Regenerative Medicine Program, Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, ON, K1H 8L6, Canada.
Skelet Muscle. 2012 Mar 6;2:5. doi: 10.1186/2044-5040-2-5.
The p38α mitogen-activated protein kinase (MAPK) is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38α is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear.
To further characterise the involvement of p38α during myoblast differentiation, we developed and applied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38α and β isoforms.
Applying the technique to p38α resulted in the identification of seven in vivo phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison with p38β revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation.
Our results suggest p38α has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38β and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling.
p38α 丝裂原活化蛋白激酶（MAPK）是肌母细胞分化的关键介质，部分通过磷酸化和调节几种转录因子和染色质重塑蛋白来实现。然而，p38α 是否参与肌发生过程中的基因调控以外的其他过程目前尚不清楚，其他 p38 同工型为什么不能弥补其缺失也不清楚。
为了进一步研究 p38α 在肌母细胞分化过程中的作用，我们开发并应用了一种简单的技术来鉴定相关的体内激酶底物及其磷酸化位点。除了鉴定一种激酶的底物外，该技术还可在体外用于比较同一实验中的多种激酶，我们利用这一技术研究了 p38α 和 β 同工型的底物特异性。
将该技术应用于 p38α，在分化肌母细胞的裂解物中鉴定出六种蛋白质上的七个体内磷酸化位点，其中四个位于细胞质中。与 p38β 的体外比较表明，底物特异性并不能区分这两种同工型，而是它们的区别特征似乎是细胞定位。
我们的结果表明，p38α 在肌发生过程中有一个新的细胞质作用，其独特的细胞定位可能是 p38β 和其他同工型不能弥补其缺失的原因。这里提出的底物发现方法也为研究存在的数百种蛋白激酶提供了必要的工具，并揭示了磷酸化依赖性细胞信号转导的更深层次机制。
