Developmental roles of tribbles protein family members

Authors

  • Leonard L. Dobens Jr.,

    Corresponding author
    1. Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri
    • Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110
    Search for more papers by this author
  • Samuel Bouyain

    1. Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri
    Search for more papers by this author

Abstract

The gene tribbles (trbl), identified 12 years ago in genetic screens for mutations that control both cell division and cell migration during embryonic Drosophila development, is the founding member of the Tribbles (Trib) family of kinase-like proteins that have diverse roles in cell signaling, tissue homeostasis, and cancer. Trib proteins share three motifs: (1) a divergent kinase region (Trib domain) with undetermined catalytic activity, (2) a COP1 site used to direct key target proteins to the proteosome for degradation, and (3) a MEK1 site that binds and modulates MAPKK kinase activity. The notion that Tribs act as scaffolding proteins to balance signaling levels in multiple pathways retains an attractive simplicity, but given recent data showing that divergent kinases act by means of novel catalytic mechanisms, the enzymatic activity of Tribs remains untested. Here, we focus on the role of Tribs during development. Developmental analysis of Drosophila trbl phenotypes reveals tissue-specific, sometimes contradictory roles. In mammals, multiple Trib isoforms exhibit overlapping and tissue-specific functions. Recent data indicate the mechanism of Trib activity is conserved and requires the Trib domain. Finally, we discuss the connections between Tribs in disease and cancer that have implications for their normal roles during organogenesis. Developmental Dynamics 241:1239–1248, 2012. © 2012 Wiley Periodicals, Inc.

INTRODUCTION

The tribbles gene was identified first in Drosophila mutational screens for genes that control cell proliferation and migration. The trbl gene sequence revealed a divergent kinase structure similar to a previously identified cDNA (C5FW, subsequently renamed Trib2) induced by mitogens in dog thyroid cells (Wilkin et al., 1996; Wilkin et al., 1997), indicating that this family of genes is conserved throughout the metazoan lineage. Because fly genes are named after their phenotype, the overproliferating mesodermal cells seen in trbl mutant embryos resembles, as it were, “Tribbles,” the fictional small, furry, and fecund animals who vexed the crew of the Enterprise in the classic “Trouble with Tribbles” episode from the original Star Trek television series.

Members of the Tribbles (Trib) gene family share a central atypical protein kinase motif along with a C-terminal conserved COP1 site that binds ubiquitin ligase. Although Tribs have been postulated to interact with a wide range of target molecules, acting as ‘molecular scaffolds' to regulate multiple pathways simultaneously, it remains unclear whether these are as well authentic kinases that act by means of a novel catalytic mechanism. Recent reviews have focused on the role of these genes in signaling, cancer and metabolism (Kiss-Toth, 2011; Yokoyama and Nakamura, 2011; Angyal and Kiss-Toth, 2012; Prudente et al., 2012), and here we focus on Tribs in animal development. We update work done in Drosophila, relate this work to vertebrate model systems, and then highlight studies in cell signaling and cancers that have special meaning for the contribution of Tribs to normal development. We conclude by posing a few questions that swirl around this enigmatic family of proteins.

DROSOPHILA Trbl HAS VARIED, TISSUE-SPECIFIC FUNCTIONS

Loss and Gain-of-Function Screens in the Year 2000: Trbl Coordinates Cell Proliferation and Migration

A total of four genetic screens published in the year 2000 identified a requirement for the tribbles gene in cell division and migration in several tissues. Two similarly designed deficiency screens identified lesions in the trbl gene that caused a failure of gastrulation (Fig. 1A,B) due to overproliferation of mesodermal cells (Grosshans and Wieschaus, 2000; Seher and Leptin, 2000). During normal gastrulation, the ventral mesoderm arrests at G2 of the cell cycle and then undergoes ingression and dorsal migration to pattern muscle and other tissue derivatives. Trbl blocks mesoderm proliferation by directing turnover of String phosphatase, preventing activation of Cyclin/cdc2, perhaps to accommodate epithelial to mesenchymal cell shape changes incompatible with cell rounding that typically occurs during division. Close examination of trbl mutants reveals that excess cell proliferation occurs both earlier at blastoderm formation and later in both the migrating germ line pole cells and cells in the head (Mata et al., 2000; Seher and Leptin, 2000), indicating that in at least three embryonic tissues Trbl regulates cell division to promote cell migration (Duncan and Su, 2004).

