Control of Cell Growth and Proliferation by the Tribbles Pseudokinase: Lessons from Drosophila

Abstract

The Tribbles (Trib) family of pseudokinase proteins regulate cell growth, proliferation, and differentiation during normal development and in response to environmental stress. Mutations in human Trib isoforms (Trib1, 2, and 3) have been associated with metabolic disease and linked to leukemia and the formation of solid tumors, including melanomas, hepatomas, and lung cancers. Drosophila Tribbles (Trbl) was the first identified member of this sub-family of pseudokinases and shares a conserved structure and similar functions to bind and direct the degradation of key mediators of cell growth and proliferation. Common Trib targets include Akt kinase (also known as protein kinase B), C/EBP (CAAT/enhancer binding protein) transcription factors, and Cdc25 phosphatases, leading to the notion that Trib family members stand athwart multiple pathways modulating their growth-promoting activities. Recent work using the Drosophila model has provided important insights into novel facets of conserved Tribbles functions in stem cell quiescence, tissue regeneration, metabolism connected to insulin signaling, and tumor formation linked to the Hippo signaling pathway. Here we highlight some of these recent studies and discuss their implications for understanding the complex roles Tribs play in cancers and disease pathologies.

Keywords: Trib protein family; cancer; growth; pseudokinase.

Figure 1

The paradigm of Trib family function. (A) Proposed structure of the Drosophila Trbl kinase-like domain core, based on the structure of Trib1. Kinase/Trib homologies are indicated (aspartic acid–leucine–lysine (DLK)/DLK, aspartic acid–phenylalanine–glycine in kinases (DFG)/SLE and valine–alanine–isoleucine–lysine in kinases (VAIK) is phenylalanine-leucine-cysteine-arginine (FLCR). SLE motif resides in a flexible domain, similar to its mammalian counterpart. Location of the MEK (Mitogen-activated protein kinase kinase) binding site and C-terminal tail (orange) is indicated. (B) Simplified representation of the bi-lobed Trbl pseudokinase structure unbound (right) and bound (left) to substrate (yellow). The C-terminal tail is released upon degron binding and the SLE motif swings from an “in” to “out” configuration [56]. MEK1 and COP1 sites on the tail are indicated. (C) The primary structure of the flexible SLE domain is part of a highly conserved region in the kinase-like domain. The residues in red indicate helical structure, the residues in blue indicate beta sheet structure. SS = secondary structure, h = helical, and e = β-sheet.

Figure 2

The Drosophila Tribbles carboxy-terminal tail is divergent from other metazoan Tribbles. The C-terminal tails of Tribbles proteins from 136 taxa were used to build a phylogenic tree rooted on the human homolog, Trib2; distances are displayed at nodes of tree. Drosophila melanogaster Trbl is highlighted in green and clusters with Drosophilidae and related fruit flies sharing a C-terminal tail bearing a “non-VP” motif distinct from other arthropods bearing a COP1 binding site similar to other metazoan (“VP”) and the three human homologs (human Trib1, 2, and 3 are each red in tree). Methods: Drosophila Tribbles protein BLAST was run against the NCBI (National Center for Biotechnology Information) database of Arthropoda (taxid:6656) and Drosophilidae (taxid:7214). The non-redundant database was used with the following settings: Expect threshold of 10, word size of 6, Matrix-BLOSUM62, Gap Costs-Existence:11 extension:1, Computational adjustments-Conditional Score matrix adjustment. Sequences were then eliminated from the data set that did not retain important defining features of the Tribbles pseudokinase core. Each sequence was then trimmed to only represent the C-terminal tail as marked by the MEK1 binding site. The gap-spaced alignment of 137 sequences was analyzed for optimal tree model using MEGA X (all sites were used in a neighbor-joining tree using the maximum likelihood statistical method). MEGA X [58,59] was used to make a maximum likelihood tree and 50x bootstrap phylogeny test, using the Jones–Taylor–Thornton substitution model [60] with gamma (n = 4) distribution and nearest-neighbor interchange. The tree was then edited in FigTree v1.4.4.

Figure 3

Trbl upstream regulation and candidate downstream targets. (A) Based on work from Gerlach et al., a comprehensive pathway of Tribbles function incorporates Hippo regulation by the bantam miRNA [108]. (B) Alignment of the Trib degron from human C/EBP proteins and the Drosophila C/EBP ortholog, Slbo, with candidate proteins from the Drosophila genome. Methods: A protein-protein BLAST search was conducted of non-redundant Drosophila sequences derived from the GenBank coding sequence (CDS) translations (including the PDB [Protein Data Bank], SwissProt, PIR [Protein Information Resource], and PRF [Protein Research Foundation]) with four sequences derived from the degron Slbo (ICEhEtSIDISAYIDPAA). The four sequences (JXXXEXTJDJXXXID, JXXXEXSJDJXXXID, LXXXEXTLDLXXXID, and IXXXEXTIDIXXXID) were chosen to exclude poorly conserved residues (X) and include where possible ambiguous amino acids (B, Z, and J) with the goal of expanding the binding sites obtained from the search. More functional data to evaluate important residues in the candidate binding sites was absent and top-ranked homologies overall are listed followed by strong homologies to subdomains of the motif.