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

Abstract

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 successes associated with targeting of cancer-associated kinases with small molecule inhibitors, it is likely that similar approaches will be useful for further evaluating the TRB pseudokinases, with the translation of this information likely to furnish new leads for drug discovery.

cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility–mass spectrometry

“…In the absence of a crystal structure, it is challenging to explain why we are unable to detect K72H nucleotide binding using standard procedures [65], especially given previous findings [56,62]. However, Lys72 of PKA is reported to interact with the α- and β-phosphates of ATP and a critical C-helix Glu residue in the closed, active PKA structure [69]; mutation of the equivalent residue in (pseudo)kinases can ablate ATP binding that can be quantified by solution DSF [47,81]. We therefore speculate that should ATP still bind to K72H PKAc, its conformation must be markedly different from that of WT and R133A PKAc, but nonetheless remain responsive to thermal stabilization by some kinase inhibitors.…”

Tribbles in the 21st Century: The Evolving Roles of Tribbles Pseudokinases in Biology and Disease

“…TRIB pseudokinases represent a subbranch of the CAMK subfamily in the human kinome [3] and, although a shared eukaryotic evolutionary origin is apparent from bioinformatic comparisons 10, 11, 12, the molecular basis for their specific evolutionary trajectory and associated cellular functions has not been evaluated in depth. Indeed, several critical questions in the TRIB field remain (see Outstanding Questions).…”

The evolving world of pseudoenzymes: proteins, prejudice and zombies

“…In our laboratories, we do of course have our particular research favourites, and these generally represent the analysis of specific human pseudokinases. For example, we think that MLKL, which controls necroptosis through a switch-like mechanism [14, 18] and the Tribbles pseudokinases, which modulate protein ubiquitination [15, 25], are of outstanding interest as models for understanding basic signalling mechanisms by pseudoenzymes. Both of these exemplify the vast and varied manner in which pseudoenzymes can modulate cell signalling outputs via roles in mediating protein interactions, including nucleating and controlling the output from multiprotein signalling complexes.…”