Structural Features Underlying the Multisite Phosphorylation of the A Domain of the NF-AT4 Transcription Factor by Protein Kinase CK1
| Autor: | Verónica A. Burzio, Flavio Meggio, Jorge E. Allende, Marco Boschetti, Oriano Marin, Catherine C. Allende, Lorenzo A. Pinna |
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| Rok vydání: | 2001 |
| Předmět: |
Time Factors
T-Lymphocytes Molecular Sequence Data Biology Models Biological Biochemistry Phosphorylation cascade Dephosphorylation chemistry.chemical_compound Cytosol Serine Animals Protein Isoforms Protein phosphorylation Amino Acid Sequence Amino Acids Phosphorylation Protein kinase A Chromatography High Pressure Liquid Zebrafish Alanine Binding Sites NFATC Transcription Factors Sequence Homology Amino Acid Kinase Nuclear Proteins Recombinant Proteins Protein Structure Tertiary Rats DNA-Binding Proteins Kinetics Liver chemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Phosphoserine Casein kinase 1 Peptides Casein Kinases Protein Kinases Gene Deletion Transcription Factors |
| Zdroj: | BIOCHEMISTRY (EASTON) Artículos CONICYT CONICYT Chile instacron:CONICYT |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi0112309 |
| Popis: | The phosphorylation and dephosphorylation of the NF-AT family of transcription factors play a key role in the activation of T lymphocytes and in the control of the immune response. The mechanistic aspects of NF-AT4 phosphorylation by protein kinase CK1 have been studied in this work with the aid of a series of 27 peptides, reproducing with suitable modifications the regions of NF-AT4 that have been reported to be phosphorylated by this protein kinase. The largest parent peptide, representing the three regions A, Z, and L spanning amino acids 173-218, is readily phosphorylated by CK1 at seryl residues belonging to the A2 segment, none of which fulfill the canonical consensus sequence for CK1. An acidic cluster of amino acids in the linker region between domains A and Z is essential for high-efficiency phosphorylation of the A2 domain, as shown by the increase in Km caused by a deletion of the linker region or a substitution of the acidic residues with glycines. Individual substitutions with alanine of each of the five serines in the A2 domain (S-177, S-180, S-181, S-184, and S-186) reduce the phosphorylation rate, the most detrimental effect being caused by Ser177 substitution which results in a 10-fold drop in Vmax. On the contrary, the replacement of Ser177 with phosphoserine triggers a hierarchical effect with a dramatic improvement in phosphorylation efficiency, which no longer depends on the linker region for optimal efficiency. These data are consistent with a two-phase phosphorylation mechanism of NF-AT4 by CK1, initiated by the linker region which provides a functional docking site for CK1 and allows the unorthodox phosphorylation of Ser177; once achieved, this phosphoserine residue primes the phospho- rylation of other downstream seryl residues, according to a hierarchical mechanism typically exploited by CK1. The large number of protein kinases in eukaryotes, with over 800 genes found in the human genome (1), raises multiple questions as to the function and specificity of these important enzymes. |
| Databáze: | OpenAIRE |
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