Activation Loop Phosphorylation Modulates Bruton’s Tyrosine Kinase (Btk) Kinase Domain Activity

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• 作者：Laura Lin*^‡ ; ; Robert Czerwinski^§ ; ; Kerry Kelleher^‡ ; ; Marshall M. Siegel ; Paul Wu ; Ron Kriz^‡ ; ; Ann Aulabaugh
  
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：March 10, 2009
• 年：2009
• 卷：48
• 期：9
• 页码：2021-2032
• 全文大小：227K
• 年卷期：v.48,no.9(March 10, 2009)
• ISSN：1520-4995

文摘

Bruton’s tyrosine kinase (Btk) plays a central role in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. A number of cell signaling studies clearly show that Btk is activated by Lyn, a Src family kinase, through phosphorylation on activation loop tyrosine 551 (Y⁵⁵¹). However, the detailed molecular mechanism regulating Btk activation remains unclear. In particular, we do not fully understand the correlation of kinase activity with Y⁵⁵¹ phosphorylation, and the role of the noncatalytic domains of Btk in the activation process. Insect cell expressed full-length Btk is enzymatically active, but a truncated version of Btk, composed of only the kinase catalytic domain, is largely inactive. Further characterization of both forms of Btk by mass spectrometry showed partial phosphorylation of Y⁵⁵¹ of the full-length enzyme and none of the truncated kinase domain. To determine whether the lack of activity of the kinase domain was due to the absence of Y⁵⁵¹ phosphorylation, we developed an in vitro method to generate Y⁵⁵¹ monophosphorylated Btk kinase domain fragment using the Src family kinase Lyn. Detailed kinetic analyses demonstrated that the in vitro phosphorylated Btk kinase domain has a similar activity as the full-length enzyme while the unphosphorylated kinase domain has a very low k_cat and is largely inactive. A divalent magnesium metal dependence study established that Btk requires a second magnesium ion for activity. Furthermore, our analysis revealed significant differences in the second metal-binding site among the kinase domain and the full-length enzyme that likely account for the difference in their catalytic profile. Taken together, our study provides important mechanistic insights into Btk kinase activity and phosphorylation-mediated regulation.