Figure 1.

Tribbles is required for proper tissue infolding in Drosophila and Xenopus. A: Ventral gastrulation in stage 6 wild-type (WT) Drosophila embryos, visualized by in situ hybridization using probe to the single-minded gene (sim, blue). At stage 6, invagination is nearly complete. B: In trbl mutant embryo, black arrows highlight the delay in defective gastrulation at a comparable stage. C,D: In Xenopus, antisense morpholino knockdown of Xtrib2 (D) leads to defects in neural tube closure, with delayed infolding (black arrows, D) documented by expression of the Xenopus Slug gene (XSlu) when compared with normal gastrulation following injection of a mutated morpholino (C). (A,B) from Mata et al. (2000) and (C,D) from Saka and Smith (2004), with permissions, with permissions from the respective publishers.

Two genetic interactions shed light on trbl activity during gastrulation. First, double mutants for trbl and string invaginate normally, suggesting that the main role of Trbl at this stage is to block String phosphatase activity (Grosshans and Wieschaus, 2000). And second, while injection of wild-type Trbl is sufficient to block excess cell divisions, injection into snail mutant embryos does not, suggesting that snail activates expression of a Trbl co-activator, whose identity and mechanism of activity remains undetermined (Grosshans and Wieschaus, 2000). Notably, during mouse gastrulation cell proliferation is regulated by Snail, and knockdown of Trib3 in tumor cells leads to down-regulation of Twist and Snail, increased E-cadherin and inhibition of migration (Hua et al., 2011). Thus, in quite diverse organisms Twist, Snail, and Trbl may act as a genetic cassette to control cell migration events (Ciruna and Rossant, 2001; Ip and Gridley, 2002).

Two gain-of-function screens conducted in the fly ovary identified additional effects of trbl mutations on cell migration and division. Trbl misexpression in the germ line triggered an extra round of cell division during oocyte differentiation (Mata et al., 2000). Complementarily, trbl mutant ovaries exhibit a reduction in germ cell number and failure of oocyte differentiation (Huynh and St Johnston, 2004). Trbl misexpression induces transit amplification of the male gamete precursors during spermatogenesis suggesting that Trbl spurs cell division in the germ line, opposite to its role in the embryonic mesoderm (Schulz et al., 2004).

In a second screen conducted in the ovarian follicle cell epithelium, which surrounds the oocyte, Trbl misexpression specifically blocks migration of the border cells, a cell cluster that constructs features of the mature eggshell (Rorth et al., 2000). A mechanism for this was defined: Trbl binds to and promotes proteosome degradation of the fly C/EBP (CAAT enhancer binding protein) homolog encoded by the gene slow border cells (slbo), a key promoter of border cell migration. Subsequent work indicates that this interaction is highly conserved with Trib1 and Trib 2 in several mammalian tissues: (1) in acute myelogenous leukemia (AML) tumors, Trib1 accelerates degradation of C/EBPα; (2) up-regulation of C/EBPβ occurs in Trib1 knockout mice (Yamamoto et al., 2007; Keeshan et al., 2008); (3) overexpression of Trib2 reduces C/EBPα levels in a proteasome-dependent manner, resulting in myeloid differentiation (Keeshan et al., 2006); and (3) Trib2 (but not Trib3) increases degradation of C/EBPβ, effectively suppressing differentiation of 3T3-L1 preadipocytes (Naiki et al., 2007).

2000-Present: Trbl Controls Cell Division During Tissue Patterning

Since this early work, trbl has been identified in several genetic screens as either an antagonist or promoter of cell division, depending on the context. In some tissues, Trbl blocks cell proliferation: (1) in the eye, Trbl misexpression enhanced while conversely, trbl mutants suppressed, misexpression of Dwee1, a kinase that phosphorylates and blocks Cdc2/Cdk activity (Price et al., 2002); (2) in S2 cells, RNAi knockdown of Trbl increases the number of S2 cells in G1 with no effect on cell size (Bettencourt-Dias et al., 2004); and (3) trbl mutations suppress eye overgrowth phenotypes resulting from overexpression of the Jak/Stat pathway ligand Unpaired (Mukherjee et al., 2006).

