MIG-10/RIAM/lamellipodin (MRL) proteins link activated Ras-GTPases with actin regulatory Ena/VASP proteins to induce local changes in cytoskeletal dynamics and cell motility. MRL proteins alter monomeric (G):filamentous (F) actin ratios, but the impact of these changes had not been fully appreciated. This study reports here that the Drosophila MRL ortholog, pico, is required for tissue and organismal growth. Reduction in pico levels resulted in reduced cell division rates, growth retardation, increased G:F actin ratios and lethality. Conversely, pico overexpression reduced G:F actin ratios and promoted tissue overgrowth in an epidermal growth factor (EGF) receptor (EGFR)-dependent manner. Consistently, in HeLa cells, lamellipodin was required for EGF-induced proliferation. pico and lamellipodin share the ability to activate serum response factor (SRF), a transcription factor that responds to reduced G:F-actin ratios via its co-factor Mal. Genetics data indicate that mal/SRF levels are important for pico-mediated tissue growth. It is proposed that MRL proteins link EGFR activation to mitogenic SRF signaling via changes in actin dynamics (Lyulcheva, 2008).

The construction of properly sized and functional tissues and organs during animal development requires tight control of cell growth, proliferation, differentiation, and death. Networks of intracellular signal transduction pathways that respond to various secreted ligands and cell surface proteins coordinate these processes. Elucidating the nature of the intracellular signaling networks that connect extracellular stimuli to basic cellular machinery controlling proliferation, growth, and morphology is not only critical for the understanding of tissue size regulation during normal development, but is also important for the identification of aberrant events underlying numerous disease processes, including cancer (Lyulcheva, 2008).

A number of pathways regulating cellular development are initiated by ligation of transmembrane receptor tyrosine kinases (RTKs), such as the epidermal growth factor (EGF) receptor (EGFR). One of the key mediators of RTK signaling is the Ras GTPase, capable of activating proteins harboring Ras association (RA) domains to initiate downstream signaling pathways, such as the mitogen-activated protein kinase (MAPK) cascade, and ultimately resulting in changes in gene transcription. The Ras/MAPK and other canonical RTK signaling pathways have been well characterized, yet they cannot account for all of the observed effects of their respective extracellular signals (Lyulcheva, 2008).

The MIG-10/Rap1-GTP-interacting adaptor molecule (RIAM)/lamellipodin (Lpd) (MRL) proteins are a family of recently identified molecular adaptors, harboring an RA, pleckstrin homology (PH), and several proline-rich domains (Krause, 2004; Lafuente, 2004). Several lines of evidence indicate that MRL proteins act downstream of Ras-like GTPases and transduce extracellular signals to changes in the actin cytoskeleton, cell motility, and adhesion. In particular, Lpd interacts with active Ras and RIAM with active Rap1. Consistent with this, only RIAM is required for Rap1-induced cell adhesion (Lafuente, 2004; Rodriguez-Viciana, 2004). Lpd also binds to PI(3,4)P2 via its PH domain, which is sufficient for membrane targeting after platelet-derived growth factor stimulation (Krause, 2004). Both Lpd and RIAM utilize their proline-rich motifs to directly interact with the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) actin regulators, known to regulate lamellipodia formation and cell migration (Jenzora, 2005; Krause, 2004; Lafuente, 2004). In addition, Lpd knockdown impairs lamellipodia formation, whereas Lpd overexpression increases speed of lamellipodia protrusion in an Ena/VASP-dependent manner (Krause, 2004). Finally, both Lpd and RIAM have been shown to alter the cellular ratio between monomeric (G) and filamentous (F) actin (Krause, 2004; Lafuente, 2004), suggesting a wider role in regulating cell metabolism. Indeed, control of the G:F actin ratio is an essential way for cells to regulate gene transcription via the transcription factor serum response factor (SRF), and has been linked to changes in proliferation, migration, and differentiation (Lyulcheva, 2008).

This study reports the characterization of the Drosophila MRL ortholog, which has been named pico on the basis of the retarded growth phenotype resulting from pico knockdown or loss-of-function mutant. Reduction in pico levels results in reduced rates of cell growth and proliferation, whereas ectopic expression of pico promotes coordinated cell growth and proliferation, leading to tissue overgrowth. pico's effect on cell proliferation is conserved in its mammalian ortholog, Lpd. Evidence is presented that pico and Lpd link extracellular signaling to tissue growth via changes in actin dynamics and SRF activation. This is the first time that MRL proteins have been implicated in controlling cell proliferation and tissue growth (Lyulcheva, 2008).

