Rembutsu Makoto, Soutar Marc P M, Van Aalten Lidy, Gourlay Robert, Hastie C James, McLauchlan Hilary, Morrice Nicholas A, Cole Adam R, Sutherland Calum
Division of Pathology and Neurosciences, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, Scotland, United Kingdom.
Biochemistry. 2008 Feb 19;47(7):2153-61. doi: 10.1021/bi702030w. Epub 2008 Jan 26.
The identification of phosphorylation state-dependent interacting proteins provides clues as to the function of the phosphorylation. Techniques such as yeast two hybrid and co-immunoprecipitation do not employ a single species of fully phosphorylated proteins. This is a particular problem for substrates of glycogen synthase kinase-3 (GSK3), where multiple Ser/Thr residues can be targeted, almost always subsequent to a priming phosphorylation by an alternative kinase. We previously identified the brain enriched collapsin response mediator proteins (CRMP2 and CRMP4) as physiological substrates of GSK3. Cdk5 phosphorylates CRMP2 at Ser522, priming for subsequent phosphorylation at three residues by GSK3 in vitro and in vivo. It is clear that phosphorylation of CRMP2 influences axonal growth; however, the molecular processes underlying this action are not fully established. In addition, the role of phosphorylation in other actions of CRMPs has not been elucidated. We developed a novel procedure to isolate CRMP2 and CRMP4 fully phosphorylated at four sites, namely, Ser522 (by CDK5), Ser518, Thr514, and Thr509 (by GSK3). These phosphoproteins were then used to identify binding partners in rat brain lysates in direct comparison with the non-phosphorylated isoforms. We validated the approach by confirming that a previously reported interaction with tubulin-beta is regulated by phosphorylation. We also show that CRMPs (CRMP1, CRMP2, and CRMP4) form heteromers and found that these complexes may also be regulated by phosphorylation. We identified DYRK and Pin1 as novel CRMP4 binding proteins with DYRK interacting preferentially with dephospho-CRMP4 and Pin1 with phospho-CRMP4. Finally, we used this approach to identify the mitochondrial protein ANT as a novel CRMP2 and CRMP4 binding protein. We believe that this approach could be applied generally to the study of phosphorylation-dependent interactions.
磷酸化状态依赖性相互作用蛋白的鉴定为磷酸化功能提供了线索。酵母双杂交和免疫共沉淀等技术并不使用单一物种的完全磷酸化蛋白。对于糖原合酶激酶-3（GSK3）的底物来说，这是一个特别的问题，因为多个丝氨酸/苏氨酸残基都可能成为靶点，几乎总是在被另一种激酶进行引发磷酸化之后。我们之前鉴定出大脑富集的塌陷反应介导蛋白（CRMP2和CRMP4）是GSK3的生理底物。细胞周期蛋白依赖性激酶5（Cdk5）在丝氨酸522位点使CRMP2磷酸化，为随后在体外和体内被GSK3在三个位点进行磷酸化做好准备。很明显，CRMP2的磷酸化会影响轴突生长；然而，这一作用背后的分子过程尚未完全明确。此外，磷酸化在CRMPs其他作用中的角色也尚未阐明。我们开发了一种新方法来分离在四个位点完全磷酸化的CRMP2和CRMP4，即丝氨酸522（由Cdk5催化）、丝氨酸518、苏氨酸514和苏氨酸509（由GSK3催化）。然后将这些磷酸化蛋白用于直接与非磷酸化异构体比较，以鉴定大鼠脑裂解物中的结合伴侣。我们通过证实先前报道的与β-微管蛋白的相互作用受磷酸化调节来验证了该方法。我们还表明CRMPs（CRMP1、CRMP2和CRMP4）形成异源二聚体，并发现这些复合物也可能受磷酸化调节。我们鉴定出双重特异性酪氨酸磷酸化调节激酶（DYRK）和肽基脯氨酰顺反异构酶NIMA相关激酶1（Pin1）是新的CRMP4结合蛋白，其中DYRK优先与去磷酸化的CRMP4相互作用，而Pin1与磷酸化的CRMP4相互作用。最后，我们使用这种方法鉴定出线粒体蛋白腺嘌呤核苷酸转运体（ANT）是一种新的CRMP2和CRMP4结合蛋白。我们相信这种方法可普遍应用于磷酸化依赖性相互作用的研究。