In other tissues, Trbl promotes cell division. In one instance, Trbl suppresses a block in cell division caused by a dominant negative version of the Rho kinase Pebble (Gregory et al., 2007). As mentioned above, Trbl misexpression in male spermatagonia increases transit amplification of stem cells (Schulz et al., 2004); subsequently, it was shown that trbl RNA was down-regulated by the Prospero transcription factor, a negative regulator of spermatogonial stem cell proliferation (Choksi et al., 2006).

Finally, Trbl has been identified as a key link between the cell cycle and cell fate determination during bristle lineage formation. During normal patterning of the external sensilla, a precursor cell pI divides once in the plane of the epithelium to give daughter cells pIIa and pIIb, which divide again to give rise to four differentiated terminal cell types: neurone, sheath, socket, and bristle shaft. In mutant screens for defects in macrochatae, loss of bristles was observed both in trbl mutations (Norga et al., 2003) and following Trbl misexpression (Abdelilah-Seyfried et al., 2000; Fichelson and Gho, 2004). Gene markers revealed that the invariant cell lineage that forms the bristles is disrupted following Trbl misexpression resulting in a loss of pIIa cell fates. Notably, co-expression of an activated version of the Notch receptor suppresses this phenotype indicating that Trbl reduces Notch signaling during bristle patterning. Collectively, these phenotypes are shared by several members of the ubiquitin-proteasome pathway and assign Trbl to a class of cell cycle inhibitors that modulate Notch signaling pathway during the step-wise assignment of cell fate during cell division (Abdelilah-Seyfried et al., 2000).

In fully differentiated brain cells, Trbl was identified in a screen for genes that affect memory (LaFerriere et al., 2008). trbl mutations reduce ‘place memory’ and elevated olfactory memory yet show no ethanol sensitivity, placing it in a novel category of genes that disconnect memory from the ethanol response. Because proper brain development requires oriented cell migration linked to cell division, this memory defect may be connected to an earlier role for trbl in neurogenesis, but that remains speculative.

TRIBS IN VERTEBRATE DEVELOPMENT

Mouse Knockouts: Do Overlapping Trib Functions Explain Lack of Phenotypes?

All three mouse Trib isoforms—Trib1/C8FW/SKIP1, Trib2/C5FW/SKIP2/SINK and Trib3/NIPK/SKIP3 (Wilkin et al., 1996, 1997; Mayumi-Matsuda et al., 1999)—are reported dispensable for development (Okamoto et al., 2007; Yamamoto et al., 2007; Takasato et al., 2008). Trib3 knock-in lacZ expression occurs in the liver and adipose tissues, however, the mutant show neither liver defects, nor a change in the phosphorylation state of Akt (Okamoto et al., 2007). Trib2 is reported dispensable for kidney and mouse development (Takasato et al., 2008). Trib1 knockout mice are reported to have some perinatal mortality with inflammation of white adipose tissue in neonates (Satoh, unpublished data, cited in Ostertag et al., 2010) and are infertile (cited in Yamamoto et al., 2007). While work is ongoing to analyze these knockouts in more detail, the expectation is that redundant roles for Trib isoforms likely exist.

Xenopus: A Conserved Function for Trbl in Cell Division and Migration

Injection into Xenopus embryos of a morpholino to the Trib2 homolog Xtrb2 results in dorsal mesoderm involution defects, a phenotype that resembles gastrulation invagination defects seen in Drosophila trbl mutants (Fig. 1A,B). Rapid embryonic cell divisions are slowed at the Xenopus mid-blastula transition (MBT), likely because cell rounding during cytokinesis is incompatible with actin cytoskeletal remodeling during migration (reviewed in Duncan and Su, 2004). Consistent with this finding, experimental manipulations that prevent the MBT delay, such as the injection of morpholinos that deplete maternal Wee1 or overexpression of Cdc25/string, also result in gastrulation defects (Murakami et al., 2004).

