了不同程度的磷酸化。在过表达CAKβ的细胞中酪氨酸磷酸化水平更高，并且有额外的含磷酸酪氨酸的蛋白被特异性免疫沉淀。此外，用钒酸盐处理过表达CAKβ的鸡胚细胞（而非过表达pp125（粘着斑激酶）的细胞）会诱导出深刻的形态变化，这可能是所观察到的底物磷酸化差异的结果。