Phylogenetic analysis has shown that pico (CG11940) encodes the only member of the MRL family of proteins in Drosophila. Two transcripts that were identified are generated from alternative transcription start sites of the pico transcription unit: pico and pico-L. pico-L encodes a 1159 amino acid protein that is identical to the protein encoded by pico, except for the presence of an additional 128 N-terminal residues. Both pico proteins contain RA and PH domains and proline-rich Ena/VASP binding sites characteristic of the MRL proteins (Lyulcheva, 2008).

This study shows that pico, which encodes the only Drosophila member of the MRL family of proteins, and its mammalian ortholog, Lpd, have a conserved role in the regulation of cellular proliferation. Reduced pico or Lpd levels result in reduced rates of cellular proliferation, but do not impair cell survival. Too much pico promotes coordinated growth and proliferation, leading to larger tissues with more normal-sized cells. In this respect, the effect of pico is distinct from that of many known Drosophila growth drivers. Growth regulators, such as Drosophila S6K, cause cells to accumulate mass faster than they can divide, primarily due to effects on translation, leading to cellular hypertrophy. Other regulators, such as E2F, can drive cell division without stimulating cell growth, leading to hyperplastic cellular hypotrophy and/or apoptosis (Lyulcheva, 2008).

Attenuating EGFR signaling abrogates the effect of ectopic pico on both F-actin accumulation and tissue growth. pico acts cell autonomously and is therefore unlikely to act upstream of Egfr by affecting the level of EGFR ligands. To rule out that pico regulates levels of EGFR, receptor levels and distribution was examined in wing imaginal discs overexpressing pico or pico^IR. EGFR levels and distribution in these genetic backgrounds resembled wild-type. Another possibility is that pico regulates EGFR activity. Although suitable reagents were not available to directly monitor EGFR activity levels in wing discs, effects on extracellular signal-regulated kinase (ERK) activation, which provides a molecular readout for EGFR/Ras/Raf signaling, were measured. Diphosphorylated (dp) ERK levels were not affected by ectopic pico. These data suggest that, rather than being upstream of EGFR, Pico needs to be activated by EGFR or a downstream component of EGFR signaling, such as activated Ras. Consistently, both Lpd and Pico bind to activated, but not wild-type, Ras. Furthermore, pico knockdown partially suppresses the effects of ectopic Egfr and activated Ras; in addition, Lpd knockdown impairs the EGF-induced increase in proliferation. Taken together, these data suggest that pico and Lpd are downstream effectors of EGFR (Lyulcheva, 2008).

Ena/VASP has been reported to act downstream of MRL proteins. Correspondingly, it was found that pico-mediated wing overgrowth and F-actin accumulation are sensitive to the levels of ena. Importantly, ena is also sufficient to cause overgrowth and F-actin accumulation when overexpressed. Changes in actin dynamics induced by Ena/VASP proteins can activate SRF-dependent gene expression in mammalian cells. Similarly, it was found that Pico and Lpd can activate SRF activity. Like pico, ectopic mal or bs/SRF in flies are sufficient to cause wing overgrowth. Pico-mediated overgrowth is sensitive to the levels of bs/SRF, but mal-induced overgrowth could not be suppressed by pico knockdown, suggesting that Mal/SRF may act downstream of pico in flies. Collectively, these data suggest that MRL proteins may exert their mitogenic effects by specifically interacting with Ena/VASP proteins and inducing SRF-responsive transcription. Interactions between EGFR, MRL proteins, Ena/VASP, and Mal may provide a mechanism linking growth factor signaling and Mal-mediated SRF activation (Lyulcheva, 2008).

Are MRL proteins uniquely able to stimulate Mal/SRF-mediated tissue growth? Although other actin regulators are known to activate Mal/SRF (Posern, 2006), there is currently little data to indicate that they play a role in proliferation control. This might be explained if different transcriptional responses occur at different Mal-dependent SRF activation thresholds, leading to diverse cellular outcomes. Alternatively, other actin regulators might influence processes that limit net tissue growth. For instance, Rho activates Mal/SRF in mammalian cells, but increased Rho activity in flies is associated with loss of epithelial integrity and cell extrusion, which may negate any potential mal-mediated growth-promoting effects. These issues warrant further study in both flies and mammals. Future studies are also needed to characterize transcriptional targets of Drosophila SRF and resolve the contribution of SRF targets to MRL-mediated growth and proliferation (Lyulcheva, 2008).

Lpd expression appears to be differentially regulated in cancer compared to normal tissues (Dahl, 2005; Eppert, 2005; Ginestier, 2006). The data, showing a conserved role for MRL proteins in proliferation control, may provide a potential mechanistic explanation for these observations. In this regard, it is interesting that loss of pico or Lpd can abrogate the effects of EGFR/Erb signaling, deregulation of which has also been implicated in cancer progression. Collectively, these data suggest that MRL proteins might play a role in the pathogenesis of certain cancers and may therefore represent novel molecular targets for therapeutic intervention (Lyulcheva, 2008).