However, close inspection of Xtrb2 knockdown finds this treatment delays, rather than accelerates progression through mitosis, an embryonic phenotype opposite to that of the fly gastrulation mutant defect (Saka and Smith, 2004). At later stages, Xtrb2 morpholinos led to (1) a block to neural crest delamination and migration associated with a disruption of G1/S; and (2) disruption in neural tube closure (Fig. 1C,D), somite segmentation, and eye development, suggesting an important role for Xtrb2 in these cell movements (Saka and Smith, 2004).

Adipocyte Differentiation and Hematopoiesis: Clues to Trib Roles in Cell Differentiation

As discussed above, Drosophila trbl coordinates precursor cell division and differentiation during neuronal patterning. In a similar manner, mammalian Tribs appear to regulate differentiation linked to cell division during (1) hematopoiesis (Lin et al., 2007; Eder et al., 2008; Sathyanarayana et al., 2008), (2) myogenesis (Kato and Du, 2007; Sung et al., 2007), (3) vascular smooth muscle formation (Chan et al., 2010), (4) lymphocyte differentiation (Selim et al., 2007), and (5) adipogenesis (described below).

In preadipocytes, expression of Trib3 and Trib2 is transiently suppressed before mitotic clone expansion (MCE), a period of growth arrest leading to a synchronous reentry into the cell cycle followed by ordered expression of genes that produce the mature adipocyte phenotype. This down-regulation of Tribs is critical to fat cell formation as on the one hand, continuous expression of Trib3 or Trib2 effectively blocks proliferation and adipogenesis, while on the other, knockdown leads to premature MCE and differentiation (Bezy et al., 2007; Naiki et al., 2007). How Tribs regulate the cell cycle in preadipocytes is not clear, but this block to differentiation is exerted in several ways: (1) repression of the activity of key transcription factors including C/EBPβ, C/EBPα (Bezy et al., 2007) and PPARγ2 (Takahashi et al., 2008), (2) inhibition of Akt phosphorylation of Foxo1, which must be inactivated to allow fat cell development to proceed, and (3) degradation of acetyl coenzyme A carboxylase (ACC), the rate-limiting enzyme in fatty acid synthesis (Qi et al., 2006). In addition, Trib1 binds LAP, the activating isoform of C/EBPβ, indicating that each Trib isoform contributes to the control of adipogenesis (Naiki et al., 2007).

TRIB STRUCTURE AND LOCALIZATION SUGGEST DIVERSE DEVELOPMENTAL FUNCTIONS

While our knowledge of their roles in vertebrate development is limited, extensive work in tissue culture reveals that each Trib isoform has specific (1) functions, (2) expression patterns, and (3) subcellular localizations that collectively suggest tissue-, stage-, and cell compartment-specific roles during development.

Subcellular Localization of Tribs

Depending on the cell type studied, Trib proteins localize variously to the cytoplasm, nuclear compartment, and cell cortex. In the cytoplasm, Tribs direct the proteosomal degradation of key signaling proteins, including ACC1 (Qi et al., 2006), SMURF1, FoxO (Matsumoto et al., 2006) and C/EBP (Rorth et al., 2000; Naiki et al., 2007; Selim et al., 2007; Dedhia et al., 2010; Keeshan et al., 2010; Grandinetti et al., 2011). Also in the cytoplasm, Tribs bind to inactivate LAP (Naiki et al., 2007), MKKs (Kiss-Toth et al., 2004; Wang et al., 2011), and Akt (Du et al., 2003).

Whereas they have no DNA binding capacity themselves, in the nucleus Tribs are tightly associated with target promoters and exhibit transcriptional co-activator and co-repressor functions. Trib3 binds (1) PPARγ2, where it blocks target gene expression (Takahashi et al., 2008); (2) CTIP, the CtBP interacting protein (Xu et al., 2007); (3) CHOP (Ohoka et al., 2005); and (4) ATF4 (Jousse et al., 2007). Trib1 associates with inflammation responsive promoter elements in white adipose tissue (Ostertag et al., 2010) and acts as a retinoic acid receptor RAR co-repressor bound to target genes (Imajo and Nishida, 2010).

