The Tribbles 2 (TRB2) pseudokinase binds to ATP and autophosphorylates in a metal-independent manner

作者：Fiona P. Bailey, Dominic P. Byrne, Krishnadev Oruganty, Claire E. Eyers, Christopher J. Novotny, Kevan M. Shokat, Natarajan Kannan, Patrick A. Eyers · 发表于：Biochemical Journal · 年份：2015 · DOI：10.1042/bj20141441 · 被引用次数：82 · 研究领域：Endoplasmic Reticulum Stress and Disease、RNA regulation and disease、Autophagy in Disease and Therapy

The human Tribbles (TRB)-related pseudokinases are CAMK (calcium/calmodulin-dependent protein kinase)-related family members that have evolved a series of highly unusual motifs in the 'pseudocatalytic' domain. In canonical kinases, conserved amino acids bind to divalent metal ions and align ATP prior to efficient phosphoryl-transfer to substrates. However, in pseudokinases, atypical residues give rise to diverse and often unstudied biochemical and structural features that are thought to be central to cellular functions. TRB proteins play a crucial role in multiple signalling networks and overexpression confers cancer phenotypes on human cells, marking TRB pseudokinases out as a novel class of drug target. In the present paper, we report that the human pseudokinase TRB2 retains the ability to both bind and hydrolyse ATP weakly in vitro. Kinase activity is metal-independent and involves a catalytic lysine residue, which is conserved in TRB proteins throughout evolution alongside several unique amino acids in the active site. A similar low level of autophosphorylation is also preserved in the closely related human TRB3. By employing chemical genetics, we establish that the nucleotide-binding site of an 'analogue-sensitive' (AS) TRB2 mutant can be targeted with specific bulky ligands of the pyrazolo-pyrimidine (PP) chemotype. Our analysis confirms that TRB2 retains low levels of ATP binding and/or catalysis that is targetable with small molecules. Given the significant clinical s...