Cell Biology and Stem Cells Unit, Center for Cooperative Research in Biosciences (CIC bioGUNE), Bizkaia Technology Park, 48160 Derio, Spain.
Biol Direct. 2011 Jan 24;6:4. doi: 10.1186/1745-6150-6-4.
Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine (Ser/Thr) kinase comprising two isoforms, GSK-3α and GSK-3β. Both enzymes are similarly inactivated by serine phosphorylation (GSK-3α at Ser21 and GSK-3β at Ser9) and activated by tyrosine phosphorylation (GSK-3α at Tyr279 and GSK-3β at Tyr216). Antibodies raised to phosphopeptides containing the sequences around these phosphorylation sites are frequently used to provide an indication of the activation state of GSK-3 in cell and tissue extracts. These antibodies have further been used to determine the subcellular localisation of active and inactive forms of GSK-3, and the results of those studies support roles for GSK-3 phosphorylation in diverse cellular processes. However, the specificity of these antibodies in immunocytochemistry has not been addressed in any detail.
Taking advantage of gene silencing technology, we examined the specificity of several commercially available anti-phosphorylated GSK-3 antibodies. We show that antibodies raised to peptides containing the phosphorylated Ser21/9 epitope crossreact with unidentified antigens that are highly expressed by mitotic cells and that mainly localise to spindle poles. In addition, two antibodies raised to peptides containing the phosphorylated Tyr279/216 epitope recognise an unidentified protein at focal contacts, and a third antibody recognises a protein found in Ki-67-positive cell nuclei. While the phosphorylated Ser9/21 GSK-3 antibodies also recognise other proteins whose levels increase in mitotic cells in western blots, the phosphorylated Tyr279/216 antibodies appear to be specific in western blotting. However, we cannot rule out the posssibility that they recognise very large or very small proteins that might not be detected using a standard western blotting approach.
Our findings indicate that care should be taken when examining the subcellular localisation of active or inactive GSK-3 and, furthermore, suggest that the role of GSK-3 phosphorylation in some cellular processes be reassessed.
糖原合酶激酶-3（GSK-3）是一种广泛表达的丝氨酸/苏氨酸（Ser/Thr）激酶，由两种同工酶组成，GSK-3α 和 GSK-3β。两种酶都通过丝氨酸磷酸化（GSK-3α 在 Ser21 和 GSK-3β 在 Ser9）失活，通过酪氨酸磷酸化（GSK-3α 在 Tyr279 和 GSK-3β 在 Tyr216）激活。针对包含这些磷酸化位点周围序列的磷酸肽的抗体常被用来提供细胞和组织提取物中 GSK-3 激活状态的指示。这些抗体还被用于确定 GSK-3 的活性和非活性形式的亚细胞定位，这些研究的结果支持 GSK-3 磷酸化在各种细胞过程中的作用。然而，这些抗体在免疫细胞化学中的特异性尚未得到详细研究。
利用基因沉默技术，我们检查了几种市售的抗磷酸化 GSK-3 抗体的特异性。我们表明，针对含有磷酸化 Ser21/9 表位的肽的抗体与有丝分裂细胞中高度表达的未鉴定抗原发生交叉反应，这些抗原主要定位于纺锤体极。此外，针对含有磷酸化 Tyr279/216 表位的肽的两种抗体识别黏着斑中的未鉴定蛋白，第三种抗体识别 Ki-67 阳性细胞核中的蛋白。虽然磷酸化 Ser9/21 GSK-3 抗体也在 Western blot 中识别有丝分裂细胞中水平升高的其他蛋白，但磷酸化 Tyr279/216 抗体似乎在 Western blot 中是特异性的。然而，我们不能排除它们识别非常大和非常小的蛋白的可能性，这些蛋白可能无法使用标准的 Western blot 方法检测到。
我们的研究结果表明，在检查活性或非活性 GSK-3 的亚细胞定位时应谨慎，并且进一步表明需要重新评估 GSK-3 磷酸化在一些细胞过程中的作用。