The subcellular localization of Trib3 undergoes dynamic changes to promote both bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFβ) signaling. In BMP-responsive osteoblasts, Trib3 is bound to the intracellular portion of the BMPRII receptor at the cell cortex and is released upon ligand binding. In the cytoplasm, Trib3 binds and degrades SMURF1, an E3 ubiquitin ligase specific for the BMP mediator Smad, effectively stabilizing Smads (Chan et al., 2007). In TGFβ-responsive HepG2 cells, Trib3 binds the MH domain of Smad3, enters the nucleus, and acts as a co-factor to modulate target gene expression (Hua et al., 2011). Thus, Tribs act in the cortex, cytoplasm and nucleus at each step of the pathway to potentiate TGFβ/BMP signaling.

Conserved Features of Tribs: A Swiss Army Knife of Cell Signaling

The canonical Trib protein has at least three defined domains that contribute to function: (1) a nonconserved N-terminal domain, (2) a central kinase-like structure (the Trib domain), and (3) a C-terminal region including conserved COP1 and MEK1 binding sites, and each domain contributes to function (Fig. 2A,B). In the N-terminus, mutations fail to induce AML in a Hox9 coexpression assay, while mutations in the COP1 binding site bind but fail to degrade C/EBP and also compromise the leukemogenic activity of Trib1 and Trib2. The Trib MEK1 motif ILLHPWF is thought to mediate interactions with multiple MAPkinase kinases (MAPKKs), including (1) Trib3 binding MLK3 in pancreatic β-cells (Humphrey et al., 2010); (2) Trib2 binding and inactivation of MKK7 and MEK1, involved in Erk and Jnk signaling, respectively (Eder et al., 2008); and (3) Trib1 binding and enhancing phosphorylation of ERK1/2, important for its leukemogenic activity (Yokoyama et al., 2010). Interdependence of these domains is suggested by Trib1 mutants that lack the MEK1 binding motif and fail to degrade C/EBPα, suggesting the COP1 function of Tribs is dependent on activation of the MEK1/ERK pathway (Keeshan et al., 2010).

Figure 2.

The structural homology and diverse functions of the Tribbles protein family. A: Clustal alignment of Drosophila Trbl with mammalian Tribs reveals conserved central kinase-like domain (red) with key features boxed. B: Conserved motifs (common identical residues in black, conserved residues shaded) in the catalytic region (red), MEK-1 (blue) and Cop-1 (green) binding sites. Key residues found in Trbl family catalytic region are contrasted with those found in canonical kinase (bottom line). Cop-1 binding site alignment follows that of Yokoyama and Nakamura (2011). C: Schematic of some Trib functions. From left to right: Trbl/Trib2 recruits COP1 to C/EBP in the border cells and adipocytes, respectively, to modulate cell differentiation; Tribl recruits COP1 to Cdc25 to block cell division in the Drosophila mesoderm; Trib1 interacts with MEK1 and enhances phosphorylation of ERK1/2, which promotes cell proliferation and suppresses apoptosis in pancreatic β-cells; Trib3 binds Akt to block Foxo phosphorylation to block cell growth in several insulin target tissues (see text for references).

Because they lack key features thought required for catalytic activity, Tribs were initially deemed pseudokinases. Functional kinase domains exhibit three highly conserved sequence motifs (Fig. 2B): (1) an N-terminal VAIK domain that interacts with the α and β phosphates of bound ATP, (2) a central HRD domain in which the catalytic aspartic acid residue functions as a base acceptor to achieve protein transfer, and (3) a C-terminal DFG domain in which the aspartic acid binds Mg2+ to coordinate the β and γ phosphates of ATP in the binding cleft (reviewed in Boudeau et al., 2006). In contrast, the Trib domain includes a degenerate LRD motif (HRD) in the potential catalytic site but completely lacks the VAIK and DFG domains (Hegedus et al., 2006; Fig. 2B). Thus, an outstanding question is whether Tribs act as nonfunctional pseudokinases, decoy kinases that require an intact Trib domain, or as true kinases that function by a novel mechanism.

