McDoom Issam, Ma Xianyue, Kirabo Annet, Lee Kuang-Yung, Ostrov David A, Sayeski Peter P
Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Biochemistry. 2008 Aug 12;47(32):8326-34. doi: 10.1021/bi800867d. Epub 2008 Jul 18.
Jak2 is a 130 kDa tyrosine kinase that is important in a number of cellular signaling pathways. Its function is intrinsically regulated by the phosphorylation of a handful of its 49 tyrosines. Here, we report that tyrosine 972 (Y972) is a novel site of Jak2 phosphorylation and, hence, autoregulation. Specifically, we found that Y972 is phosphorylated and confirmed that this residue resides on the surface of the protein. Using expression plasmids that expressed either wild-type Jak2 or a full-length Jak2 cDNA containing a single Y972F substitution mutation, we investigated the consequences of losing Y972 phosphorylation on Jak2 function. We determined that the loss of Y972 phosphorylation significantly reduced the levels of both Jak2 total tyrosine phosphorylation and phosphorylation of Y1007/Y1008. Additionally, Y972 phosphorylation was shown to be important for maximal kinase function. Interestingly, in response to classical cytokine activation, the Jak2 Y972F mutant exhibited a moderately impaired level of activation when compared to the wild-type protein. However, when Jak2 was activated via a GPCR ligand, the ability of the Y972F mutant to be activated was completely lost, therefore suggesting a differential role of Y972 in Jak2 activation. Finally, we found that phosphorylation of Y972 enhances Jak2 kinase function via a mechanism that appears to stabilize the active conformation of the protein. Collectively, our results suggest that Y972 is a novel site of Jak2 phosphorylation and plays an important differential role in ligand-dependent Jak2 activation via a mechanism that involves stabilization of the Jak2 active conformation.
Jak2是一种130 kDa的酪氨酸激酶，在许多细胞信号通路中起重要作用。其功能由其49个酪氨酸中少数几个的磷酸化进行内在调节。在此，我们报告酪氨酸972（Y972）是Jak2磷酸化及自调节的一个新位点。具体而言，我们发现Y972被磷酸化，并证实该残基位于蛋白质表面。使用表达野生型Jak2或含有单个Y972F替代突变的全长Jak2 cDNA的表达质粒，我们研究了Y972磷酸化缺失对Jak2功能的影响。我们确定Y972磷酸化的缺失显著降低了Jak2总酪氨酸磷酸化水平以及Y1007/Y1008的磷酸化水平。此外，Y972磷酸化对最大激酶功能很重要。有趣的是，与野生型蛋白相比，在经典细胞因子激活反应中，Jak2 Y972F突变体的激活水平有适度受损。然而，当Jak2通过GPCR配体激活时，Y972F突变体的激活能力完全丧失，因此表明Y972在Jak2激活中具有不同作用。最后，我们发现Y972的磷酸化通过一种似乎能稳定蛋白质活性构象的机制增强Jak2激酶功能。总体而言，我们的结果表明Y972是Jak2磷酸化的一个新位点，并且通过一种涉及稳定Jak2活性构象的机制，在配体依赖性Jak2激活中发挥重要的不同作用。
