Structural Features Underlying the Multisite Phosphorylation of the A Domain of the NF-AT4 Transcription Factor by Protein Kinase CK1

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• 作者：Oriano Marin ; Veronica Burzio ; Marco Boschetti ; Flavio Meggio ; Catherine C. Allende ; Jorge E. Allende ; and Lorenzo A. Pinna
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：January 15, 2002
• 年：2002
• 卷：41
• 期：2
• 页码：618 - 627
• 全文大小：128K
• 年卷期：v.41,no.2(January 15, 2002)
• ISSN：1520-4995

文摘

The phosphorylation and dephosphorylation of the NF-AT family of transcription factors playa key role in the activation of T lymphocytes and in the control of the immune response. The mechanisticaspects of NF-AT4 phosphorylation by protein kinase CK1 have been studied in this work with the aidof a series of 27 peptides, reproducing with suitable modifications the regions of NF-AT4 that have beenreported to be phosphorylated by this protein kinase. The largest parent peptide, representing the threeregions A, Z, and L spanning amino acids 173-218, is readily phosphorylated by CK1 at seryl residuesbelonging to the A2 segment, none of which fulfill the canonical consensus sequence for CK1. An acidiccluster of amino acids in the linker region between domains A and Z is essential for high-efficiencyphosphorylation of the A2 domain, as shown by the increase in K_m caused by a deletion of the linkerregion or a substitution of the acidic residues with glycines. Individual substitutions with alanine of eachof the five serines in the A2 domain (S-177, S-180, S-181, S-184, and S-186) reduce the phosphorylationrate, the most detrimental effect being caused by Ser177 substitution which results in a 10-fold drop inV_max. On the contrary, the replacement of Ser177 with phosphoserine triggers a hierarchical effect witha dramatic improvement in phosphorylation efficiency, which no longer depends on the linker region foroptimal efficiency. These data are consistent with a two-phase phosphorylation mechanism of NF-AT4by CK1, initiated by the linker region which provides a functional docking site for CK1 and allows theunorthodox phosphorylation of Ser177; once achieved, this phosphoserine residue primes the phosphorylation of other downstream seryl residues, according to a hierarchical mechanism typically exploited byCK1.