Some data indicate that the Trib domain does not contribute to function: (1) mutations in the Trib domain of fly Tribbles did not affect its ability to block cell proliferation in the embryo (Grosshans and Wieschaus, 2000), (2) Trib3 binding to CHOP was unaffected by a K to R catalytic site mutant (Ohoka et al., 2005), and (3) the Trib2 K/R mutation had no effect on the protein's pro-apoptotic functions (Lin et al., 2007).

Other data suggests that the Trib domain is critical for some functions: (1) mutations interfere with its ability to direct degradation of C/EBPα (Keeshan et al., 2010), (2) it is required for interaction between Trib2 and Smad3 to mediate TGFβ signaling (Hua et al., 2011), (3) it is sufficient to block MAPKK activity (Kiss-Toth et al., 2006), and (4) Q to R mutations in this domain act as Trib3 gain-of-function alleles to decrease Akt phosphorylation and predispose populations bearing this change to insulin resistance associated with type 2 diabetes (Prudente et al., 2009).

It remains unresolved whether the Trib domain acts catalytically to sponsor the transference of a phosphate group to a substrate. In this light, it is notable that mutations in the Trib domain that make Trib2 better resemble a tyrosine kinase actually disrupt its ability to direct C/EBP turnover, suggesting that however it functions, the highly conserved structure of the Trib domain must be intact for activity (Keeshan et al., 2010).

Overlapping and Opposing Activities of Trib Isoforms

While flies have one copy of the Trbl gene, multiple isoforms in vertebrates suggest a diversification of functions. In support of this, introduction of Xtrb2 (Saka and Smith, 2004), or mouse Trib1 and Trib2 (Venessa Masoner and L. Dobens, unpublished data), into transgenic flies cannot rescue tribbles mutant phenotypes. Nevertheless, mouse knockouts of Trib1, Trib2, or Trib3 are viable, indicative of overlapping roles as well, and work in tissue culture supports the notion that the three isoforms have both common and unique roles.

Examples of functional overlap can be seen in hematopoietic stem cells, where both Trib1 and Trib2 effectively inhibit and degrade C/EBPα, correlating with their ability to produce leukemia, while Trib3 had none of these activities. In fasting or diabetic mice Trib3 levels were increased, while both Trib3 and Trib2 were sufficient to block insulin dependent phosphorylation of Akt when misexpressed (Du et al., 2003). In adipocytes, each isoform contributes to fat cell differentiation: (1) Trib1 and Trib2 degrade C/EBPβ, whereas Trib3 blocks transactivation but does not degrade C/EBPβ (Bezy et al., 2007; Naiki et al., 2007); (2) Trib3 and not Trib1 or Trib2 degrades ACC and the Trib3 COP1 binding site uniquely can confer this property (Dedhia et al., 2010); and (3) Trib3 and Trib2 inhibit Akt activation, but Trib2 less effectively (Naiki et al., 2007).

Tissue-specific regulation of Trib isoform expression is hinted at by some results from tissue culture. As mentioned above, Trib2 and Trib3, but not Trib1, are down-regulated during adipocyte differentiation (Naiki et al., 2007). Posttranscriptional regulation of Trib3 variants is suggested by the occurrence of upstream open reading frames (Ord et al., 2009). Finally, even the levels of one isoform may exert distinct influences: low levels of Trib3 are reported to inhibit p38 and enhance Jnk and ERK activation, while at higher levels of Trib3, all three kinases are inhibited (Kiss-Toth et al., 2004). This outcome implies that Trib isoform levels relative to the levels of key partners may dictate tissue-specific effects.

Finally, subcellular localization varies among isoforms: GFP fusions reveal a nuclear localization for mouse Trib1 and Trib3 while mouse Trib2-GFP is cytoplasmic (Kiss-Toth et al., 2006). Of interest, GFP-tagged Xtrb2 expressed in injected embryos was transiently associated with mitotic spindles during cell division, suggesting that interactions with subcellular structures are regulated and functionally significant (Saka and Smith, 2004).

DEVELOPMENTAL IMPLICATIONS OF TRIBS IN CANCER AND INSULIN SIGNALING

During development, cells must integrate signals to grow and proliferate in a coordinated manner to build properly proportioned tissues, organs, and appendages. The role demonstrated for Tribs in cell migration, cell growth and the cell cycle makes it unsurprising that these genes have been connected to cancers and defects in insulin-regulated cell growth.

Tribs and Cell Proliferation

As noted above, in different fly tissues trbl either promotes or impedes cell division, and mammalian Tribs as well exhibit opposing effects on cell proliferation, cell differentiation, and cell death, depending on the cell line or tumor type examined.

Tribs as oncogenes.

Trib2 is up-regulated in acute myelogenous leukemia tumors (AML) and misexpression leads to growth advantages and transplantable tumors (Keeshan et al., 2006). While Trib2 directs turnover of C/EBPα in these tumor cells, C/EBPα mutants do not result in AML, making it likely that Trib2 has more targets (Keeshan et al., 2006). In melanomas, Trib2 facilitates growth and survival by down-regulating FOXO activity (Zanella et al., 2010). Trib levels are increased in lung cancers (Grandinetti et al., 2011), breast cancers (Wennemers et al., 2011a, b), B-cell chronic lymphocytic leukemia (B-CLL Johansson et al., 2010), and cancers of the esophagus and colon (Miyoshi et al., 2009). Because Trib3 is induced in tumors experiencing stress/starvation conditions, it may contribute to the ability of cells to grow under the nutrient limiting conditions typical within a tumor (Bowers et al., 2003; Schwarzer et al., 2006). In atherosclerotic arteries, Trib1 controls smooth muscle cell proliferation by regulating Jnk kinase signaling (Sung et al., 2007).

Tribs as tumor suppressors.

The ability of Tribs to promote AML noted above was contradicted by microarrays showing that Trib1 and Trib2 are down-regulated in these tumors and both effectively inhibit Jnk-mediated growth (Gilby et al., 2010). Thus, their role as either oncogene or tumor suppressor of AML remains unresolved. Trib3 mediates the tumor suppressing effects homocystein in endothelial cell model (Zou et al., 2009), cannabinoid in hepatocellular carcinomas (Vara et al., 2011), tetradecylthioacetic acid (TTA) in colon cancer cells (Lundemo et al., 2011), and dehydroxymethylepoxyquinomicin (DHMEQ) in liver cancer cells (Lampiasi et al., 2009).

Tribs promote apoptosis.

In the hematopoietic cells starved of the cytokine GMCSF, increased Trib2 levels precede apoptosis. While knockdown suppresses the effect of GMCSF withdrawal, overexpression of Trib2 stimulates apoptosis by means of a caspase, not the proteosome (Lin et al., 2007). Trib3 also promotes apoptosis in macrophages (Shang et al., 2009), gliomas (Salazar et al., 2009), pancreatic β-cells (Humphrey et al., 2010), and chondrocytes (Cravero et al., 2009).

Tribs and Cell Growth

Under starvation conditions, Trib3 is up-regulated in the liver and binds and blocks insulin stimulation of the activity of Akt (PKB), ERK1/2 and IRS1 kinases to spur glucose production (Du et al., 2003; Liu et al., 2010). Complementarily, Trib3 binds and directs ACC to the proteosome to promote lipolysis (Qi et al., 2006). These findings have unleashed a torrent of studies focused on Trib3 as an endogenous antagonist of insulin signaling modulating glucose metabolism and fatty acid oxidation during tissue homeostasis. Consistent with this, diabetic mice have increased levels of Trib3 levels and experimental misexpression of Trib3 mimics the disorder with hyperglycemia and glucose intolerance (Du et al., 2003). A Q84R missense mutation in Trib3 in human populations predisposes the carrier to insulin resistance and cardiovascular risk (Prudente et al., 2005) and the mutation has dominant properties including increased Akt binding (Liew et al., 2010) and stimulation of ras-MAPK-MEK leading to, respectively, reduced cell growth and increased cell proliferation (Formoso et al., 2011).

During an intrinsic developmental program, growth rate and cell proliferation must be harmonized with available nutrients. As well, genetically programmed differences in cell growth rate lead to changes in organ size between related species. An example of this is digit length resulting from chondrocyte proliferation, which accounts for the dramatic difference in length between a bat's fingers, which form the wing, and those of their mouse-like ancestors. The impact of Tribs on insulin, ras-MAPK and hormone receptor signaling makes it is reasonable to assume that these proteins influence the balance of cell growth and proliferation to pattern the final size of organs.

PERSPECTIVES

Fly studies indicate that Trbl plays a critical role in coordinating the cell cycle with cell migration to fine-tune development, yet a more confusing picture emerges when examined in detail: in migrating cells, Trbl either restricts (follicle cells) or promotes (mesoderm) cell migration; in proliferating cells, Trbl either blocks (mesoderm and blastoderm) or promotes (female and male germ line) the cell cycle. In mammalian systems as well, Tribs either potentiate or block cell division, apoptosis, and growth, depending on the tissue or cell line examined. The ability of Tribs to bind key tissue-specific regulatory proteins to block their activity (Akt), promote their activity (Smad3), or direct proteosomal degradation (i.e., C/EBP) likely represent mechanisms to explain these context-dependent differences (Fig. 2C).

One key question is whether Tribs are essential genes? While published work on mouse knockouts of individual isoforms reveal no notable developmental defects, reduction of Xenopus Xtrb2 does; thus, it is likely that double and triple Trib mouse knockouts will reveal informative phenotypes. Fly trbl mutant alleles exhibit variable defects in mesoderm internalization yet result in weakly fertile escaper adults (Grosshans and Wieschaus, 2000) leading to the conclusion that trbl has a nonessential role to confer precision on a rapidly unfolding developmental program (reviewed in Johnston, 2000; Leptin, 2005). In support of this notion, Trbl turnover of String protein during mesoderm invagination is augmented by activity of the Held-out wing (How(l)) protein, which binds and degrades string RNA (Nabel-Rosen et al., 2005). With two posttranscriptional controls to limit String phosphatase activity in this tissue, the loss of Trbl might be thought inconsequential. However, the original alleles of trbl were not protein null, and our own data indicates that a complete deletion of the trbl gene is embryonic lethal, suggesting an essential developmental function (Rahul Das and L. Dobens, unpublished data).

Finally, what is the role of the conserved, divergent kinase (Trib) domain? Pseudokinases represent 10% of sequenced kinase-like molecules and several proteins initially classified as nonfunctional pseudokinases—e.g., Wnk and Prpk, which lack the VAIK motif, and Haspins, Titins and Ryk/Drl, which lack an identifiable DFG—have been demonstrated to have kinase activity (Boudeau et al., 2006). In flies, a mutation in the Trib domain retains wild-type activity to block cell division, which would indicate this domain does not catalyze phosphate transfer. However, this mutation made was in lysine K268 of the proposed catalytic loop (Grosshans and Wieschaus, 2000) rather than the highly conserved K266, where catalytic null mutations are typically made. When the corresponding mutation was made in the catalytic loop of the Trib domain of Trib3, its function was abrogated (Naiki et al., 2007). Because kinases typically autophosphorylate, it is notable that Trib2 and Drosophila Trbl are phosphoproteins (Wilkin et al., 1997; Bodenmiller et al., 2008). Thus, it will be important to ascertain if they remain autophosphorylated when the catalytic loop of kinase domain is mutated. While the issue will be resolved ultimately by the identification of bonafide Trib substrates, we must allow for the possibility that Tribs work as active kinases in some contexts, and passive adaptor proteins in others.

Acknowledgements

We thank Flybase curators whose ongoing work to identify and catalog the fly literature provided critical new information about tribbles unavailable in typical online searches. We thank Dr. Endre Kiss-Toth and members of the Dobens lab for helpful comments on the manuscript. We thank Dr. Yasushi Saka for kindly sending the high quality panels of his data used in Figure 1. L.L.D. thanks Kenneth Merrifield for early assistance in this project.

Ancillary